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Sample records for highly immunogenic subunit

  1. Production of a highly immunogenic subunit ISCOM vaccine against Bovine Viral Diarrhea Virus

    DEFF Research Database (Denmark)

    Kamstrup, Søren; Roensholt, L.; Jensen, M.Holm

    1999-01-01

    by Vaccination of the dam. We describe in this report the production and initial testing of an inactivated subunit vaccine against BVDV. The vaccine is based on production of antigen in primary bovine cell cultures, extraction of antigens from infected cells with detergent, chromatographic purification......, concentration, and insertion of antigens into immune stimulating complexes (ISCOMs). Vaccines based on two different Danish strains of BVDV were injected into calves and the antisera produced were tested for neutralising activity against a panel of Danish BVDV strains. The two vaccines induced different...... neutralisation responses, which seem to partly complement each other. The implication of these observations for successful Vaccination against BVDV is discussed....

  2. Safety and immunogenicity of an MF59™-adjuvanted subunit influenza vaccine in elderly Chinese subjects

    Directory of Open Access Journals (Sweden)

    Pellegrini Michele

    2008-02-01

    Full Text Available Abstract Background The safety and immunogenicity of an MF59™-adjuvanted subunit influenza vaccine (Sub/MF59™; FLUAD®, Novartis Vaccines was evaluated among elderly Chinese subjects (≥ 60 years of age. After a preliminary Phase I, open-label study (n = 25 to assess safety 1–14 days post-vaccination, a comparative observer-blind, randomised, controlled clinical trial (n = 600 was performed to assess safety and immunogenicity versus a non-adjuvanted subunit influenza vaccine (Subunit; Agrippal®, Novartis Vaccines. Subjects were randomised (2:1 to receive Sub/MF59™ or Subunit. Results Both vaccines were well tolerated, with no vaccine-related serious adverse events reported during the Phase I trial. During the observer-blind study, local and systemic reactions were generally similar for both vaccines 1–22 days post-vaccination; however, injection-site induration was more frequent among the Subunit group (P Conclusion MF59™-adjuvanted subunit influenza vaccine is well tolerated by elderly Chinese subjects and induces a higher level of immunogenicity than a non-adjuvanted subunit influenza vaccine in this population that is at high risk of influenza-related complications. Clinical trial registry http://www.clinicaltrials.gov, NCT00310648

  3. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates.

    Science.gov (United States)

    Govindarajan, Dhanasekaran; Meschino, Steven; Guan, Liming; Clements, David E; ter Meulen, Jan H; Casimiro, Danilo R; Coller, Beth-Ann G; Bett, Andrew J

    2015-08-07

    We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 μg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 μg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 μg groups and all but one in the DEN4-80EZip 100 μg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 μg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 μg) and high (50, 50, 50, 100 μg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose. Copyright © 2015 Elsevier Ltd. All rights

  4. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

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    Xu, Juan [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Microbiology and Immunology, Nanjing Medical University (China); Wang, Shixia [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States); Gan, Weihua [Department of Pediatrics, The Second Affiliated Hospital, Nanjing Medical University (China); Zhang, Wenhong [Department of Infectious Diseases, Huashan Hospital, Fudan University (China); Ju, Liwen [School of Public Health, Fudan University (China); Huang, Zuhu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Lu, Shan, E-mail: shan.lu@umassmed.edu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

  5. Safety and Immunogenicity of an Inactivated Whole Cell Plus Recombinant B Subunit (WC/RBS) Cholera Vaccine in Healthy Adult Peruvian Military Volunteers.

    Science.gov (United States)

    1992-11-30

    AD-A260 586 IFB0 919931 MIPR NO: 92MM2532W TITLE: SAFETY AND IMMUNOGENICITY OF AN INACTIVATED WHOLE CELL PLUS RECOMBINANT B SUBUNIT (WCIRBS) COLERA ...NUMBERS Safety and Immunogenicity of an Inactivated Whole MIPR No. Cell Plus Recombinant B Subunit (WC/RBS) Colera 92MM2532 Vaccine in Healthy Adult

  6. Expression and immunogenic characterization of recombinant gp350 for developing a subunit vaccine against Epstein-Barr virus.

    Science.gov (United States)

    Wang, Man; Jiang, Shuai; Han, Zhenwei; Zhao, Bing; Wang, Li'ao; Zhou, Zhixia; Wang, Yefu

    2016-02-01

    Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is linked to the development of various malignancies. There is an urgent need for effective vaccines against EBV. EBV envelope glycoprotein gp350 is an attractive candidate for a prophylactic vaccine. This study was undertaken to produce the truncated (codons 1-443) gp350 protein (gp350(1-443)) in Pichia pastoris and evaluate its immunogenicity. The gp350(1-443) protein was expressed as a secretory protein with an N-terminal His-tag in P. pastoris and purified through Ni-NTA chromatography. Immunization with the recombinant gp350(1-443) could elicit high levels of gp350(1-443)-specific antibodies in mice. Moreover, gp350(1-443)-immunized mice developed strong lymphoproliferative and Th1/Th2 cytokine responses. Furthermore, the recombinant gp350(1-443) could stimulate CD4(+) and CD8(+) T cell responses in vaccinated mice. Collectively, these findings demonstrated that the yeast-expressed gp350(1-443) retained strong immunogenicity. This study will provide a useful source for developing EBV subunit vaccine candidates.

  7. Modulation of the immunogenicity of virus-like particles composed of mutant hepatitis B virus envelope subunits.

    Science.gov (United States)

    Cheong, Wan-Shoo; Hyakumura, Michiko; Yuen, Lilly; Warner, Nadia; Locarnini, Stephen; Netter, Hans J

    2012-02-01

    Virus-like particles (VLPs) are non-infectious subviral protein complexes, which possess structural features identical or closely related to infectious virions. They are utilized as delivery tools for immunologically relevant antigenic sequences. In order to investigate whether mutant subunits can modulate the VLP immunogenicity, comparative immunization studies with wild-type and non-native VLPs were performed. To determine whether disulfide bonding impacts on the immunogenicity of hepatitis B virus envelope proteins (HBsAg), mutant HBsAg subunits with single, double and triple cysteine residue substitutions were generated. The mutant proteins were expressed in cell culture, secretion competent non-native VLPs generated, followed by immunization studies in mice to measure the cellular immune response. The reduced ability of mutant HBsAg proteins to form disulfide bonds does not interfere with their ability to assemble into secretion competent VLPs. Depending on specific cysteine to alanine changes, VLPs could be generated with or without an increased ratio of monomeric versus dimeric/oligomeric subunits compared to wild-type VLPs. The utilization of non-native VLPs resulted in enhanced cellular immune responses and does not seem to depend on the ratio between monomeric or dimeric/oligomeric subunits. Comparative immunization studies strongly indicate that changes in the disulfide bonding modulate the VLP immunogenicity most likely due to structural changes. We hypothesize that structural features have evolved with reduced immunogenicity to evade the constraints imposed by the immune system. Altering VLP conformation may represent an attractive strategy to modulate antigen processing resulting in an enhanced immune response and/or a changed hierarchy of epitope presentation. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Immunogenicity and protection efficacy of subunit-based smallpox vaccines using variola major antigens.

    Science.gov (United States)

    Sakhatskyy, Pavlo; Wang, Shixia; Zhang, Chuanyou; Chou, Te-Hui; Kishko, Michael; Lu, Shan

    2008-02-05

    The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on VARV antigen sequences to induce immunity against poxvirus infection.

  9. Fc-based delivery system enhances immunogenicity of a tuberculosis subunit vaccine candidate consisting of the ESAT-6:CFP-10 complex.

    Science.gov (United States)

    Farsiani, Hadi; Mosavat, Arman; Soleimanpour, Saman; Sadeghian, Hamid; Akbari Eydgahi, Mohammad Reza; Ghazvini, Kiarash; Sankian, Mojtaba; Aryan, Ehsan; Jamehdar, Saeid Amel; Rezaee, Seyed Abdolrahim

    2016-06-21

    Tuberculosis (TB) remains a major global health threat despite chemotherapy and Bacilli Calmette-Guérin (BCG) vaccination. Therefore, a safer and more effective vaccine against TB is urgently needed. This study evaluated the immunogenicity of a recombinant fusion protein consisting of early secreted antigenic target protein 6 kDa (ESAT-6), culture filtrate protein 10 kDa (CFP-10) and the Fc-domain of mouse IgG2a as a novel subunit vaccine. The recombinant expression vectors (pPICZαA-ESAT-6:CFP-10:Fcγ2a and pPICZαA-ESAT-6:CFP-10:His) were transferred into Pichia pastoris. After SDS-PAGE and immunoblotting, the immunogenicity of the recombinant proteins was evaluated in mice. When both recombinant proteins (ESAT-6:CFP-10:Fcγ2a and ESAT-6:CFP-10:His) were used for vaccination, Th1-type cellular responses were induced producing high levels of IFN-γ and IL-12. However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a small increase in IL-4 as compared to the BCG and ESAT-6:CFP-10:His groups. Moreover, mice primed with BCG and then supplemented with ESAT-6:CFP-10:Fcγ2a produced the highest levels of IFN-γ and IL-12 in immunized groups. The findings indicate that when Fcγ2a is fused to the ESAT-6:CFP-10 complex, as a delivery vehicle, there could be an increase in the immunogenicity of this type of subunit vaccine. Therefore, additional investigations are necessary for the development of appropriate Fc-based tuberculosis vaccines.

  10. Safety and immunogenicity of two subunit influenza vaccines in healthy children, adults and the elderly: a randomized controlled trial in China.

    Science.gov (United States)

    Zhu, Feng Cai; Zhou, Weizhong; Pan, Hongxing; Lu, Lily; Gerez, Lisya; Nauta, Jos; Giezeman, Katinka; de Bruijn, Iris

    2008-08-18

    The burden of influenza is well known in the elderly and at-risk patients, but also in children. Especially in those under 5 years old, influenza may cause severe morbidity and mortality. Influenza infections and complications can be reduced by vaccination. In a randomized, endpoint-blinded, parallel group trial the immunogenicity and safety was studied of two trivalent inactivated surface antigen (subunit) influenza vaccines Influvac and Agrippal in healthy children as well as in adults and the elderly. An open safety part in 30 children aged 3-12 years and 30 adults aged 18-60 years vaccinated with Influvac was followed by an endpoint-blind, parallel group part in 300 healthy children aged 3-12 years, 300 healthy adults aged 18-59 years, and 240 healthy elderly persons aged 60 years or over, in which subjects were randomized 2:1 to vaccination with either Influvac or Agrippal. The primary immunogenicity endpoint was the geometric mean titer (GMT) 4 weeks after vaccination. Both Influvac and Agrippal induced high anti-hemagglutinin antibody titers in the children and in the adult and elderly subjects. Seroprotection rates were >85% and seroconversion rates >70% for both vaccines in all three age groups for all three-virus strains. The GMT ratios after vaccination indicated that the immunogenicity of Influvac was at least comparable with that of Agrippal in all three age groups. Both vaccines were well tolerated and safe. In this trial, Influvac and Agrippal were immunogenic, safe and well tolerated in healthy children as well as in adults and elderly people.

  11. Immunogenicity of Mycobacterium avium subsp. paratuberculosis specific peptides for inclusion in a subunit vaccine against paratuberculosis

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Tollefsen, S.; Olsen, I.

    Paratuberculosis in ruminants is caused by an infection with Mycobacterium avium subspecies paratuberculosis (MAP) and is a chronic disease characterized by granulomatous enteritis. Available vaccines against paratuberculosis consist of variations of whole bacteria with adjuvant showing various...... efficacies. The main problem with available vaccines is their interference with surveillance and diagnosis of bovine tuberculosis and paratuberculosis. Our ultimate aim is to develop a subunit vaccine consisting of selected MAP peptides, which allow differentiation of infected from vaccinated animals. Here...... full blood IFN-γ release assay and ELISPOT measuring IFN-γ release of PBMCs. A number of peptides resulted in high T cell proliferative responses in T-cell lines and some peptides induced IFN-γ production measured by ELISPOT. This indicates that some of the peptides in the panel contain T cell epitopes...

  12. Improved immunogenicity of novel baculovirus-derived Theileria parva p67 subunit antigens

    NARCIS (Netherlands)

    Kaba, S.A.; Schaap, D.; Roode, E.C.; Nene, V.; Musoke, A.J.; Vlak, J.M.; Oers, van M.M.

    2004-01-01

    East Coast fever (ECF) in cattle is caused by the tick-borne protozoan parasite Theileria parva. The major sporozoite surface antigen of T parva (p67) is an important candidate for inclusion in a subunit vaccine. Recently, we reported the expression and production of different parts of p67 as

  13. Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice.

    Science.gov (United States)

    Alvarez, M Lucrecia; Pinyerd, Heidi L; Crisantes, Jason D; Rigano, M Manuela; Pinkhasov, Julia; Walmsley, Amanda M; Mason, Hugh S; Cardineau, Guy A

    2006-03-24

    Yersinia pestis, the causative agent of plague, is an extremely virulent bacterium but there are no approved vaccines for protection against it. Our goal was to produce a vaccine that would address: ease of delivery, mucosal efficacy, safety, rapid scalability, and cost. We developed a novel production and delivery system for a plague vaccine of a Y. pestis F1-V antigen fusion protein expressed in tomato. Immunogenicity of the F1-V transgenic tomatoes was confirmed in mice that were primed subcutaneously with bacterially-produced F1-V and boosted orally with transgenic tomato fruit. Expression of the plague antigens in fruit allowed producing an oral vaccine candidate without protein purification and with minimal processing technology.

  14. Safety and immunogenicity of a parenterally administered rotavirus VP8 subunit vaccine in healthy adults.

    Science.gov (United States)

    Fix, Alan D; Harro, Clayton; McNeal, Monica; Dally, Len; Flores, Jorge; Robertson, George; Boslego, John W; Cryz, Stanley

    2015-07-17

    The P2-VP8 subunit vaccine for the prevention of rotavirus gastroenteritis is comprised of a truncated VP8 subunit protein from the rotavirus Wa strain (G1[P8]) fused to the tetanus toxin P2 epitope, and adsorbed on aluminum hydroxide for intramuscular administration. Three groups of 16 adults were randomized to receive three injections of P2-VP8 (12) or placebo (4) at doses of 10, 30 or 60 μg of vaccine. IgG and IgA antibodies to P2-VP8 were assessed by ELISA in serum and lymphocyte supernatant (ALS). Serum samples were tested for neutralizing antibodies to homologous and heterologous strains of rotavirus. The vaccine was well-tolerated. All vaccine recipients demonstrated significant IgA responses and all but one demonstrated IgG responses; in the 60 μg cohort, geometric mean titers (GMTs) rose 70- and 80-fold for IgA and IgG, respectively. Homologous neutralizing antibody responses were observed in about half of participants in all three dose cohorts; in the 60 μg cohort, GMTs against Wa rose from 128 to 992. Neutralizing antibody responses were robust to P[8] strains, moderate to P[4] strains and negligible to P[6] strains. ALS IgA responses were dose dependent. The P2-VP8 subunit vaccine was well tolerated and evoked promising immune responses. NCT01764256. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Lactogenic immunity to transmissible gastroenteritis virus induced by a subunit immunogen.

    Science.gov (United States)

    Gough, P M; Frank, C J; Moore, D G; Sagona, M A; Johnson, C J

    1983-12-01

    A subunit prepared from transmissible gastroenteritis (TGE) virus and used to immunize 24 gilts prior to farrowing induced production of specific antibody in the serum and milk. Challenge of pigs, two to seven days of age and suckling on the vaccinated gilts, with the Illinois strain of TGE virus resulted in morbidity of 28% and mortality of 4% as compared with 100 and 73%, respectively, for control piglets. Piglets nursing on a sow which had been immunized approximately 10 months previously were not protected, indicating that lactogenic immunity may be of short duration. Revaccination of this animal resulted in an anamnestic response.

  16. Cloning, Expression, and Immunogenicity of Fimbrial-F17A Subunit Vaccine against Escherichia coli Isolated from Bovine Mastitis

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-01-01

    Full Text Available There is a need to identify and select new promising immunodominant antigens that have the ability to provide protective immunity against E. coli causing bovine mastitis. Recently we showed that f17a was found to be the most prevalent and crucial virulent factor among the pathogenic E. coli isolated from bovine mastitis. Here, in this report, the recombinant F17A based subunit vaccine adjuvant with MF59 was tested for immunogenicity against E. coli in a murine model. The vaccinated mice did not show any abnormal behavioral changes and histopathological lesions after vaccination. The specific antibody level against F17A was significantly higher in MF59-adjuvant-group, and also lasted for longer duration with a significant (P<0.01 production level of IgG1 and IgG2a. Moreover, we noted higher survival rate in mice injected with F17A-MF59-adjuvant group after challenging with the clinical E. coli strain. Our findings of bacterial clearance test revealed that elimination rate from liver, spleen, and kidney in MF59-adjuvant-group was significantly higher than the control group. Finally, the proportion of CD4+T cells was increased, while CD8+ was decreased in MF59-adjuvant group. In conclusion, the current study reveals the capability of F17A-MF59 as a potential vaccine candidate against pathogenic E. coli causing mastitis in dairy animals.

  17. [Immunogenicity of inactivated subunit adsorbed monovalent vaccine against influenza A/California/7/2009 (H1N1) strain].

    Science.gov (United States)

    Zverev, V V; Kostinov, M P; Mikhailova, N A; Zhirova, S N; Mironov, A N; Terkacheva, O A; Romanova, A A; Cherdantsev, A P

    2011-01-01

    The immunogenicity of Pandeflu subunit vaccine against influenza A/California/7/2009 (H1N1) was evaluated in 70 healthy volunteers aged 18 to 60 years. The vaccine was intramuscularly injected twice at an interval of 28 days. Each dose (0.5 ml) contains A(HIN1) influenza virus hemagglutinin (15 +/- 2.2 microg), aluminum hydroxide (Denmark) (0.475 +/- 0.075 microg), and the preservative thiomerosal (merthiolate) (50 +/- 7.5 microg). The level of antibodies was determined in the microneutralization assay. After administration of two doses of the vaccine at a 28-day interval, the geometric mean antibody titer (GMAT) reached 1:21.1 with a further increase to 1:30 (the baseline GMAT) was 1:6.1). The frequencies of seroconversion and seroprotection were 71.4 and 59.2%, respectively; the antibody increase factor was 4.92, which meets the CPMP criteria. The administration of the vaccine did not result in adverse reactions in the postvaccination period.

  18. Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines.

    Science.gov (United States)

    Wen, Xiaobo; Wen, Ke; Cao, Dianjun; Li, Guohua; Jones, Ronald W; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka; Yuan, Lijuan

    2014-07-31

    Currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4(+) T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 μg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 μg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4(+) T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or

  19. Evaluation of the Immunogenicity of an Experimental Subunit Vaccine That Allows Differentiation between Infected and Vaccinated Animals against Bluetongue Virus Serotype 8 in Cattle

    Science.gov (United States)

    Hägglund, Sara; Bréard, Emmanuel; Comtet, Loic; Lövgren Bengtsson, Karin; Pringle, John; Zientara, Stéphan

    2013-01-01

    Bluetongue virus (BTV), the causative agent of bluetongue in ruminants, is an emerging virus in northern Europe. The 2006 outbreak of BTV serotype 8 (BTV-8) in Europe was marked by an unusual teratogenic effect and a high frequency of clinical signs in cattle. Conventional control strategies targeting small ruminants were therefore extended to include cattle. Since cattle were not routinely vaccinated before 2006, the immune responses to BTV have not been studied extensively in this species. With the aims of developing a subunit vaccine against BTV-8 for differentiation between infected and vaccinated animals based on viral protein 7 (VP7) antibody detection and of improving the current understanding of the immunogenicity of BTV proteins in cattle, the immune responses induced by recombinant VP2 (BTV-8) and nonstructural protein 1 (NS1) and NS2 (BTV-2) were studied. Cows were immunized twice (with a 3-week interval) with the experimental vaccine, a commercial inactivated vaccine, or a placebo. The two vaccines induced similar neutralizing antibody responses to BTV-8. Furthermore, the antibody responses detected against VP2, NS1, and NS2 were strongest in the animals immunized with the experimental vaccine, and for the first time, a serotype cross-reactive antibody response to NS2 was shown in cattle vaccinated with the commercial vaccine. The two vaccines evoked measurable T cell responses against NS1, thereby supporting a bovine cross-reactive T cell response. Finally, VP7 seroconversion was observed after vaccination with the commercial vaccine, as in natural infections, but not after vaccination with the experimental vaccine, indicating that the experimental vaccine may allow the differentiation of vaccinated animals from infected animals regardless of BTV serotype. The experimental vaccine will be further evaluated during a virulent challenge in a high-containment facility. PMID:23720365

  20. Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice.

    Science.gov (United States)

    Du, Shouwen; Wang, Yuhang; Liu, Cunxia; Wang, Maopeng; Zhu, Yilong; Tan, Peng; Ren, Dayong; Li, Xiao; Tian, Mingyao; Yin, Ronglan; Li, Chang; Jin, Ningyi

    2015-07-01

    No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT(-CCMp24) was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT(-CCMp24). And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs

    Science.gov (United States)

    Liao, Chih-Ming; Huang, Chienjin; Hsuan, Shih-Ling; Chen, Zeng-Weng; Lee, Wei-Cheng; Liu, Cheng-I; Winton, James R.; Chien, Maw-Sheng

    2006-01-01

    Three short fragments of recombinant subunit Pasteurella multocida toxin (rsPMT) were constructed for evaluation as candidate vaccines against progressive atrophic rhinitis (PAR) of swine. PMT-specific antibody secreting cells and evidence of cellular immunity were detected in rsPMT-immunized pigs following authentic PMT challenge or homologous antigen booster. Piglets immunized with rsPMT fragments containing either the N-terminal or the C-terminal portions of PMT developed high titers of neutralizing antibodies. Pregnant sows immunized with rsPMT had higher levels of maternal antibodies in their colostrum than did those immunized with a conventional PAR-toxoid vaccine. Offspring from rsPMT vaccinated sows had better survival after challenge with a five-fold lethal dose of authentic PMT and had better growth performance after challenge with a sublethal dose of toxin. Our findings indicate these non-toxic rsPMT proteins are attractive candidates for development of a subunit vaccine against PAR in pigs.

  2. Multi-subunit BCG booster vaccine GamTBvac: Assessment of immunogenicity and protective efficacy in murine and guinea pig TB models.

    Science.gov (United States)

    Tkachuk, A P; Gushchin, V A; Potapov, V D; Demidenko, A V; Lunin, V G; Gintsburg, A L

    2017-01-01

    New innovative vaccines are highly needed to combat the global threat posed by tuberculosis. Efficient components-antigens and adjuvants-are crucial for development of modern recombinant TB vaccines. This study describes a new vaccine (GamTBvac) consisting of two mycobacterial antigen fusions (Ag85A and ESAT6-CFP10)-with dextran-binding domain immobilized on dextran and mixed with an adjuvant consisting of DEAE-dextran core, and with CpG oligodeoxynucleotides (TLR9 agonists). GamTBvac and its components were assessed for immunogenicity and protective efficacy in GamTBvac-prime/boost and BCG-prime/ GamTBvac-boost in murine and guinea pig TB models. Results show that in both infectious models, GamTBvac has a strong immunogenicity and significant protective effect against Mycobacterium tuberculosis strain H37Rv under aerosol and intravenous challenges. GamTBvac showed a particularly strong protective effect as a BCG booster vaccine.

  3. Immunogenicity and Safety of the HZ/su Adjuvanted Herpes Zoster Subunit Vaccine in Adults Previously Vaccinated With a Live Attenuated Herpes Zoster Vaccine.

    Science.gov (United States)

    Grupping, Katrijn; Campora, Laura; Douha, Martine; Heineman, Thomas C; Klein, Nicola P; Lal, Himal; Peterson, James; Vastiau, Ilse; Oostvogels, Lidia

    2017-12-12

    Protection against herpes zoster (HZ) induced by the live attenuated zoster vaccine Zostavax (ZVL) wanes within 3-7 years. Revaccination may renew protection. We assessed whether (re)vaccination with the adjuvanted HZ subunit vaccine candidate (HZ/su) induced comparable immune responses in previous ZVL recipients and ZVL-naive individuals (HZ-NonVac). In an open-label, multicenter study, adults ≥65 years of age, vaccinated with ZVL ≥5 years previously (HZ-PreVac), were matched to ZVL-naive adults (HZ-NonVac). Participants received 2 doses of HZ/su 2 months apart. The primary objective of noninferiority of the humoral immune response 1 month post-dose 2 was considered demonstrated if the upper limit of the 95% confidence interval (CI) of the adjusted anti-glycoprotein E geometric mean concentration (GMC) ratio of HZ-NonVac over HZ-PreVac was <1.5. HZ/su cellular immunogenicity, reactogenicity, and safety were also assessed. In 430 participants, humoral immune response to HZ/su was noninferior in HZ-PreVac compared with HZ-NonVac (adjusted GMC ratio, 1.04 [95% CI, .92-1.17]). Cellular immunogenicity, reactogenicity, and safety appeared to be comparable between groups. HZ/su was well-tolerated, with no safety concerns raised within 1 month post-dose 2. HZ/su induces a strong immune response irrespective of prior vaccination with ZVL, and may be an attractive option to revaccinate prior ZVL recipients. NCT02581410.

  4. Inferior immunogenicity and efficacy of respiratory syncytial virus fusion protein-based subunit vaccine candidates in aged versus young mice.

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    Corinne Cayatte

    Full Text Available Respiratory syncytial virus (RSV is recognized as an important cause of lower and upper respiratory tract infections in older adults, and a successful vaccine would substantially lower morbidity and mortality in this age group. Recently, two vaccine candidates based on soluble purified glycoprotein F (RSV F, either alone or adjuvanted with glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE, failed to reach their primary endpoints in clinical efficacy studies, despite demonstrating the desired immunogenicity profile and efficacy in young rodent models. Here, one of the RSV F vaccine candidates (post-fusion conformation, RSV post-F, and a stabilized pre-fusion form of RSV F (RSV pre-F, DS-Cav1 were evaluated in aged BALB/c mice. Humoral and cellular immunogenicity elicited after immunization of naïve, aged mice was generally lower compared to young animals. In aged mice, RSV post-F vaccination without adjuvant poorly protected the respiratory tract from virus replication, and addition of GLA-SE only improved protection in the lungs, but not in nasal turbinates. RSV pre-F induced higher neutralizing antibody titers compared to RSV post-F (as previously reported but interestingly, RSV F-specific CD8 T cell responses were lower compared to RSV post-F responses regardless of age. The vaccines were also tested in RSV seropositive aged mice, in which both antigen forms similarly boosted neutralizing antibody titers, although GLA-SE addition boosted neutralizing activity only in RSV pre-F immunized animals. Cell-mediated immune responses in the aged mice were only slightly boosted and well below levels induced in seronegative young mice. Taken together, the findings suggest that the vaccine candidates were not able to induce a strong anti-RSV immune response in recipient mice with an aged immune system, in agreement with recent human clinical trial results. Therefore, the aged mouse model could be a useful tool to evaluate improved vaccine

  5. Immunogenicity and Safety of an Adjuvanted Herpes Zoster Subunit Vaccine Coadministered With Seasonal Influenza Vaccine in Adults Aged 50 Years or Older.

    Science.gov (United States)

    Schwarz, Tino F; Aggarwal, Naresh; Moeckesch, Beate; Schenkenberger, Isabelle; Claeys, Carine; Douha, Martine; Godeaux, Olivier; Grupping, Katrijn; Heineman, Thomas C; Fauqued, Marta Lopez; Oostvogels, Lidia; Van den Steen, Peter; Lal, Himal

    2017-12-12

    The immunogenicity and safety of an adjuvanted herpes zoster subunit (HZ/su) vaccine when coadministered with a quadrivalent seasonal inactivated influenza vaccine (IIV4) was investigated in a phase 3, open-label, randomized clinical trial in adults aged ≥50 years. Subjects were randomized 1:1 to receive either HZ/su (varicella zoster virus glycoprotein E; AS01B Adjuvant System) and IIV4 at day 0 followed by a second HZ/su dose at month 2 (coadministration group), or IIV4 at month 0 and HZ/su at months 2 and 4 (control group). The primary objectives were the HZ/su vaccine response rate in the coadministration group and the noninferiority of the antibody responses to HZ/su and IIV4 in the coadministration compared with the control group. Safety information was collected throughout the duration of the study. A total of 413 subjects were vaccinated in the coadministration group and 415 in the control group. The HZ/su vaccine response rate in the coadministration group was 95.8% (95% confidence interval, 93.3%-97.6%) and the anti-glycoprotein E GMCControl/Coadmin ratio was 1.08 (.97-1.20). The primary noninferiority objectives were met. No safety concerns were observed. No interference in the immune responses to either vaccine was observed when the vaccines were coadministered, and no safety concerns were identified. NCT01954251.

  6. Safety and immunogenicity of a recombinant Staphylococcus aureus α-toxoid and a recombinant Panton-Valentine leukocidin subunit, in healthy adults.

    Science.gov (United States)

    Landrum, Michael L; Lalani, Tahaniyat; Niknian, Minoo; Maguire, Jason D; Hospenthal, Duane R; Fattom, Ali; Taylor, Kimberly; Fraser, Jamie; Wilkins, Kenneth; Ellis, Michael W; Kessler, Paul D; Fahim, Rafaat E F; Tribble, David R

    2017-04-03

    We conducted a randomized, double-blind, placebo-controlled dose-escalation study in healthy adults to evaluate the safety and immunogenicity of recombinant Staphylococcus aureus candidate vaccine antigens, recombinant α-toxoid (rAT) and a sub-unit of Panton-Valentine leukocidin (rLukS-PV). 176 subjects were enrolled and randomized within 1 of 11 treatment cohorts: monovalent rAT or rLukS-PV dosages of 10, 25, 50, and 100 μg; bivalent rAT:rLukS dosages of 10:10, 25:25, and 50:50 μg; and alum or saline placebo. All subjects were assessed at Days 0, 7, 14, 28, and 84. Subjects in the 50:50 μg bivalent cohort received a second injection on Day 84 and were assessed on Days 98 and 112. Incidence and severity of reactogenicity and adverse events (AEs) were compared. Geometric mean serum concentrations (GMC) and neutralizing activity of anti-rAT and anti-rLukS-PV IgG were assessed. Reactogenicity incidence was significantly higher in vaccine than placebo recipients (77% versus 55%, respectively; p = 0.006). However, 77% of reactogenicity events were mild and 19% were moderate in severity. The AE incidence and severity were similar between the cohorts. All monovalent and bivalent rAT dosages resulted in a significant increase in the anti-rAT IgG and anti- rLukS-PV GMCs between day 0 and 28 compared with placebo, and persisted through Day 84. Exploratory subgroup analyses suggested a higher GMC and neutralizing antibody titers for the 50 μg monovalent or bivalent rAT and rLukS-PV dose as compared to the other doses. No booster effect was observed after administration of the second dose. We conclude that the rAT and rLukS-PV vaccine formulations were well-tolerated and had a favorable immunogenicity profile, producing antibody with neutralizing activity through day 84. There was no benefit observed with a booster dose of the vaccine.

  7. Expression of the multimeric and highly immunogenic Brucella spp. lumazine synthase fused to bovine rotavirus VP8d as a scaffold for antigen production in tobacco chloroplasts

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    Edgardo Federico Alfano

    2015-12-01

    Full Text Available Lumazine synthase from Brucella spp. (BLS is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity.The inner core domain (VP8d of VP8 spike protein from bovine rotavirus (BRV is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination.In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (TSP (4.85 mg/g fresh tissue. BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines.

  8. [Study on the immunogenicity and safety of recombinant B-subunit/whole cell cholera vaccine infused with antacids in healthy population at ages of 2-6 years].

    Science.gov (United States)

    Huang, T; Li, R C; Liu, D P

    2017-09-06

    Objective: To assess the immunogenicity and safety of recombinant B-subunit/whole cell cholera vaccine (rBS/WC) oral cholera vaccine (Ora Vacs) infused with antacids in healthy population at ages of 2-6 years. Methods: Between December 2009 and January 2010, we recruited 900 volunteers aged 2-6 years od through giving out recruitment notice for the eligible children's parents from different vaccination clinics of Chongzuo city in Guangxi Zhuang Autonomous Region. This study was a randomized, double-blind, placebo-controlled trial, and subjects were randomly (2∶1) assigned to receive Cholera vaccine infused with antacids or placebo, and observed for safety. Serum samples of 300 subjects in immunogenicity subgroups (200 for vaccine groups, 100 for control groups) before the 1st dose and 49 d (±3 d) after immunization were collected, and determined for antibody levels against the cholera toxin (anti-CT) and cholera vibriocidal (anti-Vab) with Enzyme-linked immunosorbent assays (ELISA), based on which the GMT was calculated. There were 266 cases paired with the serum samples before and after immunization (177 for vaccine groups, 89 for control groups). The comparison of subjects' age at enrollment and the level of GMT before and after immunization between groups were analyzed by t test. The superiority test for the difference between seroconversion rates of vaccine groups and control groups were analyzed by χ(2) test. Results: Of 900 subjects enrolled, the number of males and females were 503 and 397 respectively (vaccine groups 335 vs. 265, control groups 168 vs. 132), the average ages of vaccine groups and control groups at enrollment were (4.8±1.2) years and (4.9±1.2) years respectively. There were no significant differences between groups in terms of gender and age (χ(2)=0.00, P=1.000; t=0.55, P=0.585). The 2 times increase rates of anti-CT and anti-Vab in vaccine groups after inoculation were 90.96% and 57.63% respectively, which were superiority to those

  9. Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs

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    Yinglin eQi

    2015-03-01

    Full Text Available Rabies remains an important worldwide public health threat, so safe, effective and affordable vaccines are still being sought. Virus-like particle (VLP-based vaccines targeting various viral pathogens have been successfully produced, licensed and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs containing rabies virus (RABV glycoprotein (G, matrix (M protein, and membrane-anchored flagellin (EVLP-F or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNA responses and elicited greater numbers of CD4+ and CD8+ T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin and LTB possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine candidate for animals.

  10. Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

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    Mariana Souza Costa

    2013-02-01

    Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

  11. Immunogenicity and safety of an adjuvanted herpes zoster subunit candidate vaccine in adults ≥ 50 years of age with a prior history of herpes zoster: A phase III, non-randomized, open-label clinical trial.

    Science.gov (United States)

    Godeaux, Olivier; Kovac, Martina; Shu, Daniel; Grupping, Katrijn; Campora, Laura; Douha, Martine; Heineman, Thomas C; Lal, Himal

    2017-05-04

    This phase III, non-randomized, open-label, multi-center study (NCT01827839) evaluated the immunogenicity and safety of an adjuvanted recombinant subunit herpes zoster (HZ) vaccine (HZ/su) in adults aged ≥ 50 y with prior physician-documented history of HZ. Participants (stratified by age: 50-59, 60-69 and ≥ 70 y) received 2 doses of HZ/su 2 months apart and were followed-up for another 12 months. Anti-glycoprotein E (gE) antibodies were measured by enzyme-linked immunosorbent assay before vaccination and 1 month after the second dose (Month 3). Solicited local and general adverse events (AEs) were recorded for 7 d and unsolicited AEs for 30 d after each vaccination. Serious AEs were recorded until study end. The primary immunogenicity objective was met if the lower limit of the 95% confidence interval (CI) of the vaccine response rate (VRR), defined as a 4-fold increase in anti-gE over baseline, at Month 3 was ≥ 60%. 96 participants (32/age group) were enrolled. The primary immunogenicity objective was met, as the VRR at Month 3 was 90.2% (95% CI: 81.7-95.7). Geometric mean anti-gE antibody concentrations at Month 3 were similar across age groups. 77.9% and 71.6% of participants reported local and general solicited AEs, respectively. The most frequent solicited AEs were pain at injection site, fatigue, headache, myalgia and shivering. The HZ/su vaccine was immunogenic in adults aged ≥ 50 y with a physician-documented history of HZ, and no safety concerns were identified.

  12. N-Trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination : Biological properties and immunogenicity in a mouse model

    NARCIS (Netherlands)

    Amidi, Maryam; Romeijn, Stefan G.; Verhoef, J. Coos; Junginger, Hans E.; Bungener, Laura; Huckriede, Anke; Crommelin, Daan J. A.; Jiskoot, Wim

    2007-01-01

    In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine was investigated. The antigen-loaded nanoparticles were prepared by mixing a solution containing TMC and monovalent influenza A subunit H3N2

  13. Immunogenicity and efficacy of intramuscular replication-defective and subunit vaccines against herpes simplex virus type 2 in the mouse genital model.

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    Simon Delagrave

    Full Text Available Herpes simplex virus type 2 (HSV-2 is a sexually transmitted virus that is highly prevalent worldwide, causing a range of symptoms that result in significant healthcare costs and human suffering. ACAM529 is a replication-defective vaccine candidate prepared by growing the previously described dl5-29 on a cell line appropriate for GMP manufacturing. This vaccine, when administered subcutaneously, was previously shown to protect mice from a lethal vaginal HSV-2 challenge and to afford better protection than adjuvanted glycoprotein D (gD in guinea pigs. Here we show that ACAM529 given via the intramuscular route affords significantly greater immunogenicity and protection in comparison with subcutaneous administration in the mouse vaginal HSV-2 challenge model. Further, we describe a side-by-side comparison of intramuscular ACAM529 with a gD vaccine across a range of challenge virus doses. While differences in protection against death are not significant, ACAM529 protects significantly better against mucosal infection, reducing peak challenge virus shedding at the highest challenge dose by over 500-fold versus 5-fold for gD. Over 27% (11/40 of ACAM529-immunized animals were protected from viral shedding while 2.5% (1/40 were protected by the gD vaccine. Similarly, 35% (7/20 of mice vaccinated with ACAM529 were protected from infection of their dorsal root ganglia while none of the gD-vaccinated mice were protected. These results indicate that measuring infection of the vaginal mucosa and of dorsal root ganglia over a range of challenge doses is more sensitive than evaluating survival at a single challenge dose as a means of directly comparing vaccine efficacy in the mouse vaginal challenge model. The data also support further investigation of ACAM529 for prophylaxis in human subjects.

  14. NY-ESO-1 cancer testis antigen demonstrates high immunogenicity in triple negative breast cancer.

    Science.gov (United States)

    Ademuyiwa, Foluso O; Bshara, Wiam; Attwood, Kristopher; Morrison, Carl; Edge, Stephen B; Karpf, Adam R; James, Smith A; Ambrosone, Christine B; O'Connor, Tracey L; Levine, Ellis G; Miliotto, Anthony; Ritter, Erika; Ritter, Gerd; Gnjatic, Sacha; Odunsi, Kunle

    2012-01-01

    NY-ESO-1 cancer testis (CT) antigen is an attractive candidate for immunotherapy as a result of its high immunogenicity. The aim of this study was to explore the potential for NY-ESO-1 antigen directed immunotherapy in triple negative breast cancer (TNBC) by determining the frequency of expression by immunohistochemistry (IHC) and the degree of inherent immunogenicity to NY-ESO-1. 168 TNBC and 47 ER+/HER2- primary breast cancer specimens were used to determine NY-ESO-1 frequency by IHC. As previous studies have shown that patients with a robust innate humoral immune response to CT antigens are more likely to develop CD8 T-cell responses to NY-ESO-1 peptides, we evaluated the degree to which patients with NY-ESO-1 expression had inherent immunogenicity by measuring antibodies. The relationship between NY-ESO-1 expression and CD8+ T lymphocytes was also examined. The frequency of NY-ESO-1 expression in the TNBC cohort was 16% versus 2% in ER+/HER2- patients. A higher NY-ESO-1 score was associated with a younger age at diagnosis in the TNBC patients with NY-ESO-1 expression (p = 0.026). No differences in OS (p = 0.278) or PFS (p = 0.238) by NY-ESO-1 expression status were detected. Antibody responses to NY-ESO-1 were found in 73% of TNBC patients whose tumors were NY-ESO-1 positive. NY-ESO-1 positive patients had higher CD8 counts than negative patients (p = 0.018). NY-ESO-1 is expressed in a substantial subset of TNBC patients and leads to a high humoral immune response in a large proportion of these individuals. Given these observations, patients with TNBC may benefit from targeted therapies directed against NY-ESO-1.

  15. Safety and immunogenicity of a parenteral P2-VP8-P[8] subunit rotavirus vaccine in toddlers and infants in South Africa: a randomised, double-blind, placebo-controlled trial.

    Science.gov (United States)

    Groome, Michelle J; Koen, Anthonet; Fix, Alan; Page, Nicola; Jose, Lisa; Madhi, Shabir A; McNeal, Monica; Dally, Len; Cho, Iksung; Power, Maureen; Flores, Jorge; Cryz, Stanley

    2017-08-01

    Efficacy of live oral rotavirus vaccines is reduced in low-income compared with high-income settings. Parenteral non-replicating rotavirus vaccines might offer benefits over oral vaccines. We assessed the safety and immunogenicity of the P2-VP8-P[8] subunit rotavirus vaccine at different doses in South African toddlers and infants. This double-blind, randomised, placebo-controlled, dose-escalation trial was done at a single research unit based at a hospital in South Africa in healthy HIV-uninfected toddlers (aged 2 to placebo injection. The two highest tolerated doses were then assessed in an expanded cohort (in a 1:1:1 ratio). Parents of participants and clinical, data, and laboratory staff were masked to treatment assignment. P2-VP8-P[8] vaccine versus placebo was assessed first in toddlers (single injection) and then in infants (three injections 4 weeks apart). The primary safety endpoints were local and systemic reactions within 7 days after each injection, adverse events within 28 days after each injection, and all serious adverse events, assessed in toddlers and infants who received at least one dose. In infants receiving all study injections, primary immunogenicity endpoints were anti-P2-VP8-P[8] IgA and IgG and neutralising antibody seroresponses and geometric mean titres 4 weeks after the third injection. This trial is registered at ClinicalTrials.gov, number NCT02109484. Between March 17, 2014, and Sept 29, 2014, 42 toddlers (36 to vaccine and six to placebo) and 48 infants (36 to vaccine and 12 to placebo) were enrolled in the dose-escalation phase, in which the 30 μg and 60 μg doses where found to be the highest tolerated doses. A further 114 infants were enrolled in the expanded cohort between Nov 3, 2014, and March 20, 2015, and all 162 infants (12 assigned to 10 μg, 50 to 30 μg, 50 to 60 μg, and 50 to placebo) were included in the safety analysis. Serum IgA seroresponses were observed in 38 (81%, 95% CI 67-91) of 47 infants in the 30 μg group

  16. The immunogenicity of colorectal cancers with high-degree microsatellite instability

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    Dorudi Sina

    2005-05-01

    Full Text Available Abstract Background High-degree microsatellite instability (MSI-H is a feature of approximately 15% of sporadic colorectal cancers. Patients with MSI-H cancers have been reported to have a better prognosis than those with non-MSI-H cancers. The MSI-H subset is also characterised by a dense infiltrate of intra-epithelial lymphocytes and the hypothesis that the latter represents an efficacious immune response contributing to improved outcome is very attractive. Methods Data for this review were identified by searches of MEDLINE, PubMed, and cross references from relevant articles using the search terms 'microsatellite instability', 'colorectal cancer' and 'immunology', 'immune response' or 'immunogenicity'. Results A total of 38 articles were identified by the search criteria and a further 95 articles by cross-referencing. The relevance of the articles to be interviewed was established by hand searching. Out of a total of 133 articles identified, 47 articles were rejected due to lack of relevance. A total of 86 articles were included in the review, pertaining to microsatellite instability in colorectal cancer, and immune mechanisms in colorectal cancer. Conclusion It is suggested that this distinct group of colorectal cancers may have inherent immunogenic properties and that further elucidation of these may be invaluable to the development of successful immunotherapy.

  17. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

    Science.gov (United States)

    Salvat, Regina S.; Parker, Andrew S.; Kirsch, Jack R.; Brooks, Seth A.

    2017-01-01

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide–MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening. PMID:28607051

  18. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    Science.gov (United States)

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

  19. High resolution solution structure of the 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

    Science.gov (United States)

    Reddy, D V; Shenoy, B C; Carey, P R; Sönnichsen, F D

    2000-03-14

    Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. Within the multi-subunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier and also binds the other two subunits to assist in the overall assembly of the enzyme. The 1.3S subunit is a 123 amino acid polypeptide (12.6 kDa) to which biotin is covalently attached at Lys 89. The three-dimensional solution structure of the full-length holo-1.3S subunit of TC has been solved by multidimensional heteronuclear NMR spectroscopy. The C-terminal half of the protein (51-123) is folded into a compact all-beta-domain comprising of two four-stranded antiparallel beta-sheets connected by short loops and turns. The fold exhibits a high 2-fold internal symmetry and is similar to that of the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase, but lacks an extension that has been termed "protruding thumb" in BCCP. The first 50 residues, which have been shown to be involved in intersubunit interactions in the intact enzyme, appear to be disordered in the isolated 1.3S subunit. The molecular surface of the folded domain has two distinct surfaces: one side is highly charged, while the other comprises mainly hydrophobic, highly conserved residues.

  20. Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins.

    Science.gov (United States)

    Barro, Francisco; Iehisa, Julio C M; Giménez, María J; García-Molina, María D; Ozuna, Carmen V; Comino, Isabel; Sousa, Carolina; Gil-Humanes, Javier

    2016-03-01

    Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α-, γ-, ω-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ- and α-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω-gliadins and γ-gliadins, and three of these also silenced α-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α- and ω-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. High hydrostatic pressure induces immunogenic cell death in human tumor cells.

    Science.gov (United States)

    Fucikova, Jitka; Moserova, Irena; Truxova, Iva; Hermanova, Ivana; Vancurova, Irena; Partlova, Simona; Fialova, Anna; Sojka, Ludek; Cartron, Pierre-Francois; Houska, Milan; Rob, Lukas; Bartunkova, Jirina; Spisek, Radek

    2014-09-01

    Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high-mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP-treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA-DR and the release of interleukin IL-6, IL-12p70 and TNF-α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific T cells. DCs pulsed with HHP-treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress-mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase-8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells. © 2014 UICC.

  2. Expression and immunogenic analysis of recombinant polypeptides derived from capsid protein VP1 for developing subunit vaccine material against hepatitis A virus.

    Science.gov (United States)

    Jang, Kyoung Ok; Park, Jong-Hwa; Lee, Hyun Ho; Chung, Dae Kyun; Kim, Wonyong; Chung, In Sik

    2014-08-01

    Three recombinant polypeptides, VP1-His, VP1-3N-His, and 3D2-His, were produced by Escherichia coli expression system. Recombinant VP1-His, VP1-3N-His, and 3D2-His were expressed as bands with molecular weights of 32, 38, and 30 kDa, respectively. These were purified by affinity chromatography using Ni-NTA Fast-flow resin and/or ion-exchange chromatography using DEAE-Sepharose Fast-flow resin. Intraperitoneal immunizations of recombinant polypeptides successfully elicited the productions of VP1-His, VP1-3N-His, and 3D2-His specific IgG antibodies (IgG subclass distribution of IgG1>IgG2a>IgG2b>IgG3) in sera and induced the secretions of cytokines IFN-γ and IL-6 in spleen cells. Sera from recombinant VP1-His-, VP1-3N-His-, and 3D2-His-immunized mice neutralized the propagation of HAV. The highest neutralizing activity was shown in sera from recombinant VP1-3N-His-immunized mice. These results suggest that recombinant VP1-3N-His can be a useful source for developing hepatitis A virus (HAV) subunit vaccine candidates. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  4. High resolution structure of the large ribosomal subunit from a Mesophilic Eubacterium

    Energy Technology Data Exchange (ETDEWEB)

    Harms, Joerg; Schluenzen, Frank; Zarivach, Raz; Bashan, Anat; Gat, Sharon; Agmon, Ilana; Bartels, Heike; Franceschi, Francois; Yonath, Ada (Weizmann Inst Israel); (Mac Planck Germany); (Max Planck Germany)

    2009-10-07

    We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

  5. Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling.

    OpenAIRE

    Felix Geeraedts; Nadege Goutagny; Veit Hornung; Martina Severa; Aalzen de Haan; Judith Pool; Jan Wilschut; Katherine A Fitzgerald; Anke Huckriede

    2008-01-01

    In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (...

  6. Cytokine-modified VSV is attenuated for neural pathology, but is both highly immunogenic and oncolytic.

    Science.gov (United States)

    Miller, James; Bidula, Sarah M; Jensen, Troels M; Reiss, Carol Shoshkes

    2009-12-01

    Vesicular stomatitis virus (VSV), an enveloped, nonsegmented, negative-stranded RNA virus, is being tested by several laboratories as an antitumor agent. Unfortunately, viral infection of the central nervous system (CNS) has been observed by many groups following administration to tumor-bearing animals. In rodents, VSV encephalitis is characterized by weight-loss, paralysis, and high mortality. In order to provide protection from VSV infection of the CNS after therapeutic administration, we have attenuated VSV by the introduction of the gene encoding the proinflammatory cytokine interleukin (IL)-23, and designated the new virus VSV23. We hypothesize that while VSV23 is replicating within tumors, resulting in tumor destruction, the expression of IL-23 will enhance host antitumor and antiviral immune responses. In the event that the virus escapes from the tumor, the host's immune system will be activated and the virus will be rapidly cleared from healthy tissue. Experimental VSV23 infection of the CNS is characterized by decreased viral replication, morbidity, and mortality. VSV23 is capable of stimulating the enhanced production of nitric oxide in the CNS, which is critical for elimination of VSV from infected neurons. Intraperitoneal administration of VSV23 stimulates both nonspecific natural killer cell, virus-specific cytolytic T lymphocyte and memory virus-specific proliferative T cell responses against wild-type VSV in splenocytes. Furthermore, VSV23 is able to replicate in, and induce apoptosis of tumor cells in vitro. These data indicate that VSV23 is immunogenic, attenuated and suitable for testing as an efficacious and safe oncolytic agent.

  7. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce...... been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly...

  8. PCPP-Adjuvanted Respiratory Syncytial Virus (RSV) sF Subunit Vaccine: Self-Assembled Supramolecular Complexes Enable Enhanced Immunogenicity and Protection.

    Science.gov (United States)

    Cayatte, Corinne; Marin, Alexander; Rajani, Gaurav Manohar; Schneider-Ohrum, Kirsten; Snell Bennett, Angie; Marshall, Jason D; Andrianov, Alexander K

    2017-07-03

    PCPP, a well-defined polyphosphazene macromolecule, has been studied as an immunoadjuvant for a soluble form of the postfusion glycoprotein of respiratory syncytial virus (RSV sF), which is an attractive vaccine candidate for inducing RSV-specific immunity in mice and humans. We demonstrate that RSV sF-PCPP formulations induce high neutralization titers to RSV comparable to alum formulations even at a low PCPP dose and protect animals against viral challenge both in the lung and in the upper respiratory tract. PCPP formulations were also characterized by Th1-biased responses, compared to Th2-biased responses that are more typical for RSV sF alone or RSV sF-alum formulations, suggesting an inherent immunostimulating activity of the polyphosphazene adjuvant. We defined these immunologically active RSV sF-PCPP formulations as self-assembled water-soluble protein-polymer complexes with distinct physicochemical parameters. The secondary structure and antigenicity of the protein in the complex were fully preserved during the spontaneous aqueous self-assembly process. These findings further advance the concept of polyphosphazene immunoadjuvants as unique dual-functionality adjuvants integrating delivery and immunostimulating modalities in one water-soluble molecule.

  9. The novel oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in 2 vaccine presentations.

    Science.gov (United States)

    Kirkpatrick, Beth D; Tenney, Katherine M; Larsson, Catherine J; O'Neill, J Patrick; Ventrone, Cassandra; Bentley, Matthew; Upton, Anthony; Hindle, Zoe; Fidler, Christine; Kutzko, Deborah; Holdridge, Regan; Lapointe, Casey; Hamlet, Sandra; Chatfield, Steven N

    2005-08-01

    M01ZH09 (Salmonella enterica serovar Typhi [Ty2 aroC(-) ssaV(-)] ZH9) is a live oral-dose typhoid vaccine candidate. M01ZH09 was rationally modified with 2 independently attenuating mutations, including a novel mutation in Salmonella pathogenicity island (SPI)-2. We demonstrate that M01ZH09, in a single oral dose, is well tolerated and prompts broad immune responses, regardless of whether prevaccination with a bicarbonate buffer is given. Thirty-two healthy adult subjects were randomized and given 5x109 cfu of M01ZH09, with (presentation 1) or without (presentation 2) prevaccination with a bicarbonate buffer. Immunogenicity data included Salmonella Typhi lipopolysaccharide (LPS)-specific immunoglobulin (Ig) A antibody-secreting cells (enzyme-linked immunospot [ELISPOT] assay), IgG serologic responses to Salmonella Typhi LPS, lymphocyte proliferation, and interferon (IFN)- gamma production. The vaccine was well tolerated; adverse events after vaccination were mild. No fever or prolonged vaccine shedding occurred. Immunogenicity data demonstrated that 88% and 93% of subjects who received presentation 1 and presentation 2, respectively, had a positive response by ELISPOT assay; 81% of subjects in both groups underwent IgG seroconversion on day 14. Both groups had similar cellular immune responses to presentation 1 and presentation 2; lymphocyte proliferation to Salmonella Typhi flagellin occurred in 63% and 67% of subjects, respectively, and 69% and 73% of subjects, respectively, had an increase in IFN- gamma production. The oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in a single oral dose, with and without prevaccination with a bicarbonate buffer. Field studies to demonstrate protective efficacy are planned.

  10. A Large Size Chimeric Highly Immunogenic Peptide Presents Multistage Plasmodium Antigens as a Vaccine Candidate System against Malaria

    Directory of Open Access Journals (Sweden)

    José Manuel Lozano

    2017-11-01

    Full Text Available Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of PfCSP, STARP; MSA1 and Pf155/RESA from pre- and erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei-ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems.

  11. Further progress on defining highly conserved immunogenic epitopes for a global HIV vaccine

    DEFF Research Database (Denmark)

    De Groot, Anne S; Levitz, Lauren; Ardito, Matthew T

    2012-01-01

    of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs......Two major obstacles confronting HIV vaccine design have been the extensive viral diversity of HIV-1 globally and viral evolution driven by escape from CD8(+) cytotoxic T-cell lymphocyte (CTL)-mediated immune pressure. Regions of the viral genome that are not able to escape immune response...... and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context...

  12. Dicarbonyl Induced Structural Perturbations Make Histone H1 Highly Immunogenic and Generate an Auto-Immune Response in Cancer.

    Directory of Open Access Journals (Sweden)

    Abdul Rouf Mir

    Full Text Available Increased oxidative stress under hyperglycemic conditions, through the interaction of AGEs with RAGE receptors and via activation of interleukin mediated transcription signalling, has been reported in cancer. Proteins modifications are being explored for their roles in the development and progression of cancer and autoantibody response against them is gaining interest as a probe for early detection of the disease. This study has analysed the changes in histone H1 upon modification by methylglyoxal (MG and its implications in auto-immunopathogenesis of cancer. Modified histone showed modifications in the aromatic residues, changed tyrosine microenvironment, intermolecular cross linking and generation of AGEs. It showed masking of hydrophobic patches and a hypsochromic shift in the in ANS specific fluorescence. MG aggressively oxidized histone H1 leading to the accumulation of reactive carbonyls. Far UV CD measurements showed di-carbonyl induced enhancement of the alpha structure and the induction of beta sheet conformation; and thermal denaturation (Tm studies confirmed the thermal stability of the modified histone. FTIR analysis showed amide I band shift, generation of a carboxyethyl group and N-Cα vibrations in the modified histone. LCMS analysis confirmed the formation of Nε-(carboxyethyllysine and electron microscopic studies revealed the amorphous aggregate formation. The modified histone showed altered cooperative binding with DNA. Modified H1 induced high titre antibodies in rabbits and the IgG isolated form sera of rabbits immunized with modified H1 exhibited specific binding with its immunogen in Western Blot analysis. IgG isolated from the sera of patients with lung cancer, prostate cancer, breast cancer and cancer of head and neck region showed better recognition for neo-epitopes on the modified histone, reflecting the presence of circulating autoantibodies in cancer. Since reports suggest a link between AGE-RAGE axis and

  13. High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

    Directory of Open Access Journals (Sweden)

    Gilbert Greub

    Full Text Available BACKGROUND: With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. METHODS/PRINCIPAL FINDINGS: We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. CONCLUSIONS/SIGNIFICANCE: This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

  14. Highly Active, Nonprecious Electrocatalyst Comprising Borophene Subunits for the Hydrogen Evolution Reaction.

    Science.gov (United States)

    Chen, Yanli; Yu, Guangtao; Chen, Wei; Liu, Yipu; Li, Guo-Dong; Zhu, Pinwen; Tao, Qiang; Li, Qiuju; Liu, Jingwei; Shen, Xiaopeng; Li, Hui; Huang, Xuri; Wang, Dejun; Asefa, Tewodros; Zou, Xiaoxin

    2017-09-13

    Developing nonprecious hydrogen evolution electrocatalysts that can work well at large current densities (e.g., at 1000 mA/cm2: a value that is relevant for practical, large-scale applications) is of great importance for realizing a viable water-splitting technology. Herein we present a combined theoretical and experimental study that leads to the identification of α-phase molybdenum diboride (α-MoB2) comprising borophene subunits as a noble metal-free, superefficient electrocatalyst for the hydrogen evolution reaction (HER). Our theoretical finding indicates, unlike the surfaces of Pt- and MoS2-based catalysts, those of α-MoB2 can maintain high catalytic activity for HER even at very high hydrogen coverage and attain a high density of efficient catalytic active sites. Experiments confirm α-MoB2 can deliver large current densities in the order of 1000 mA/cm2, and also has excellent catalytic stability during HER. The theoretical and experimental results show α-MoB2's catalytic activity, especially at large current densities, is due to its high conductivity, large density of efficient catalytic active sites and good mass transport property.

  15. Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent.

    Science.gov (United States)

    Thiéry, Odile; Vasar, Martti; Jairus, Teele; Davison, John; Roux, Christophe; Kivistik, Paula-Ann; Metspalu, Andres; Milani, Lili; Saks, Ülle; Moora, Mari; Zobel, Martin; Öpik, Maarja

    2016-06-01

    Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra-organism genetic variation. However, information about intra- vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra-isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12-40 clones per isolate. Intra-isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut-off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next-generation sequencing; and its ease of amplification in single-step PCR. © 2016 John Wiley & Sons Ltd.

  16. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  17. Optimized subunit vaccine protects against experimental leishmaniasis.

    Science.gov (United States)

    Bertholet, Sylvie; Goto, Yasuyuki; Carter, Lauren; Bhatia, Ajay; Howard, Randall F; Carter, Darrick; Coler, Rhea N; Vedvick, Thomas S; Reed, Steven G

    2009-11-23

    Development of a protective subunit vaccine against Leishmania spp. depends on antigens and adjuvants that induce appropriate immune responses. We evaluated a second generation polyprotein antigen (Leish-110f) in different adjuvant formulations for immunogenicity and protective efficacy against Leishmania spp. challenges. Vaccine-induced protection was associated with antibody and T cell responses to Leish-110f. CD4 T cells were the source of IFN-gamma, TNF, and IL-2 double- and triple-positive populations. This study establishes the immunogenicity and protective efficacy of the improved Leish-110f subunit vaccine antigen adjuvanted with natural (MPL-SE) or synthetic (EM005) Toll-like receptor 4 agonists.

  18. Expression of Cathepsin S in BCG converts it into a pro-apoptotic and highly immunogenic strain.

    Science.gov (United States)

    Lau, Alice; Singh, Vijender; Soualhine, Hafid; Hmama, Zakaria

    2017-04-11

    -type strain, resulting in a highly immunogenic TB vaccine. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  19. Unexpected high digestion rate of cooked starch by the Ct-maltase-glucoamylase small intestine mucosal α-glucosidase subunit.

    Science.gov (United States)

    Lin, Amy Hui-Mei; Nichols, Buford L; Quezada-Calvillo, Roberto; Avery, Stephen E; Sim, Lyann; Rose, David R; Naim, Hassan Y; Hamaker, Bruce R

    2012-01-01

    For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

  20. Unexpected high digestion rate of cooked starch by the Ct-maltase-glucoamylase small intestine mucosal α-glucosidase subunit.

    Directory of Open Access Journals (Sweden)

    Amy Hui-Mei Lin

    Full Text Available For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM and sucrase-isomaltase (SI of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

  1. Unexpected High Digestion Rate of Cooked Starch by the Ct-Maltase-Glucoamylase Small Intestine Mucosal α-Glucosidase Subunit

    Science.gov (United States)

    Lin, Amy Hui-Mei; Nichols, Buford L.; Quezada-Calvillo, Roberto; Avery, Stephen E.; Sim, Lyann; Rose, David R.; Naim, Hassan Y.; Hamaker, Bruce R.

    2012-01-01

    For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies. PMID:22563462

  2. Highly immunogenic prime–boost DNA vaccination protects chickens against challenge with homologous and heterologous H5N1 virus

    Directory of Open Access Journals (Sweden)

    Anna Stachyra

    2014-01-01

    Full Text Available Highly pathogenic avian influenza viruses (HPAIVs cause huge economic losses in the poultry industry because of high mortality rate in infected flocks and trade restrictions. Protective antibodies, directed mainly against hemagglutinin (HA, are the primary means of protection against influenza outbreaks. A recombinant DNA vaccine based on the sequence of H5 HA from the H5N1/A/swan/Poland/305-135V08/2006 strain of HPAIV was prepared. Sequence manipulation included deletion of the proteolytic cleavage site to improve protein stability, codon usage optimization to improve translation and stability of RNA in host cells, and cloning into a commercially available vector to enable expression in animal cells. Naked plasmid DNA was complexed with a liposomal carrier and the immunization followed the prime–boost strategy. The immunogenic potential of the DNA vaccine was first proved in broilers in near-to-field conditions resembling a commercial farm. Next, the protective activity of the vaccine was confirmed in SPF layer-type chickens. Experimental infections (challenge experiments indicated that 100% of vaccinated chickens were protected against H5N1 of the same clade and that 70% of them were protected against H5N1 influenza virus of a different clade. Moreover, the DNA vaccine significantly limited (or even eliminated transmission of the virus to contact control chickens. Two intramuscular doses of DNA vaccine encoding H5 HA induced a strong protective response in immunized chicken. The effective protection lasted for a minimum 8 weeks after the second dose of the vaccine and was not limited to the homologous H5N1 virus. In addition, the vaccine reduced shedding of the virus.

  3. Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant.

    Science.gov (United States)

    Holmgren, J; Bourgeois, L; Carlin, N; Clements, J; Gustafsson, B; Lundgren, A; Nygren, E; Tobias, J; Walker, R; Svennerholm, A-M

    2013-05-07

    A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. A whole virus pandemic influenza H1N1 vaccine is highly immunogenic and protective in active immunization and passive protection mouse models.

    Science.gov (United States)

    Kistner, Otfried; Crowe, Brian A; Wodal, Walter; Kerschbaum, Astrid; Savidis-Dacho, Helga; Sabarth, Nicolas; Falkner, Falko G; Mayerhofer, Ines; Mundt, Wolfgang; Reiter, Manfred; Grillberger, Leopold; Tauer, Christa; Graninger, Michael; Sachslehner, Alois; Schwendinger, Michael; Brühl, Peter; Kreil, Thomas R; Ehrlich, Hartmut J; Barrett, P Noel

    2010-02-23

    The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.

  5. A whole virus pandemic influenza H1N1 vaccine is highly immunogenic and protective in active immunization and passive protection mouse models.

    Directory of Open Access Journals (Sweden)

    Otfried Kistner

    Full Text Available The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.

  6. Identification of immunogenic HLA-B7 "Achilles' heel" epitopes within highly conserved regions of HIV

    DEFF Research Database (Denmark)

    De Groot, Anne S; Rivera, Daniel S; McMurry, Julie A

    2008-01-01

    Genetic polymorphisms in class I human leukocyte antigen molecules (HLA) have been shown to determine susceptibility to HIV infection as well as the rate of progression to AIDS. In particular, the HLA-B7 supertype has been shown to be associated with high viral loads and rapid progression to dise...

  7. Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

    Science.gov (United States)

    Davis, John N.

    2013-01-01

    Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3′ end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12′GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12′GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12′GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12′GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12′GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12′GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12′GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential. PMID:23135719

  8. Highly attenuated recombinant vesicular stomatitis virus VSV-12'GFP displays immunogenic and oncolytic activity.

    Science.gov (United States)

    van den Pol, Anthony N; Davis, John N

    2013-01-01

    Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3' end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12'GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12'GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12'GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12'GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12'GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12'GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12'GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential.

  9. Cytokine-modified VSV is attenuated for neural pathology, but is both highly immunogenic and oncolytic

    OpenAIRE

    Miller, James; Bidula, Sarah M; Jensen, Troels M; Reiss, Carol Shoshkes

    2009-01-01

    Vesicular stomatitis virus (VSV), an enveloped, nonsegmented, negative-stranded RNA virus, is being tested by several laboratories as an antitumor agent. Unfortunately, viral infection of the central nervous system (CNS) has been observed by many groups following administration to tumor-bearing animals. In rodents, VSV encephalitis is characterized by weight-loss, paralysis, and high mortality. In order to provide protection from VSV infection of the CNS after therapeutic administration, we h...

  10. Production of highly immunogenic virus-like particles of bovine papillomavirus type 6 in silkworm pupae.

    Science.gov (United States)

    Watanabe, Satoko; Iizuka, Tetsuya; Hatama, Shinichi; Kanno, Toru; Mase, Masaji; Shibahara, Tomoyuki

    2017-10-13

    Bovine papillomaviruses (BPVs) are the causative agent of bovine teat papillomatosis, which can lead to severe economic losses in dairy cattle. Among the 14 identified BPV genotypes, BPV type 6 (BPV6) is the most frequently detected in teat papilloma lesions, and is therefore thought to play a major role in teat papillomatosis. To develop an effective vaccine against BPV6 infection, we produced virus-like particles of BPV6 (BPV6-VLP) in silkworm (Bombyx mori) pupae and purified these by heparin affinity chromatography using a single column. About 0.7mg purified BPV6-VLP was obtained from one pupa. BPV6-VLP-immunized mice produced a specific IgG to BPV6 that recognized BPV6 antigen with high sensitivity in an immunohistochemical analysis. Thus, silkworm pupae are a useful bioreactor for the production of BPV6-VLP, which can potentially be used as a vaccine for bovine teat papillomatosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens.

    Science.gov (United States)

    Awasthi, Sita; Mahairas, Gregory G; Shaw, Carolyn E; Huang, Meei-Li; Koelle, David M; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M

    2015-08-01

    We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4(+) and CD8(+) T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and

  12. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic.

    OpenAIRE

    Kirnbauer, R.; Booy, F; Cheng, N.; Lowy, D R; Schiller, J T

    1992-01-01

    Infection by certain human papillomavirus types is regarded as the major risk factor in the development of cervical cancer, one of the most common cancers of women worldwide. Analysis of the immunogenic and structural features of papillomavirus virions has been hampered by the inability to efficiently propagate the viruses in cultured cells. For instance, it has not been established whether the major capsid protein L1 alone is sufficient for virus particle assembly. In addition, it is not kno...

  13. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.

    Science.gov (United States)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J P; Holstein, Thomas W; Gründer, Stefan

    2010-04-16

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.

  14. Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus.

    Science.gov (United States)

    Czarnota, Anna; Tyborowska, Jolanta; Peszyńska-Sularz, Grażyna; Gromadzka, Beata; Bieńkowska-Szewczyk, Krystyna; Grzyb, Katarzyna

    2016-04-13

    Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2-3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412-425) was inserted into the hydrophilic loop of sHBsAg. The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412-425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes. For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new

  15. Genetic characteristic of high molecular weight glutenin subunits in somatic hybrid wheat lines -- potential application to wheat breeding.

    Science.gov (United States)

    Heng, Liu; Lei, Shi; Junsheng, Zhao; Guangmin, Xia

    2006-07-12

    Analysis of 17 derivatives from a somatic fusion between common wheat (Triticum aestivum) and tall wheat grass (Thinopyrum ponticum) showed a diversity of high molecular weight glutenin subunit (HMW-GS) compositions. On the basis of the inheritance of HMW-GS patterns, the derivatives were either (i) bred true over four successive generations, (ii) generated a few novel HMW-GS combinations at each generation, or (iii) showed highly unstable HMW-GS compositions. HMW-GS analysis of F(5) seed and each single seed-generated F(6) progenies further revealed that most of the HMW-GS had genetic stability. The variations of HMW-GS were inferred to occur in early generations and were maintained thereafter. Low molecular weight glutenin subunits (LMW-GS) in hybrid lines with high or low bread-making quality, classified into the first pattern, were compared. The result showed that hybrid lines with the uniform HMW-GS patterns also have identical LMW-GS patterns. The Glu-1 quality score was inferred to be relatively significant to the sodium dodecyl dulfate sedimentation value, as well as to correlate with the gluten exponent and contents of dry gluten and proteins. Sexual hybridization between high-quality somatic hybrid progeny II-12 and Chinese Spring (CS) showed that these high-quality HMW-GS genes could entail progenies. There was not subunit variation in the progenies of II-12 x CS. Therefore, sexual hybridization between somatic hybrid line and cultivars can transfer novel high-quality HMW-GS of somatic hybrids and benefit wheat breeding.

  16. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D

    2010-01-01

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated...... by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore...... properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new...

  17. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  18. Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

    Directory of Open Access Journals (Sweden)

    Alan eNeely

    2014-06-01

    Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

  19. Safety and immunogenicity of high-dose trivalent inactivated influenza vaccine in adults 50-64 years of age.

    Science.gov (United States)

    DiazGranados, Carlos A; Saway, William; Gouaux, James; Baron, Mira; Baker, Jeffrey; Denis, Martine; Jordanov, Emilia; Landolfi, Victoria; Yau, Eddy

    2015-12-16

    Individuals 50-64 years of age have reduced immune responses to influenza vaccines. The current study examined whether a high-dose inactivated trivalent influenza vaccine (IIV3-HD) might improve immune responses over a standard-dose inactivated influenza vaccine (IIV3-SD) in this age group. This was a multicenter, observer-blinded, randomized, active-controlled phase II trial. Adults 50-64 years of age were randomized 1:1 to receive IIV3-HD or IIV3-SD. Hemagglutination inhibition titers were measured before and 28 days after vaccination. Reactogenicity was recorded for 7 days after vaccination and adverse events for 28 days. 148 participants received IIV3-HD and 152 received IIV3-SD. For all vaccine strains, day 28 geometric mean hemagglutination inhibition titers were significantly higher in the IIV3-HD group than in the IIV3-SD group (geometric mean titer ratio [95% confidence interval (CI)]=1.43 [1.04-1.97] for A/H1N1, 1.65 [1.21-2.25] for A/H3N2, and 1.60 [1.23-2.08] for B). Seroconversion rates were significantly higher in the IIV3-HD group than in the IIV3-SD group for strains A/H3N2 and B but not A/H1N1 (difference [95% CI]=13.5% [4.76-22.0] for A/H3N2, 23.1% [11.7-33.6] for B, and -0.2% [-9.66 to 9.18] for A/H1N1). The post-vaccination seroprotection rate was significantly higher in the IIV3-HD group than in the IIV3-SD group for strain B but not for strains A/H1N1 or A/H3N2 (difference=9.1% [2.95-15.7] for B, 2.0% [-0.907 to 5.68] for A/H1N1, and 0.6% [-3.14 to 4.43] for A/H3N2). Reactogenicity was higher in the IIV3-HD group than in the IIV3-SD group, but reactions were mostly of low intensity, transient, and self-limited. Rates of unsolicited adverse events were similar between groups. No serious AEs, AEs leading to early withdrawal, or deaths were reported. The study suggests that in adults 50-64 years of age, IIV3-HD may improve immunogenicity compared to IIV3-SD while maintaining an acceptable safety profile. Copyright © 2015 The Authors. Published

  20. A multi-peptide, dual-adjuvant telomerase vaccine (GX301) is highly immunogenic in patients with prostate and renal cancer.

    Science.gov (United States)

    Fenoglio, Daniela; Traverso, Paolo; Parodi, Alessia; Tomasello, Laura; Negrini, Simone; Kalli, Francesca; Battaglia, Florinda; Ferrera, Francesca; Sciallero, Stefania; Murdaca, Giuseppe; Setti, Maurizio; Sobrero, Alberto; Boccardo, Francesco; Cittadini, Giuseppe; Puppo, Francesco; Criscuolo, Domenico; Carmignani, Giorgio; Indiveri, Francesco; Filaci, Gilberto

    2013-06-01

    Anti-tumor vaccination is a new frontier in cancer treatment applicable to immunogenic neoplasms such as prostate and renal cancers. GX301 is a vaccine constituted by four telomerase peptides and two adjuvants, Montanide ISA-51 and Imiquimod. The aim of this study was to analyze safety and tolerability of GX301 in an open-label, phase I/II trial. Immunological and clinical responses were also evaluated as secondary endpoints. GX301 was administered by intradermally injecting 500 μg of each peptide (dissolved in Montanide ISA-51) in the skin of the abdomen. Imiquimod was applied as a cream at the injection sites. The protocol included 8 administrations at days 1, 3, 5, 7, 14, 21, 35, 63. Eligible patients were affected with stage IV prostate or renal cancer resistant to conventional treatments. Patients were clinically and immunologically monitored up to 6 months from the first immunization. No grade 3-4 adverse events were observed. Evidence of vaccine-specific immunological responses was detected in 100 % of patients. Disease stabilization occurred in 4 patients. Prolonged progression-free survival and overall survival were observed in patients showing a full pattern of vaccine-specific immunological responses. GX301 demonstrated to be safe and highly immunogenic. Further studies are needed to determine its clinical efficacy.

  1. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

    Science.gov (United States)

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  2. Supramolecular peptide hydrogel adjuvanted subunit vaccine elicits protective antibody responses against West Nile virus.

    Science.gov (United States)

    Friedrich, Brian M; Beasley, David W C; Rudra, Jai S

    2016-11-04

    A crucial issue in vaccine development is to balance safety with immunogenicity. The low immunogenicity of most subunit antigens warrants a search for adjuvants able to stimulate both cell-mediated and humoral immunity. In recent years, successful applications of nanotechnology and bioengineering in the field of vaccine development have enabled the production of novel adjuvant technologies. In this work, we investigated totally synthetic and supramolecular peptide hydrogels as novel vaccine adjuvants in conjunction with the immunoprotective envelope protein domain III (EIII) of West Nile virus as an immunogen in a mouse model. Our results indicate that, compared to the clinically approved adjuvant alum, peptide hydrogel adjuvanted antigen elicited stronger antibody responses and conferred significant protection against mortality after virus challenge. The high chemical definition and biocompatibility of self-assembling peptide hydrogels makes them attractive as immune adjuvants for the production of subunit vaccines against viral and bacterial infections where antibody-mediated protection is desirable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Effect of high molecular weight glutenin subunit composition in common wheat on dough properties and steamed bread quality.

    Science.gov (United States)

    Zhang, Pingping; Jondiko, Tom O; Tilley, Michael; Awika, Joseph M

    2014-10-01

    Steamed bread is a popular staple food in Asia with different flour quality requirements from pan bread. Little is known about how glutenin characteristics affect steamed bread quality. This work investigated how deletions of high-molecular-weight glutenin subunits (HMW-GS) influence gluten properties and Chinese steamed bread quality using 16 wheat lines grown in Texas. Although similar in protein content (134-140 mg g⁻¹), gluten composition and dough properties differed widely among the lines. Compared with non-deletion lines, deletion lines had lower (P bread quality (score, 60.8-65.0) with good elasticity and crumb structure. Deletion at Glu-B1y and/or Glu-D1y loci in high-strength hard wheat produced good dough properties for steamed bread. This suggests that wheat functionality for steamed bread can be improved by manipulating HMW-GS composition. © 2014 Society of Chemical Industry.

  4. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

    Science.gov (United States)

    Feng, Hao; Hu, Gui-qiu; Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  5. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

    Directory of Open Access Journals (Sweden)

    Hao Feng

    Full Text Available The VP2 structural protein of parvovirus can produce virus-like particles (VLPs by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+ and CD8(+ T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  6. Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell-driven rejection of high-grade glioma.

    Science.gov (United States)

    Garg, Abhishek D; Vandenberk, Lien; Koks, Carolien; Verschuere, Tina; Boon, Louis; Van Gool, Stefaan W; Agostinis, Patrizia

    2016-03-02

    The promise of dendritic cell (DC)-based immunotherapy has been established by two decades of translational research. Of the four malignancies most targeted with clinical DC immunotherapy, high-grade glioma (HGG) has shown the highest susceptibility. HGG-induced immunosuppression is a roadblock to immunotherapy, but may be overcome by the application of T helper 1 (T(H)1) immunity-biased, next-generation, DC immunotherapy. To this end, we combined DC immunotherapy with immunogenic cell death (ICD; a modality shown to induce T(H)1 immunity) induced by hypericin-based photodynamic therapy. In an orthotopic HGG mouse model involving prophylactic/curative setups, both biologically and clinically relevant versions of ICD-based DC vaccines provided strong anti-HGG survival benefit. We found that the ability of DC vaccines to elicit HGG rejection was significantly blunted if cancer cell-associated reactive oxygen species and emanating danger signals were blocked either singly or concomitantly, showing hierarchical effect on immunogenicity, or if DCs, DC-associated MyD88 signal, or the adaptive immune system (especially CD8(+) T cells) were depleted. In a curative setting, ICD-based DC vaccines synergized with standard-of-care chemotherapy (temozolomide) to increase survival of HGG-bearing mice by ~300%, resulting in ~50% long-term survivors. Additionally, DC vaccines also induced an immunostimulatory shift in the brain immune contexture from regulatory T cells to T(H)1/cytotoxic T lymphocyte/T(H)17 cells. Analysis of the The Cancer Genome Atlas glioblastoma cohort confirmed that increased intratumor prevalence of T(H)1/cytotoxic T lymphocyte/T(H)17 cells linked genetic signatures was associated with good patient prognosis. Therefore, pending final preclinical checks, ICD-based vaccines can be clinically translated for glioma treatment. Copyright © 2016, American Association for the Advancement of Science.

  7. Assessing the Immunogenicity of Biopharmaceuticals.

    Science.gov (United States)

    Pineda, Carlos; Castañeda Hernández, Gilberto; Jacobs, Ira A; Alvarez, Daniel F; Carini, Claudio

    2016-06-01

    Biopharmaceuticals have the potential to raise an immunogenic response in treated individuals, which may impact the efficacy and safety profile of these drugs. As a result, it is essential to evaluate immunogenicity throughout the different phases of the clinical development of a biopharmaceutical, including post-marketing surveillance. Although rigorous evaluation of biopharmaceutical immunogenicity is required by regulatory authorities, there is a lack of uniform standards for the type, quantity, and quality of evidence, and for guidance on experimental design for immunogenicity assays or criteria to compare immunogenicity of biopharmaceuticals. Moreover, substantial technological advances in methods to assess immune responses have yielded higher immunogenicity rates with modern assays, and limit comparison of immunogenicity of biopharmaceuticals outside of head-to-head clinical trials. Accordingly, research programs, regulatory agencies, and clinicians need to keep pace with continuously evolving analyses of immunogenicity. Here, we review factors associated with immunogenicity of biopharmaceuticals, potential clinical ramifications, and current regulatory guidance for evaluating immunogenicity, and discuss methods to assess immunogenicity in non-clinical and clinical studies. We also describe special considerations for evaluating the immunogenicity of biosimilar candidates.

  8. Superior protection conferred by inactivated whole virus vaccine over subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Xu, Cheng; Mutoloki, Stephen; Evensen, Øystein

    2012-06-06

    Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime-boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log(10) reduction of virus titer in heart for the IWV group. Prime-boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime-boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against Middle East respiratory syndrome coronavirus.

    Science.gov (United States)

    Zhang, Naru; Channappanavar, Rudragouda; Ma, Cuiqing; Wang, Lili; Tang, Jian; Garron, Tania; Tao, Xinrong; Tasneem, Sumaiya; Lu, Lu; Tseng, Chien-Te K; Zhou, Yusen; Perlman, Stanley; Jiang, Shibo; Du, Lanying

    2016-03-01

    Middle East respiratory syndrome (MERS), an emerging infectious disease caused by MERS coronavirus (MERS-CoV), has garnered worldwide attention as a consequence of its continuous spread and pandemic potential, making the development of effective vaccines a high priority. We previously demonstrated that residues 377-588 of MERS-CoV spike (S) protein receptor-binding domain (RBD) is a very promising MERS subunit vaccine candidate, capable of inducing potent neutralization antibody responses. In this study, we sought to identify an adjuvant that optimally enhanced the immunogenicity of S377-588 protein fused with Fc of human IgG (S377-588-Fc). Specifically, we compared several commercially available adjuvants, including Freund's adjuvant, aluminum, Monophosphoryl lipid A, Montanide ISA51 and MF59 with regard to their capacity to enhance the immunogenicity of this subunit vaccine. In the absence of adjuvant, S377-588-Fc alone induced readily detectable neutralizing antibody and T-cell responses in immunized mice. However, incorporating an adjuvant improved its immunogenicity. Particularly, among the aforementioned adjuvants evaluated, MF59 is the most potent as judged by its superior ability to induce the highest titers of IgG, IgG1 and IgG2a subtypes, and neutralizing antibodies. The addition of MF59 significantly augmented the immunogenicity of S377-588-Fc to induce strong IgG and neutralizing antibody responses as well as protection against MERS-CoV infection in mice, suggesting that MF59 is an optimal adjuvant for MERS-CoV RBD-based subunit vaccines.

  10. A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

    OpenAIRE

    Kistner, Otfried; Crowe, Brian A.; Wodal, Walter; Kerschbaum, Astrid; Savidis-Dacho, Helga; Sabarth, Nicolas; Falkner, Falko G.; Mayerhofer, Ines; Mundt, Wolfgang; Reiter, Manfred; Grillberger, Leopold; Tauer, Christa; Graninger, Michael; Sachslehner, Alois; Schwendinger, Michael

    2010-01-01

    The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus c...

  11. GABAA/Benzodiazepine receptor binding in patients with schizophrenia using [11C]Ro15-4513, a radioligand with relatively high affinity for alpha5 subunit.

    Science.gov (United States)

    Asai, Yoshiyuki; Takano, Akihiro; Ito, Hiroshi; Okubo, Yoshiro; Matsuura, Masato; Otsuka, Akihiko; Takahashi, Hidehiko; Ando, Tomomichi; Ito, Shigeo; Arakawa, Ryosuke; Asai, Kunihiko; Suhara, Tetsuya

    2008-02-01

    Dysfunction of the GABA system is considered to play a role in the pathology of schizophrenia. Individual subunits of GABA(A)/Benzodiazepine (BZ) receptor complex have been revealed to have different functional properties. alpha5 subunit was reported to be related to learning and memory. Changes of alpha5 subunit in schizophrenia were reported in postmortem studies, but the results were inconsistent. In this study, we examined GABA(A)/BZ receptor using [(11)C]Ro15-4513, which has relatively high affinity for alpha5 subunit, and its relation to clinical symptoms in patients with schizophrenia. [(11)C]Ro15-4513 bindings of 11 patients with schizophrenia (6 drug-naïve and 5 drug-free) were compared with those of 12 age-matched healthy control subjects using positron emission tomography. Symptoms were assessed using the Positive and Negative Syndrome Scale. [(11)C]Ro15-4513 binding was quantified by binding potential (BP) obtained by the reference tissue model. [(11)C]Ro15-4513 binding in the prefrontal cortex and hippocampus was negatively correlated with negative symptom scores in patients with schizophrenia, although there was no significant difference in BP between patients and controls. GABA(A)/BZ receptor including alpha5 subunit in the prefrontal cortex and hippocampus might be involved in the pathophysiology of negative symptoms of schizophrenia.

  12. Immunogenicity and safety of a high-dose hepatitis B vaccine among patients receiving methadone maintenance treatment: A randomized, double-blinded, parallel-controlled trial.

    Science.gov (United States)

    Shi, Jing; Feng, Yongliang; Gao, Linying; Feng, Dan; Yao, Tian; Shi, Shan; Zhang, Yawei; Liang, Xiaofeng; Wang, Suping

    2017-04-25

    To explore whether the immunization with high-dose (60μg) hepatitis B vaccines in patients receiving methadone maintenance treatment (MMT) could yield a superior protection against hepatitis B infection than did the standard dose (20μg). We conducted a randomized, double-blinded, parallel-controlled trial in MMT patients. Patients with serologically negative hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) were randomized in a ratio of 1:1 to receive three intramuscular injections of 20μg or 60μg recombinant hepatitis B vaccine at months 0, 1, and 6. Serum HBsAg and anti-HBs were measured at months 7 and 12 post-vaccination to assess the immunogenicity. A total of 196 MMT patients were randomized and 195 received at least one injection (98 and 97 in 20 and 60μg vaccine groups, respectively). The 60μg vaccine group showed a seroconversion of anti-HBs of 87.3%, high-level response rate of 56.3%, and GMC of 742.9mIU/mL at month 7. While these results were numerically higher than the 20μg group, a statistical difference was not found. HIV infection and concomitant drug abuse were negatively associated with the robust immune responses. 7.7% of MMT patients receiving at least one dose of vaccine reported solicited adverse reactions within 7days after vaccination, 2.6% reported unsolicited adverse reactions within 28days after vaccination. None of the MMT patients reported serious adverse events or became HBsAg positive during the follow-up. The three-dose regimen of 60μg recombinant hepatitis B vaccine at months 0, 1, and 6 can yield a similar immunogenicity among MMT patients as compared to the 20μg vaccine. ClinicalTrials.gov identifier: NCT02991599. Copyright © 2017. Published by Elsevier Ltd.

  13. Structure-guided Design and Immunological Characterization of Immunogens Presenting the HIV-1 gp120 V3 Loop on a CTB Scaffold

    Energy Technology Data Exchange (ETDEWEB)

    M Totrov; X Jiang; X Kong; S Cohen; C Krachmarov; A Salomon; C Williams; M Seaman; R Abagyan; et al.

    2011-12-31

    V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.

  14. A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice.

    Science.gov (United States)

    Koraka, Penelope; Bosch, Berend-Jan; Cox, Manon; Chubet, Rick; Amerongen, Geert van; Lövgren-Bengtsson, Karen; Martina, Byron E E; Roose, Jouke; Rottier, Peter J M; Osterhaus, Albert D M E

    2014-08-06

    Rabies is a disease characterized by an invariably lethal encephalitis of viral origin that can be controlled by preventive vaccination programs of wildlife, domestic animals and humans in areas with a high risk of exposure. Currently available vaccines are expensive, cumbersome to produce and require intensive immunization and booster schemes to induce and maintain protective immunity. In the present study, we describe the development of candidate recombinant subunit rabies vaccines based on the glycoprotein G of the prototype rabies virus (RABV-G) expressed either as a monomer (RABV-mG) or in its native trimeric configuration (RABV-tG), with or without Matrix-M™ adjuvant. Immunogenicity and protective efficacy of the respective candidate vaccines were tested in outbred NIH Swiss albino mice. The RABV-tG candidate vaccine proved to be superior to the RABV-mG vaccine candidate both in terms of immunogenicity and efficacy. The relatively poor immunogenicity of the RABV-mG vaccine candidate was greatly improved by the addition of the adjuvant. A single, low dose of RABV-tG in combination with Matrix-M™ induced high levels of high avidity neutralizing antibodies and protected all mice against challenge with a lethal dose of RABV. Consequently RABV-tG used in combination with Matrix-M™ is a promising vaccine candidate that overcomes the limitations of currently used vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Redox thermodynamics of high-spin and low-spin forms of chlorite dismutases with diverse subunit and oligomeric structures.

    Science.gov (United States)

    Hofbauer, Stefan; Bellei, Marzia; Sündermann, Axel; Pirker, Katharina F; Hagmüller, Andreas; Mlynek, Georg; Kostan, Julius; Daims, Holger; Furtmüller, Paul G; Djinović-Carugo, Kristina; Oostenbrink, Chris; Battistuzzi, Gianantonio; Obinger, Christian

    2012-11-27

    Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus "Nitrospira defluvii" (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°' values for free and cyanide-bound proteins were determined to be -119 and -397 mV for NwCld and -113 and -404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.

  16. Humoral Immunogenicity of an HIV-1 Envelope Residue 649-684 Membrane-proximal Region Peptide Fused to the Plague Antigen F1-V

    Science.gov (United States)

    Matoba, Nobuyuki; Shah, Namrata Rahul; Mor, Tsafrir S

    2011-01-01

    The membrane-proximal region spanning residues 649-684 of the HIV-1 envelope protein gp41 (MPR649-684) is an attractive vaccine target for humoral immunity that blocks viral transcytosis across the mucosal epithelia. However, induction of high-titer MPR649-684-specific antibodies remains a challenging task. To explore potential solutions for this challenge, we tested a new translational fusion protein comprising the plague F1-V antigen and MPR649-684 (F1-V-MPR649-684). We employed systemic immunization for initial feasibility analyses. Despite strong immunogenicity demonstrated for the immunogen, repeated systemic immunizations of mice with F1-V-MPR649-684 hardly induced MPR649-684-specific IgG. In contrast, a single immunization with F1-V-MPR649-684 mounted a significant anti-MPR649-684 IgG response in animals that were primed with another MPR649-684 fusion protein based on the cholera toxin B subunit. Additional boost immunizations with F1-V-MPR649-684 recalled and maintained the antibody response and expanded the number of specific antibody-secreting B cells. Thus, while F1-V-MPR649-684 alone was not sufficiently immunogenic to induce detectable levels of MPR649-684-specific antibodies, these results suggest that prime-boost immunization using heterologous antigen-display platforms may overcome the poor humoral immunogenicity of MPR649-684 for the induction of durable humoral immunity. Further studies are warranted to evaluate the feasibility of this strategy in mucosal immunization. Lastly, our findings add to a growing body of evidence in support of this strategy for immunogen design for poorly immunogenic epitopes besides the MPR of HIV-1's transmembrane envelope protein. PMID:21693158

  17. Immunogenicity of autoantigens.

    Science.gov (United States)

    Backes, Christina; Ludwig, Nicole; Leidinger, Petra; Harz, Christian; Hoffmann, Jana; Keller, Andreas; Meese, Eckart; Lenhof, Hans-Peter

    2011-07-04

    Autoantibodies against self-antigens have been associated not only with autoimmune diseases, but also with cancer and are even found in healthy individuals. The mechanism causing the autoantibody response remains elusive for the majority of the immunogenic antigens. To deepen the understanding of autoantibody responses, we ask whether natural-occurring, autoimmunity-associated and tumor-associated antigens have structural or biological features related to the immune response. To this end, we have carried out the most comprehensive in-silicio study of different groups of autoantigens including large antigen sets identified by our groups combined with publicly available antigen sets. We found evidence for an enrichment of genes with a larger exon length increasing the probability of the occurrence of potential immunogenic features such as mutations, SNPs, immunogenic sequence patterns and structural epitopes, or alternative splicing events. While SNPs seem to play a more central role in autoimmunity, somatic mutations seem to be stronger enriched in tumor-associated antigens. In addition, antigens of autoimmune diseases are different from other antigen sets in that they appear preferentially secreted, have frequently an extracellular location, and they are enriched in pathways associated with the immune system. Furthermore, for autoantibodies in general, we found enrichment of sequence-based properties including coiled-coils motifs, ELR motifs, and Zinc finger DNA-binding motifs. Moreover, we found enrichment of proteins binding to proteins or nucleic acids including RNA and enrichment of proteins that are part of ribosome or spliceosome. Both, homologies to proteins of other species and an enrichment of ancient protein domains indicate that immunogenic proteins are evolutionary conserved and that mimicry might play a central role. Our results provide evidence that proteins which i) are evolutionary conserved, ii) show specific sequence motifs, and iii) are part of

  18. Immunogenicity of autoantigens

    Directory of Open Access Journals (Sweden)

    Keller Andreas

    2011-07-01

    Full Text Available Abstract Background Autoantibodies against self-antigens have been associated not only with autoimmune diseases, but also with cancer and are even found in healthy individuals. The mechanism causing the autoantibody response remains elusive for the majority of the immunogenic antigens. To deepen the understanding of autoantibody responses, we ask whether natural-occurring, autoimmunity-associated and tumor-associated antigens have structural or biological features related to the immune response. To this end, we have carried out the most comprehensive in-silicio study of different groups of autoantigens including large antigen sets identified by our groups combined with publicly available antigen sets. Results We found evidence for an enrichment of genes with a larger exon length increasing the probability of the occurrence of potential immunogenic features such as mutations, SNPs, immunogenic sequence patterns and structural epitopes, or alternative splicing events. While SNPs seem to play a more central role in autoimmunity, somatic mutations seem to be stronger enriched in tumor-associated antigens. In addition, antigens of autoimmune diseases are different from other antigen sets in that they appear preferentially secreted, have frequently an extracellular location, and they are enriched in pathways associated with the immune system. Furthermore, for autoantibodies in general, we found enrichment of sequence-based properties including coiled-coils motifs, ELR motifs, and Zinc finger DNA-binding motifs. Moreover, we found enrichment of proteins binding to proteins or nucleic acids including RNA and enrichment of proteins that are part of ribosome or spliceosome. Both, homologies to proteins of other species and an enrichment of ancient protein domains indicate that immunogenic proteins are evolutionary conserved and that mimicry might play a central role. Conclusions Our results provide evidence that proteins which i are evolutionary conserved

  19. Comparison of neutralizing antibody responses elicited from highly diverse polyvalent heterotrimeric HIV-1 gp140 cocktail immunogens versus a monovalent counterpart in rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Emma J Bowles

    Full Text Available Eliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a major goal of HIV-1 vaccine design. The challenge is that envelopes (Envs of circulating viruses are almost certainly different from any Env used in a vaccine. A novel immunogen composed of a highly diverse set of gp140 Envs including subtypes A, B, C, D and F was developed to stimulate a more cross-neutralizing antibody response. Env heterotrimers composed of up to 54 different gp140s were produced with the aim of focusing the response to the conserved regions of Env while reducing the dominance of any individual hypervariable region. Heterotrimeric gp140 Envs of inter- and intra-subtype combinations were shown to bind CD4 and a panel of neutralizing monoclonal antibodies with similar affinity to monovalent UG37 gp140. Macaques immunized with six groups of heterotrimer mixtures showed slightly more potent neutralizing antibody responses in TZM-BL tier 1 and A3R5 tier 2 pseudovirus assays than macaques immunized with monovalent Env gp140, and exhibited a marginally greater focus on the CD4-binding site. Carbopol enhanced neutralization when used as an adjuvant instead of RIBI in combination with UG37 gp140. These data indicate that cross-subtype heterotrimeric gp140 Envs may elicit some improvement of the neutralizing antibody response in macaques compared to monovalent gp140 Env.

  20. Assessing the Immunogenicity of Biopharmaceuticals

    OpenAIRE

    Pineda, Carlos; Casta?eda Hern?ndez, Gilberto; Jacobs, Ira A.; Alvarez, Daniel F.; Carini, Claudio

    2016-01-01

    Biopharmaceuticals have the potential to raise an immunogenic response in treated individuals, which may impact the efficacy and safety profile of these drugs. As a result, it is essential to evaluate immunogenicity throughout the different phases of the clinical development of a biopharmaceutical, including post-marketing surveillance. Although rigorous evaluation of biopharmaceutical immunogenicity is required by regulatory authorities, there is a lack of uniform standards for the type, qua...

  1. Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

    Science.gov (United States)

    Postler, Thomas S.; Martinez-Navio, José M.; Yuste, Eloísa

    2012-01-01

    The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called “Kennedy epitope,” that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia

  2. Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco.

    Directory of Open Access Journals (Sweden)

    Igor Kolotilin

    Full Text Available Post-weaning diarrhea (PWD in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc, engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc. The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R, and inhibition of the F4-possessing (F4+ ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD.

  3. Improved immunogenicity of high-dose influenza vaccine compared to standard-dose influenza vaccine in adult oncology patients younger than 65 years receiving chemotherapy: A pilot randomized clinical trial.

    Science.gov (United States)

    Jamshed, Saad; Walsh, Edward E; Dimitroff, Lynda J; Santelli, Jeanine Seguin; Falsey, Ann R

    2016-01-27

    Patients undergoing chemotherapy often fail to develop robust responses to influenza vaccination. Compared to standard-dose influenza vaccine (SD), high-dose influenza vaccine (HD) has shown improved immunogenicity and protection against influenza illness in adults 65 years and older. This study compared the immunogenicity and tolerability of HD to SD in adults younger than 65 years of age receiving chemotherapy. This double-blind study randomized patients receiving chemotherapy to vaccination with either SD or HD influenza vaccine. Hemagglutination inhibition assays (HAI) were performed prior to and 4 weeks after vaccination. HAI were summarized as geometric mean titers (GMT), seroconversion rates, and seroprotection rates. A total of 105 subjects were enrolled in the trial (51 received SD and 54 received HD). Subjects were well matched for demographic and medical conditions. Both vaccines were well tolerated with no SAEs. Of the 100 subjects with evaluable data, seroconversion rates for all 3 influenza antigens & post-vaccination GMTs for H3N2 & B strains were significantly improved with HD compared to SD. Seroprotection was excellent and equivalent in both groups. Trivalent high-dose influenza vaccine can be safely administered to patients receiving chemotherapy with improved immunogenicity and seroconversion compared to standard-dose vaccine. Post-vaccination seroprotection rates were similar in both groups. A larger study is needed to show clinical benefits with HD in this population. This study was registered at ClinicalTrials.gov as NCT01666782. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

    Science.gov (United States)

    For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

  5. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH:200) and their application in the detection of current amoebic infection.

    Science.gov (United States)

    Sengupta, K; Das, P; Johnson, T M; Chaudhuri, P P; Das, D; Nair, G B

    1993-01-01

    Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.

  7. Immunogenicity and safety of high-dose trivalent inactivated influenza vaccine compared to standard-dose vaccine in children and young adults with cancer or HIV infection.

    Science.gov (United States)

    Hakim, Hana; Allison, Kim J; Van de Velde, Lee-Ann; Tang, Li; Sun, Yilun; Flynn, Patricia M; McCullers, Jonathan A

    2016-06-08

    Approaches to improve the immune response of immunocompromised patients to influenza vaccination are needed. Children and young adults (3-21 years) with cancer or HIV infection were randomized to receive 2 doses of high-dose (HD) trivalent influenza vaccine (TIV) or of standard-dose (SD) TIV. Hemagglutination inhibition (HAI) antibody titers were measured against H1, H3, and B antigens after each dose and 9 months later. Seroconversion was defined as ≥4-fold rise in HAI titer comparing pre- and post-vaccine sera. Seroprotection was defined as a post-vaccine HAI titer ≥1:40. Reactogenicity events (RE) were solicited using a structured questionnaire 7 and 14 days after each dose of vaccine, and adverse events by medical record review for 21 days after each dose of vaccine. Eighty-five participants were enrolled in the study; 27 with leukemia, 17 with solid tumor (ST), and 41 with HIV. Recipients of HD TIV had significantly greater fold increase in HAI titers to B antigen in leukemia group and to H1 antigen in ST group compared to SD TIV recipients. This increase was not documented in HIV group. There were no differences in seroconversion or seroprotection between HD TIV and SD TIV in all groups. There was no difference in the percentage of solicited RE in recipients of HD TIV (54% after dose 1 and 38% after dose 2) compared to SD TIV (40% after dose 1 and 20% after dose 2, p=0.27 and 0.09 after dose 1 and 2, respectively). HD TIV was more immunogenic than SD TIV in children and young adults with leukemia or ST, but not with HIV. HD TIV was safe and well-tolerated in children and young adults with leukemia, ST, or HIV. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. High immunogenic potential of p53 mRNA-transfected dendritic cells in patients with primary breast cancer

    DEFF Research Database (Denmark)

    Met, Özcan; Balslev, Eva; Flyger, Henrik

    2011-01-01

    it will help to identify likely responders to TAAs among patients who qualify and may benefit from this form of immune therapy. This study aimed to determine pre-existent T-cell reactivity against the tumor suppressor protein p53 in breast cancer patients (BCP) at the time point of primary diagnosis. After...... a short-term stimulation with autologous wt p53 mRNA-transfected DCs, IFN-¿ enzyme-linked immunosorbent spot (ELISPOT) analysis revealed p53-reactive T cells in the peripheral blood of more than 40% (15 of 36) of the tested patients. Both CD4(+) and CD8(+) p53-specific T cells secreted IFN-¿ after...... stimulation with p53-transfected DCs. Interestingly, more than 72% (13 of 18) of patients with high p53 (p53(high)) expression in tumors were able to mount a p53-specific IFN-¿ T-cell response, in contrast to only 10% (1 of 10) of healthy donors and 11% (2 of 18) of patients with low or absent p53 (p53(low...

  9. Effect of shading from jointing to maturity on high molecular weight glutenin subunit accumulation and glutenin macropolymer concentration in grain of winter wheat

    DEFF Research Database (Denmark)

    Li, X.; Cai, J.; Li, H.

    2012-01-01

    the accumulation and concentration of HMW-GS in the grains. Consequently, S1 reduced falling number and SDS-sedimentation volume, while shortened dough development time (DDT) and dough stability time (DST). In contrast, S2 and S3 increased falling number, wet-gluten concentration and SDS-sedimentation volume......Accumulations of high molecular weight glutenin subunits (HMW-GS) and glutenin macropolymer (GMP) in wheat grains are important indicators of grain quality. Two wheat cultivars, Yangmai 158 (shading tolerant) and Yangmai 11 (shading intolerant) which contains the same subunit pairs of 7 + 8 and 2...... + 12, were used to evaluate the impacts of shading on HMW-GS accumulation and GMP concentration in the grain. Three shading levels were implemented from jointing to maturity, i.e. S1, S2 and S3, in which the plants received 8 %, 15 % and 23 % less radiation of the control (S0), respectively...

  10. SagE induces highly effective protective immunity against Streptococcus iniae mainly through an immunogenic domain in the extracellular region.

    Science.gov (United States)

    Sun, Yun; Sun, Li; Xing, Ming-qing; Liu, Chun-sheng; Hu, Yong-hua

    2013-11-12

    Streptococcus iniae is a Gram-positive bacterium and a severe pathogen of a wide range of farmed fish. S. iniae possesses a virulence-associated streptolysin S cluster composed of several components, one of which is SagE. SagE a transmembrane protein with one major extracellular region named ECR. This study aimed to develop a SagE-based DNA candidate vaccine against streptococcosis and examine the immunoprotective mechanism of the vaccine. We constructed a DNA vaccine, pSagE, based on the sagE gene and examined its immunological property in a Japanese flounder (Paralichthys olivaceus) model. The results showed that at 7 days post-vaccination, expression of SagE at transcription and translation levels was detected in the tissues of the vaccinated fish. After challenge with S. iniae at one and two months post-vaccination, pSagE-vaccinated fish exhibited relative percent survival (RPS) of 95% and 88% respectively. Immunological analysis showed that (i) pSagE significantly upregulated the expression of a wide range of immune genes, (ii) pSagE induced the production of specific serum antibodies that bound whole-cell S. iniae, and (iii) treatment of S. iniae with pSagE-induced antibodies blocked bacterial invasion of host cells. To localize the immunoprotective domain of SagE, the ECR-expressing DNA vaccine pSagEECR was constructed. Immunization analysis showed that flounder vaccinated with pSagEECR exhibited a RPS of 68%, and that pSagEECR induced serum antibody production and immune gene expression in a manner similar to, though to lower magnitudes than, those induced by pSagE. We in this study developed a DNA vaccine, pSagE, which induces highly protective immunity against S. iniae. The protective effect of pSagE is probably due to its ability to elicit systemic immune response, in particular that of the humoral branch, which leads to production of specific serum antibodies that impair bacterial infection. These results add insights to the immunoprotective mechanism

  11. A multi-subunit Chlamydia vaccine inducing neutralizing antibodies and strong IFN-γ(+) CMI responses protects against a genital infection in minipigs

    DEFF Research Database (Denmark)

    Bøje, Sarah; Olsen, Anja Weinreich; Erneholm, Karin

    2016-01-01

    Chlamydia is the most widespread sexually transmitted bacterial disease and a prophylactic vaccine is highly needed. Ideally, this vaccine is required to induce a combined response of Th1 cell-mediated immune (CMI) response in concert with neutralizing antibodies. Using a novel Göttingen minipig...... animal model, we evaluated the immunogenicity and efficacy of a multi-subunit vaccine formulated in the strong Th1-inducing adjuvant CAF01. We evaluated a mixture of two fusion proteins (Hirep1 and CTH93) designed to promote either neutralizing antibodies or cell-mediated immunity, respectively. Hirep1...... is a novel immunogen based on the variant domain (VD) 4 region from major outer membrane protein (MOMP) serovar (Sv) D, SvE and SvF, and CTH93 is a fusion molecule of three antigens (CT043, CT414 and MOMP). Pigs were immunized twice intramuscularly with either Hirep1+CTH93/CAF01, UV-inactivated Chlamydia...

  12. The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks.

    Science.gov (United States)

    Bermek, Oya; Weller, Sandra K; Griffith, Jack D

    2017-09-22

    During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Meta-analysis of the immunogenicity and tolerability of pandemic influenza A 2009 (H1N1 vaccines.

    Directory of Open Access Journals (Sweden)

    Lamberto Manzoli

    Full Text Available BACKGROUND: Although the 2009 (H1N1 influenza pandemic officially ended in August 2010, the virus will probably circulate in future years. Several types of H1N1 vaccines have been tested including various dosages and adjuvants, and meta-analysis is needed to identify the best formulation. METHODS: We searched MEDLINE, EMBASE, and nine clinical trial registries to April 2011, in any language for randomized clinical trials (RCTs on healthy children, adolescents, adults and the elderly. Primary outcome was the seroconversion rate according to hemagglutinination-inhibition (HI; secondary outcomes were adverse events. For the primary outcome, we used head-to-head meta-analysis and multiple-treatments meta-analysis. RESULTS: Eighteen RCTs could be included in all primary analyses, for a total of 76 arms (16,725 subjects. After 2 doses, all 2009 H1N1 split/subunit inactivated vaccines were highly immunogenic and overcome CPMP seroconversion criteria. After 1 dose only, all split/subunit vaccines induced a satisfactory immunogenicity (> = 70% in adults and adolescents, while only some formulations showed acceptable results for children and elderly (non-adjuvanted at high-doses and oil-in-water adjuvanted vaccines. Vaccines with oil-in-water adjuvants were more immunogenic than both nonadjuvanted and aluminum-adjuvanted vaccines at equal doses and their immunogenicity at doses < = 6 µg (even with as little as 1.875 µg of hemagglutinin antigen was not significantly lower than that achieved after higher doses. Finally, the rate of serious vaccine-related adverse events was low for all 2009 H1N1 vaccines (3 cases, resolved in 10 days, out of 22826 vaccinated subjects. However, mild to moderate adverse reactions were more (and very frequent for oil-in-water adjuvanted vaccines. CONCLUSIONS: Several one-dose formulations might be valid for future vaccines, but 2 doses may be needed for children, especially if a low-dose non-adjuvanted vaccine is

  14. Stoichiometry of δ subunit containing GABAA receptors

    Science.gov (United States)

    Patel, B; Mortensen, M; Smart, T G

    2014-01-01

    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  15. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

    Directory of Open Access Journals (Sweden)

    Andrea Pecora

    2015-03-01

    Full Text Available Bovine viral diarrhea virus (BVDV is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2 was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

  16. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    Science.gov (United States)

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  17. Murine immunization with CS21 pili or LngA major subunit of enterotoxigenic Escherichia coli (ETEC) elicits systemic and mucosal immune responses and inhibits ETEC gut colonization.

    Science.gov (United States)

    Zhang, Chengxian; Iqbal, Junaid; Gómez-Duarte, Oscar G

    2017-04-01

    CS21 pili of enterotoxigenic Escherichia coli (ETEC) is one of the most prevalent ETEC colonization factors. CS21 major subunit, LngA, mediates ETEC adherence to intestinal cells, and contributes to ETEC pathogenesis in a neonatal mouse infection model. The objectives of this work were to evaluate LngA major subunit purified protein and CS21 purified pili on immunogenicity and protection against ETEC colonization of mice intestine. Recombinant LngA purified protein or purified CS21 pili from E9034A ETEC strain were evaluated for immunogenicity after immunization of C57BL/6 mice. Specific anti-LngA antibodies were detected from mice serum, feces, and intestine fluid samples by ELISA assays. Protection against gut colonization was evaluated on immunized mice orally challenged with wild type E9034A ETEC strain and by subsequent quantification of bacterial colony forming units (CFU) recovered from feces. Recombinant LngA protein and CS21 pili induced specific humoral and mucosal anti-LngA antibodies in the mouse model. CS21 combined with CT delivered intranasally as well as LngA combined with incomplete Freund adjuvant delivered intraperitoneally inhibited ETEC gut colonization in a mouse model. In conclusion, both LngA purified protein and CS21 pili from ETEC are highly immunogenic and may inhibit ETEC intestinal shedding. Our data on immunogenicity and immunoprotection indicates that CS21 is a suitable vaccine candidate for a future multivalent vaccine against ETEC diarrhea. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Mosaic HIV envelope immunogenic polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette T. M.; Gnanakaran, S.; Perkins, Simon; Sodroski, Joseph; Haynes, Barton

    2018-01-02

    Disclosed herein are mosaic HIV envelope (Env) polypeptides that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). Also disclosed are sets of the disclosed mosaic Env polypeptides, which include two or more (for example, three) of the polypeptides. Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in a subject comprising administering to the subject at least one (such as two, three, or more) of the immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.

  19. Design of a hyperstable 60-subunit protein icosahedron

    Science.gov (United States)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  20. A Plasmodium falciparum 48/45 single epitope R0.6C subunit protein elicits high levels of transmission blocking antibodies

    DEFF Research Database (Denmark)

    Singh, Susheel K; Roeffen, Will; Andersen, Gorm

    2015-01-01

    The sexual stage Pfs48/45 antigen is a well-established lead candidate for a transmission blocking (TB) vaccine because of its critical role in parasite fertilization. We have recently produced the carboxy-terminal 10C-fragment of Pfs48/45 containing three known epitopes for TB antibodies......-units (6C). All constructs harbor the major epitope I for TB antibodies. One of these sub-units (R0.6Cc), produced high yields of correctly folded conformers, which could be purified by a simple 2-step procedure. Purified R0.6Cc was stable and elicits high titer TB antibodies in rats. The yield, purity...

  1. Immunogenicity and safety of high-dose hepatitis B vaccine among drug users: A randomized, open-labeled, blank-controlled trial.

    Science.gov (United States)

    Feng, Yongliang; Shi, Jing; Gao, Linying; Yao, Tian; Feng, Dan; Luo, Dan; Li, Zhansheng; Zhang, Yawei; Wang, Fuzhen; Cui, Fuqiang; Li, Li; Liang, Xiaofeng; Wang, Suping

    2017-06-03

    Due to the low uptake, adherence, and completion of vaccination among drug users, and their compromised immune responses to hepatitis B vaccination, the current practice of hepatitis B vaccination may not provide optimal protection. The aim of this study was to evaluate the immunogenicity and safety of 60 µg and 20 µg hepatitis B vaccines among drug users. A randomized, open-labeled, blank-controlled trial was conducted among drug users at 2 drug rehabilitation centers in China. The eligible participants were drug users who were serologically negative for the hepatitis B surface antigen (HBsAg) and the hepatitis B surface antibody (anti-HBs). Participants were randomized in a ratio of 1:1:1 to receive 20 µg (IM20 group) or 60 µg (IM60 group) of hepatitis B vaccine or blank control at months 0, 1, and 6, and followed at months 6, 7, and 12. Seroconversion rates of 94.7% and 92.6% were observed in IM20 and IM60 groups at month 7, and correspondingly decreased to 89.5% and 91.7% respectively at month 12. The IM60 group showed significantly higher geometric mean concentrations (GMCs) of anti-HBs (2022.5 and 676.7 mIU mL-1) than the IM20 group did (909.6 and 470.5 mIU mL-1) at months 7 and 12 (P B vaccines showed good immunogenicity among the drug users.

  2. Safety and immunogenicity of H1/IC31®, an adjuvanted TB subunit vaccine, in HIV-infected adults with CD4+ lymphocyte counts greater than 350 cells/mm3: a phase II, multi-centre, double-blind, randomized, placebo-controlled trial.

    Directory of Open Access Journals (Sweden)

    Klaus Reither

    Full Text Available Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1 formulated with the adjuvant IC31, was evaluated in HIV-infected adults.HIV-infected adults with CD4+ T cell counts >350/mm3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5:1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay.47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015. Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells.H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response.Pan African Clinical Trials Registry (PACTR PACTR201105000289276.

  3. Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

    Science.gov (United States)

    Silva, Wellington M; Berger, Madeleine; Bass, Chris; Williamson, Martin; Moura, Danielle M N; Ribeiro, Lílian M S; Siqueira, Herbert A A

    2016-07-01

    The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in

  4. Vaccine profile of herpes zoster (HZ/su) subunit vaccine.

    Science.gov (United States)

    Cunningham, Anthony L; Heineman, Thomas

    2017-07-01

    Herpes zoster (HZ) causes an often severe and painful rash in older people and may be complicated by prolonged pain (postherpetic neuralgia; PHN) and by dissemination in immune-compromised patients. HZ results from reactivation of latent varicella-zoster virus (VZV) infection, often associated with age-related or other causes of decreased T cell immunity. A live attenuated vaccine boosts this immunity and provides partial protection against HZ, but this decreases with age and declines over 8 years. Areas covered: A new HZ subunit (HZ/su) vaccine combines a key surface VZV glycoprotein (E) with a T cell-boosting adjuvant system (AS01 B ) and is administered by two intramuscular injections two months apart. Expert commentary: HZ/su showed excellent efficacy of ~90% in immunocompetent adults ≥50 and ≥70 years of age, respectively, in the ZOE-50 and ZOE-70 phase III controlled trials. Efficacy was unaffected by advancing age and persisted for >3 years. Approximately 9.5% of subjects had severe, but transient (1-2 days) injection site pain, swelling or redness. Compliance with both vaccine doses was high (95%). The vaccine will have a major impact on HZ management. Phase I-II trials showed safety and immunogenicity in severely immunocompromised patients. Phase III trial results are expected soon.

  5. Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling.

    Directory of Open Access Journals (Sweden)

    Felix Geeraedts

    2008-08-01

    Full Text Available In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV or subunit (SU vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR. The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.

  6. Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling.

    Science.gov (United States)

    Geeraedts, Felix; Goutagny, Nadege; Hornung, Veit; Severa, Martina; de Haan, Aalzen; Pool, Judith; Wilschut, Jan; Fitzgerald, Katherine A; Huckriede, Anke

    2008-08-29

    In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.

  7. Cloning and characterization of hypoxia-inducible factor-1 subunits from Ascaris suum - a parasitic nematode highly adapted to changes of oxygen conditions during its life cycle.

    Science.gov (United States)

    Goto, Miho; Amino, Hisako; Nakajima, Mikage; Tsuji, Naotoshi; Sakamoto, Kimitoshi; Kita, Kiyoshi

    2013-03-01

    The parasitic nematode Ascaris suum successfully adapts to a significant decrease in oxygen availability during its life cycle by altering its metabolic system dramatically. However, little is known about the regulatory mechanisms of adaptation to hypoxic environments in A. suum. In multicellular organisms, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits, is a master regulator of genes involved in adaptation to hypoxia. In the present study, cDNAs encoding HIF-1α and HIF-1β were cloned from A. suum and characterized. The full-length A. suum hif-1α and hif-1β cDNAs contain open reading frames encoding proteins with 832 and 436 amino acids, respectively. In the deduced amino acid sequences of A. suum HIF-1α and HIF-1β, functional domains essential for DNA-binding, dimerization, and oxygen-dependent prolyl hydroxylation were conserved. The interaction between A. suum HIF-1α and HIF-1β was confirmed by the yeast two-hybrid assay. Both A. suum hif-1α and hif-1β mRNAs were expressed at all stages examined (fertilized eggs, third-stage larvae, lung-stage larvae, young adult worms, and adult muscle tissue), and most abundantly in the aerobic free-living third-stage larvae, followed by a gradual decrease after infection of the host. hif-1 mRNA transcription was not sensitive to the oxygen environment in either third-stage larvae or adult worms (muscle tissue), and was regulated in a stage-specific manner. High expression of hif-1 mRNAs in third-stage larvae suggests its contribution to pre-adaptation to a hypoxic environment after infection of their host. Sequence analysis of 5'-upstream regions of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) genes, which show stage-specific expression and play an important role in oxygen adaptation during the life cycle, revealed that all subunits except for the adult-type flavoprotein subunit (Fp) possess putative hypoxia

  8. Further progress on defining highly conserved immunogenic epitopes for a global HIV vaccine: HLA-A3-restricted GAIA vaccine epitopes.

    Science.gov (United States)

    De Groot, Anne S; Levitz, Lauren; Ardito, Matthew T; Skowron, Gail; Mayer, Kenneth H; Buus, Soren; Boyle, Christine M; Martin, William D

    2012-07-01

    Two major obstacles confronting HIV vaccine design have been the extensive viral diversity of HIV-1 globally and viral evolution driven by escape from CD8(+) cytotoxic T-cell lymphocyte (CTL)-mediated immune pressure. Regions of the viral genome that are not able to escape immune response and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs of HIV-infected donors from Providence, Rhode Island. Validation of these HLA-A3 epitopes conserved across time, clades, and geography supports the hypothesis that epitopes such as these would be candidates for inclusion in our globally relevant GAIA HIV vaccine constructs.

  9. allelic variation of hmw glutenin subunits of ethiopian bread wheat ...

    African Journals Online (AJOL)

    journal

    reduced subunits of glutenin proteins bands are separated: the high molecular weight (HMW) and low molecular weight (LMW) subunits (Payne et al.,1980; Jackson et al., 1983). The HMW glutenin subunits (GS) of wheat protein are quantitatively minor, but functionally an important group of gluten proteins in the process of ...

  10. Properties and expression of Na+/K+-ATPase α-subunit isoforms in the brain of the swamp eel, Monopterus albus, which has unusually high brain ammonia tolerance.

    Directory of Open Access Journals (Sweden)

    Xiu L Chen

    Full Text Available The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l(-1 and accumulate ammonia to high concentrations in its brain (4.5 µmol g(-1. Na(+/K(+-ATPase (Nka is an essential transporter in brain cells, and since NH4(+ can substitute for K(+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K(+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4(+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na(+/K(+-ATPase and Na(+/NH4(+-ATPase activities over a range of K(+/NH4(+ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l(-1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na(+/NH4(+-ATPase activities were significantly lower than the Na(+/K(+-ATPase activities assayed at various NH4(+/K(+ concentrations. Furthermore, the effectiveness of NH4(+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1 lack of nkaα2 expression, (2 high K(+ specificity of K(+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3 down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.

  11. Subunit mass analysis for monitoring antibody oxidation.

    Science.gov (United States)

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  12. Key points in evaluating immunogenicity of pandemic influenza vaccines: A lesson from immunogenicity studies of influenza A(H1N1)pdm09 vaccine.

    Science.gov (United States)

    Ohfuji, Satoko; Kobayashi, Masayuki; Ide, Yuichiro; Egawa, Yumi; Saito, Tomoko; Kondo, Kyoko; Ito, Kazuya; Kase, Tetsuo; Maeda, Akiko; Fukushima, Wakaba; Hirota, Yoshio

    2017-09-18

    Immunogenicity studies on pandemic influenza vaccine are necessary to inform rapid development and implementation of a vaccine during a pandemic. Thus, strategies for immunogenicity assessment are required. To identify essential factors to consider when evaluating the immunogenicity of pandemic influenza vaccines using the experience in Japan with the influenza A(H1N1)pdm09 vaccine. We conducted a search of observational studies using PubMed and IchushiWeb. Search terms included "influenza vaccine AND (immunogenicity OR immune response) AND Japan AND (2009 OR pdm09) NOT review," and was limited to studies conducted in humans. A total of 33 articles were identified, of which 16 articles met the inclusion criteria. Immunogenicity of the commercially available influenza A(H1N1)pdm09 vaccine satisfied the international criteria for influenza vaccine immunogenicity in all study populations. The most remarkable immune response was observed in junior high school students, while the lowest immune response was observed in hematological malignancy patients. Similar to immunogenicity studies on seasonal influenza vaccines, factors such as patient background (e.g., age, underlying condition, pre-vaccination titer, body mass index, etc.) and study procedure (e.g., concurrent measurement of pre- and post-vaccination antibody titer, effects of infection during the study period) may have affected the assessment of immunogenicity to the influenza A(H1N1)pdm09 vaccine. In addition, prior vaccination with the seasonal influenza vaccine may inhibit antibody induction by the influenza A(H1N1)pdm09 vaccine. This review discusses factors and strategies that must be considered and addressed during immunogenicity assessments of pandemic influenza vaccines, which may provide useful information for future influenza pandemics. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  13. High frequency stimulation alters motor maps, impairs skilled reaching performance and is accompanied by an upregulation of specific GABA, glutamate and NMDA receptor subunits.

    Science.gov (United States)

    Henderson, A K; Pittman, Q J; Teskey, G C

    2012-07-26

    High frequency stimulation (HFS) has the potential to interfere with learning and memory. HFS and motor skill training both lead to potentiation of the stimulated network and alter motor map expression. However, the extent to which HFS can interfere with the learning and performance of a skilled motor task and the resulting effect on the representation of movement has not been examined. Moreover, the molecular mechanisms associated with HFS and skilled motor training on the motor cortex are not known. We hypothesized that HFS would impair performance on a skilled reaching task, and would be associated with alterations in motor map expression and protein levels compared to non-stimulated and untrained controls. Long Evans Hooded rats were chronically implanted with stimulating and recording electrodes in the corpus callosum and frontal neocortex, respectively. High frequency theta burst stimulation or sham stimulation was applied once daily for 20 sessions. The rats were divided into five groups: control, HFS and assessed at 1 week post stimulation, HFS and assessed 3 weeks post stimulation, reach trained, and HFS and reach trained. A subset of rats from each group was assessed with either intracortical microstimulation (ICMS) to examine motor map expression or Western blot techniques to determine protein expression of several excitatory and inhibitory receptor subunits. Firstly, we found that HFS resulted in larger and reorganized motor maps, and lower movement thresholds compared to controls. This was associated with an up-regulation of the GABA(A)α1 and NR1 receptor subunits 3 weeks after the last stimulation session only. Stimulation affected skilled reaching performance in a subset of all stimulated rats. Rats that were poor performers had larger rostral forelimb areas, higher proximal and lower distal movement thresholds compared to rats that were good performers after stimulation. Reach training alone was associated with an up-regulation of GABA(A)α1, α2

  14. Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB Affect Protein Function.

    Directory of Open Access Journals (Sweden)

    Scott H Millen

    Full Text Available Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.

  15. In a randomized trial, the live attenuated tetravalent dengue vaccine TV003 is well-tolerated and highly immunogenic in subjects with flavivirus exposure prior to vaccination

    Science.gov (United States)

    Whitehead, Stephen S.; Durbin, Anna P.; Pierce, Kristen K.; Elwood, Dan; McElvany, Benjamin D.; Fraser, Ellen A.; Carmolli, Marya P.; Tibery, Cecilia M.; Hynes, Noreen A.; Jo, Matthew; Lovchik, Janece M.; Larsson, Catherine J.; Doty, Elena A.; Dickson, Dorothy M.; Luke, Catherine J.; Subbarao, Kanta; Kirkpatrick, Beth D.

    2017-01-01

    Infection caused by the four serotypes of dengue virus (DENV-1-4) is a leading cause of mosquito-borne disease. Clinically-severe dengue disease is more common when secondary dengue infection occurs following prior infection with a heterologous dengue serotype. Other flaviviruses such as yellow fever virus, Japanese encephalitis virus, and Zika virus, can also elicit antibodies which are cross-reactive to DENV. As candidate dengue vaccines become available in endemic settings and for individuals who have received other flavivirus vaccines, it is important to examine vaccine safety and immunogenicity in these flavivirus-experienced populations. We performed a randomized, controlled trial of the National Institutes of Health live attenuated tetravalent dengue vaccine candidate (TV003) in fifty-eight individuals with prior exposure to flavivirus infection or vaccine. As in prior studies of this vaccine in flavivirus-naive volunteers, flavivirus-experienced subjects received two doses of vaccine six months apart and were followed closely for clinical events, laboratory changes, viremia, and neutralizing antibody titers. TV003 was well tolerated with few adverse events other than rash, which was predominately mild. Following one dose, 87% of vaccinees had an antibody response to all four serotypes (tetravalent response), suggesting a robust immune response. In addition, 76% of vaccinees were viremic; mean peak titers ranged from 0.68–1.1 log10 PFU/mL and did not differ by serotype. The second dose of TV003 was not associated with viremia, rash, or a sustained boost in antibody titers indicating that a single dose of the vaccine is likely sufficient to prevent viral replication and thus protect against disease. In comparison to the viremia and neutralizing antibody response elicited by TV003 in flavivirus-naïve subjects from prior studies, we found that subjects who were flavivirus-exposed prior to vaccination exhibited slightly higher DENV-3 viremia, higher

  16. Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

    Science.gov (United States)

    Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

    2015-04-01

    mouse model while retaining the required immunogenicity needed for subsequent protection against infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

    Science.gov (United States)

    Tiner, Bethany L.; Kirtley, Michelle L.; Erova, Tatiana E.; Popov, Vsevolod L.; Baze, Wallace B.; van Lier, Christina J.; Ponnusamy, Duraisamy; Andersson, Jourdan A.; Motin, Vladimir L.; Chauhan, Sadhana

    2015-01-01

    mouse model while retaining the required immunogenicity needed for subsequent protection against infection. PMID:25605764

  18. Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.

    Science.gov (United States)

    Thakur, Dipak; Rejtar, Tomas; Karger, Barry L; Washburn, Nathaniel J; Bosques, Carlos J; Gunay, Nur S; Shriver, Zachary; Venkataraman, Ganesh

    2009-11-01

    With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for

  19. High-molecular-weight glutenin subunit-deficient mutants induced by ion beam and the effects of Glu-1 loci deletion on wheat quality properties.

    Science.gov (United States)

    Zhang, Lujun; Chen, Qiufang; Su, Mingjie; Yan, Biao; Zhang, Xiangqi; Jiao, Zhen

    2016-03-15

    High-molecular-weight glutenin subunits (HMW-GSs) play a critical role in determining the viscoelastic properties of wheat. Mutations induced by ion beam radiation have been applied to improve the yield and quality of crop. In this study, HMW-GS-deficient mutant lines were selected and the effects of Glu-1 loci deletion on wheat quality properties were illustrated according to the analysis of dry seeds of common wheat (Triticum aestivum L.) Xiaoyan 81 treated with a nitrogen ion beam. Three HMW-GS-deficient mutant lines were obtained and then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Large-chromosome-fragment deletion resulted in specific deficiencies, and the deleted region sizes were determined using molecular markers. Agronomic characters, quantity and proportion of glutenins and dough microstructure of the deletion lines all proved to be quite different from those of wild-type Xiaoyan 81. Analysis of quality properties suggested that GluA1(-) had superior property parameters, while GluB1(-) and GluD1(-) both showed a significant decrease in quality properties compared with Xiaoyan 81. The effects of the three Glu-1 loci on flour and dough quality-related parameters should be Glu-D1 > Glu-B1 > Glu-A1. Ion beam radiation can be used as a mutagen to create new crop mutants. © 2015 Society of Chemical Industry.

  20. Variation in concentrations of high-molecular-weight glutenin subunits and macropolymers in wheat grains of a recombinant inbred lines population and in two contrasting eco-sites in China

    DEFF Research Database (Denmark)

    Li, Xiangnan; Cai, Jian; Liu, Fulai

    2012-01-01

    BACKGROUND: Concentrations of high-molecular-weight glutenin subunits andmacropolymers in wheat grains are important indicators of grain quality, which are genetically determined and affected by environmental factors. The 6 VS·6AL translocation chromosome segment is reported to own high powdery m...... wheat cultivars for high resistence to powdery mildew and yellow rust with less risk of undesirable effects on grain quality. c 2012 Society of Chemical Industry Supporting information may be found in the online version of this article.......BACKGROUND: Concentrations of high-molecular-weight glutenin subunits andmacropolymers in wheat grains are important indicators of grain quality, which are genetically determined and affected by environmental factors. The 6 VS·6AL translocation chromosome segment is reported to own high powdery...... mildew and yellow rust resistance genes of Pm21 and Yr26. This study investigated the variation in concentrations of high-molecular-weight glutenin subunits (HMW-GS) and gluteninmacropolymer (GMP) in response to the 6 VS·6AL translocation segment and the two contrasting sites. RESULTS: Large variations...

  1. Coexpression of the high molecular weight glutenin subunit 1Ax1 and puroindoline improves dough mixing properties in durum wheat (Triticum turgidum L. ssp. durum).

    Science.gov (United States)

    Li, Yin; Wang, Qiong; Li, Xiaoyan; Xiao, Xin; Sun, Fusheng; Wang, Cheng; Hu, Wei; Feng, Zhijuan; Chang, Junli; Chen, Mingjie; Wang, Yuesheng; Li, Kexiu; Yang, Guangxiao; He, Guangyuan

    2012-01-01

    Wheat end-use quality mainly derives from two interrelated characteristics: the compositions of gluten proteins and grain hardness. The composition of gluten proteins determines dough rheological properties and thus confers the unique viscoelastic property on dough. One group of gluten proteins, high molecular weight glutenin subunits (HMW-GS), plays an important role in dough functional properties. On the other hand, grain hardness, which influences the milling process of flour, is controlled by Puroindoline a (Pina) and Puroindoline b (Pinb) genes. However, little is known about the combined effects of HMW-GS and PINs on dough functional properties. In this study, we crossed a Pina-expressing transgenic line with a 1Ax1-expressing line of durum wheat and screened out lines coexpressing 1Ax1 and Pina or lines expressing either 1Ax1 or Pina. Dough mixing analysis of these lines demonstrated that expression of 1Ax1 improved both dough strength and over-mixing tolerance, while expression of PINA detrimentally affected the dough resistance to extension. In lines coexpressing 1Ax1 and Pina, faster hydration of flour during mixing was observed possibly due to the lower water absorption and damaged starch caused by PINA expression. In addition, expression of 1Ax1 appeared to compensate the detrimental effect of PINA on dough resistance to extension. Consequently, coexpression of 1Ax1 and PINA in durum wheat had combined effects on dough mixing behaviors with a better dough strength and resistance to extension than those from lines expressing either 1Ax1 or Pina. The results in our study suggest that simultaneous modulation of dough strength and grain hardness in durum wheat could significantly improve its breadmaking quality and may not even impair its pastamaking potential. Therefore, coexpression of 1Ax1 and PINA in durum wheat has useful implications for breeding durum wheat with dual functionality (for pasta and bread) and may improve the economic values of durum

  2. Coexpression of the high molecular weight glutenin subunit 1Ax1 and puroindoline improves dough mixing properties in durum wheat (Triticum turgidum L. ssp. durum.

    Directory of Open Access Journals (Sweden)

    Yin Li

    Full Text Available Wheat end-use quality mainly derives from two interrelated characteristics: the compositions of gluten proteins and grain hardness. The composition of gluten proteins determines dough rheological properties and thus confers the unique viscoelastic property on dough. One group of gluten proteins, high molecular weight glutenin subunits (HMW-GS, plays an important role in dough functional properties. On the other hand, grain hardness, which influences the milling process of flour, is controlled by Puroindoline a (Pina and Puroindoline b (Pinb genes. However, little is known about the combined effects of HMW-GS and PINs on dough functional properties. In this study, we crossed a Pina-expressing transgenic line with a 1Ax1-expressing line of durum wheat and screened out lines coexpressing 1Ax1 and Pina or lines expressing either 1Ax1 or Pina. Dough mixing analysis of these lines demonstrated that expression of 1Ax1 improved both dough strength and over-mixing tolerance, while expression of PINA detrimentally affected the dough resistance to extension. In lines coexpressing 1Ax1 and Pina, faster hydration of flour during mixing was observed possibly due to the lower water absorption and damaged starch caused by PINA expression. In addition, expression of 1Ax1 appeared to compensate the detrimental effect of PINA on dough resistance to extension. Consequently, coexpression of 1Ax1 and PINA in durum wheat had combined effects on dough mixing behaviors with a better dough strength and resistance to extension than those from lines expressing either 1Ax1 or Pina. The results in our study suggest that simultaneous modulation of dough strength and grain hardness in durum wheat could significantly improve its breadmaking quality and may not even impair its pastamaking potential. Therefore, coexpression of 1Ax1 and PINA in durum wheat has useful implications for breeding durum wheat with dual functionality (for pasta and bread and may improve the economic

  3. Oral immunogenicity of the plant proteinase bromelain.

    Science.gov (United States)

    Hale, Laura P; Fitzhugh, David J; Staats, Herman F

    2006-12-20

    Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

  4. Immunogenicity of HIV-1LAI gp160 and env peptides in squirrel monkey Saimiri sciureus using alumine and experimental adjuvants.

    Science.gov (United States)

    Perraut, R; Chouteau, P; Moog, C; Bonnemains, B; Kieny, M P

    1996-12-01

    Since the identification of the HIV virus, important advances have been achieved in the definition of potential subunit vaccines. We investigated the immunogenicity of a recombinant gp160 antigen and of two gp41 peptides from HIV-1LAI associated with seven different adjuvant formulations in squirrel monkeys. All animals were immunized twice with gp160 and then with a gp41 peptide using the same formulation. All adjuvants used led to a subsequent antibody response against gp160, and 55% of the animals immunized developed anti-gp160 antibodies that could neutralize the virus in vitro. Specific anti-gp41 antibody response was also observed. Results obtained underlined the key role of the adjuvant formulation in the antibody response against a given part of the immunogen, and indicate that such immunogenicity-related investigation can be carried out conveniently in the squirrel monkey Saimiri sciureus.

  5. Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Bert Lagrain

    Full Text Available The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS, the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC, and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%, the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and

  6. An Approach to Identify and Characterize a Subunit Candidate Shigella Vaccine Antigen.

    Science.gov (United States)

    Pore, Debasis; Chakrabarti, Manoj K

    2016-01-01

    Shigellosis remains a serious issue throughout the developing countries, particularly in children under the age of 5. Numerous strategies have been tested to develop vaccines targeting shigellosis; unfortunately despite several years of extensive research, no safe, effective, and inexpensive vaccine against shigellosis is available so far. Here, we illustrate in detail an approach to identify and establish immunogenic outer membrane proteins from Shigella flexneri 2a as subunit vaccine candidates.

  7. Starch characteristics of transgenic wheat (Triticum aestivum L.) overexpressing the Dx5 high molecular weight glutenin subunit are substantially equivalent to those in nonmodified wheat.

    Science.gov (United States)

    Beckles, Diane M; Tananuwong, Kanitha; Shoemaker, Charles F

    2012-04-01

    The effects of engineering higher levels of the High Molecular Weight Glutenin Dx5 subunit on starch characteristics in transgenic wheat (Triticum aestivum L.) grain were evaluated. This is important because of the interrelationship between starch and protein accumulation in grain, the strong biotechnological interest in modulating Dx5 levels and the increasing likelihood that transgenic wheat will be commercialized in the U.S. Unintended effects of Dx5 overexpression on starch could affect wheat marketability and therefore should be examined. Two controls with native levels of Dx5 were used: (i) the nontransformed Bobwhite cultivar, and (ii) a transgenic line (Bar-D) expressing a herbicide resistant (bar) gene, and they were compared with 2 transgenic lines (Dx5G and Dx5J) containing bar and additional copies of Dx5. There were few changes between Bar-D and Dx5G compared to Bobwhite. However, Dx5J, the line with the highest Dx5 protein (×3.5) accumulated 140% more hexose, 25% less starch and the starch had a higher frequency of longer amylopectin chains. These differences were not of sufficient magnitude to influence starch functionality, because granule morphology, crystallinity, amylose-to-amylopectin ratio, and the enthalpy of starch gelatinization and the amylose-lipid complex melting were similar to the control (P > 0.05). This overall similarity was borne out by Partial Least Squares-Discriminant Function Analysis, which could not distinguish among genotypes. Collectively our data imply that higher Dx5 can affect starch accumulation and some aspects of starch molecular structure but that the starches of the Dx5 transgenic wheat lines are substantially equivalent to the controls. Transgenic manipulation of biochemical pathways is an effective way to enhance food sensory quality, but it can also lead to unintended effects. These spurious changes are a concern to Government Regulatory Agencies and to those Industries that market the product. In this study we

  8. Mitotic illegitimate recombination is a mechanism for novel changes in high-molecular-weight glutenin subunits in wheat-rye hybrids.

    Directory of Open Access Journals (Sweden)

    Zhongwei Yuan

    Full Text Available Wide hybrids can have novel traits or changed expression of a quantitative trait that their parents do not have. These phenomena have long been noticed, yet the mechanisms are poorly understood. High-molecular-weight glutenin subunits (HMW-GS are seed storage proteins encoded by Glu-1 genes that only express in endosperm in wheat and its related species. Novel HMW-GS compositions have been observed in their hybrids. This research elucidated the molecular mechanisms by investigating the causative factors of novel HMW-GS changes in wheat-rye hybrids. HMW-GS compositions in the endosperm and their coding sequences in the leaves of F(1 and F(2 hybrids between wheat landrace Shinchunaga and rye landrace Qinling were investigated. Missing and/or additional novel HMW-GSs were observed in the endosperm of 0.5% of the 2078 F(1 and 22% of 36 F(2 hybrid seeds. The wildtype Glu-1Ax null allele was found to have 42 types of short repeat sequences of 3-60 bp long that appeared 2 to 100 times. It also has an in-frame stop codon in the central repetitive region. Analyzing cloned allele sequences of HMW-GS coding gene Glu-1 revealed that deletions involving the in-frame stop codon had happened, resulting in novel ∼1.8-kb Glu-1Ax alleles in some F(1 and F(2 plants. The cloned mutant Glu-1Ax alleles were expressed in Escherichia coli, and the HMW-GSs produced matched the novel HMW-GSs found in the hybrids. The differential changes between the endosperm and the plant of the same hybrids and the data of E. coli expression of the cloned deletion alleles both suggested that mitotic illegitimate recombination between two copies of a short repeat sequence had resulted in the deletions and thus the changed HMW-GS compositions. Our experiments have provided the first direct evidence to show that mitotic illegitimate recombination is a mechanism that produces novel phenotypes in wide hybrids.

  9. Stoichiometry of δ subunit containing GABA(A) receptors.

    Science.gov (United States)

    Patel, B; Mortensen, M; Smart, T G

    2014-02-01

    Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. © 2013 The British Pharmacological Society.

  10. Phase I immunotherapeutic trial with long peptides spanning the E6 and E7 sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patients shows low toxicity and robust immunogenicity

    NARCIS (Netherlands)

    Kenter, Gemma G.; Welters, Marij J. P.; Valentijn, A. Rob P. M.; Lowik, Margriet J. G.; Berends-van der Meer, Dorien M. A.; Vloon, Annelies P. G.; Drijfhout, Jan W.; Wafelman, Amon R.; Oostendorp, Jaap; Fleuren, Gert Jan; Offringa, Rienk; van der Burg, Sjoerd H.; Melief, Cornelis J. M.

    2008-01-01

    PURPOSE: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. EXPERIMENTAL DESIGN: Three groups of end-stage cervical cancer patients (in total n = 35) were s.c. vaccinated with

  11. Using Fitness Landscapes for Rational Hepatitis C Immunogen Design

    Science.gov (United States)

    Hart, Gregory; Ferguson, Andrew

    2015-03-01

    Hepatitis C virus afflicts 170 million people worldwide, 2-3% of the global population. Prophylactic vaccination offers the most realistic and cost effective hope of controlling this epidemic, particularly in the developing world where expensive drug therapies are unavailable. Despite 20 years of research, the high mutability of the virus, and lack of knowledge of what constitutes effective immune responses, have impeded development of an effective vaccine. Coupling data mining of sequence databases with the Potts model, we have developed a computational approach to systematically identify viral vulnerabilities and perform rational design of vaccine immunogens. We applied our approach to the nonstructural proteins NS3, NSA, NSA, and NSB which are crucial for viral replication.The predictions of our model are in good accord with experimental measurements and clinical observations, and we have used our model to design immunogen candidates to elicit T-cell responses against vulnerable regions of theseviral proteins.

  12. A multi-subunit Chlamydia vaccine inducing neutralizing antibodies and strong IFN-γ(+) CMI responses protects against a genital infection in minipigs

    DEFF Research Database (Denmark)

    Bøje, Sarah; Olsen, Anja Weinreich; Erneholm, Karin

    2016-01-01

    animal model, we evaluated the immunogenicity and efficacy of a multi-subunit vaccine formulated in the strong Th1-inducing adjuvant CAF01. We evaluated a mixture of two fusion proteins (Hirep1 and CTH93) designed to promote either neutralizing antibodies or cell-mediated immunity, respectively. Hirep1......Chlamydia is the most widespread sexually transmitted bacterial disease and a prophylactic vaccine is highly needed. Ideally, this vaccine is required to induce a combined response of Th1 cell-mediated immune (CMI) response in concert with neutralizing antibodies. Using a novel Göttingen minipig...... trachomatis SvD bacteria (UV-SvD/CAF01) or CAF01. The Hirep1+CTH93/CAF01 vaccine induced a strong CMI response against the vaccine antigens and high titers of antibodies, particularly against the VD4 region of MOMP. Sera from Hirep1+CTH93/CAF01 immunized pigs neutralized C. trachomatis SvD and SvF infectivity...

  13. Cholera vaccine candidate 638: intranasal immunogenicity and expression of a foreign antigen from the pulmonary pathogen Coccidioides immitis.

    Science.gov (United States)

    Silva, Anisia J; Mohan, Archana; Benitez, Jorge A

    2003-12-01

    Vibrio cholerae strain 638 is a live genetically attenuated candidate cholera vaccine in which the CTXPhi prophage encoding cholera toxin has been deleted and hapA, encoding an extracellular Zn-dependent metalloprotease, was insertionally inactivated. Strain 638 was highly immunogenic when inoculated to adult Swiss mice by the intranasal route as judged by the induction of a strong serum vibriocidal antibody response. A side-by-side comparison of strain 638 with its isogenic hapA(+) precursor (strain 81) in the above model indicated that inactivation of hapA does not affect immunogenicity. The spherule-associated antigen 2/proline-rich antigen (Ag2/PRA) of Coccidioides immitis has been shown to protect mice against coccidioidomycosis to an extent dependent on the modes of antigen presentation and challenge with C. immitis arthrospores. In this work, we demonstrate the use of a live genetically attenuated V. cholerae strain to deliver Ag2/PRA. Ag2/PRA was expressed in 638 as a fusion protein with the Escherichia coli heat labile toxin B subunit leader peptide using the strong Tac promoter. The recombinant Ag2/PRA was efficiently expressed, processed and secreted to the periplasmic space. Intranasal immunizations of adult mice with strain 638 expressing Ag2/PRA induced serum vibriocidal antibody response to the vector strain and serum total IgG response to Ag2/PRA. Strain 638 expressing PRA could be recovered from trachea and lung up to 20h after immunization but was effectively cleared 72h post-inoculation.

  14. A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Goujani, Goli; Amani, Jafar; Ahangari, Ghasem; Akhavian, Asal; Jafari, Mahyat

    2017-07-01

    Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  15. Microarray-based method for screening of immunogenic proteins from bacteria

    Directory of Open Access Journals (Sweden)

    Hoppe Sebastian

    2012-03-01

    Full Text Available Abstract Background Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures. Results Four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168. Conclusions The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and

  16. Tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the Middle East respiratory coronavirus (MERS-CoV receptor-binding domain as an antigen.

    Directory of Open Access Journals (Sweden)

    Jiaming Lan

    Full Text Available The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV pandemic. Some studies have indicated the receptor-binding domain (RBD protein of MERS-CoV spike (S is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m. with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum alone, alum and polyriboinosinic acid (poly I:C or alum and cysteine-phosphate-guanine (CpG oligodeoxynucleotides (ODN. The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA and CpG ODN by a subcutaneous (s.c. route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production. Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting

  17. An experimental subunit vaccine based on Bluetongue virus 4 VP2 protein fused to an antigen-presenting cells single chain antibody elicits cellular and humoral immune responses in cattle, guinea pigs and IFNAR(-/-) mice.

    Science.gov (United States)

    Legisa, D M; Perez Aguirreburualde, M S; Gonzalez, F N; Marin-Lopez, A; Ruiz, V; Wigdorovitz, A; Martinez-Escribano, J A; Ortego, J; Dus Santos, M J

    2015-05-21

    Bluetongue virus (BTV), the causative agent of bluetongue disease (BT) in domestic and wild ruminants, is worldwide distributed. A total of 27 serotypes have been described so far, and several outbreaks have been reported. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional ones. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the antigen presenting cell homing (APCH) molecule, in the baculovirus insect cell expression system. The immunogenicity of both proteins was evaluated in guinea pigs and cattle. Titers of specific neutralizing antibodies in guinea pigs and cattle immunized with VP2 or APCH-VP2 were high and similar to those induced by a conventional inactivated vaccine. The immunogenicity of recombinant proteins was further studied in the IFNAR(-/-) mouse model where the fusion of VP2 to APCH enhanced the cellular immune response and the neutralizing activity induced by VP2. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Subunit Vaccines Consisting of Antigens from Dormant and Replicating Bacteria Show Promising Therapeutic Effect against Mycobacterium Bovis BCG Latent Infection.

    Science.gov (United States)

    Li, F; Kang, H; Li, J; Zhang, D; Zhang, Y; Dannenberg, A M; Liu, X; Niu, H; Ma, L; Tang, R; Han, X; Gan, C; Ma, X; Tan, J; Zhu, B

    2017-06-01

    To screen effective antigens as therapeutic subunit vaccines against Mycobacterium latent infection, we did bioinformatics analysis and literature review to identify effective antigens and evaluated the immunogenicity of five antigens highly expressed in dormant bacteria, which included Rv2031c (HspX), Rv2626c (Hrp1), Rv2007c (FdxA), Rv1738 and Rv3130c. Then, several fusion proteins such as Rv2007c-Rv2626c (F6), Rv2031c-Rv1738-Rv1733c (H83), ESAT6-Rv1738-Rv2626c (LT40), ESAT6-Ag85B-MPT64 -Mtb8.4 (EAMM), and EAMM-Rv2626c (LT70) were constructed and their therapeutic effects were evaluated in pulmonary Mycobacterium bovis Bacilli Calmette-Guérin (BCG) - latently infected rabbit or mouse models. The results showed that EAMM and F6 plus H83 had therapeutic effect against BCG latent infection in the rabbit model, respectively, and that the combination of EAMM with F6 plus H83 significantly reduced the bacterial load. In addition, the fusion proteins LT40 and LT70 consisting of multistage antigens showed promising therapeutic effects in the mouse model. We conclude that subunit vaccines consisting of both latency and replicating-associated antigens show promising therapeutic effects in BCG latent infection animal models. © 2017 The Foundation for the Scandinavian Journal of Immunology.

  19. Human papillomavirus L1 protein expressed in Escherichia coli self-assembles into virus-like particles that are highly immunogenic.

    Science.gov (United States)

    Chen, Yumei; Liu, Yunchao; Zhang, Gaiping; Wang, Aiping; Dong, Ziming; Qi, Yanhua; Wang, Jucai; Zhao, Baolei; Li, Ning; Jiang, Min

    2016-07-15

    HPV vaccines based on L1 virus-like particles (VLPs) provided a high degree of protection against HPVs infection. In this study, the codon optimized HPV16 L1 gene were sub-cloned into five procaryotic expression vectors (pET-28a, pET-32a, pGEX-4T-2, pE-sumo and pHSIE), and fused with different protein tags. No recombinant proteins were expressed in pET-28a-L1 and pHSIE-L1, and the proteins expressed by pET-32a-L1 plasmid with TRX-tag were in the form of inclusion body. Only SUMO-tagged and GST-tagged L1 proteins expressed by pE-Sumo-L1 or pGEX-4T-L1 were soluble. The yield of SUMO-L1 protein reached 260mg/L fermentation medium in shake flask. After SUMO tags were eliminated, a 90% purity of L1 proteins was generated by ion-exchange and Ni-NTA affinity chromatography. The purified HPV16 L1 protein self-assembled into virus-like particles (VLPs) and showed a haemagglutination activity. High titers specific and neutralizing antibodies were detected in HPV 16 L1VLPs vaccinated mice. Cytokines such as IFN-γ and IL-2 showed significant higher in VLPs vaccinated mice compared with negative control (p<0.05, p=0.055). Thus, the expression of recombinant HPV16 L1 VLPs in Escherichia coli was feasible, which could potentially be used for a VLP-based HPV vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Newcastle Disease Virus-Vectored Rabies Vaccine Is Safe, Highly Immunogenic, and Provides Long-Lasting Protection in Dogs and Cats ▿

    Science.gov (United States)

    Ge, Jinying; Wang, Xijun; Tao, Lihong; Wen, Zhiyuan; Feng, Na; Yang, Songtao; Xia, Xianzhu; Yang, Chinglai; Chen, Hualan; Bu, Zhigao

    2011-01-01

    Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 109.8 50% egg infective doses (EID50)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 108.3 EID50 completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies. PMID:21632762

  1. Dendritic cell-based in vitro assays for vaccine immunogenicity.

    Science.gov (United States)

    Vandebriel, Rob; Hoefnagel, Marcel M N

    2012-09-01

    Dendritic cells (DC) are pivotal in the induction of adaptive immune responses because they can activate naive T-cells. Moreover, they steer these adaptive immune responses by integrating various stimuli, such as from different pathogen associated molecular patterns and the cytokine milieu. Immature DC are very well capable of ingesting protein antigens, whereas mature DC are efficient presenters of peptides to naive T cells. Human DC can be readily cultured from peripheral blood mononuclear cells, which are isolated from human blood. There is a strong need to monitor in a high-throughput fashion the immunogenicity of candidate vaccines during the process of vaccine development. Furthermore, regulators require efficacy and safety testing for batch release. For some vaccines, these tests require animal testing, causing pain and discomfort, which cannot be contested because it would interfere with the test results. With the aims of promoting vaccine development and reducing the number of animals for batch release testing, we propose to use more broadly human DC for vaccine immunogenicity testing. In this commentary, this proposition is illustrated by several examples in which the maturation of human DC was successfully used to test for vaccine and adjuvant immunogenicity.

  2. Immunogenic Apoptosis as a Novel Tool for Anticancer Vaccine Development

    Directory of Open Access Journals (Sweden)

    Barbara Montico

    2018-02-01

    Full Text Available Immunogenic apoptosis, or more appropriately called immunogenic cell death (ICD, is a recently described form of apoptosis induced by a specific set of chemotherapeutic drugs or by physical therapeutic modalities, such as ionizing irradiation and photodynamic therapy. The peculiar characteristic of ICD is the ability to favor recognition and elimination of dying tumor cells by phagocytes in association with the release of pro-inflammatory molecules (such as cytokines and high-mobility group box-1. While in vitro and animal models pointed to ICD as one of the molecular mechanisms mediating the clinical efficacy of some anticancer agents, it is hard to clearly demonstrate its contribution in cancer patients. Clinical evidence suggests that the induction of ICD alone is possibly not sufficient to fully subvert the immunosuppressive tumor microenvironment. However, interesting results from recent studies contemplate the exploitation of ICD for improving the immunogenicity of cancer cells to use them as an antigen cargo in the development of dendritic cell (DC vaccines. Herein, we discuss the effects of danger signals expressed or released by cancer cells undergoing ICD on the maturation and activation of immature and mature DC, highlighting the potential added value of ICD in adoptive immunotherapy protocols.

  3. Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

    Directory of Open Access Journals (Sweden)

    Roland Züst

    Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

  4. Attenuation and Immunogenicity of a Live High Pathogenic PRRSV Vaccine Candidate with a 32-Amino Acid Deletion in the nsp2 Protein

    Directory of Open Access Journals (Sweden)

    Wenhui Lu

    2014-01-01

    Full Text Available A porcine reproductive and respiratory syndrome virus (PRRSV QY1 was serially passed on Marc-145 cells. Virulence of different intermediate derivatives of QY1 (P5, P60, P80, and P100 were determined. The study found that QY1 had been gradually attenuated during the in vitro process. Pathogenicity study showed that pigs inoculated with QY1 P100 and P80 did not develop any significant PRRS clinic symptoms. However, mild-to-moderate clinical signs and acute HP-PRRSV symptoms of infection were observed in pigs inoculated with QY1 P60 and P5, respectively. Furthermore, we determined the whole genome sequences of these four intermediate viruses. The results showed that after 100 passages, compared to QY1 P5, a total of 32 amino acid mutations were found. Moreover, there were one nucleotide deletion and a unique 34-amino acid deletion found at 5′UTR and in nsp2 gene during the attenuation process, respectively. Such deletions were genetically stable in vivo. Following PRRSV experimental challenge, pigs inoculated with a single dose of QY1 P100 developed no significant clinic symptoms and well tolerated lethal challenge, while QY1 P80 group still developed mild fever in the clinic trial after challenge. Thus, we concluded that QY1 P100 was a promising and highly attenuated PRRSV vaccine candidate.

  5. Diversity of heterotrimeric G-protein γ subunits in plants

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    Trusov Yuri

    2012-10-01

    Full Text Available Abstract Background Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. Results After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX. According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. Conclusion Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

  6. High loading of graphene oxide/multi-walled carbon nanotubes into PDLLA: A route towards the design of osteoconductive, bactericidal and non-immunogenic 3D porous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zanin, Hudson [Laboratory of Biomedical Nanotechnology (NANOBIO), Institute of Research and Development - IP& D, University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, Sao Paulo (Brazil); Laboratory of Energy Storage & Supply - ES& S, Institute of Research and Development - IP& D, University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, CEP: 12.244-000, Sao Paulo (Brazil); Rodrigues, Bruno Vinícius Manzolli [Laboratory of Biomedical Nanotechnology (NANOBIO), Institute of Research and Development - IP& D, University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, Sao Paulo (Brazil); Ribeiro Neto, Wilson Alves; Bretas, Rosario Elida Suman [Department of Materials Engineering, Federal University of Sao Carlos, Rodovia Washington Luis, km 235 – SP-310, Sao Carlos, Sao Paulo (Brazil); Da-Silva, Newton Soares [Laboratory of Cell Biology and Tissue, Institute of Research and Development - IP& D, University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, CEP: 12244-000, Sao Paulo (Brazil); Marciano, Fernanda Roberta [Laboratory of Biomedical Nanotechnology (NANOBIO), Institute of Research and Development - IP& D, University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, Sao Paulo (Brazil); Oliveira Lobo, Anderson, E-mail: aolobo@pq.cnpq.br [Laboratory of Biomedical Nanotechnology (NANOBIO), Institute of Research and Development - IP& D, University of Vale do Paraiba, Av. Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, Sao Paulo (Brazil)

    2016-07-01

    We have prepared a novel 3D porous biomaterial combining poly (DL-lactic acid) (PDLLA) and graphene and multi-walled carbon nanotubes oxides (MWCNTO-GO) composite. PDLLA as control and a high loading of PDLLA/MWCNTO-GO (50/50 w/w) bioscaffolds were prepared and functionalized. MWCNTs were exfoliated to form MWCNTO-GO by oxygen plasma etching. The later was also applied to enhance the scaffolds wettability, attaching oxygen-containing groups on their surfaces. This approach produced a porous architecture observed by scanning electron microscopy and semi-quantified by electrochemical analysis. The later also indicated a notable increase on the conductivity of PDLLA/MWCNTO-GO scaffold compared to MWCNTO-GO free PDLLA (about 5 orders of magnitudes at low frequencies). Thermogravimetric analysis showed that the MWCNTO-GO acted protecting the PDLLA matrix, enhancing its thermal stability. The PDLLA/MWCNTO-GO scaffolds had significant cellular adhesion, did not present cytotoxicity effect, besides reduced bactericidal proliferation and produced mineralized tissues in SBF media. The metallic MWCNTO-GO powder held together by PDLLA polymer opens a whole new branch of applications, including bioelectroanalyses, drug delivery systems and tissue engineering. - Highlights: • We produced a novel 3D porous material from PDLLA, graphene oxide and MWCNT oxide. • MWCNTO-GO loading (50/50 w/w) increased notably the conductivity of PDLLA scaffold. • MWCNTO-GO acted protecting the PDLLA matrix, enhancing its thermal stability. • PDLLA/MWCNTO-GO scaffolds did not present cytotoxicity effect. • PDLLA/MWCNTO-GO scaffolds presented bioactivity properties.

  7. Comparative immunogenicity in mice of rotavirus VP6 tubular structures and virus-like particles.

    Science.gov (United States)

    Lappalainen, Suvi; Tamminen, Kirsi; Vesikari, Timo; Blazevic, Vesna

    2013-09-01

    Rotavirus (RV) is the most important cause of severe gastroenteritis in children worldwide. Current live RV vaccines are efficacious but show lower efficacy in developing countries, as well as a low risk of intussusception. This has led to the development of parenteral non-live candidate vaccines against RV. RV capsid VP6 protein is highly conserved and the most abundant RV protein forming highly immunogenic oligomeric structures with multivalent antigen expression. Both recombinant VP6 (rVP6) or double-layered (dl) 2/6-virus-like particles (VLPs), might be considered as the simplest RV subunit vaccine candidates. Human rVP6 protein and dl2/6-VLPs were produced in Sf9 insect cells by baculovirus expression system. Formation of rVP6 tubules and VLPs were confirmed by electron microscopy. BALB/c mice were immunized intramuscularly, and immune responses were analyzed. Both rVP6 and dl2/6-VLPs induced a balanced Th1-type and Th2-type response and high levels of serum IgG antibodies with cross-reactivity against different RV strains (Wa, SC2, BrB, 69M, L26, WC3, and RRV). In addition, mucosal VP6-specific IgG and IgA antibodies were detected in feces and vaginal washes (VW) of immunized animals. Importantly, VWs of immunized mice inhibited RV Wa and RRV infection in vitro. Immunization with either protein preparation induced a similar level of VP6-specific, interferon-γ secreting CD4(+) T cells in response to different RVs or the 18-mer peptide (AA 242-259), a VP6-specific CD4(+) T cell epitope. RV rVP6 and dl2/6-VLPs induced equally strong humoral and cellular responses against RV in mice and therefore, may be considered as non-live vaccine candidates against RV.

  8. Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease.

    Science.gov (United States)

    Matsui, Takeshi; Takita, Eiji; Sato, Toshio; Aizawa, Michie; Ki, Misa; Kadoyama, Yumiko; Hirano, Kenji; Kinjo, Satoko; Asao, Hiroshi; Kawamoto, Keiko; Kariya, Haruko; Makino, Sou-Ichi; Hamabata, Takashi; Sawada, Kazutoshi; Kato, Ko

    2011-08-01

    Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

  9. Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults.

    Science.gov (United States)

    Moutschen, Michel; Léonard, Philippe; Sokal, Etienne M; Smets, Françoise; Haumont, Michèle; Mazzu, Pasqualina; Bollen, Alex; Denamur, Francoise; Peeters, Pascal; Dubin, Gary; Denis, Martine

    2007-06-11

    Two double-blind randomised controlled studies (phase I and I/II) were performed to assess for the first time the safety and immunogenicity of a recombinant subunit gp350 Epstein-Barr virus (EBV) vaccine in 148 healthy adult volunteers. All candidate vaccine formulations had a good safety profile and were well tolerated, with the incidence of solicited and unsolicited symptoms within a clinically acceptable range. One serious adverse event was reported in the phase I trial which was considered to be of suspected relationship to vaccination. The gp350 vaccine formulations were immunogenic and induced gp350-specific antibody responses (including neutralising antibodies).

  10. Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease.

    Science.gov (United States)

    Rapoport, Basil; Aliesky, Holly A; Chen, Chun-Rong; McLachlan, Sandra M

    2015-06-01

    The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease. This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits. Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms. The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers.

  11. Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin

    Directory of Open Access Journals (Sweden)

    Peiwu Li

    2017-05-01

    Full Text Available Research about antibody specificity spectra was conducted to develop single-specific antibodies or broad-specific antibodies. Aflatoxins, as one class of high-toxicity mycotoxins, were selected as the research targets to investigate the effect of the immunogen dose on antibody specificity spectra. For this aim, 16 monoclonal antibodies were induced by low or high doses of aflatoxin B1-BSA, and 34 monoclonal antibodies were induced by low or high doses of aflatoxin M1-BSA. The specificities of the antibodies induced, whether by aflatoxin B1 conjugate or aflatoxin M1 conjugate, indicated that the low dose of the immunogen induced a narrow spectrum of antibody specificity, while the high dose of the immunogen showed an advantage to form a broad spectrum of antibody specificity. Therefore, this report provides important information for the development of new antibodies against small molecules like aflatoxins.

  12. Live attenuated Shigella dysenteriae type 1 vaccine strains overexpressing shiga toxin B subunit.

    Science.gov (United States)

    Wu, Tao; Grassel, Christen; Levine, Myron M; Barry, Eileen M

    2011-12-01

    Shigella dysenteriae serotype 1 (S. dysenteriae 1) is unique among the Shigella species and serotypes in the expression of Shiga toxin which contributes to more severe disease sequelae and the ability to cause explosive outbreaks and pandemics. S. dysenteriae 1 shares characteristics with other Shigella species, including the capability of causing clinical illness with a very low inoculum (10 to 100 CFU) and resistance to multiple antibiotics, underscoring the need for efficacious vaccines and therapeutics. Following the demonstration of the successful attenuating capacity of deletion mutations in the guaBA operon in S. flexneri 2a vaccine strains in clinical studies, we developed a series of S. dysenteriae 1 vaccine candidates containing the fundamental attenuating mutation in guaBA. All strains are devoid of Shiga toxin activity by specific deletion of the gene encoding the StxA subunit, which encodes enzymatic activity. The StxB subunit was overexpressed in several derivatives by either plasmid-based constructs or chromosomal manipulation to include a strong promoter. All strains are attenuated for growth in vitro in the HeLa cell assay and for plaque formation and were safe in the Serény test and immunogenic in the guinea pigs. Each strain induced robust serum and mucosal anti-S. dysenteriae 1 lipopolysaccharide (LPS) responses and protected against wild-type challenge. Two strains engineered to overexpress StxB induced high titers of Shiga toxin neutralizing antibodies. These candidates demonstrate the potential for a live attenuated vaccine to protect against disease caused by S. dysenteriae 1 and potentially to protect against the toxic effects of other Shiga toxin 1-expressing pathogens.

  13. Live Attenuated Shigella dysenteriae Type 1 Vaccine Strains Overexpressing Shiga Toxin B Subunit

    Science.gov (United States)

    Wu, Tao; Grassel, Christen; Levine, Myron M.; Barry, Eileen M.

    2011-01-01

    Shigella dysenteriae serotype 1 (S. dysenteriae 1) is unique among the Shigella species and serotypes in the expression of Shiga toxin which contributes to more severe disease sequelae and the ability to cause explosive outbreaks and pandemics. S. dysenteriae 1 shares characteristics with other Shigella species, including the capability of causing clinical illness with a very low inoculum (10 to 100 CFU) and resistance to multiple antibiotics, underscoring the need for efficacious vaccines and therapeutics. Following the demonstration of the successful attenuating capacity of deletion mutations in the guaBA operon in S. flexneri 2a vaccine strains in clinical studies, we developed a series of S. dysenteriae 1 vaccine candidates containing the fundamental attenuating mutation in guaBA. All strains are devoid of Shiga toxin activity by specific deletion of the gene encoding the StxA subunit, which encodes enzymatic activity. The StxB subunit was overexpressed in several derivatives by either plasmid-based constructs or chromosomal manipulation to include a strong promoter. All strains are attenuated for growth in vitro in the HeLa cell assay and for plaque formation and were safe in the Serény test and immunogenic in the guinea pigs. Each strain induced robust serum and mucosal anti-S. dysenteriae 1 lipopolysaccharide (LPS) responses and protected against wild-type challenge. Two strains engineered to overexpress StxB induced high titers of Shiga toxin neutralizing antibodies. These candidates demonstrate the potential for a live attenuated vaccine to protect against disease caused by S. dysenteriae 1 and potentially to protect against the toxic effects of other Shiga toxin 1-expressing pathogens. PMID:21969003

  14. Evaluation of Recombinant Multi-Epitope Outer Membrane Protein-Based Klebsiella pneumoniae Subunit Vaccine in Mouse Model

    Directory of Open Access Journals (Sweden)

    Litty Babu

    2017-09-01

    Full Text Available Safety and protective efficacy of recombinant multi-epitope subunit vaccine (r-AK36 was evaluated in a mouse model. Recombinant AK36 protein comprised of immunodominant antigens from outer membrane proteins (Omp’s of Klebsiella pneumoniae namely OmpA and OmpK36. r-AK36 was highly immunogenic and the hyperimmune sera reacted strongly with native OmpA and OmpK36 proteins from different K. pneumoniae strains. Hyperimmune sera showed cross-reactivity with Omp’s of other Gram-negative organisms. Humoral responses showed a Th2-type polarized immune response with IgG1 being the predominant antibody isotype. Anti-r-AK36 antibodies showed antimicrobial effect during in vitro testing with MIC values in the range of 25–50 μg/ml on different K. pneumoniae strains. The recombinant antigen elicited three fold higher proliferation of splenocytes from immunized mice compared to those with sham-immunized mice. Anti-r-AK36 antibodies also exhibited in vitro biofilm inhibition property. Subunit vaccine r-AK36 immunization promoted induction of protective cytokines IL-2 and IFN-γ in immunized mice. When r-AK36-immunized mice were challenged with 3 × LD100 dose, ∼80% of mice survived beyond the observation period. Passive antibody administration to naive mice protected them (67% against the lethal challenge. Since the targeted OMPs are conserved among all K. pneumoniae serovars and due to the strong nature of immune responses, r-AK36 subunit vaccine could be a cost effective candidate against klebsiellosis.

  15. Potentiation of Taishan Pinus massoniana pollen polysaccharide on the immune response and protection elicited by a highly pathogenic porcine reproductive and respiratory syndrome virus glycoprotein 5 subunit in pigs.

    Science.gov (United States)

    Peng, Jun; Yuan, Yanmei; Du, Yijun; Wu, Jiaqiang; Li, Baoquan; Li, Jun; Yu, Jiang; Hu, Liping; Shen, Si; Wang, Jinbao; Zhu, Ruiliang

    2016-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) heavily affects the global pork industry. Current available vaccine strategies have inherent drawbacks. In this work, the immune enhancement from Taishan Pinus massoniana pollen polysaccharide (TPPPS) and Freund's adjuvant on the efficacy of a PRRSV subunit vaccine were examined. Titers of specific anti-highly pathogenic PRRSV (HP-PRRSV) ELISA antibody and neutralizing antibody were significantly higher in pigs from the groups inoculated with medium- and high-dose TPPPS (mTPPPS, hTPPPS) adjuvant co-administered with a recombinant HP-PRRSV glycoprotein 5 subunit (GP5) than those from other groups (P 0.05). The ratio between CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocyte subpopulations indicated the inoculums of GP5 + mTPPPS and GP5 + hTPPPS induced consistently higher CD3(+)CD4(+) T lymphocyte subpopulations than other inoculums (P 0.05). The low-dose TPPPS (lTPPPS) adjuvant also exhibited enhancement effects on humoral immune and T lymphocyte proliferation responses but these were significantly lower than the mTPPPS and hTPPPS doses (P < 0.05). Pigs challenged with HP-PRRSV from the GP5 + mTPPPS, GP5 + hTPPPS, and GP5 + Freund's adjuvant groups showed lower viremia, fewer clinical signs, and fewer pathological lung lesions compared with the groups of GP5-alone and GP5 + lTPPPS (P < 0.05). There were significant differences between the GP5-alone and GP5 + lTPPPS groups in detection indexes after viral challenge (P < 0.05). In conclusion, moderate doses of TPPPS as an adjuvant with GP5 show promise as a candidate for a HP-PRRSV subunit vaccine to efficiently prevent and control HP-PRRSV. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Stabilization of a recombinant ricin toxin A subunit vaccine through lyophilization.

    Science.gov (United States)

    Hassett, Kimberly J; Cousins, Megan C; Rabia, Lilia A; Chadwick, Chrystal M; O'Hara, Joanne M; Nandi, Pradyot; Brey, Robert N; Mantis, Nicholas J; Carpenter, John F; Randolph, Theodore W

    2013-10-01

    Lyophilization was used to prepare dry, glassy solid vaccine formulations of recombinant ricin toxin A-chain containing suspensions of colloidal aluminum hydroxide adjuvant. Four lyophilized formulations were prepared by using combinations of rapid or slow cooling during lyophilization and one of two buffers, histidine or ammonium acetate. Trehalose was used as the stabilizing excipient. Aggregation of the colloidal aluminum hydroxide suspension was reduced in formulations processed with a rapid cooling rate. Aluminum hydroxide particle size distributions, glass transition temperatures, water contents, and immunogenicities of lyophilized vaccines were independent of incubation time at 40 °C for up to 15 weeks. Mice immunized with reconstituted ricin toxin subunit A (RTA) vaccines produced RTA-specific antibodies and toxin-neutralizing antibodies (TNAs) regardless of the length of high temperature vaccine storage or the degree of aluminum adjuvant aggregation that occurred during lyophilization. In murine studies, lyophilized formulations of vaccines conferred protection against exposure to lethal doses of ricin, even after the lyophilized formulations had been stored at 40 °C for 4 weeks. A corresponding liquid formulation of vaccine stored at 40 °C elicited RTA-specific antibody titers but failed to confer immunity during a ricin challenge. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Secukinumab, a novel anti–IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity

    Science.gov (United States)

    Karle, Anette; Spindeldreher, Sebastian; Kolbinger, Frank

    2016-01-01

    ABSTRACT Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4+ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence. PMID:26817498

  18. Exploiting the immunogenic potential of cancer cells for improved dendritic cell vaccines

    Directory of Open Access Journals (Sweden)

    Lien eVandenberk

    2016-01-01

    Full Text Available Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies like dendritic cell (DC vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments and heat shock and five potent inducers of immunogenic cell death (radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses and (hypericin-based photodynamic therapy on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

  19. Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines.

    Science.gov (United States)

    Vandenberk, Lien; Belmans, Jochen; Van Woensel, Matthias; Riva, Matteo; Van Gool, Stefaan W

    2015-01-01

    Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

  20. Role of fused Mycobacterium tuberculosis immunogens and adjuvants in modern tuberculosis vaccines

    Directory of Open Access Journals (Sweden)

    Ana Paula eJunqueira-Kipnis

    2014-04-01

    Full Text Available Several approaches have been developed to improve or replace the only available vaccine for tuberculosis (TB, BCG (Bacille Calmette Guerin. The development of subunit protein vaccines is a promising strategy because it combines specificity and safety. In addition, subunit protein vaccines can be designed to have selected immune epitopes associated with immunomodulating components to drive the appropriate immune response. However, the limited antigens present in subunit vaccines reduce their capacity to stimulate a complete immune response compared with vaccines composed of live attenuated or killed microorganisms. This deficiency can be compensated by the incorporation of adjuvants in the vaccine formulation. The fusion of adjuvants with Mycobacterium tuberculosis (Mtb proteins or immune epitopes has the potential to become the new frontier in the TB vaccine development field. Researchers have addressed this approach by fusing the immune epitopes of their vaccines with molecules such as interleukins, lipids, lipoproteins, and immune stimulatory peptides, which have the potential to enhance the immune response. The fused molecules are being tested as subunit vaccines alone or within live attenuated vector contexts. Therefore, the objectives of this review are to discuss the association of Mtb fusion proteins with adjuvants; Mtb immunogens fused with adjuvants; and cytokine fusion with Mtb proteins and live recombinant vectors expressing cytokines. The incorporation of adjuvant molecules in a vaccine can be complex, and developing a stable fusion with proteins is a challenging task. Overall, the fusion of adjuvants with Mtb epitopes, despite the limited number of studies, is a promising field in vaccine development.

  1. Immunogenicity of a chimeric hepatitis A virus (HAV) carrying the HIV gp41 epitope 2F5.

    Science.gov (United States)

    Kusov, Yuri Y; Zamjatina, Natalja A; Poleschuk, Valentina F; Michailov, Michail I; Morace, Graziella; Eberle, Josef; Gauss-Müller, Verena

    2007-02-01

    Its stable particle structure combined with its high immunogenicity makes the hepatitis A virus (HAV) a perfect carrier to expose foreign epitopes to the host immune system. In an earlier report [Beneduce, F., Kusov, Y., Klinger, M., Gauss-Müller, V., Morace, G., 2002. Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface. Antiviral Res. 55, 369-377] chimeric virus-like particles (HAV-gp41) were described that carried at their surface the dominant gp41 epitope 2F5 (2F5e) of the human immunodeficiency virus HIV-1. Extending this work, we now report that chimeric virus HAV-gp41 replicates in HAV-susceptible cells as well as in non-human primates. Infected marmosets developed both an anti-HAV and anti-2F5 epitope immune response. Furthermore, an HIV-neutralizing antibody response was elicited in guinea pigs immunized with HAV-gp41 chimeric particles. The results demonstrate that the replication-competent chimeric HAV-gp41 can serve as either a live or a subunit vaccine for eliciting of antibodies directed against a foreign antigenic epitope.

  2. Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes Efeito das subunidades de glutenina de alto e baixo peso molecular e das subunidades de gliadina sobre os parâmetros físico-químicos de diferentes genótipos de trigo

    Directory of Open Access Journals (Sweden)

    Mariana Souza Costa

    2013-02-01

    Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.A identificação das propriedades funcionais de farinhas de trigo através de testes específicos possibilita selecionar genótipos de trigo com características adequadas a cada uso industrial. O principal objetivo dos programas de melhoramento genético é melhorar a qualidade do banco de germoplasma para que seja possível desenvolver trigos com força de glúten e extensibilidade adequadas para produção de produtos panificáveis. O objetivo deste estudo foi avaliar 16 genótipos de trigo correlacionando as subunidades de gluteninas de alto e baixo peso

  3. Structures and functions of calcium channel beta subunits.

    Science.gov (United States)

    Birnbaumer, L; Qin, N; Olcese, R; Tareilus, E; Platano, D; Costantin, J; Stefani, E

    1998-08-01

    Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.

  4. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...... the loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the catalytic...... alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit...

  5. Immunogenicity of therapeutic proteins: influence of aggregation.

    Science.gov (United States)

    Ratanji, Kirsty D; Derrick, Jeremy P; Dearman, Rebecca J; Kimber, Ian

    2014-01-01

    The elicitation of anti-drug antibodies (ADA) against biotherapeutics can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immunogenicity of biotherapeutics is, therefore, an important issue. There is evidence that protein aggregation can result in enhanced immunogenicity; however, the precise immunological and biochemical mechanisms responsible are poorly defined. In the context of biotherapeutic drug development and safety assessment, understanding the mechanisms underlying aggregate immunogenicity is of considerable interest. This review provides an overview of the phenomenon of protein aggregation, the production of unwanted aggregates during bioprocessing, and how the immune response to aggregated protein differs from that provoked by non-aggregated protein. Of particular interest is the nature of the interaction of aggregates with the immune system and how subsequent ADA responses are induced. Pathways considered here include 'classical' activation of the immune system involving antigen presenting cells and, alternatively, the breakdown of B-cell tolerance. Additionally, methods available to screen for aggregation and immunogenicity will be described. With an increased understanding of aggregation-enhanced immune responses, it may be possible to develop improved manufacturing and screening processes to avoid, or at least reduce, the problems associated with ADA.

  6. Immunogenicity of novel sulfadimethoxide conjugates | Chen ...

    African Journals Online (AJOL)

    SDM antibodies are useful for the detection of residual SDM in foods, feeds and biological fluids by ELISA. In this study, we show that SDM is immunogenic in rabbits when it is conjugated with soy 11S globulin or with β- amylase. Rabbit ...

  7. Immunogenicity of toxins during Staphylococcus aureus infection

    NARCIS (Netherlands)

    N.J. Verkaik (Nelianne); O. Dauwalder (Olivier); K. Antri (Kenza); I. Boubekri (Ilhem); C.P. de Vogel (Corné); C. Badiou (Cédric); M. Bes (Michèle); F. Vandenesch (François); M. Tazir (Mohammed); H. Hooijkaas (Herbert); H.A. Verbrugh (Henri); A.F. van Belkum (Alex); J. Etienne (Jerome); G. Lina (Gérard); N. Ramdani-Bouguessa (Nadjia); W.J.B. van Wamel (Willem)

    2010-01-01

    textabstractAB - BACKGROUND: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. METHODS: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects

  8. Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China.

    Science.gov (United States)

    Schnittger, L; Yin, H; Jianxun, L; Ludwig, W; Shayan, P; Rahbari, S; Voss-Holtmann, A; Ahmed, J S

    2000-05-01

    A fatal disease of sheep and goats in the northwestern part of China has been reported to be due to Theileria lestoquardi (syn. T. hirci). However, some characteristics of the causative agent are not in accordance with attributes ascribed to this parasite. We therefore determined the nucleotide sequence of the small-subunit ribosomal RNA (srRNA) gene of T. lestoquardi and the parasite identified in China and compared it with that of other Theileria and Babesia species. In the inferred phylogenetic tree the srRNA sequence of the Chinese parasite was found to be most closely related to T. buffeli and clearly divergent from T. lestoquardi, suggesting that it is an as yet unrecognized Theileria species. Extensive structural similarities were observed between the srRNA sequences of T. lestoquardi and T. annulata, revealing a close phylogenetic relationship between these two Theileria species. On the basis of the srRNA nucleotide sequence, polymerase chain reaction (PCR) primers were designed that specifically amplified genomic DNA of the Chinese Theileria species. These primers may be valuable tools in future epidemiology studies.

  9. Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.

    Science.gov (United States)

    Ahmadi, Maryam; Bryson, Christine J; Cloake, Edward A; Welch, Katie; Filipe, Vasco; Romeijn, Stefan; Hawe, Andrea; Jiskoot, Wim; Baker, Matthew P; Fogg, Mark H

    2015-04-01

    Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

  10. Group B streptococcal type II and III conjugate vaccines: physicochemical properties that influence immunogenicity.

    Science.gov (United States)

    Michon, Francis; Uitz, Catherine; Sarkar, Arun; D'Ambra, Anello J; Laude-Sharp, Maryline; Moore, Samuel; Fusco, Peter C

    2006-08-01

    Recent efforts toward developing vaccines against group B streptococci (GBS) have focused on increasing the immunogenicity of GBS polysaccharides by conjugation to carrier proteins. However, partial depolymerization of GBS polysaccharides for the production of vaccines is a difficult task because of their acid-labile, antigenically critical sialic acids. Here we report a method for the partial depolymerization of type II and III polysaccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-d-mannose residues. Through the free aldehydes of their newly formed end groups, the fragments were conjugated to tetanus toxoid by reductive amination. The resulting conjugates stimulated the production in animals of high-titer type II- and III-specific antibodies which induced opsonophagocytic killing of type II and III strains of group B streptococci. For the type II conjugates, immunogenicity increased as oligosaccharide size decreased, whereas for type III conjugates, the size of the oligosaccharides did not significantly influence immunogenicity. When oligosaccharides of defined size were conjugated through sialic acid residues, the resulting cross-linkages were shown to affect immunogenicity. When oligosaccharides were conjugated through terminal aldehyde groups generated by deamination, modification of the exocyclic chain of sialic acid did not influence immunogenicity.

  11. Infective and inactivated filamentous phage as carriers for immunogenic peptides.

    Science.gov (United States)

    Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

    2012-07-01

    The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Genetic Variability of the High-affinity IgE Receptor α Subunit (Fc ε RI α is Related to Total Serum IgE levels in Allergic Subjects

    Directory of Open Access Journals (Sweden)

    Marek Sanak

    2007-01-01

    Full Text Available Known susceptibility genes to atopy and asthma have been identified by linkage or associations with clinical phenotypes, including total serum IgE levels. IgE-mediated sensitivity reactions require a high-affinity IgE receptor (FcεRI, which immobilizes the immunoglobulin on the surface of the effector cells, mostly mast cells and basophils. In this mini-review, recent findings are presented on genetic variation of this receptor, as related to atopy. Transcription of FCER1A gene encoding the receptor α subunit can be initiated from two separate promoters, the proximal one and the distal one, which results in a transcript containing two novel untranslated exons (1A, 2A. Our knowledge on the role of this mechanism in allergic diseases is still at an infancy stage. Within regulatory elements of FCER1A some common single nucleotide polymorphisms have functional associations, which were recently reported and replicated in different ethnical groups. Interestingly, these associations do not confer susceptibility to allergic diseases, but rather modulate serum concentrations of IgE. Similarly to the previously investigated β subunit of the receptor, FCER1A is a good candidate for a quantitative trait locus (QTL in allergic diseases, and appears to participate in the systemic regulation of IgE levels.

  13. High-resolution mapping of the [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster on chromosome 15q11-q13, and localization of breakpoints in two Angelman syndrome patients

    Energy Technology Data Exchange (ETDEWEB)

    Sinnett, D.; Wagstaff, J.; Woolf, E. (Children' s Hospital, Boston, MA (United States) Harvard Medical School, Boston, MA (United States)); Glatt, K. (Children' s Hospital, Boston, MA (United States)); Kirkness, E.J. (National Inst. of Alcohol Abuse and Alcoholism, Rockville, MD (United States))Lalande, M. (Children' s Hospital, Boston, MA (United States) Harvard Medical School, Boston, MA (United States) Howard Hughes Medical Inst., Boston, MA (United States))

    1993-06-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptors are a family of ligand-gated chloride channels constituting the major inhibitory neurotransmitter receptors in the nervous system. In order to determine the genomic organization of the GABA[sub A] receptor [beta]3 subunit gene (GABRB3) and [alpha]5 subunit gene (GABRA5) in chromosome 15q11-q13, the authors have constructed a high-resolution physical map using the combined techniques of field-inversion gel electrophoresis and phage genomic library screening. This map, which covers nearly 1.0 Mb, shows that GABRB3 and GABRA5 are separated by less than 100 kb and are arranged in a head-to-head configuration. GABRB3 encompasses approximately 250 kb, while GABRA5 is contained within 70 kb. This difference in size is due in large part to an intron of 150 kb within GABRB3. The authors have also identified seven putative CpG islands within a 600-kb interval. Chromosomal rearrangement breakpoints -- in one Angelman syndrome (AS) patient with an unbalanced translocation and in another patient with a submicroscopic deletion -- are located within the large GABRB3 intron. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the DNA rearrangements in these AS patients and will be valuable for mapping new genes to the AS chromosomal region. 64 refs., 6 figs., 2 tabs.

  14. Proteomic and immunoproteomic characterization of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae

    Directory of Open Access Journals (Sweden)

    Maas Alexander

    2011-04-01

    Full Text Available Abstract Background Protection of pigs by vaccination against Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is hampered by the presence of 15 different serotypes. A DIVA subunit vaccine comprised of detergent-released proteins from A. pleuropneumoniae serotypes 1, 2 and 5 has been developed and shown to protect pigs from clinical symptoms upon homologous and heterologous challenge. This vaccine has not been characterized in-depth so far. Thus we performed i mass spectrometry in order to identify the exact protein content of the vaccine and ii cross-serotype 2-D immunoblotting in order to discover cross-reactive antigens. By these approaches we expected to gain results enabling us to argue about the reasons for the efficacy of the analyzed vaccine. Results We identified 75 different proteins in the vaccine. Using the PSORTb algorithm these proteins were classified according to their cellular localization. Highly enriched proteins are outer membrane-associated lipoproteins like OmlA and TbpB, integral outer membrane proteins like FrpB, TbpA, OmpA1, OmpA2, HgbA and OmpP2, and secreted Apx toxins. The subunit vaccine also contained large amounts of the ApxIVA toxin so far thought to be expressed only during infection. Applying two-dimensional difference gel electrophoresis (2-D DIGE we showed different isoforms and variations in expression levels of several proteins among the strains used for vaccine production. For detection of cross-reactive antigens we used detergent released proteins of serotype 7. Sera of pigs vaccinated with the detergent-released proteins of serotypes 1, 2, and 5 detected seven different proteins of serotype 7, and convalescent sera of pigs surviving experimental infection with serotype 7 reacted with 13 different proteins of the detergent-released proteins of A. pleuropneumoniae serotypes 1, 2, and 5. Conclusions A detergent extraction-based subunit vaccine of A. pleuropneumoniae was

  15. Microbial Proteases in Baked Goods: Modification of Gluten and Effects on Immunogenicity and Product Quality

    Science.gov (United States)

    Heredia-Sandoval, Nina G.; Valencia-Tapia, Maribel Y.; Calderón de la Barca, Ana M.; Islas-Rubio, Alma R.

    2016-01-01

    Gluten-related diseases are a range of inflammatory disorders of the small intestine, characterized by an adverse response to gluten ingestion; therefore, the treatment is a gluten withdrawal. In spite of the increased market of gluten-free products, widely available breads with high acceptability are still missing due to the technological challenge of substituting the special gluten properties. Instead of using alternative ingredients for baking, some attempts have been done to decrease gluten immunogenicity by its enzymatic degradation with microbial proteases. Although the gluten immunogenicity reduction has been reached to an acceptable level, some quality parameters of the products are affected. This review focus on the use of microbial peptidases to prepare less immunogenic baked goods and their effect on product quality. PMID:28231153

  16. Microbial Proteases in Baked Goods: Modification of Gluten and Effects on Immunogenicity and Product Quality

    Directory of Open Access Journals (Sweden)

    Nina G. Heredia-Sandoval

    2016-08-01

    Full Text Available Gluten-related diseases are a range of inflammatory disorders of the small intestine, characterized by an adverse response to gluten ingestion; therefore, the treatment is a gluten withdrawal. In spite of the increased market of gluten-free products, widely available breads with high acceptability are still missing due to the technological challenge of substituting the special gluten properties. Instead of using alternative ingredients for baking, some attempts have been done to decrease gluten immunogenicity by its enzymatic degradation with microbial proteases. Although the gluten immunogenicity reduction has been reached to an acceptable level, some quality parameters of the products are affected. This review focus on the use of microbial peptidases to prepare less immunogenic baked goods and their effect on product quality.

  17. Empirical fitness models for hepatitis C virus immunogen design.

    Science.gov (United States)

    Hart, Gregory R; Ferguson, Andrew L

    2015-11-24

    Hepatitis C virus (HCV) afflicts 170 million people worldwide, 2%-3% of the global population, and kills 350 000 each year. Prophylactic vaccination offers the most realistic and cost effective hope of controlling this epidemic in the developing world where expensive drug therapies are not available. Despite 20 years of research, the high mutability of the virus and lack of knowledge of what constitutes effective immune responses have impeded development of an effective vaccine. Coupling data mining of sequence databases with spin glass models from statistical physics, we have developed a computational approach to translate clinical sequence databases into empirical fitness landscapes quantifying the replicative capacity of the virus as a function of its amino acid sequence. These landscapes explicitly connect viral genotype to phenotypic fitness, and reveal vulnerable immunological targets within the viral proteome that can be exploited to rationally design vaccine immunogens. We have recovered the empirical fitness landscape for the HCV RNA-dependent RNA polymerase (protein NS5B) responsible for viral genome replication, and validated the predictions of our model by demonstrating excellent accord with experimental measurements and clinical observations. We have used our landscapes to perform exhaustive in silico screening of 16.8 million T-cell immunogen candidates to identify 86 optimal formulations. By reducing the search space of immunogen candidates by over five orders of magnitude, our approach can offer valuable savings in time, expense, and labor for experimental vaccine development and accelerate the search for a HCV vaccine. HCV-hepatitis C virus, HLA-human leukocyte antigen, CTL-cytotoxic T lymphocyte, NS5B-nonstructural protein 5B, MSA-multiple sequence alignment, PEG-IFN-pegylated interferon.

  18. Empirical fitness models for hepatitis C virus immunogen design

    Science.gov (United States)

    Hart, Gregory R.; Ferguson, Andrew L.

    2015-12-01

    Hepatitis C virus (HCV) afflicts 170 million people worldwide, 2%-3% of the global population, and kills 350 000 each year. Prophylactic vaccination offers the most realistic and cost effective hope of controlling this epidemic in the developing world where expensive drug therapies are not available. Despite 20 years of research, the high mutability of the virus and lack of knowledge of what constitutes effective immune responses have impeded development of an effective vaccine. Coupling data mining of sequence databases with spin glass models from statistical physics, we have developed a computational approach to translate clinical sequence databases into empirical fitness landscapes quantifying the replicative capacity of the virus as a function of its amino acid sequence. These landscapes explicitly connect viral genotype to phenotypic fitness, and reveal vulnerable immunological targets within the viral proteome that can be exploited to rationally design vaccine immunogens. We have recovered the empirical fitness landscape for the HCV RNA-dependent RNA polymerase (protein NS5B) responsible for viral genome replication, and validated the predictions of our model by demonstrating excellent accord with experimental measurements and clinical observations. We have used our landscapes to perform exhaustive in silico screening of 16.8 million T-cell immunogen candidates to identify 86 optimal formulations. By reducing the search space of immunogen candidates by over five orders of magnitude, our approach can offer valuable savings in time, expense, and labor for experimental vaccine development and accelerate the search for a HCV vaccine. Abbreviations: HCV—hepatitis C virus, HLA—human leukocyte antigen, CTL—cytotoxic T lymphocyte, NS5B—nonstructural protein 5B, MSA—multiple sequence alignment, PEG-IFN—pegylated interferon.

  19. Listeriolysin o is strongly immunogenic independently of its cytotoxic activity.

    Directory of Open Access Journals (Sweden)

    Javier A Carrero

    Full Text Available The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000-7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10-100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.

  20. Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

    Science.gov (United States)

    Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L.; van Lier, Christina J.; Sha, Jian; Yeager, Linsey A.; Chopra, Ashok K.; Rao, Venigalla B.

    2013-01-01

    Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines. PMID:23853602

  1. Identification of immunogenic and virulence-associated Campylobacter jejuni proteins.

    Science.gov (United States)

    Nielsen, Lene N; Luijkx, Thomas A; Vegge, Christina S; Johnsen, Christina Kofoed; Nuijten, Piet; Wren, Brendan W; Ingmer, Hanne; Krogfelt, Karen A

    2012-02-01

    With the aim of identifying proteins important for host interaction and virulence, we have screened an expression library of NCTC 11168 Campylobacter jejuni genes for highly immunogenic proteins. A commercial C. jejuni open reading frame (ORF) library consisting of more than 1,600 genes was transformed into the Escherichia coli expression strain BL21(DE3), resulting in 2,304 clones. This library was subsequently screened for immunogenic proteins using antibodies raised in rabbit against a clinical isolate of C. jejuni; this resulted in 52 highly reactive clones representing 25 different genes after sequencing. Selected candidate genes were inactivated in C. jejuni NCTC 11168, and the virulence was examined using INT 407 epithelial cell line and motility, biofilm, autoagglutination, and serum resistance assays. These investigations revealed C. jejuni antigen 0034c (Cj0034c) to be a novel virulence factor and support the usefulness of the method. Further, several antigens were tested as vaccine candidates in two mouse models, in which Cj0034c, Cj0404, and Cj0525c resulted in a reduction of invasion in spleen and liver after challenge.

  2. Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications.

    Science.gov (United States)

    Kuriakose, Anshu; Chirmule, Narendra; Nair, Pradip

    2016-01-01

    Today, potential immunogenicity can be better evaluated during the drug development process, and we have rational approaches to manage the clinical consequences of immunogenicity. The focus of the scientific community should be on developing sensitive diagnostics that can predict immunogenicity-mediated adverse events in the small fraction of subjects that develop clinically relevant anti-drug antibodies. Here, we discuss the causes of immunogenicity which could be product-related (inherent property of the product or might be picked up during the manufacturing process), patient-related (genetic profile or eating habits), or linked to the route of administration. We describe various posttranslational modifications (PTMs) and how they may influence immunogenicity. Over the last three decades, we have significantly improved our understanding about the types of PTMs of biotherapeutic proteins and their association with immunogenicity. It is also now clear that all PTMs do not lead to clinical immunogenicity. We also discuss the mechanisms of immunogenicity (which include T cell-dependent and T cell-independent responses) and immunological tolerance. We further elaborate on the management of immunogenicity in preclinical and clinical setting and the unique challenges raised by biosimilars, which may have different immunogenic potential from their parent biotherapeutics.

  3. Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications

    Directory of Open Access Journals (Sweden)

    Anshu Kuriakose

    2016-01-01

    Full Text Available Today, potential immunogenicity can be better evaluated during the drug development process, and we have rational approaches to manage the clinical consequences of immunogenicity. The focus of the scientific community should be on developing sensitive diagnostics that can predict immunogenicity-mediated adverse events in the small fraction of subjects that develop clinically relevant anti-drug antibodies. Here, we discuss the causes of immunogenicity which could be product-related (inherent property of the product or might be picked up during the manufacturing process, patient-related (genetic profile or eating habits, or linked to the route of administration. We describe various posttranslational modifications (PTMs and how they may influence immunogenicity. Over the last three decades, we have significantly improved our understanding about the types of PTMs of biotherapeutic proteins and their association with immunogenicity. It is also now clear that all PTMs do not lead to clinical immunogenicity. We also discuss the mechanisms of immunogenicity (which include T cell-dependent and T cell-independent responses and immunological tolerance. We further elaborate on the management of immunogenicity in preclinical and clinical setting and the unique challenges raised by biosimilars, which may have different immunogenic potential from their parent biotherapeutics.

  4. The Positive Correlation of the Enhanced Immune Response to PCV2 Subunit Vaccine by Conjugation of Chitosan Oligosaccharide with the Deacetylation Degree

    Directory of Open Access Journals (Sweden)

    Guiqiang Zhang

    2017-07-01

    Full Text Available Chitosan oligosaccharides (COS, the degraded products of chitosan, have been demonstrated to have versatile biological functions. In primary studies, it has displayed significant adjuvant effects when mixed with other vaccines. In this study, chitosan oligosaccharides with different deacetylation degrees were prepared and conjugated to porcine circovirus type 2 (PCV2 subunit vaccine to enhance its immunogenicity. The vaccine conjugates were designed by the covalent linkage of COSs to PCV2 molecules and administered to BALB/c mice three times at two-week intervals. The results indicate that, as compared to the PCV2 group, COS–PCV2 conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting lymphocyte proliferation and initiating a mixed T-helper 1 (Th1/T-helper 2 (Th2 response, including raised levels of PCV2-specific antibodies and an increased production of inflammatory cytokines. Noticeably, with the increasing deacetylation degree, the stronger immune responses to PCV2 were observed in the groups with COS-PCV2 vaccination. In comparison with NACOS (chitin oligosaccharides–PCV2 and LCOS (chitosan oligosaccharides with low deacetylation degree–PCV2, HCOS (chitosan oligosaccharides with high deacetylation degree–PCV2 showed the highest adjuvant effect, even comparable to that of PCV2/ISA206 (a commercialized adjuvant group. In summary, COS conjugation might be a viable strategy to enhance the immune response to PCV2 subunit vaccine, and the adjuvant effect was positively correlated with the deacetylation degree of COS.

  5. Examination of some biological properties of glycoprotein subunits of PHY-LMV.42 strain of Newcastle disease virus

    Directory of Open Access Journals (Sweden)

    Milić Nenad

    2015-01-01

    Full Text Available The objective of our work was to investigate some biological characteristics of purified glycoprotein subunits of Newcastle disease virus strain PHY-LMV.42 isolated from pigeons for the purpose of vaccine production. PHY-LMV.42 strain of Newcastle disease virus was multiplied by successive passages in embryonated eggs and identified by the methods of Reverse transcriptase PCR and Real-Time PCR along with F gene sequencing. Proving the presence of HN and F antigene in the virus subunits samples was carried out by hemagglutination inhibition method with referent immune sera. Biochemical characterization of glycoprotein subunits was performed by SDS-PAGE method as well as liquid chromatography with mass spectrometry (LC ESI-TOF-MS/MS. Testing for the virus subunits immunogenicity was carried out in biological experiment on 75 laying hens Tetra-SSL and 25 chickens Isa Brown by inducing an artificial infection with Hertz 33 strain of the virus. Low concentrations of the virus antigens of 0.36 mg/ml along with glycoprotein fractions of 77 i 58 kDa manifested a strong hemagglutination activity of 4096 HJ/0,1ml. The subunit vaccines of 256 and 128 HJ/0.5 ml induced a protective immune response in all the vaccinated animals. Based on the obtained results it can be concluded that low concentrations of purified virus subunits of PHY-LMV.42 strain can be used for preparing of effective vaccines. [Projekat Ministarstva nauke Republike Srbije, br. TR 31008: Development and application of molecular methods based on polymerase chain reaction (PCR in quick and direct identification of Newcastle disease virus strains and investigation of immunogenicity of subunit vaccine prepared of their antigens

  6. Identification of immunogenic and virulence-associated Campylobacter jejuni proteins

    DEFF Research Database (Denmark)

    Nielsen, Lene Nørby; Luijkx, Thomas A.; Vegge, Christina Skovgaard

    2012-01-01

    With the aim of identifying proteins important for host interaction and virulence, we have screened an expression library of NCTC 11168 Campylobacter jejuni genes for highly immunogenic proteins. A commercial C. jejuni open reading frame (ORF) library consisting of more than 1,600 genes was trans...

  7. Immunogenicity and tolerability of inactivated flu vaccine In high risk and healthy children Inmunogenicidad y tolerancia de la vacuna inactivada anti-influenza en niños en alto riesgo y en controles sanos

    Directory of Open Access Journals (Sweden)

    Maria Luisa Avila Aguero

    2007-08-01

    Full Text Available We conducted this open study to evaluate the immunogenicity and safety of the inactivated influenza vaccine, Imovax Gripe® in 154 children between 6 and 36 months of age at high risk of influenza- related complications, and in a reference group of 64 healthy children. The study was conducted over two flu seasons, in which the vaccine contained the same A strains but different B strains. The results for the A/H3N2 and A/H1N1 strains from the two flu seasons were pooled, but those for the B strains were not. Anti-hemagglutinin (HA antibody titers were determined before, and one month after each vaccination, and safety was evaluated based on diary card reporting any adverse event observed, either included or not in the list of "solicited events". Within each group of vaccines, the seroconversion rates, seroprotection rates, and ratio of post- to prevaccination geometric mean titers (GMTR for the A/H3N2 and the A/H1N1 strains fulfilled all requirements of the criteria of the European Union Committee for Proprietary Medicinal Products (CPMP. The immune responses in high-risk and in healthy children were similar, and consistent with those observed in previous studies conducted in healthy children. The vaccine was equally well tolerated by all study groups. Reactogenicity was low and similar in both high-risk and healthy children. Overall from 9.5% to 15.4% of at-risk children and 12% of healthy children reported a solicited local reaction; 23.0 to 28.8% of high-risk and 25.3% of healthy children reported a solicited systemic reaction. The study results provide support for vaccination of children at high-risk of influenza related complications.Se realizó un estudio clínico abierto para evaluar la inmunogenícidad y la seguridad de la vacuna inactivada anti-influenza, Imovax Gripe®, en 154 niños entre 6 y 36 meses de edad con alto riesgo de complicaciones ligadas a la influenza, y en un grupo de referencia de 64 niños sanos. El estudio fue

  8. Immunogenicity of Anti-TNF-α Biotherapies

    DEFF Research Database (Denmark)

    Bendtzen, Klaus

    2015-01-01

    -necrosis factor-α (TNF). Emphasis will be on commonly used methods for detection of ADA in human serum including issues that question the clinical applicability of these methodologies. The use of dubious assays for ADA in a clinical context may not only contribute to confusion as to the importance of drug...... immunogenicity but may also prevent development of safe and cost-effective ways of using biological TNF-antagonists....

  9. IMMUNOGENICITY OF ADJUVANT INFLUENZA VACCINE FOR PREGNANT WOMEN

    Directory of Open Access Journals (Sweden)

    M. P. Kostinov

    2017-01-01

    Full Text Available Recent epidemiological events showed that pregnant women are the most vulnerable part of population if there is the flu in the country and they die much more often than the rest part of people. That is why influenza vaccination of population including pregnant women is one of the priorities of public health service in our state. Worldwide experience of influenza vaccination of either adults or children by new adjuvant vaccine has caused our research of its efficiency among pregnant women. The aim of the study was to investigate the level of antibodies to influenza virus strain A/H1N1/v, A/H3N2 and B in pregnant women vaccinated adjuvant trivalent subunit vaccine. Our research is randomized and comparative on parallel groups. It was carried out within the demands of Russian Federation and International ethic norms adapted to such kind of researches. Evaluation of the immunogenicity of the vaccine was conducted in 27 pregnant women in the II trimester of gestation, and in 23 pregnant women in the III trimester of gestation, 19 non-pregnant women was in the control group. The level of antibodies in the serum was determined using a reaction of hemagglutination inhibition before and 1, 3, 6, 9 and 12 months after the vaccination. Revealed that influenza vaccination of pregnant women in the II and III trimester, causes the increase in titers of antibodies to vaccine influenza strains A and B, to fully meet the required criteria CPMP, and does not differ from the nonpregnant group. In a month after vaccination the level of seroprotective against A/H1N1/v was 77.0%, A/H3N2 — 88.9%, B — 85.2% after vaccination in II trimester, and 87.0; 87.0; 91.35% in III trimester of gestation. The factor of seroconversion after vaccination in II trimester for A/H1N1/v was equal to 6.5, A/H3N2 — 7.2, B — 6.5, after vaccination in III trimester of pregnancy: 7.1, 6.5 and 5.1 correspondingly. At the same time revealed accelerated decline in antibody titer against

  10. Fused Mycobacterium tuberculosis multi-stage immunogens with an Fc-delivery system as a promising approach for the development of a tuberculosis vaccine.

    Science.gov (United States)

    Mosavat, Arman; Soleimanpour, Saman; Farsiani, Hadi; Sadeghian, Hamid; Ghazvini, Kiarash; Sankian, Mojtaba; Jamehdar, Saeid Amel; Rezaee, Seyed Abdolrahim

    2016-04-01

    Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guérin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272 pg/mL, p<0.001) and IL-12 (191 pg/mL, p<0.001). However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low level of IL-4 (10 pg/mL) compared to the CFP-10:HspX:His group. The production of IFN-γ to CFP-10:HspX:Fcγ2a was markedly consistent and showed an increasing trend for IL-12 compared with the BCG or CFP-10:HspX:His primed and boosted groups. Findings revealed that CFP-10:Hspx:Fcγ2a fusion protein can elicit strong Th1 antigen-specific immune responses in favor of protective immunity in mice and could provide new insight for introducing an effective multi-stage subunit vaccine against TB. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

  12. Subunit mass analysis for monitoring antibody oxidation

    National Research Council Canada - National Science Library

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-01-01

    ... (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit...

  13. Nucleosomal histone proteins of L. donovani: a combination of recombinant H2A, H2B, H3 and H4 proteins were highly immunogenic and offered optimum prophylactic efficacy against Leishmania challenge in hamsters.

    Science.gov (United States)

    Baharia, Rajendra K; Tandon, Rati; Sahasrabuddhe, Amogh A; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    The present study includes cloning and expression of recombinant Leishmania donovani histone proteins (rLdH2B, rLdH3, rLdH2A and rLdH4), assessment of their immunogenicity in Leishmania infected cured patients/endemic contacts as well as in cured hamsters and finally evaluation of their prophylactic efficacy in hamsters against L. donovani challenge. All recombinant proteins were expressed and purified from the heterologous bacterial host system. Leishmania infected cured patients/endemic contacts as well as cured hamsters exhibited significantly higher proliferative responses to individual recombinant histones and their pooled combination (rLdH2B+rLdH3+rLdH2A+rLdH4) than those of L.donovani infected hosts. The L.donovani soluble antigens (SLD) stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLdH2B, rLdH3, rLdH2A, rLdH4 and pooled combination (rLdH2-4) stimulated the production of Th1 cytokines IFN-γ, IL-12 and TNF-α but not Th2 cytokines IL-4 or IL-10. The immunogenicity of these histone proteins along with their combination was also checked in cured hamsters where they stimulated higher lymphoproliferation and Nitric oxide production in lymphocytes of cured hamsters than that of infected controls. Moreover, significantly increased IgG2 response, an indicative of cell mediated immunity, was observed in cured hamsters against these individual proteins and their combination as compared to infected hamsters. Further, it was demonstrated that rLdH2B, rLdH3, rLdH2A and rLdH4 and pooled combination were able to provide considerable protection for hamsters against L. donovani challenge. The efficacy was supported by the increased inducible Nitric Oxide Synthase (iNOS) mRNA transcripts and Th1-type cytokines--IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-β. Hence, it is inferred that pooled rLdH2-4 elicits Thl-type of immune responses exclusively and confer considerable protection

  14. Multiple heat and drought events affect grain yield and accumulations of high molecular weight glutenin subunits and glutenin macropolymers in wheat

    DEFF Research Database (Denmark)

    Zhang, Xiaxiang; Cai, Jian; Wollenweber, Bernd

    2013-01-01

    Spring wheat plants were subjected to water deficit and/or high temperature episodes at spikelet initiation, anthesis or both stages. The stresses modified the early dough stage and maturity, shortened the kernel desiccation period and caused grain yield loss. Plants subjected to stress...

  15. The human [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster in chromosome 15q11-q13 is rich in highly polymorphic (CA)[sub n] repeats

    Energy Technology Data Exchange (ETDEWEB)

    Glatt, K.; Lalande, M. (Howard Hughes Medical Institute, Boston, MA (United States)); Sinnett, D. (Harvard Medical School, Boston, MA (United States))

    1994-01-01

    The [gamma]-aminobutyric acid (GABA[sub A]) receptor [beta]33 (GABRB3) and [alpha]5 (GABRA5) subunit genes have been localized to the Angelman and Prader-Willi syndrome region of chromosome 15q11-q13. GABRB3, which encompasses 250 kb, is located 100 kb proximal of GABRA5, with the two genes arranged in head-to-head transcriptional orientation. In screening 135 kb of cloned DNA within a 260-kb interval extending from within GABRB3 to the 5[prime] end of GABRA5, 10 new (CA), repeats have been identified. Five of these have been analyzed in detail and found to be highly polymorphic, with the polymorphism information content (PIC) ranging from 0.7 to 0.85 and with heterozygosities of 67 to 94%. In the clones from GABRB3/GABRA5 region, therefore, the frequency of (CA)[sub n] with PICs [ge] 0.7 is 1 per 27 kb. Previous estimates of the density of (CA)[sub n] with PICs [ge] 0.7 in the human genome have been approximately 10-fold lower. The GABRB3/GABRA5 region appears, therefore, to be enriched for highly informative (CA)[sub n]. This set of closely spaced, short tandem repeat polymorphisms will be useful in the molecular analyses of Prader-Willi and Angelman syndromes and in high-resolution studies of genetic recombination within this region. 21 refs., 2 figs., 1 tab.

  16. Immunogenicity of the hTERT540-548 peptide in cancer

    DEFF Research Database (Denmark)

    Wenandy, L.; Sorensen, R.B.; Sengelov, L.

    2008-01-01

    Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is an attractive target antigen for cancer immunotherapy due to its expression in the vast majority of human tumors. The first immunogenic peptide described from hTERT was the HLA-A2-restricted peptide hTERT540...... (ILAKFLHWL). However, much discrepancy exists about the processing and presentation of this epitope on the surface of neoplastic cells. Originally, it was described that specific CTL can be generated in vitro and that such cells are able to kill a range of hTERT(+) tumor cell lines and primary tumors...... in a peptide-specific, HLA-A2-restricted fashion. Furthermore, it was described that vaccination of cancer patients with hTERT540 introduced functional antitumor CD8(+) Tcells in patients. More recently, it was described that most patients with cancer have circulating hTERT540-specific CD8(+) T lymphocytes...

  17. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies.......cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  18. Investigation of the immunogenicity of different types of aggregates of a murine monoclonal antibody in mice.

    Science.gov (United States)

    Freitag, Angelika J; Shomali, Maliheh; Michalakis, Stylianos; Biel, Martin; Siedler, Michael; Kaymakcalan, Zehra; Carpenter, John F; Randolph, Theodore W; Winter, Gerhard; Engert, Julia

    2015-02-01

    The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures. Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated. In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures. This study shows that not all protein drug aggregates are equally immunogenic.

  19. Tetraspanins displayed in retrovirus-derived virus-like particles and their immunogenicity.

    Science.gov (United States)

    Soares, H R; Castro, R; Tomás, H A; Rodrigues, A F; Gomes-Alves, P; Bellier, B; Klatzmann, D; Carrondo, M J T; Alves, P M; Coroadinha, A S

    2016-03-18

    Virus-like particles (VLPs) are a particular subset of subunit vaccines which are currently explored as safer alternatives to live attenuated or inactivated vaccines. VLPs derived from retrovirus (retroVLPs) are commonly used as scaffolds for vaccine candidates due to their ability to incorporate heterologous envelope proteins. Pseudotyping retroVLPs is however not a selective process therefore, host cellular proteins such as tetraspanins are also included in the membrane. The contribution of these host-proteins to retrovirus immunogenicity remains unclear. In this work, human cells silenced and not silenced for tetraspanin CD81 were used to produce CD81(-) or CD81(+) retroVLPs. We first analyzed mice immune response against human CD81. Despite effective silencing of CD81 in retroVLP producing cells, both humoral and cellular immune responses showed persistent anti-CD81 immunogenicity, suggesting cross reactivity to related antigens. We thus compared the incorporation of related tetraspanins in retroVLPs and showed that decreased CD81 incorporation in CD81(-) retro-VLPs is compensated by an increased incorporation of CD9 and CD63 tetraspanins. These results highlight the dynamic nature of host-derived proteins incorporation in retroVLPs membrane, which should be considered when retrovirus-based biopharmaceuticals are produced in xenogeneic cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Assessment of the potential utility of different regions of Streptococcus uberis adhesion molecule (SUAM) for mastitis subunit vaccine development.

    Science.gov (United States)

    Perrig, Melina Soledad; Veaute, Carolina; Renna, María Sol; Pujato, Nazarena; Calvinho, Luis; Marcipar, Iván; Barbagelata, María Sol

    2017-04-01

    Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Cellular immunogenicity of novel gene immunogens in mice monitored by in vivo imaging.

    Science.gov (United States)

    Starodubova, Elizaveta; Krotova, Olga; Hallengärd, David; Kuzmenko, Yulia; Engström, Gunnel; Legzdina, Diana; Latyshev, Oleg; Eliseeva, Olesja; Karin Maltais, Anna; Tunitskaya, Vera; Karpov, Vadim; Bråve, Andreas; Isaguliants, Maria

    2012-01-01

    The efficient cell-mediated immune response clears cells expressing deoxyribonucleic acid (DNA) immunogens, but there are no methods to monitor this in vivo. We hypothesized that immune-mediated clearance can be monitored in vivo if DNA immunogens are coexpressed with reporter(s). To test this, we designed genes encoding human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) fused via its N- or C-terminus to 30-amino acid-long Gly-Ala-repeat of Epstein-Barr virus nuclear antigen 1 or via the N-terminus to the transport signal of invariant chain/Ii or inserted between the cytoplasmic and luminal domains of lysosome-associated membrane protein I (LAMP). DNA immunogens mixed with luciferase gene were injected into BALB/c mice with subsequent electroporation. Reporter expression seen as luminescence was monitored by in vivo imaging. When luminescence faded, mice were sacrificed, and their splenocytes were stimulated with RT-derived antigens. Fading of luminescence correlated with the RT-specific secretion of interferon-γ and interleukin-2. Both immune and in vivo imaging techniques concordantly demonstrated an enhanced immunogenicity of RT-LAMP and of the N-terminal Gly-Ala-RT fusion genes. In vivo imaging performed as an animal-sparing method to estimate the overall performance of DNA immunogens, predicting it early in the experiment. So far, in vivo imaging cannot be a substitute for conventional immune assays, but it is supplementary to them. Further experiments are needed to identify which arms of cellular immune response in vivo imaging monitors best.

  2. The Comparative Immunogenicity Of Three Lentogenic Brands Of ...

    African Journals Online (AJOL)

    The comparative immunogenicity of a new lentogenic viscerotropic Newcastle disease vaccine, NDvac-1 (VG/GA strain) and two other existing proprietary pneumotropic lentogenic Newcastle disease vaccines in Nigeria, NDvac-2 (R2B) and NDvac-3 (LaSota) were studied. Immunogenicity was assessed on the basis of ...

  3. Effects of Post-Anthesis Drought and Waterlogging on Accumulation of High-Molecular-Weight Glutenin Subunits and Glutenin Macropolymers Content in Wheat Grain

    DEFF Research Database (Denmark)

    Jiang, D; Yue, H; Wollenweber, B

    2009-01-01

    wheat cultivar Yumai 34 with high grain protein content (GPC) and Yangmai 9 with low GPC were studied. At maturity, GPC was higher under drought and lower under waterlogging compared to the control, while contents of GMP and HMW-GS were reduced by the two water-stress treatments. The contents of both...... to protein ratios were also depressed under the two water-stress events, while the HMW-GS to GMP ratio was very close between the three treatments in Yumai 34 and much higher under the control than the drought and waterlogging treaments in Yangmai 9. It is concluded that the variation in GMP content...... with various water-stress treatments is attributed to the changing in accumulation of HMW-GS in the grain....

  4. Posttranslational Modifications and the Immunogenicity of Biotherapeutics

    Directory of Open Access Journals (Sweden)

    Roy Jefferis

    2016-01-01

    Full Text Available Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs, including quality control (QC in the endoplasmic reticulum (ER and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA; aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs, a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

  5. Anticancer metal drugs and immunogenic cell death.

    Science.gov (United States)

    Terenzi, Alessio; Pirker, Christine; Keppler, Bernhard K; Berger, Walter

    2016-12-01

    Conventional chemotherapeutics, but also innovative precision anticancer compounds, are commonly perceived to target primarily the cancer cell compartment. However, recently it was discovered that some of these compounds can also exert immunomodulatory activities which might be exploited to synergistically enhance their anticancer effects. One specific phenomenon of the interplay between chemotherapy and the anticancer immune response is the so-called "immunogenic cell death" (ICD). ICD was discovered based on a vaccination effect exerted by cancer cells dying from pretreatment with certain chemotherapeutics, termed ICD inducers, in syngeneic transplantation mouse models. Interestingly, only a minority of drugs is able to trigger ICD without a clear-cut relation to chemical structures or their primary modes-of-action. Nevertheless, generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress are clearly linked to ICD. With regard to metal drugs, oxaliplatin but not cisplatin is considered a bona fide ICD inducer. Taken into account that several experimental metal compounds are efficient ROS and ER stress mediators, presence of potent ICD inducers within the plethora of novel metal complexes seems feasible and has occasionally been reported. In the light of recent successes in cancer immunotherapy, here we review existing literature regarding anticancer metal drugs and ICD induction. We recommend a more profound investigation of the immunogenic features of experimental anticancer metal drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice.

    Science.gov (United States)

    Inoguchi, Noriko; Mizuno, Nobuhiro; Baba, Seiki; Kumasaka, Takashi; Natarajan, Chandrasekhar; Storz, Jay F; Moriyama, Hideaki

    2017-01-01

    Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.

  7. Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice

    Energy Technology Data Exchange (ETDEWEB)

    Inoguchi, Noriko; Mizuno, Nobuhiro; Baba, Seiki; Kumasaka, Takashi; Natarajan, Chandrasekhar; Storz, Jay F.; Moriyama, Hideaki; Permyakov, Eugene A.

    2017-03-31

    Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.

  8. Expression of an immunogenic LTB-based chimeric protein targeting Zaire ebolavirus epitopes from GP1 in plant cells.

    Science.gov (United States)

    Ríos-Huerta, Regina; Monreal-Escalante, Elizabeth; Govea-Alonso, Dania O; Angulo, Carlos; Rosales-Mendoza, Sergio

    2017-02-01

    An antigenic protein targeting two epitopes from the Zaire ebolavirus GP1 protein was expressed in plant cells rendering an antigen capable of inducing humoral responses in mouse when administered subcutaneously or orally. The 2014 Ebola outbreak made clear that new treatments and prophylactic strategies to fight this disease are needed. Since vaccination is an intervention that could achieve the control of this epidemic disease, exploring the production of new low-cost vaccines is a key path to consider; especially in developing countries. In this context, plants are attractive organisms for the synthesis and delivery of subunit vaccines. This study aimed at producing a chimeric protein named LTB-EBOV, based on the B subunit of the Escherichia coli heat-labile enterotoxin as an immunogenic carrier and two epitopes from the Zaire ebolavirus GP1 protein recognized by neutralizing antibodies. The LTB-EBOV protein was expressed in plant tissues at levels up to 14.7 µg/g fresh leaf tissue and proven to be immunogenic in BALB/c mice when administered by either subcutaneous or oral routes. Importantly, IgA and IgG responses were induced following the oral immunization. The potential use of the plant-made LTB-EBOV protein against EBOV is discussed.

  9. Efficacy, safety and immunogenicity of biosimilars in inflammatory bowel diseases: A systematic review.

    Science.gov (United States)

    Martelli, Laura; Peyrin-Biroulet, Laurent

    2016-10-14

    Anti-tumor necrosis factor (anti-TNF) monoclonal antibodies have revolutionized the treatment of inflammatory bowel diseases (IBD). However, because of their complexity, their production is expensive contributing to their high price. As the patent protection of these therapies has expired in several countries, biosimilars have been developed to reduce the healthcare costs. The aim of this article is to review the literature on the safety, efficacy and immunogenicity of biosimilars in IBD. A PubMed literature search was performed using the following terms until May 2016: 'biosimilars', 'CT-P13', 'infliximab', 'Crohn's disease', 'ulcerative colitis', 'inflammatory bowel diseases', 'efficacy', 'safety', 'immunogenicity'. Additionally, abstracts from international meetings were also reviewed. A total of eleven studies in IBD patients provided real-world evidence on the efficacy, safety and immunogenicity profile of biosimilars in IBD patients. Based on available evidence, CT-P13 is efficacious and well tolerated in IBD patients in a real-life setting. The vast majority of studies only included IBD patients who had never received biological therapies. Information regarding the interchangeability between CT-P13 and its originator is currently being investigated in the NOR-SWITCH trial. Otherwise, the immunogenicity profile of CT-P13 seems to be similar to the originator. The infliximab biosimilar seems to be efficacious, safe and with a similar immunogenicity profile as the originator in IBD. Large prospective post-marketing studies are needed to assess the long-term safety profile of CT-P13. The use of infliximab biosimilars may lead to major healthcare cost savings.

  10. Safety and immunogenicity of a CRM or TT conjugated meningococcal vaccine in healthy toddlers.

    Science.gov (United States)

    Bona, Gianni; Castiglia, Paolo; Zoppi, Giorgio; de Martino, Maurizio; Tasciotti, Annaelisa; D'Agostino, Diego; Han, Linda; Smolenov, Igor

    2016-06-17

    MenACWY-CRM (Menveo(®); GlaxoSmithKline) and MenACWY-TT (Nimenrix(®); Pfizer) are two meningococcal vaccines licensed in the European Union for use in both children and adults. While both vaccines target meningococcal serogroups A, C, W and Y, immunogenicity and reactogenicity of these quadrivalent meningococcal conjugate vaccines may differ due to differences in formulation processes and chemical structure. Yet data on the comparability of these two vaccines are limited. The reactogenicity and immunogenicity of one dose of either MenACWY-CRM or MenACWY-TT were evaluated in healthy toddlers aged 12-15 months. Immunogenicity was assessed using serum bactericidal antibody assays (SBA) with human (hSBA) and rabbit (rSBA) complement. A total of 202 children aged 12-15 months were enrolled to receive one dose of MenACWY-CRM or MenACWY-TT. Similar numbers of subjects reported solicited reactions within 7 days following either vaccination. Tenderness at the injection site was the most common local reaction. Systemic reactions reported were similar for both vaccines and mostly mild to moderate in severity: irritability, sleepiness and change in eating habits were most commonly reported. Immunogenicity at 1 month post-vaccination was generally comparable for both vaccines across serogroups. At 6 months post-vaccination antibody persistence against serogroups C, W, and Y was substantial for both vaccines, as measured by both assay methodologies. For serogroup A, hSBA titers declined in both groups, while rSBA titers remained high. Despite differences in composition, the MenACWY-CRM and MenACWY-TT vaccines have comparable reactogenicity and immunogenicity profiles. Immediate immune responses and short-term antibody persistence were largely similar between groups. Both vaccines were well-tolerated and no safety concerns were identified. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Expression of recombinant Newcastle disease virus F protein in Pichia pastoris and its immunogenicity using flagellin as the adjuvant.

    Science.gov (United States)

    Kang, Xilong; Wang, Jing; Jiao, Yang; Tang, Peipei; Song, Li; Xiong, Dan; Yin, Yuelan; Pan, Zhiming; Jiao, Xinan

    2016-12-01

    Newcastle disease (ND), a highly contagious, acute, and potent infectious disease caused by Newcastle disease virus (NDV), has a considerable impact on the global poultry industry. Although both live attenuated and inactivated vaccines are used to prevent and control the spread of ND among chickens, the increasing number of ND outbreaks in commercial poultry flocks worldwide indicates that routine vaccinations are insufficient to control ND. Hence, efforts are being invested into developing alternative and more effective vaccination strategies. In this study, we focus on F protein, the neutralizing and protective antigen of NDV, and flagellin (FliC), a toll-like receptor 5 (TLR5) agonist that is an effective inducer of innate immune responses. We amplified F gene from velogenic NDV strain F48E8. The recombinant histidine (His)-tagged F protein was efficiently expressed in a Pichia pastoris (P. pastoris) eukaryotic system and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The conditions for F protein expression in P. pastoris were optimal. The immunogenicity of F protein with FliC as the adjuvant was evaluated in a C3H/HeJ mouse model. FliC was found to enhance both F-specific and NDV-specific IgG responses and F-specific cellular immune responses following intraperitoneal co-administration with F protein. Thus, the recombinant F protein expressed by P. pastoris when used with flagellin as the adjuvant has potential as a subunit vaccine candidate. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus

    Science.gov (United States)

    Maddux, Sarah; Choi, K. Yeon; McGregor, Alistair

    2015-01-01

    Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene

  13. Influence of high-molecular-weight glutenin subunit composition at Glu-A1 and Glu-D1 loci on secondary and micro structures of gluten in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Li, Xuejun; Liu, Tianhong; Song, Lijun; Zhang, Heng; Li, Liqun; Gao, Xin

    2016-12-15

    As one of critical gluten proteins, high-molecular-weight glutenin subunits (HMW-GS) mainly affect the rheological behaviour of wheat dough. The influence of HMW-GS variations at the Glu-A1 and Glu-D1 loci on both secondary and micro structures of gluten and rheological properties of wheat dough was investigated in this study. Results showed that the Amide I bands of the three near-isogenic lines (NILs) shifted slightly, but the secondary structures differed significantly. The micro structure of gluten in NIL 4 (Ax null) showed bigger apertures and less connection, compared to that in Xinong 1330 (Ax1). The micro structure of gluten in NIL 5 (Dx5+Dy10) showed more compact than that in Xinong 1330 (Dx2+Dy12). Correlation analysis demonstrated that the content of β-sheets and disulfide bonds in gluten has a significant relationship with dough properties. The secondary structures of native gluten are suggested to be used as predictors of wheat quality. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Allelic variation of HMW glutenin subunits of Ethiopian bread wheat ...

    African Journals Online (AJOL)

    High molecular weight glutenins are often effective in identifying wheat (Triticum aestivum) genotypes with good baking quality. The high molecular weight glutenin subunit composition of Ethiopian cultivars and advanced lines was investigated to determine their influence on quality. Three alleles at Glu-A1, five at Glu-B1 ...

  15. A bioinformatic and computational study of myosin phosphatase subunit diversity.

    Science.gov (United States)

    Dippold, Rachael P; Fisher, Steven A

    2014-08-01

    Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. Copyright © 2014 the American Physiological Society.

  16. Phase III, randomized controlled trial to evaluate lot consistency of a trivalent subunit egg-based influenza vaccine in adults.

    Science.gov (United States)

    Rivera, Luis; Mazara, Sonia; Vargas, Maria; Fragapane, Elena; Casula, Daniela; Groth, Nicola

    2012-07-27

    Vaccination is the most effective preventive strategy to control influenza. The demonstration of lot-to-lot consistency to confirm the reliability of the manufacturing process has become a mandatory step in vaccine development. This phase III, observer-blind, controlled trial assessed lot-to-lot consistency, immunogenicity, and safety of a subunit trivalent influenza vaccine (Agrippal®, Novartis Vaccines and Diagnostics) in healthy adults aged 18-49 years. The immunogenicity and safety profile of Agrippal was compared with a control vaccine (Fluvirin®, Novartis Vaccines and Diagnostics). A total of 1507 subjects were randomized 2:2:2:1 to receive one vaccination of one of the three lots of influenza vaccine or control vaccine. Antibody levels were measured by hemagglutination inhibition assay on days 1 and 22. Adverse reactions were solicited via diary cards for 7 days after vaccination, and unsolicited adverse events were collected throughout the study period. Equivalence of day 22 immune responses to the three lots was shown for each of the three strains. Robust immunogenic responses after one dose were observed for all vaccine groups, and both Center for Biologics Evaluation and Research criteria for licensure of influenza vaccines were met for all three virus strains. Both vaccines exhibited a robust safety profile and were well tolerated, with no differences in local and systemic solicited reactions or in unsolicited adverse events. The demonstration of consistency between manufacturing lots confirms for purposes of clinical development the reliability of the production process. The robust immunogenic responses and favorable safety profiles further support the use of trivalent subunit influenza vaccines Agrippal and Fluvirin for active immunization against influenza. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    Science.gov (United States)

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

  18. Glycan bioengineering in immunogen design for tumor T antigen immunotargeting

    DEFF Research Database (Denmark)

    Sendra, Victor G; Zlocowski, Natacha; Ditamo, Yanina

    2009-01-01

    MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope...... to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens...

  19. Immunogenicity and thermal stability of a combined vaccine against Haemophilus influenzae type b and Neisseria meningitidis serogroup C diseases.

    Science.gov (United States)

    Saydam, Manolya; Burkin, Karena; Care, Rory; Rigsby, Peter; Bolgiano, Barbara; Mawas, Fatme

    2010-08-31

    The immunogenicity, structure and stability of a combined conjugate vaccine against Haemophilus influenzae type b and meningococcal serogroup C (Hib/MenC) were investigated. A rat model for immunogenicity showed that antibody responses to Hib and MenC in the combined vaccine were similar to or higher than those of individual conjugates given alone, or concomitantly at separate sites. At elevated temperatures, the combination vaccine was slightly more stable than a monovalent Hib-TT vaccine, with respect to molecular size, which could be attributed to differences in the formulations. Following 5 weeks incubation at 56 degrees C, there was some dissociation of high molecular weight conjugate without significant loss of saccharide integrity; however, this did not significantly affect the vaccine immunogenicity, demonstrating the stability of this lyophilized vaccine. (c) 2010 Elsevier Ltd. All rights reserved.

  20. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT, which consists of two subunits: the A subunit (CTA and the B subunit (CTB. CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  1. Golimumab in the treatment of inflammatory diseases: A role of immunogenicity

    Directory of Open Access Journals (Sweden)

    D. E. Karateev

    2015-01-01

    Full Text Available The review considers the specific features of golimumab (GLM, a representative of a group of tumor necrosis factor-α inhibitors primarily by comparing its immunogenicity parameters with other drugs in this group (infliximab, adalimumab, certolizumab pegol, etanercept. Despite its fundamental similarity with other biologicals from a category of monoclonal antibodies, GLM is shown to be characterized by a significantly lower detection rate for antibodies to the drug and by its high serum concentration stabilities and a sustained clinical response.

  2. Recombinant α-actinin subunit antigens of Trichomonas vaginalis as potential vaccine candidates in protecting against trichomoniasis.

    Science.gov (United States)

    Xie, Yi-Ting; Gao, Jiang-Mei; Wu, Ya-Ping; Tang, Petrus; Hide, Geoff; Lai, De-Hua; Lun, Zhao-Rong

    2017-02-16

    Human trichomoniasis caused by Trichomonas vaginalis is one of the most common sexually transmitted diseases with more than 200 million cases worldwide. It has caused a series of health problems to patients. For prevention and control of infectious diseases, vaccines are usually considered as one of the most cost-efficient tools. However, until now, work on the development of T. vaginalis vaccines is still mainly focused on the screening of potential immunogens. Alpha-actinin characterized by high immunogenicity in T. vaginalis was suggested as a promising candidate. Therefore, the purpose of this study was to evaluate the protective potency of recombinant α-actinin against T. vaginalis infection in a mouse intraperitoneal model. Two selected coding regions of α-actinin (ACT-F, 14-469 aa and ACT-T, 462-844 aa) amplified from cDNA were cloned into pET-32a (+) expression vector and transfected into BL21 cells. After induction with IPTG and purification with electroelution, the two recombinant fusion proteins were emulsified in Freund's adjuvant (FA) and used to immunize BALB/C mice. Following intraperitoneal inoculation with T. vaginalis, the survival rate of mice was monitored for the assessment of protective potency. After immunization, the antibody level in mouse serum was assessed by ELISA, splenocyte proliferation response was detected with CCK8 and cytokines in the supernatant of splenocytes were quantified with a cytometric bead-based assay. We successfully obtained purified ACT-F (70.33 kDa) and ACT-T (61.7kDa). Both recombinant proteins could provide significant protection against T. vaginalis challenge, especially ACT-T (with 100% protection within one month). Meanwhile, high levels of specific total IgG and subtypes (IgG1 > IgG2a) were detected in sera from the immunized mice. Our results also revealed a statistically significant increase in splenocyte proliferation and related cytokine (IFN-γ, IL-6, IL-17A and IL-10) production after repeated

  3. Subunit heterogeneity in the lima bean lectin.

    Science.gov (United States)

    Roberts, D D; Etzler, M E; Goldstein, I J

    1982-08-10

    Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.

  4. Immunogenicity and safety of a cell culture-derived inactivated trivalent influenza vaccine (NBP607): A randomized, double-blind, multi-center, phase 3 clinical trial.

    Science.gov (United States)

    Song, Joon Young; Cheong, Hee Jin; Lee, Jacob; Woo, Heung Jeong; Wie, Seong-Heon; Lee, Jin-Soo; Kim, Shin Woo; Noh, Ji Yun; Choi, Won Suk; Kim, Hun; Kim, Kyung-Ho; Kim, Woo Joo

    2015-10-05

    Cell culture-derived influenza vaccines (CCIVs) have several important advantages over egg-based influenza vaccines, including shorter production time, better preservation of wild-type virus antigenicity and large-scale production capacity. A randomized, double-blind, phase 3 trial was undertaken to evaluate the immunogenicity and safety of a novel cell culture-derived inactivated, subunit, trivalent influenza vaccine (NBP607, SK Chemicals, Seongnam, Korea) compared to the control vaccine (AgrippalS1, Novartis Vaccines and Diagnostics Srl, Siena, Italy) among healthy adults aged 19 years or older (Clinical trial Number-NCT02344134). Immunogenicity was determined at pre-vaccination, 1 month and 6 month post-vaccination by the hemagglutination inhibition assay. Solicited and unsolicited adverse events were assessed after vaccination. A total of 1156 healthy subjects were recruited. NBP607 met all of the criteria of Committee for Medicinal Products for Human Use (CHMP) at 21 days post-vaccination. Contrary to NBP607, the control vaccine did not satisfy the seroconversion criteria for influenza B irrespective of age. Although the geometric mean titer for each influenza subtype declined gradually, seroprotection rate still remained ≥80% for all subtypes up to six month after NBP607 administration. NBP607 recipients met the seroprotection criteria for all three influenza subtypes up to 6 month post-vaccination. There was no significant difference in the occurrence of adverse events between the NBP607 and control groups. NBP607, a novel CCIV, showed excellent immunogenicity that lasted ≥6 months after vaccination and had tolerable safety profiles. In particular, NBP607 was more immunogenic against influenza B compared to the control, an egg-based subunit vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Immunogenicity and safety of an acellular pertussis, diphtheria ...

    African Journals Online (AJOL)

    Immunogenicity and safety of an acellular pertussis, diphtheria, tetanus, inactivated poliovirus, Hib-conjugate combined vaccine (Pentaxim™) and monovalent hepatitis B vaccine at 6, 10 and 14 weeks of age in infants in South Africa.

  6. Subunit compensation and plasticity of synaptic GABAA receptors induced by ethanol in α4 subunit knockout mice

    Directory of Open Access Journals (Sweden)

    Asha eSuryanarayanan

    2011-09-01

    Full Text Available There is considerable evidence that ethanol (EtOH potentiates γ-aminobutyric acid type A receptor (GABAAR action, but only GABAARs containing δ subunits appear sensitive to low mM EtOH. The α4 and δ subunits co-assemble into GABAARs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABAAR function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABAARs. Based on this hypothesis, any highly EtOH-sensitive GABAARs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABAARs in mice with a global deletion of the α4 subunit (KO. The dentate granule cells (DGCs of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABAARs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p. revealed increased intracellular fraction of the α1, α4 and δ, but not α2, α5 or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 μM, requiring α1/α2, inactive at α4/5 GABAARs than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice

  7. A next-generation, serum-free, highly purified Vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab® when administered according to a post-exposure regimen in healthy children and adults in China.

    Science.gov (United States)

    Li, Rongcheng; Huang, Lirong; Li, Jia; Mo, Zhaojun; He, Bin; Wang, Yunpeng; Wu, Xiaohong; Minutello, Maria; Guinet-Morlot, Françoise; Pichon, Sylvie

    2013-12-05

    As an evolution of its currently licensed rabies vaccine Verorab(®), Sanofi Pasteur has developed a next-generation, serum-free, highly purified Vero rabies vaccine (PVRV-NG). Through this Phase III clinical trial, we aimed to demonstrate the non-inferiority of PVRV-NG over Verorab when administered according to a post-exposure regimen and to assess its clinical safety. A total of 816 healthy subjects aged ≥10 years were randomized according to a 2:1 ratio to receive PVRV-NG or Verorab. Half of the subjects were aged 10-17 years, the other half were aged ≥18 years. All subjects were to receive 5 injections on days 0, 3, 7, 14 and 28. Three blood samples were taken for rabies virus neutralizing antibodies (RVNA) assessment, at baseline, on day 14 and day 42. Solicited adverse reactions (between injections 1, 2 and 3, and within 7 days post-injections 4 and 5) and adverse events (up to 28 days after the last injection) were collected for clinical safety assessment; serious adverse events were reported up to 6-months after the last injection. The proportion of subjects with an RVNA titer ≥0.5 IU/mL after the third injection of PVRV-NG was non-inferior to the proportion of those who received Verorab. PVRV-NG was shown to be as immunogenic as Verorab in each age range in the per-protocol and full analysis sets. PVRV-NG induced a strong immune response in both age ranges, with high RVNA levels and increased geometric mean titers compared to baseline after each measured time point. PVRV-NG had a satisfactory safety profile after each injection, similar to Verorab with regards to the nature, frequency, duration and severity of adverse events. Two serious adverse events were reported, none was related to vaccination. This trial demonstrated the immunogenic non-inferiority of PVRV-NG over Verorab and showed that both vaccines have similar safety profiles. This trial is registered at ClinicalTrials.gov (NCT01339312). This manuscript is the first full report of the

  8. Translating HIV sequences into quantitative fitness landscapes predicts viral vulnerabilities for rational immunogen design

    Science.gov (United States)

    Ferguson, Andrew L.; Mann, Jaclyn K.; Omarjee, Saleha; Ndung’u, Thumbi; Walker, Bruce D.; Chakraborty, Arup K.

    2013-01-01

    Summary A prophylactic or therapeutic vaccine offers the best hope to curb the HIV-AIDS epidemic gripping sub-Saharan Africa, but remains elusive. A major challenge is the extreme viral sequence variability among strains. Systematic means to guide immunogen design for highly variable pathogens like HIV are not available. Using computational models, we have developed an approach to translate available viral sequence data into quantitative landscapes of viral fitness as a function of the amino acid sequences of its constituent proteins. Predictions emerging from our computationally defined landscapes for the proteins of HIV-1 clade B Gag were positively tested against new in vitro fitness measurements, and were consistent with previously defined in vitro measurements and clinical observations. These landscapes chart the peaks and valleys of viral fitness as protein sequences change, and inform the design of immunogens and therapies that can target regions of the virus most vulnerable to selection pressure. PMID:23521886

  9. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  10. Accurate prediction of immunogenic T-cell epitopes from epitope sequences using the genetic algorithm-based ensemble learning.

    Science.gov (United States)

    Zhang, Wen; Niu, Yanqing; Zou, Hua; Luo, Longqiang; Liu, Qianchao; Wu, Weijian

    2015-01-01

    T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system. In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA)-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve) on the training set. Two datasets named 'IMMA2' and 'PAAQD' are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant. The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.

  11. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W., E-mail: mcho@iastate.edu

    2016-03-15

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; {sup 671}NWFDITNWLWYIK{sup 683}) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. - Highlights: • Four gp41 MPER-based immunogens that resemble fusion intermediates were generated. • C-terminal region of MPER that contains 4E10/10E8 epitopes was highly immunogenic. • Altering 6HB structure can influence immunogenic properties of the MPER. • Induced antibodies targeted multiple residues critical for 4E10/10E8 binding. • Development of immunogens based on fusion intermediates is a promising strategy.

  12. Hydroxyl radical modification of collagen type II increases its arthritogenicity and immunogenicity.

    Science.gov (United States)

    Shahab, Uzma; Ahmad, Saheem; Moinuddin; Dixit, Kiran; Habib, Safia; Alam, Khursheed; Ali, Asif

    2012-01-01

    The oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA). Collagen induced arthritis (CIA) in rodents (rats and mice) is an accepted experimental model for RA. Hydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII) was modified by •OH radical (CII-OH) and analysed by ultraviolet-visible (UV-VIS), fluorescence and circular dichroism (CD) spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA). The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatory markers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group. Neo-antigenic epitopes were generated on (•)OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA.

  13. Reappraisal of the Immunogenicity and Safety of Three Hepatitis A Vaccines in Adolescents.

    Science.gov (United States)

    Yoon, Seo Hee; Kim, Han Wool; Ahn, Jong Gyun; Kim, In Tae; Kim, Jong-Hyun; Kong, Kyoung Ae; Kim, Kyung-Hyo

    2016-01-01

    Although the overall incidence of hepatitis A in Korea has been decreasing, adolescents remain highly vulnerable to its outbreaks. This study was conducted to compare the immunogenicity and safety of three hepatitis A vaccines in Korean adolescents. Healthy anti-hepatitis A virus seronegative subjects aged 13 to 19 yr were randomized in three equal groups to receive two doses of Avaxim™, Epaxal®, or Havrix®, 6 to 12 months apart. Seroconversion rates one month after the first dose were 98%, 95%, and 93% for Avaxim™, Epaxal®, and Havrix®, respectively. Seroconversion rates reached 100% for all vaccine groups one month after the second dose. Anti-HAV geometric mean concentrations (GMCs) were 7,207.7 mIU/mL (95% CI, 6023.1-8684.7), 1,750.5 mIU/mL (95% CI, 1362.9-2248.3), and 1,953.5 mIU/mL (95% CI, 1459.4-2614.7) after two doses of Avaxim™, Epaxal®, and Havrix® respectively. Avaxim™ was significantly more immunogenic than Epaxal® and Havrix®, whereas there were no significant differences in antibody responses between Epaxal® and Havrix®. Local and systemic solicited adverse events (AEs) were mostly of mild-to-moderate intensity and resolved within 5 days. No serious AEs were reported. In conclusion, all three vaccines are highly immunogenic and well-tolerated in Korean adolescents. (Clinical Trial Registry NCT00483470).

  14. Hydroxyl radical modification of collagen type II increases its arthritogenicity and immunogenicity.

    Directory of Open Access Journals (Sweden)

    Uzma Shahab

    Full Text Available BACKGROUND: The oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA. Collagen induced arthritis (CIA in rodents (rats and mice is an accepted experimental model for RA. METHODOLOGY/PRINCIPAL FINDINGS: Hydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII was modified by •OH radical (CII-OH and analysed by ultraviolet-visible (UV-VIS, fluorescence and circular dichroism (CD spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA. The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatory markers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group. CONCLUSIONS/SIGNIFICANCE: Neo-antigenic epitopes were generated on (•OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA.

  15. Stable transfection of Eimeria intestinalis and investigation of its life cycle, reproduction and immunogenicity

    Directory of Open Access Journals (Sweden)

    Tuanyuan eShi

    2016-05-01

    Full Text Available Rabbit coccidiosis, caused by infection of Eimeria spp. is one of the most severe parasitic diseases in rabbits. E. intestinalis is one of the most immunogenic species in rabbit coccidia. Due to the lack of genomic information and unsuccessful in vitro cultivation, genetic manipulation of rabbit coccidia lagged behind other apicomplexan parasites. Using regulatory sequences from E. tenella, we obtained a transgenic line of E. intestinalis expressing yellow fluorescent protein (YFP. YFP was continuously expressed throughout the whole life cycle. Morphological features of E. intestinalis in the different developmental stages were dynamically observed with the transgenic line. Some important features in the endogenous development stages were observed. Trophozoites were found as early as 4 h post inoculation. Two-types of schizonts and merozoites were observed in first three of the four schizogonies. Beside jejunum and ileum, gametogony stage and oocysts were also found in the duodenum and vermiform appendix. In addition, the transgenic strain was highly immunogenic but less pathogenic than the wild type. Considering the high immunogenicity of E. intestinalis and amenability to transfection with foreign genes, transgenic E. intestinalis could be a promising oral eukaryotic vaccine vector.

  16. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

  17. A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine.

    Directory of Open Access Journals (Sweden)

    William P Halford

    2011-03-01

    Full Text Available Glycoprotein D (gD-2 is the entry receptor of herpes simplex virus 2 (HSV-2, and is the immunogen in the pharmaceutical industry's lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0⁻ virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain. In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0⁻ virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein.

  18. Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs.

    Science.gov (United States)

    Fiuza, Jacqueline Araújo; Santiago, Helton da Costa; Selvapandiyan, Angamuthu; Gannavaram, Sreenivas; Ricci, Natasha Delaqua; Bueno, Lilian Lacerda; Bartholomeu, Daniella Castanheira; Correa-Oliveira, Rodrigo; Nakhasi, Hira Lal; Fujiwara, Ricardo Toshio

    2013-04-03

    Zoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen(-/-) in a canine infection model and compared it to that of Leishmune(®), a commercially available recombinant vaccine. The immunogenicity of the LdCen(-/-) parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen(-/-) resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen(-/-) vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Immunogenic cell death due to a new photodynamic therapy (PDT) with glycoconjugated chlorin (G-chlorin).

    Science.gov (United States)

    Tanaka, Mamoru; Kataoka, Hiromi; Yano, Shigenobu; Sawada, Takuya; Akashi, Haruo; Inoue, Masahiro; Suzuki, Shugo; Inagaki, Yusuke; Hayashi, Noriyuki; Nishie, Hirotada; Shimura, Takaya; Mizoshita, Tsutomu; Mori, Yoshinori; Kubota, Eiji; Tanida, Satoshi; Takahashi, Satoru; Joh, Takashi

    2016-07-26

    Both the pre-apoptotic exposure to calreticulin (CRT) and the post-apoptotic release of high-mobility group box 1 protein (HMGB1) are required for immunogenic cell death. Photodynamic therapy (PDT) uses non-toxic photosensitizers and visible light at a specific wavelength in combination with oxygen to produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis and/or necrosis, shut down the tumor microvasculature, and stimulate the host immune system. We have previously shown that glycoconjugated chlorin (G-chlorin) has superior cancer cell selectivity and effectively suppresses the growth of xenograft tumors. In the present study, we evaluated the immunogenicity of PDT with G-chlorin treatment in colon cancer cells. PDT with G-chlorin suppressed CT26 (mouse colon cancer cells) tumor growth considerably more efficiently in immunocompetent mice (wild-type mice, allograft model) than in immune-deficient mice (nude mice, xenograft model), although control treatments were not different between the two. This treatment also induced CRT translocation and HMGB1 release in cells, as shown by western blot and immunofluorescence staining. To evaluate the use of PDT-treated cells as a tumor vaccine, we employed a syngeneic mouse tumor model (allograft model). Mice inoculated with PDT-treated CT26 cells were significantly protected against a subsequent challenge with live CT26 cells, and this protection was inhibited by siRNA for CRT or HMGB1. In conclusion, PDT with G-chlorin treatment induced immunogenic cell death in a mouse model, where the immunogenicity of this treatment was directed by CRT expression and HMGB1 release.

  20. Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

    Science.gov (United States)

    Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

    2015-01-01

    Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

  1. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

    Directory of Open Access Journals (Sweden)

    George Janssen

    Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

  2. Autonomic function in mice lacking alpha5 neuronal nicotinic acetylcholine receptor subunit.

    Science.gov (United States)

    Wang, Ningshan; Orr-Urtreger, Avi; Chapman, Joab; Rabinowitz, Ruth; Nachman, Rachel; Korczyn, Amos D

    2002-07-15

    Neuronal acetylcholine nicotinic receptors (nAChR) are composed of 12 subunits (alpha2-10, beta2-4), of which alpha3, alpha5, alpha7, beta2 and beta4 subunits are known to exist in the autonomic nervous system (ANS). alpha5 subunits possess unique biophysical and pharmacological properties. The present study was undertaken to examine the functional role and pharmacological properties of the nAChR alpha5 subunits in the ANS using mice lacking alpha5 nAChR subunits (alpha5-/-). These mice grew to normal size showing no obvious physical or neurological deficit. They also showed normality in thermoregulation, pupil size and resting heart rate under physiological conditions. The heart rate and rectal temperature did not differ between alpha5-/- and wild-type mice during exposure to cold stress. An impairment of cardiac parasympathetic ganglionic transmission was observed during high frequency vagal stimulation, which caused cardiac arrest in all wild-type animals while alpha5-/- mice were more resistant. Deficiency of alpha5 subunits strikingly increased the sensitivity to a low concentration of hexamethonium, leading to a nearly complete blockade of bradycardia in response to vagal stimulation. Such a concentration of hexamethonium only slightly depressed the effects of vagal stimulation in control mice. Deficiency of alpha5 subunits significantly increased ileal contractile responses to cytisine and epibatidine. These results suggest that alpha5 subunits may affect the affinity and sensitivity of agonists and antagonists in the native receptors. Previous studies revealed that alpha5 subunits form functional receptors only in combination with other alpha and beta subunits. Thus, the data presented here imply that alpha5 subunits modulate the activity of nAChR in autonomic ganglia in vivo.

  3. Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.

    Science.gov (United States)

    Sanchez, J; Svennerholm, A M; Holmgren, J

    1988-12-05

    A decapeptide highly homologous to the STa Escherichia coli heat-stable enterotoxin and to several other heat-stable enterotoxins was fused genetically to the amino-end of the B-subunit of cholera toxin (CTB) and the hybrid protein gene expressed from a tacP overexpression system. The STa-related decapeptide used, which was encoded by a synthetic oligodeoxynucleotide, contained a single mutation which substituted a disulfide-linked cysteine by alanine. After its fusion to CTB the decapeptide was able to both react with and to give rise to anti-STa antibodies. Expression of the decapeptide-CTB hybrid by non-toxigenic Vibrio cholerae resulted in its full secretion into the extracellular milieu from where it could then be readily purified by single-step affinity chromatography using immobilized GM1 ganglioside. Bacteria producing this non-toxic, immunogenic decapeptide-CTB toxoid might be useful for the development of oral vaccines against diarrhea caused by E. coli and other bacteria producing immunologically related heat-stable enterotoxins, and as a source of immunoreagents for methods used to diagnose disease caused by these bacteria.

  4. Antibodies to a full-length VAR2CSA immunogen are broadly strain-transcendent but do not cross-inhibit different placental-type parasite isolates.

    Directory of Open Access Journals (Sweden)

    Marion Avril

    Full Text Available The high molecular weight, multidomain VAR2CSA protein mediating adhesion of Plasmodium falciparum-infected erythrocytes in the placenta is the leading candidate for a pregnancy malaria vaccine. However, it has been difficult so far to generate strong and consistent adhesion blocking antibody responses against most single-domain VAR2CSA immunogens. Recent advances in expression of the full-length recombinant protein showed it binds with much greater specificity and affinity to chondroitin sulphate A (CSA than individual VAR2CSA domains. This raises the possibility that a specific CSA binding pocket(s is formed in the full length antigen and could be an important target for vaccine development. In this study, we compared the immunogenicity of a full-length VAR2CSA recombinant protein containing all six Duffy binding-like (DBL domains to that of a three-domain construct (DBL4-6 in mice and rabbits. Animals immunized with either immunogen acquired antibodies reacting with several VAR2CSA individual domains by ELISA, but antibody responses against the highly conserved DBL4 domain were weaker in animals immunized with full-length DBL1-6 recombinant protein compared to DBL4-6 recombinant protein. Both immunogens induced cross-reactive antibodies to several heterologous CSA-binding parasite lines expressing different VAR2CSA orthologues. However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species.

  5. Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications.

    Science.gov (United States)

    Lai, Guan-Hua; Lin, Ming-Kuem; Lien, Yi-Yang; Fu, Jiun-Hau; Chen, Hsi-Jien; Huang, Chi-Hung; Tzen, Jason T C; Lee, Meng-Shiou

    2013-08-13

    Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins

  6. Biotechnology approaches to produce potent, self-adjuvanting antigen-adjuvant fusion protein subunit vaccines.

    Science.gov (United States)

    Moyle, Peter Michael

    Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their

  7. All three subunits of soybean beta-conglycinin are potential food allergens.

    Science.gov (United States)

    Krishnan, Hari B; Kim, Won-Seok; Jang, Sungchan; Kerley, Monty S

    2009-02-11

    Soybeans are recognized as one of the "big 8" food allergens. IgE antibodies from soybean-sensitive patients recognize more than 15 soybean proteins. Among these proteins only the alpha-subunit of beta-conglycinin, but not the highly homologous alpha'- and beta-subunits, has been shown to be a major allergenic protein. The objective of this study was to examine if the alpha'- and beta-subunits of beta-conglycinin can also serve as potential allergens. Immunoblot analysis using sera collected from soybean-allergic patients revealed the presence of IgE antibodies that recognized several soy proteins including 72, 70, 52, 34, and 21 kDa proteins. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis of trypsin-digested 72, 70, and 52 kDa proteins indicated that these proteins were the alpha'-, alpha-, and beta-subunits of beta-conglycinin, respectively. Additionally, purified alpha'-, alpha-, and beta-subunits of beta-conglycinin were recognized by IgE antibodies present in the soybean-allergic patients. The IgE reactivity to the beta-subunit of beta-conglycinin was not abolished when this glycoprotein was either deglycosylated using glycosidases or expressed as a recombinant protein in Escherichia coli . The results suggest that in addition to the previously recognized alpha-subunit of beta-conglycinin, the alpha'- and beta-subunits of beta-conglycinin also are potential food allergens.

  8. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice

    Science.gov (United States)

    McGuire, Andrew T.; Gray, Matthew D.; Dosenovic, Pia; Gitlin, Alexander D.; Freund, Natalia T.; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B.; Glenn, Jolene; Seaman, Michael S.; Schief, William R.; Strong, Roland K.; Nussenzweig, Michel C.; Stamatatos, Leonidas

    2016-01-01

    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype. PMID:26907590

  9. Immunisation with a multivalent, subunit vaccine reduces patent infection in a natural bovine model of onchocerciasis during intense field exposure.

    Directory of Open Access Journals (Sweden)

    Benjamin L Makepeace

    Full Text Available Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund's adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia, compared with only 58% of vaccinated cattle (P = 0.015. This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate.

  10. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs

    2016-01-01

    the sum of the risks of the individual subunits. The question is how to allocate the risk capital of the group among the subunits in a fair way. In this paper we propose to use the Lorenz set as an allocation method. We show that the Lorenz set is operational and coherent. Moreover, we propose three......Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller than...... fairness tests related directly to the problem of risk capital allocation and show that the Lorenz set satisfies all three tests in contrast to other well-known coherent methods. Finally, we discuss how to deal with non-uniqueness of the Lorenz set....

  11. Immunogenic peptides comprising a T-helper epitope and a B-cell neutralizing antibody epitope

    Science.gov (United States)

    Haynes, Barton F.; Korber, Bette T.; De Lorimier, Robert M.

    2006-12-26

    The present invention relates, generally, to a polyvalent immunogen and, more particularly, to a method of inducing neutralizing antibodies against HIV and to a polyvalent immunogen suitable for use in such a method.

  12. Effective immunotherapy of weakly immunogenic solid tumours using a combined immunogene therapy and regulatory T-cell inactivation.

    LENUS (Irish Health Repository)

    Whelan, M C

    2012-01-31

    Obstacles to effective immunotherapeutic anti-cancer approaches include poor immunogenicity of the tumour cells and the presence of tolerogenic mechanisms in the tumour microenvironment. We report an effective immune-based treatment of weakly immunogenic, growing solid tumours using a locally delivered immunogene therapy to promote development of immune effector responses in the tumour microenvironment and a systemic based T regulatory cell (Treg) inactivation strategy to potentiate these responses by elimination of tolerogenic or immune suppressor influences. As the JBS fibrosarcoma is weakly immunogenic and accumulates Treg in its microenvironment with progressive growth, we used this tumour model to test our combined immunotherapies. Plasmids encoding GM-CSF and B7-1 were electrically delivered into 100 mm(3) tumours; Treg inactivation was accomplished by systemic administration of anti-CD25 antibody (Ab). Using this approach, we found that complete elimination of tumours was achieved at a level of 60% by immunogene therapy, 25% for Treg inactivation and 90% for combined therapies. Moreover, we found that these responses were immune transferable, systemic, tumour specific and durable. Combined gene-based immune effector therapy and Treg inactivation represents an effective treatment for weakly antigenic solid growing tumours and that could be considered for clinical development.

  13. Assignment of human protein phosphatase 2A regulatory subunit genes B56{alpha}, B56{beta}, B56{gamma}, B56{delta}, and B56{epsilon} (PPP2R5A-PPP2R5E), highly expressed in muscle and brain, to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.1 {r_arrow} p12

    Energy Technology Data Exchange (ETDEWEB)

    McCright, B.; Virshup, D.M.; Brothman, A.R. [Univ. of Utah School of Medicine, Salt Lake City, UT (United States)

    1996-08-15

    The activity of the major intracellular protein phosphatase, protein phosphatase 2A WPM, is determined by the nature of the associated regulatory subunit. A new family of human PP2A regulatory subunits has recently been identified. Three of these subunits, B56{beta}, B56{delta}, and B56{epsilon}, are most highly expressed in brain, while the B56{alpha} and B56{gamma} isoforms are highly expressed in cardiac and skeletal muscle. Genes PPP2R5A-PPP2R5E encoding the phosphatase regulatory proteins B56{alpha}, B56{beta}, B56{gamma}, B56{delta}, and B56{epsilon} have now been mapped by fluorescence in situ hybridization to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2-p12, respectively. 16 refs., 1 fig.

  14. Comparison of assay formats for drug-tolerant immunogenicity testing.

    Science.gov (United States)

    Butterfield, Anthony M; Chain, Jana S; Ackermann, Bradley L; Konrad, Robert J

    2010-12-01

    Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development. Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(®) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive. Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.

  15. Structural characterization of the main immunogenic region of the Torpedo acetylcholine receptor.

    Science.gov (United States)

    Morell, Stuart W; Trinh, Vu B; Gudipati, Eswari; Friend, Alexander; Page, Nelson A; Agius, Mark A; Richman, David P; Fairclough, Robert H

    2014-03-01

    To develop antigen-specific immunotherapies for autoimmune diseases, knowledge of the molecular structure of targeted immunological hotspots will guide the production of reagents to inhibit and halt production of antigen specific attack agents. To this end we have identified three noncontiguous segments of the Torpedo nicotinic acetylcholine receptor (AChR) α-subunit that contribute to the conformationally sensitive immunological hotspot on the AChR termed the main immunogenic region (MIR): α(1-12), α(65-79), and α(110-115). This region is the target of greater than 50% of the anti-AChR Abs in serum from patients with myasthenia gravis (MG) and animals with experimental autoimmune myasthenia gravis (EAMG). Many monoclonal antibodies (mAbs) raised in one species against an electric organ AChR cross react with the neuromuscular AChR MIR in several species. Probing the Torpedo AChR α-subunit with mAb 132A, a disease inducing anti-MIR mAb raised against the Torpedo AChR, we have determined that two of the three MIR segments, α(1-12) and α(65-79), form a complex providing the signature components recognized by mAb 132A. These two segments straddle a third, α(110-115), that seems not to contribute specific side chains for 132A recognition, but is necessary for optimum antibody binding. This third segment appears to form a foundation upon which the three-dimensional 132A epitope is anchored. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Immunogenicity of acrylate chemicals as assessed by antibody induction.

    Science.gov (United States)

    Bull, J E; Henderson, D C; Turk, J L

    1987-01-01

    The immunogenicities of two acrylate chemicals, trimethylolpropane triacrylate (TMPTA) and methyl acrylate (MeAc), and one related vinyl compound, 4-vinyl pyridine (4VP), were investigated by determining the in vivo induction of IgG antibodies in guinea pigs. The injection of the chemicals emulsified in Freund's complete adjuvant resulted in the induction of serum antibody responses against MeAc and 4VP but not TMPTA. However, antibody with anti-TMPTA activity was produced following immunization of guinea pigs with TMPTA conjugated to protein, which allowed comparisons to be made of the immunogenic structural features of the compounds.

  17. Identification of Dominant Immunogenic Bacteria and Bacterial Proteins in Periodontitis

    DEFF Research Database (Denmark)

    Agerbæk, Mette Rylev; Haubek, Dorte; Birkelund, Svend

    Marginal periodontitis is considered an infectious disease that triggers host inflammatory responses resulting in destruction of the periodontium. A complex biofilm of bacteria is associated with periodontitis. Some species have been identified as putative pathogens such as Porphyromonas gingivalis...... (P.g) and Actinobacillus actinomycetemcomitans (A.a), but the identity of dominate immunogens of these bacteria is poorly elucidated. The aim of the study was to identify dominant immunogenic proteins of P.g and A.a in patients suffering from chronic and aggressive periodontitis by proteomic analysis...... will be able to identify immunodominant proteins and potentially important virulence factors of putative periodontal pathogens....

  18. Expression of HIV-1 antigens in plants as potential subunit vaccines

    Directory of Open Access Journals (Sweden)

    Tanzer Fiona L

    2008-06-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24 and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant

  19. Increased intraepithelial CD3+ T-lymphocytes and high PD-L1 expression on tumor cells are associated with a favorable prognosis in esophageal squamous cell carcinoma and allow prognostic immunogenic subgrouping.

    Science.gov (United States)

    Jesinghaus, Moritz; Steiger, Katja; Slotta-Huspenina, Julia; Drecoll, Enken; Pfarr, Nicole; Meyer, Petra; Konukiewitz, Björn; Bettstetter, Marcus; Wieczorek, Kathrin; Ott, Katja; Feith, Markus; Langer, Rupert; Weichert, Wilko; Specht, Katja; Boxberg, Melanie

    2017-07-18

    Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer associated with poor prognosis and additional therapeutic strategies must be implemented to optimize ESCC treatment. Meanwhile, the important biologic role and potential prognostic and therapeutic implications of a tumors immunologic microenvironment (IM) have been recognized in various cancers.In order to investigate the contexture and the prognostic relevance of the IM in ESCC, we immunohistochemically evaluated the extent of overall/intraepithelial TILs (CD3+/CD8+) and of PD-1 / PD-L1 expression in a cohort of 125 therapy-naive ESCCs, additionally assessing PD-L1 copy number status via fluorescence in-situ hybridization.High intraepithelial CD3+ TILs (CD3ihigh) and high PD-L1 expression on tumor cells (PD-L1high) were each significantly associated with improved overall- (OS) (CD3+: p = 0.019; PD-L1: p = 0.028), disease specific- (DSS) (CD3+: p = 0.05; PD-L1: p = 0.006) and disease free survival (DFS) (CD3+: p = 0.009; PD-L1: p < 0.001). CD3ihigh- and PD-L1high cases were significantly associated with one another (p < 0.001). Subgrouping of ESCC revealed decreased OS (p = 0.031), DSS (p = 0.012) and DFS (p < 0.001) for CD3ilow/PD-L1low cancers.Our data not only associate CD3ihigh- and PD-L1high ESCC with a beneficial outcome, but also demonstrate PD-L1high- and CD3ihigh status to be closely intertwined. Furthermore, our study demarcates a prognostically unfavorable, "non-immunoreactive" CD3ilow / PD-L1low ESCC-subgroup, potentially forming the basis for an immune-based stratification of ESCC.

  20. Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers.

    Science.gov (United States)

    Ringe, Rajesh P; Ozorowski, Gabriel; Rantalainen, Kimmo; Struwe, Weston B; Matthews, Katie; Torres, Jonathan L; Yasmeen, Anila; Cottrell, Christopher A; Ketas, Thomas J; LaBranche, Celia C; Montefiori, David C; Cupo, Albert; Crispin, Max; Wilson, Ian A; Ward, Andrew B; Sanders, Rogier W; Klasse, P J; Moore, John P

    2017-08-01

    Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged.IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence

  1. Immunogenicity of heme complexes of peptides designed to mimic the heme environment of myoglobin and hemoglobin.

    Science.gov (United States)

    Atassi, M Zouhair; Childress, Catherine

    2005-01-01

    In the preceding paper (Protein J. 25, pages 37-49, 2005), we reported the preparation and oxygen-binding properties of peptides that form stable complexes with heme mimic. The design of the peptides was based on the natural environment of the heme group in myoglobin (Mb) and in the alpha- and beta-subunits of human adult hemoglobin (Hb). In the present work, the heme-peptides were each administered into mice, either as emulsions in adjuvant (both for injections and boosters) or intravenously as solutions in phosphate-buffered saline. Antibody (Ab) responses, monitored up to 14 weeks after the first administration, showed that when the heme-peptides were injected with adjuvant they stimulated Ab responses against the immunizing peptide, which in most cases bound to the correlate protein (Mb or Hb). However these heme-peptides were non-immunogenic when administered in PBS intravenously. It is concluded that heme-peptides:(a) would not trigger an adverse immune response if used for transfusion purposes.

  2. Immunological and protective effects of Bordetella bronchiseptica subunit vaccines based on the recombinant N-terminal domain of dermonecrotic toxin.

    Science.gov (United States)

    Wang, Chuanwen; Liu, Liping; Zhang, Zhen; Yan, Zhengui; Yu, Cuilian; Shao, Mingxu; Jiang, Xiaodong; Chi, Shanshan; Wei, Kai; Zhu, Ruiliang

    2015-10-01

    Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. The 131-amino-acid repeat region of the essential 39-kilodalton core protein of fowlpox virus FP9, equivalent to vaccinia virus A4L protein, is nonessential and highly immunogenic.

    Science.gov (United States)

    Boulanger, D; Green, P; Smith, T; Czerny, C P; Skinner, M A

    1998-01-01

    The immunodominant, 39,000-molecular weight core protein (39K protein) of fowlpox virus (FP9 strain), equivalent to the vaccinia virus A4L gene product, contains highly charged domains at each end of the protein and multiple copies of a 12-amino-acid serine-rich repeat sequence in the middle of the protein. Similar repeats were also detected in other fowlpox virus strains, suggesting that they might confer a selective advantage to the virus. The molloscum contagiosum virus homolog (MC107L) also contains repeats, unlike the vaccinia virus protein. The number of repeats in the fowlpox virus protein does not seem to be crucial, since some strains have a different number of repeats, as shown by the difference in the size of the protein in these strains. The repeat region could be deleted, indicating that it is not essential for replication in vitro. It was not possible to delete the entire 39K protein, indicating that it was essential (transcriptional control signals for the flanking genes were left intact). The repeat region is partly responsible for the immunodominance of the protein, but the C-terminal part of the protein also contains highly antigenic linear epitopes. A role for the 39K protein in immune system modulation is discussed.

  4. A randomized lot-to-lot immunogenicity consistency study of the candidate zoster vaccine HZ/su.

    Science.gov (United States)

    Strezova, Ana; Godeaux, Olivier; Aggarwal, Naresh; Leroux-Roels, Geert; Lopez-Fauqued, Marta; Van Damme, Pierre; Vanden Abeele, Carline; Vastiau, Ilse; Heineman, Thomas C; Lal, Himal

    2017-12-04

    The risk of developing herpes zoster (HZ) increases with age and is thought to be associated with a decrease in cell-mediated immunity in older adults. The adjuvanted varicella-zoster virus (VZV) glycoprotein E (gE) recombinant subunit vaccine (HZ/su) showed >90% efficacy in the prevention of HZ when administered in adults ≥50 years of age. Here we aim to evaluate immunogenicity consistency of 3 different HZ/su vaccine lots and to assess safety of these lots. This multicenter, phase III, double-blind, randomized study (NCT02075515), assessed lot-to-lot consistency in terms of immunogenicity of HZ/su and also assessed safety of these lots. Participants aged 50 years or older were randomized (1:1:1) to receive 2 doses of HZ/su, 2 months apart, from 1 out of 3 randomized HZ/su lots (Lots A, B and C). Humoral immunogenicity was assessed pre-vaccination and 1 month post-second vaccination by anti-gE antibody enzyme-linked immunosorbent assay. Lot-to-lot consistency was demonstrated if the 2-sided 95% confidence intervals of the anti-gE geometric mean concentration ratio between all lot pairs were within 0.67 and 1.5. Solicited symptoms were recorded within 7 days and unsolicited adverse events (AEs) within 30 days after each vaccination. Serious AEs (SAEs) and potential immune-mediated diseases (pIMDs) were reported until study end (12 months post-second vaccination). Of 651 participants enrolled in the study, 638 received both doses of the HZ/su vaccine and 634 completed the study. Humoral immune responses were robust and consistency between 3 manufacturing lots was demonstrated. The incidence of solicited symptoms, unsolicited AEs and SAEs was comparable between all lots. Three fatal SAEs, 1 in each lot, were reported, none of which were considered vaccine-related by investigator assessment. Two out of the 8 reported pIMDs were considered vaccine-related by the investigator. The three HZ/su manufacturing lots demonstrated consistent immunogenicity. No safety

  5. Testing experimental subunit furunculosis vaccines for rainbow trout

    DEFF Research Database (Denmark)

    Marana, Moonika H.; Chettri, Jiwan Kumar; Skov, Jakob

    2016-01-01

    Aeromonas salmonicida subsp. salmonicida (AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality. In this study we vaccinated rainbow...... trout with subunit vaccines containing protein antigens that were selected based on an in silico antigen discovery approach. Thus, the proteome of AS strain A449 was analyzed by an antigen discovery platform and its proteins consequently ranked by their predicted ability to evoke protective immune...... response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent...

  6. Immunogenic Response of Rabbits to Monovalent and Polyvalent ...

    African Journals Online (AJOL)

    Slide agglutination and indirect haemagglutination tests were used for detecting and measuring specific antibodies against the strains used. Antisera against polyvalent immunogens protected 83-100% of rabbits against A1, A7 and 50% of rabbits against A2 challenge while the lowest protection (16- 33%) was seen in ...

  7. Comparative immunogenicity of local and imported infectious bursal ...

    African Journals Online (AJOL)

    A comparative immunogenicity and efficacy study of local and imported infectious bursal disease (IBD) vaccines administered to chicks (cockerels) at varying regimes (10 and 18, 10 and 28, 14 and 35 days of age) was carried out. The test birds were challenged seven days after the booster dose of the IBD vaccine by ...

  8. Immunogenicity and Pathology of Formalin-Killed-Sepa Salmonella ...

    African Journals Online (AJOL)

    Bulletin of Animal Health and Production in Africa ... Intensive poultry production in Nigeria is currently on the increase with associated health challenges. ... The unimmunized control group (UC) was infected with 1x108cfu /ml Salmonella enterica Paratyphi A. The formalin-killed vaccine of SEPA was immunogenic in poultry ...

  9. Major integral membrane protein immunogens of Treponema pallidum are proteolipids

    Energy Technology Data Exchange (ETDEWEB)

    Chamberlain, N.R.; Brandt, M.E.; Erwin, A.L.; Radolf, J.D.; Norgard, M.V. (Univ. of Texas Southwestern Medical Center, Dallas (USA))

    1989-09-01

    A number of the major pathogen-specific immunogens of Treponema pallidum were characterized recently as amphiphilic, integral membrane proteins by phase partitioning with Triton X-114. In the present study, we demonstrated that the same membrane immunogens (designated as detergent phase proteins (DPPs)) become radiolabeled upon in vitro incubation of T. pallidum with various {sup 3}H-labeled fatty acids. Radioimmunoprecipitation with a monoclonal antibody confirmed that the {sup 3}H-labeled 47-kilodalton protein corresponded to the well-characterized treponemal antigen with the identical apparent molecular mass. Failure to detect {sup 3}H-labeled DPPs following incubation with erythromycin confirmed that protein acylation required de novo protein synthesis by the bacteria. When treponemes were incubated with ({sup 3}H)myristate, ({sup 3}H)palmitate, or ({sup 3}H)oleate, radiolabeled proteins corresponding to the DPPs were detected upon autoradiography. Demonstration that a number of the abundant membrane immunogens of T. pallidum are proteolipids provides information to help clarify their membrane association(s) and may serve to explain their extraordinary immunogenicity.

  10. Immunogenicity of a lowcost hepatitis Bvaccine in the South African ...

    African Journals Online (AJOL)

    Background. A low-cost, 'flash' heat-inactivated hepatitis B vaccine with enhanced immunogenicity allowing for a relatively low dose (Hepaccine B; Cheil Foods and Chemicals, Korea) was introduced into the South African Expanded Programme on Immunisation during 1995 to immunise infants against hepatitis B. To ...

  11. Immunogenicity and safety of a live attenuated varicella vaccine in ...

    African Journals Online (AJOL)

    To investigate the safety of live attenuated varicella vaccine (aka strain) and the optimal virus titre/ dose required for immunogenicity in healthy South African children. ... Six subjects who were initially seropositive maintained or increased their titres post-vaccination; 3 of the 6 showed a booster response (a ;:;, 4-fold increase ...

  12. Immunogenicity and efficacy of Hoshino strain of Mumps Vaccine in ...

    African Journals Online (AJOL)

    This report describes the immunogenicity and efficacy and long term immunity of Hoshino strain of Mumps (included in MMR Vaccine) in shahr-e-kord, Islamic Republic of Iran (I.R.Iran). A total of 338 Children aged 3-18 years were tested for Mumps IgG using enzyme-linked immunosorbent assay (ELISA). The proportion of ...

  13. Immunogenic Properties of Ricinus Communis Var Minor Seed on ...

    African Journals Online (AJOL)

    The immunogenic properties of Ricinus communis var minor seed was determined after feeding 7 healthy virgin albino white rabbits with varying doses of 0.5g – 0.9g dried ground Ricinus communis var minor seed included in their feed (5g/100g body weight). Booster doses of the same weight were further administered ...

  14. Field Evaluation of Immunogenicity of Five Commercial Vaccines ...

    African Journals Online (AJOL)

    This study was conducted to evaluate immunogenicity of five commonly used vaccines for prevention of Newcastle disease (ND) in Ibadan, the capital city of Oyo State Nigeria. Two hundred and twenty (220) blood samples were collected from apparently healthy vaccinated chickenin 8 poultry farms in suburbs of the city.

  15. Safety and immunogenicity of two Haemophilus influenzae type b ...

    African Journals Online (AJOL)

    Objectives. Haemophilus influenzae type b (Hib) infection remains a major public health problem inthe developing world. We evaluated the safety and immunogenicity of a new PRP-CRM197 conjugate Hib vaccine (Vaxem Hib, Chiron Vacdnes), compared with theHibTITER vaccine (WyethLederle Vaccines), following the ...

  16. Immunogenicity and safety of a live attenuated varicella vaccine in ...

    African Journals Online (AJOL)

    Objectives. To investigate the safety of live attenuated varicella vaccine (aka strain) and the optimal virus titre/ dose required for immunogenicity in healthy South African children. Design. Double-blind randomised clinical study using two different lots of varicella vaccine, each at two different titres. Subjects were randomly ...

  17. Safety and immunogenicity of GMZ2 - a MSP3-GLURP fusion protein malaria vaccine candidate

    DEFF Research Database (Denmark)

    Esen, Meral; Kremsner, Peter G; Schleucher, Regina

    2009-01-01

    Malaria is a major public health problem in Sub-Saharan Africa. In highly endemic regions infants, children and pregnant women are mostly affected. An effective malaria vaccine would complement existing malaria control strategies because it can be integrated in existing immunization programs easily....... Here we present the results of the first phase Ia clinical trial of GMZ2 adjuvanted in aluminium hydroxide. GMZ2 is a malaria vaccine candidate, designed upon the rationale to induce immune responses against asexual blood stages of Plasmodium falciparum similar to those encountered in semi...... is a safe and immunogenic malaria vaccine candidate suitable for further clinical development....

  18. The immunogenicity of a single dose of hepatitis A virus vaccines (Havrix® and Epaxal®) in Korean young adults.

    Science.gov (United States)

    Lim, Jiseun; Song, Yeong-Jun; Park, Woong-Sub; Sohn, Haesook; Lee, Moo-Sik; Shin, Dong-Hoon; Kim, Chun-Bae; Kim, Hwasung; Oh, Gyung-Jae; Ki, Moran

    2014-01-01

    Assessing the immunogenicity of a single dose of hepatitis A virus (HAV) vaccines is important because some people receive only a single dose. However, previous studies have shown variable results and have not examined the effects of demographic characteristics other than gender. This study was performed to examine the immunogenicity of a single dose of HAV vaccine according to the vaccine type and demographic characteristics in young adults. Seronegative medical school students were randomly allocated to receive either Havrix or Epaxal. After approximately 11 months, the seroconversion rate in 451 participants was 80.7%. In men, the Havrix group showed a significantly higher seroconversion rate (81.9%) than the Epaxal group (69.2%), whereas both vaccine groups showed similarly high immunogenicity in women (Havrix: 90.1%, Epaxal: 92.9%; P for interaction=0.062). According to the results of a multivariate analysis, Epaxal showed significantly lower immunogenicity than Havrix only in men. Age, obesity, drinking, smoking, and follow-up time did not significantly affect seroconversion in either gender. The seroconversion rate of single-dose HAV vaccines was low in men, particularly in those who received Epaxal. Our results suggest that gender effects should be considered when comparing the immunogenicity of different HAV vaccines.

  19. Soluble HIV-1 Envelope Immunogens Derived from an Elite Neutralizer Elicit Cross-Reactive V1V2 Antibodies and Low Potency Neutralizing Antibodies

    Science.gov (United States)

    Glenn, Jolene; Stamatatos, Leonidas; Sather, D. Noah

    2014-01-01

    We evaluated four gp140 Envelope protein vaccine immunogens that were derived from an elite neutralizer, subject VC10042, whose plasma was able to potently neutralize a wide array of genetically distinct HIV-1 isolates. We sought to determine whether soluble Envelope proteins derived from the viruses circulating in VC10042 could be used as immunogens to elicit similar neutralizing antibody responses by vaccination. Each gp140 was tested in its trimeric and monomeric forms, and we evaluated two gp140 trimer vaccine regimens in which adjuvant was supplied at all four immunizations or at only the first two immunizations. Interestingly, all four Envelope immunogens elicited high titers of cross-reactive antibodies that recognize the variable regions V1V2 and are potentially similar to antibodies linked with a reduced risk of HIV-1 acquisition in the RV144 vaccine trial. Two of the four immunogens elicited neutralizing antibody responses that neutralized a wide array of HIV-1 isolates from across genetic clades, but those responses were of very low potency. There were no significant differences in the responses elicited by trimers or monomers, nor was there a significant difference between the two adjuvant regimens. Our study identified two promising Envelope immunogens that elicited anti-V1V2 antibodies and broad, but low potency, neutralizing antibody responses. PMID:24466285

  20. Reappraisal of the Immunogenicity and Safety of Three Hepatitis A Vaccines in Adolescents

    Science.gov (United States)

    2016-01-01

    Although the overall incidence of hepatitis A in Korea has been decreasing, adolescents remain highly vulnerable to its outbreaks. This study was conducted to compare the immunogenicity and safety of three hepatitis A vaccines in Korean adolescents. Healthy anti-hepatitis A virus seronegative subjects aged 13 to 19 yr were randomized in three equal groups to receive two doses of Avaxim™, Epaxal®, or Havrix®, 6 to 12 months apart. Seroconversion rates one month after the first dose were 98%, 95%, and 93% for Avaxim™, Epaxal®, and Havrix®, respectively. Seroconversion rates reached 100% for all vaccine groups one month after the second dose. Anti-HAV geometric mean concentrations (GMCs) were 7,207.7 mIU/mL (95% CI, 6023.1-8684.7), 1,750.5 mIU/mL (95% CI, 1362.9-2248.3), and 1,953.5 mIU/mL (95% CI, 1459.4-2614.7) after two doses of Avaxim™, Epaxal®, and Havrix® respectively. Avaxim™ was significantly more immunogenic than Epaxal® and Havrix®, whereas there were no significant differences in antibody responses between Epaxal® and Havrix®. Local and systemic solicited adverse events (AEs) were mostly of mild-to-moderate intensity and resolved within 5 days. No serious AEs were reported. In conclusion, all three vaccines are highly immunogenic and well-tolerated in Korean adolescents. (Clinical Trial Registry NCT00483470) PMID:26770041

  1. Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis.

    Science.gov (United States)

    Dey, Ayan; Kumar, Umesh; Sharma, Pawan; Singh, Sarman

    2009-08-13

    Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-gamma and IL-2 response against kinesin (3012+/-102 and 367.5+/-8.92pg/ml) and ESAT-6 (1334+/-46.5 and 245.1+/-7.72pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-gamma and IL-2) against individual construct was lower (kinesin motor domain: 1788+/-36.48 and 341.8+/-9.801pg/ml and ESAT-6: 867.0+/-47.23 and 170.8+/-4.578pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-gamma production against chimera vaccination is statistically significant (pleishmaniasis and tuberculosis and have important implication in future vaccine design.

  2. Idiotype vaccination against murine B cell lymphoma. Humoral and cellular responses elicited by tumor-derived immunoglobulin M and its molecular subunits.

    Science.gov (United States)

    Campbell, M J; Carroll, W; Kon, S; Thielemans, K; Rothbard, J B; Levy, S; Levy, R

    1987-10-15

    C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.

  3. A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Peng, Bo; Shi, Lin; Chen, Huanchun; Li, Xiangmin

    2015-05-15

    Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17-25nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; PGM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (PGM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Over-expressions of AMPK subunits in ovarian carcinomas with significant clinical implications

    Directory of Open Access Journals (Sweden)

    Li Cuilan

    2012-08-01

    Full Text Available Abstract Background AMP-activated protein kinase (AMPK has recently been considered as a potential target for cancer therapy. However, the expression status of various subunits of the heterotrimeric AMPK in human cancers is rarely reported. We decided to determine their expressions in ovarian carcinomas and their relationships with the disease. Methods Expressions and locations of the AMPK-α1, -α2, -β1, -β2, -γ1 and -γ2 were detected by quantitative PCR (Q-PCR and immunohistochemical staining (IHC. Their expression levels in ovarian tumors were compared with normal controls and also correlated with clinicopathological parameters. Results Except AMPK-α1, expressions of the other five AMPK subunits are significantly higher in ovarian carcinomas as determined by Q-PCR. Although IHC detection of AMPK-γ1 and -γ2 were not successful, over-expressions of AMPK-α2, -β1, and -β2 were further confirmed by IHC. Over-expressions of various AMPK subunits occurred independently and were mainly detected in the cytoplasm. Interestingly, AMPK-α2 and -β1 were also detected in the nucleus and cell membrane, respectively. Clinical correlation analyses indicate that expressions of different AMPK subunits are associated with different subtypes of carcinoma. High expression of AMPK-α2 is significantly associated with endometrioid carcinomas. On the other hand, high expressions of AMPK-β and -γ subunits are associated with mucinous and serous carcinomas, respectively. Furthermore, high expressions of AMPK-β1 and -γ2 are also associated with early and late stages of disease, respectively. Finally, patients with high expression of AMPK-α2 had better prognosis. Conclusions Aberrant expressions of AMPK subunits may play important roles in ovarian carcinogenesis. Each AMPK subunit may have its own function other than just a component of the AMPK molecule. Correlations with clinical parameters suggest that expressions of AMPK subunits have different

  5. Oncogenic potential of guanine nucleotide stimulatory factor alpha subunit in thyroid glands of transgenic mice.

    OpenAIRE

    Michiels, F M; Caillou, B; Talbot, M; Dessarps-Freichey, F; Maunoury, M T; Schlumberger, M; Mercken, L; Monier, R; Feunteun, J

    1994-01-01

    Transgenic mice have been used to address the issue of the oncogenic potential of mutant guanine nucleotide stimulatory factor (Gs) alpha subunit in the thyroid gland. The expression of the mutant Arg-201-->His Gs alpha subunit transgene has been directed to murine thyroid epithelial cells by bovine thyroglobulin promoter. The transgenic animals develop hyperfunctioning thyroid adenomas with increased intracellular cAMP levels and high uptake of [125I]iodine and produced elevated levels of ci...

  6. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    Science.gov (United States)

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  7. Screening for AMPA receptor auxiliary subunit specific modulators.

    Directory of Open Access Journals (Sweden)

    Caleigh M Azumaya

    Full Text Available AMPA receptors (AMPAR are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM, respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R-CNIH3 and GluA2(Q complexes and PAM activity in GluA2(Q-GSG1L complexes.

  8. Efficacy, Immunogenicity and Safety of a Human Rotavirus Vaccine RIX4414 in Singaporean Infants.

    Science.gov (United States)

    Phua, Kong Boo; Lim, Fong Seng; Quak, Seng Hock; Lee, Bee Wah; Teoh, Yee Leong; Suryakiran, Pemmaraju V; Han, Htay Htay; Bock, Hans L

    2016-02-01

    This was the first study conducted to evaluate the efficacy of 2 oral doses of the human rotavirus vaccine, RIX4414 in Singaporean infants during the first 3 years of life. Healthy infants, 11 to 17 weeks of age were enrolled in this randomised (1:1), double-blinded, placebo-controlled study to receive 2 oral doses of RIX4414 vaccine/placebo following a 0-, 1-month schedule. Vaccine efficacy against severe rotavirus (RV) gastroenteritis (Vesikari score ≥11) caused by wild-type RV strains from a period starting from 2 weeks post-Dose 2 until 2 and 3 years of age was calculated with 95% confidence interval (CI). Immunogenicity and safety of the vaccine were also assessed. Of 6542 infants enrolled, 6466 were included in the efficacy analysis and a subset of 100 infants was included in the immunogenicity analysis. Fewer severe RV gastroenteritis episodes were reported in the RIX4414 group when compared to placebo at both 2 and 3 year follow-up periods. Vaccine efficacy against severe RV gastroenteritis at the respective time points were 93.8% (95% CI, 59.9 to 99.9) and 95.2% (95% CI, 70.5 to 99.9). One to 2 months post-Dose 2 of RIX4414, 97.5% (95% CI, 86.8 to 99.9) of infants seroconverted for anti-RV IgA antibodies. The number of serious adverse events recorded from Dose 1 until 3 years of age was similar in both groups. Two oral doses of RIX4414 vaccine was immunogenic and provided high level of protection against severe RV gastroenteritis in Singaporean children, during the first 3 years of life when the disease burden is highest.

  9. Immunogenicity of human and bovine insulin in type 1 diabetes mellitus patients.

    Science.gov (United States)

    Himanshu, M; Bhat, Chetan B; Ramchandran, Sarita; Bhat, K Geetha; Kumar, Prasanna K M

    2008-08-01

    The advantages of synthetic insulin (human insulin) over bovine insulin in the treatment of type 1 diabetes mellitus (DM) are much debated in terms of potency and purity. Immunogenicity is one of several factors that determine potency and safety. This study was designed to investigate and study the difference in immunogenicity of human and bovine insulin. We investigated anti-insulin antibody (AIAB) status in 69 type 1 DM patients receiving insulin therapy. Group 1 had 33 patients treated with bovine insulin, and group 2 had 32 patients treated with human insulin. All patients had received their respective insulin therapy for a minimum period of 1 year and had no history of change in insulin type. Forty-three subjects from the normal population were the control group. AIABs were assayed in serum samples of all subjects using a semiquantitative radioimmunoassay kit. The Kruskal-Wallis non-parametric and Mann-Whitney U tests were used to study the difference in immunogenicity of human and bovine insulins. The Kruskal-Wallis test showed that antibody titers in the three groups significantly differed (P<0.001). The Mann-Whitney U test showed no significant difference in AIAB titer between the treatment groups. AIAB titers in the two treatment groups differed significantly from that of the control group, independently (P<0.001). High titers of AIABs are present in patients receiving bovine and human insulin compared to that of the normal population. Bovine and human insulins are antigenic, and there is no significant difference in AIAB titer. Prospective studies are required to determine the long-term clinical significance of these antibodies.

  10. Protective Efficacy and Immunogenicity of an Adenoviral Vector Vaccine Encoding the Codon-Optimized F Protein of Respiratory Syncytial Virus▿

    OpenAIRE

    Kohlmann, Rebekka; Schwannecke, Sarah; Tippler, Bettina; Ternette, Nicola; Temchura, Vladimir V.; Tenbusch, Matthias; Überla, Klaus; Grunwald, Thomas

    2009-01-01

    Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the c...

  11. Distorting malaria peptide backbone structure to enable fitting into MHC class II molecules renders modified peptides immunogenic and protective.

    Science.gov (United States)

    Cifuentes, Gladys; Patarroyo, Manuel Elkin; Urquiza, Mauricio; Ramirez, Luis E; Reyes, Claudia; Rodriguez, Raul

    2003-05-22

    The conserved, nonantigenic, nonimmunogenic malaria Merozoite Surface Protein-2 peptide 1, having high affinity for red blood cells, was rendered immunogenic and protective in Aotus monkeys by specifically changing some critical residues. The NMR structure revealed a switch from classical type III' into distorted III' and III beta turns in the protective peptides. These changes may lead to a better fit into the Aotus MHC class II human HLA-DRbeta1 12 molecule equivalent, thus activating the immune system.

  12. A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly

    DEFF Research Database (Denmark)

    Schmitt, N; Schwarz, M; Peretz, A

    2000-01-01

    The LQT1 locus (KCNQ1) has been correlated with the most common form of inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and high risk of sudden death. The KCNQ1 gene encodes KvLQT1 alpha-subunits, which together with auxiliary IsK (KCNE1, minK) subunits form IK(s) K...

  13. Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

    Directory of Open Access Journals (Sweden)

    Christoph Straub

    2016-07-01

    Full Text Available Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

  14. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.

    Science.gov (United States)

    Ilca, Serban L; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M; Stuart, David I; Huiskonen, Juha T

    2015-11-04

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.

  15. Characterization of influenza vaccine immunogenicity using influenza antigen microarrays.

    Directory of Open Access Journals (Sweden)

    Jordan V Price

    Full Text Available Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.We developed influenza hemagglutinin (HA whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, q <0.2. Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, q <0.2, implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively.Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza

  16. Evaluation of Prevalence, Homology and Immunogenicity of Dispersin among Enteroaggregative Escherichia coli Isolates from Iran.

    Science.gov (United States)

    Asadi Karam, Mohammad Reza; Rezaei, Ali Akbar; Siadat, Seyed Davar; Habibi, Mehri; Bouzari, Saeid

    2017-01-01

    Diarrhea, caused by enteroaggregative Escherichia coli (EAEC), is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin (antiaggregation protein, aap) is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients. PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund's adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated. The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed. Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections.

  17. Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex*

    Science.gov (United States)

    Setiaputra, Dheva; Ross, James D.; Lu, Shan; Cheng, Derrick T.; Dong, Meng-Qiu; Yip, Calvin K.

    2015-01-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

  18. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    Science.gov (United States)

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. {alpha}' Subunit of soybean {beta}-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds.

    Science.gov (United States)

    Motoyama, Takayasu; Maruyama, Nobuyuki; Amari, Yoshiki; Kobayashi, Kanna; Washida, Haruhiko; Higasa, Takahiko; Takaiwa, Fumio; Utsumi, Shigeru

    2009-01-01

    The alpha' and beta subunits of soybean beta-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The alpha' subunit accumulated in rice seeds at a higher level than the beta subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the alpha' subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the alpha' subunit and the beta subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the beta subunit accumulated in low electron density regions in the periphery of PB-II, whereas the alpha' subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated alpha' subunits lacking one cysteine residue in the N-terminal mature region (alpha'DeltaCys1) or five cysteine residues in the pro and N-terminal mature regions (alpha'DeltaCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing alpha'DeltaCys 5 and alpha'DeltaCys1. alpha'DeltaCys5 was localized only in the low-density regions, whereas alpha'DeltaCys1 was found in both low- and high-density regions. These results suggest that the alpha' subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

  20. Structural Determinants for Functional Coupling Between the β and α Subunits in the Ca2+-activated K+ (BK) Channel

    Science.gov (United States)

    Orio, Patricio; Torres, Yolima; Rojas, Patricio; Carvacho, Ingrid; Garcia, Maria L.; Toro, Ligia; Valverde, Miguel A.; Latorre, Ramon

    2006-01-01

    High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. The most remarkable effects of β1 and β2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by α and β1 or β2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of β1 but not β2. Here we reveal the molecular regions in these β subunits that determine their differential functional coupling with the pore-forming α-subunit. We made chimeric constructs between β1 and β2 subunits, and BK channels formed by α and chimeric β subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the β1 and β2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these β subunits. Moreover, the intracellular domains of β1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the α-subunit to be the target of the modulation by the β1-subunit. PMID:16446507

  1. Effect of Vaccine Administration Modality on Immunogenicity and Efficacy

    Science.gov (United States)

    Zhang, Lu; Wang, Wei; Wang, Shixia

    2016-01-01

    Summary The many factors impacting the efficacy of a vaccine can be broadly divided into three categories: (1) features of the vaccine itself, including immunogen design, vaccine type, formulation, adjuvant, and dosing; (2) individual variations among vaccine recipients; and (3) vaccine administration-related parameters. While much literature exists related to vaccines, and recently systems biology has started to dissect the impact of individual subject variation on vaccine efficacy, few studies have focused on the role of vaccine administration-related parameters on vaccine efficacy. Parenteral and mucosal vaccinations are traditional approaches for licensed vaccines; novel vaccine delivery approaches, including needless injection and adjuvant formulations, are being developed to further improve vaccine safety and efficacy. This review provides a brief summary of vaccine administration-related factors, including vaccination approach, delivery route, and method of administration, to gain a better understanding of their potential impact on the safety and immunogenicity of candidate vaccines. PMID:26313239

  2. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  3. An Albumin-Free Formulation for Escherichia coli-Derived Interferon Beta-1b with Decreased Immunogenicity in Immune Tolerant Mice

    NARCIS (Netherlands)

    Haji Abdolvahab, Mohadeseh; Fazeli, Ahmad; Radmalekshahi, Mazda; Nejadnik, M Reza; Fazeli, Mohammad Reza; Schellekens, Huub

    2016-01-01

    Human serum albumin (HSA)-free formulation of Escherichia coli-derived human interferon beta (IFNβ-1b) with a high percentage of monomeric protein and low immunogenicity is developed and characterized in the current study. UV spectroscopy, fluorescence spectroscopy, dynamic light scattering, sodium

  4. Immunogenicity of a 7-valent pneumococcal conjugate vaccine (PCV7) and impact on carriage in Venezuelan children at risk of invasive pneumococcal diseases

    NARCIS (Netherlands)

    Rivera-Olivero, I.A.; Nogal, B. del; Fuentes, M.; Cortez, R.; Bogaert, D.; Hermans, P.W.M.; Waard, J.H. de

    2014-01-01

    BACKGROUND AND AIMS: We evaluated the immunogenicity of the 7-valent pneumococcal conjugate vaccine (PCV7), and its impact on pneumococcal carriage in Venezuelan children at high risk for invasive pneumococcal disease (IPD). METHODS: 82 children (age 2-59 months) with sickle cell anemia (n=22),

  5. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

    Science.gov (United States)

    Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

  6. Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling

    NARCIS (Netherlands)

    Geeraedts, Felix; Goutagny, Nadege; Hornung, Veit; Severa, Martina; de Haan, Aalzen; Pool, Judith; Wilschut, Jan; Fitzgerald, Katherine A.; Huckriede, Anke

    In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed

  7. Probing the effects of hapten stability on cocaine vaccine immunogenicity.

    Science.gov (United States)

    Cai, Xiaoqing; Whitfield, Timothy; Moreno, Amira Y; Grant, Yanabel; Hixon, Mark S; Koob, George F; Janda, Kim D

    2013-11-04

    Judicious hapten design has been shown to be of importance when trying to generate a viable vaccine against a drug of abuse. Hapten design has typically been predicated upon faithfully emulating the unique chemical architecture that each drug presents. However, the need for drug-hapten congruency may also compromise vaccine immunogenicity if the drug-hapten conjugate possesses chemical epitope instability. There has been no systematic study on the impact of hapten stability as it relates to vaccine immunogenicity. As a starting point, we have probed the stability of a series of cocaine haptens through varying several of its structural elements, including functionality at the C2-position, the nature of the linker, and its site of attachment. Accordingly, a hydrolytic stability profile of four cocaine haptens (GNNA, GNNS, GNE, and GNC) was produced, and these results were compared through each hapten's immunological properties, which were generated via active vaccination. From this group of four, three of the haptens, GNE, GNNA, and GNC, were further examined in an animal behavioral model, and findings here were again measured in relationship to hapten stability. We demonstrate a corresponding relationship between the half-life of the hapten and its immunogenicity, wherein haptens presenting a fully representative cocaine framework elicited higher concentrations of cocaine-specific IgG in sera and also conferred better protection against cocaine-induced locomotor activity. Our results indicate that hapten half-life plays an important role in vaccine immunogenicity and this in turn can impact animal behavioral effects when challenged with a drug of abuse.

  8. Polysorbates, peroxides, protein aggregation, and immunogenicity – a growing concern

    Directory of Open Access Journals (Sweden)

    Edward T. Maggio

    2012-06-01

    Full Text Available Aggregation can have a number of deleterious effects on biotherapeutics including the loss of efficacy, the induction of unwanted immunogenicity, altered pharmacokinetics, and reduced shelf life. Aggregation is ameliorated by the inclusion of surfactants in biotherapeutics formulations, typically non-ionic polymeric ether surfactants. The most commonly used examples are Tween® 20 (Polysorbate 20 and Tween® 80 (Polysorbate 80. Others include Triton™ X-100, Pluronic® F-68, Pluronic® F-88, Pluronic® F-127 (poloxamers, and Brij 35 (polyoxyethylene alkyl ether. The usefulness of polysorbates, in particular in preventing protein aggregation in biotherapeutic formulations, is well accepted. However, polysorbates contain ether linkages and unsaturated alkyl chains that have been shown to auto-oxidize in aqueous solution to protein-damaging peroxides and reactive aldehydes including formaldehyde and acetaldehyde. The peroxides principally affect methionine and tryptophan moieties. The aldehydes react with primary amino groups on proteins and are known to induce immunogenicity of proteins in the absence of aggregation or adjuvants. Detection of protein aggregation and prevention of aggregation using polysorbates is relatively straightforward using light scattering or size exclusion chromatography methods. Detection of oxidative damage to amino acyl moieties or increased immunogenicity resulting from the reaction of biotherapeutics with the degradation products of polysorbates is considerably more difficult and has generally been ignored in the scientific literature. As an increasing number of biotherapeutic agents come into use in common clinical practice, including both as innovator and as biosimilar products, these latter issues will come under increased scrutiny. Substitution of non-ionic, non-ether-based surfactants, could offer significant improvements in stability, reduced immunogenicity, and shelf life, and represents a significant unmet

  9. Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

    Science.gov (United States)

    Waldmann, Herman

    2014-01-01

    One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

  10. Enhancing the Immunogenicity of a Tetravalent Dengue DNA Vaccine

    Science.gov (United States)

    2016-08-01

    primates to enhance the immunogenicity of the vaccine by (a) testing different modes of delivery of dengue DNA vaccine for optimal humoral and T cell...the vaccine by (a) testing different modes of delivery of dengue DNA vaccines for optimal humoral and T cell responses, and (b) testing the optimal...will proceed once the animals are released from quarantine. Nothing to report. Nothing to report. Ichor and PharmaJet will provide training on the use

  11. Optimisation of Production of Immunogenic Malaria Surface Antigens in Plants

    OpenAIRE

    KARTIKA MAILLIKA SETYABUDI

    2017-01-01

    Vaccines against malaria could serve a useful purpose in malaria control, prevention and eradication efforts. P. falciparum merozoite surface proteins (MSP): MSP119, MSP4 and MSP5 are promising targets to establish a protective immune response against malaria. Plant-based expression system presents potential as a cost-effective, scalable and safe production platform. This study provides evidence of the feasibility of plants to produce immunogenic P. falciparum MSP119, MSP4, MSP5. The demonstr...

  12. Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death

    Directory of Open Access Journals (Sweden)

    Abhishek D Garg

    2015-11-01

    Full Text Available The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of so-called damage-associated molecular patterns (DAMPs. The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical and clinical aspects of ICD, in an attempt to capture the essence of this clinically relevant phenomenon, and identify future challenges for this rapidly expanding field of investigation.

  13. Comparative study on characterization of recombinant B subunit of E. coli heat-labile enterotoxin (rLTB) prepared from E. coli and P. patoris.

    Science.gov (United States)

    Ma, Xingyuan; Yao, Bi; Zheng, Wenyun; Li, Linfeng

    2010-03-01

    Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-l fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6x His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at 30 degrees C, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at 37 degrees C. The expression level increased dramatically to 250- 300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6x His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GM1 gangliosides. The MW of LTB-6x His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

  14. Antigenicity and Immunogenicity of Rotavirus VP6 Protein Expressed on the Surface of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    L. E. Esteban

    2013-01-01

    Full Text Available Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

  15. Immunogenicity Studies of Bivalent Inactivated Virions of EV71/CVA16 Formulated with Submicron Emulsion Systems

    Directory of Open Access Journals (Sweden)

    Chih-Wei Lin

    2014-01-01

    Full Text Available We assessed two strategies for preparing candidate vaccines against hand, foot, and mouth disease (HFMD caused mainly by infections of enterovirus (EV 71 and coxsackievirus (CV A16. We firstly design and optimize the potency of adjuvant combinations of emulsion-based delivery systems, using EV71 candidate vaccine as a model. We then perform immunogenicity studies in mice of EV71/CVA16 antigen combinations formulated with PELC/CpG. A single dose of inactivated EV71 virion (0.2 μg emulsified in submicron particles was found (i to induce potent antigen-specific neutralizing antibody responses and (ii consistently to elicit broad antibody responses against EV71 neutralization epitopes. A single dose immunogenicity study of bivalent activated EV71/CVA16 virion formulated with either Alum or PELC/CpG adjuvant showed that CVA16 antigen failed to elicit CVA16 neutralizing antibody responses and did not affect EV71-specific neutralizing antibody responses. A boosting dose of emulsified EV71/CVA16 bivalent vaccine candidate was found to be necessary to achieve high seroconversion of CVA16-specific neutralizing antibody responses. The current results are important for the design and development of prophylactic vaccines against HFMD and other emerging infectious diseases.

  16. Immunotherapeutic Potential of Oncolytic H-1 Parvovirus: Hints of Glioblastoma Microenvironment Conversion towards Immunogenicity

    Directory of Open Access Journals (Sweden)

    Assia L. Angelova

    2017-12-01

    Full Text Available Glioblastoma, one of the most aggressive primary brain tumors, is characterized by highly immunosuppressive microenvironment. This contributes to glioblastoma resistance to standard treatment modalities and allows tumor growth and recurrence. Several immune-targeted approaches have been recently developed and are currently under preclinical and clinical investigation. Oncolytic viruses, including the autonomous protoparvovirus H-1 (H-1PV, show great promise as novel immunotherapeutic tools. In a first phase I/IIa clinical trial (ParvOryx01, H-1PV was safe and well tolerated when locally or systemically administered to recurrent glioblastoma patients. The virus was able to cross the blood–brain (tumor barrier after intravenous infusion. Importantly, H-1PV treatment of glioblastoma patients was associated with immunogenic changes in the tumor microenvironment. Tumor infiltration with activated cytotoxic T cells, induction of cathepsin B and inducible nitric oxide (NO synthase (iNOS expression in tumor-associated microglia/macrophages (TAM, and accumulation of activated TAM in cluster of differentiation (CD 40 ligand (CD40L-positive glioblastoma regions was detected. These are the first-in-human observations of H-1PV capacity to switch the immunosuppressed tumor microenvironment towards immunogenicity. Based on this pilot study, we present a tentative model of H-1PV-mediated modulation of glioblastoma microenvironment and propose a combinatorial therapeutic approach taking advantage of H-1PV-induced microglia/macrophage activation for further (preclinical testing.

  17. EFFECTS OF DIFFERENT ADJUVANTS UPON IMMUNOGENICITY OF ANTI-GROUP B STREPTOCOCCAL VACCINE COMPONENTS

    Directory of Open Access Journals (Sweden)

    A. N. Suvorov

    2008-01-01

    Full Text Available Abstract. Design of an effective and safe vaccine against pathogenic streptococci is still on the agenda, in spite of numerous attempts in this area undertaken by different laboratories. In order to improve immunogenicity of recombinant vaccine preparations, a selection of effective adjuvants is necessary. Previously, two recombinant GBS polypeptides P6 and ScaAB were found to be immunogenic, and their injection in separate preparations or mixed manner boosted production of specific and protective antibodies with high affinity. Four different adjuvants (Freund adjuvant, aluminum hydroxide, Bestim and Interleukine-1β have been tested for immunization of mice with single polypeptides, or with their mixtures. As a result of vaccination, it was demonstrated that aluminum hydroxide was providing the most desirable immunological parameters of immune response among the adjuvants tested. A mixture of polypeptides containing aluminum hydroxide was found to produce specific antibodies with better opsonizing activity against group B streptococci. (Med. Immunol., 2008, vol. 10, N 2-3, pp 215-222.

  18. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    Directory of Open Access Journals (Sweden)

    Daniel O Connor

    Full Text Available Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  19. Mechanisms of the scaffold subunit in facilitating protein phosphatase 2A methylation.

    Directory of Open Access Journals (Sweden)

    Vitali Stanevich

    Full Text Available The function of the biologically essential protein phosphatase 2A (PP2A relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A and catalytic (C or PP2Ac subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A-C heterodimer than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1 stabilization of a proper protein fold and an active conformation of PP2Ac; 2 limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3 weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.

  20. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L. [Argonne National Lab., IL (United States); Maulik, P.R.; Reed, R.A.; Shipley, G. [Boston Univ., MA (United States). School of Medicine; Westbrook, E.M. [Argonne National Lab., IL (United States)]|[Northwestern Univ., Evanston, IL (United States); Scott, D.L.; Otwinowski, Z. [Yale Univ., New Haven, CT (United States)

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

  1. Regulatory role of voltage-gated sodium channel β subunits in sensory neurons

    Directory of Open Access Journals (Sweden)

    Mohamed eChahine

    2011-11-01

    Full Text Available Voltage-gated Na+ channels are transmembrane-bound proteins incorporating aqueous conduction pores that are highly selective for Na+. The opening of these channels results in the rapid influx of Na+ ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na+-selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary β subunits (β1, β2 were first described. Subsequent cloning and heterologous expression studies revealed that the  subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, ten isoforms of the Na+ channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four β subunits (β1-β4 and two splice variants (β1A, β1B have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of β subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.

  2. Subunit dimers in sheep spleen apoferritin. The effect on iron storage.

    Science.gov (United States)

    Mertz, J R; Theil, E C

    1983-10-10

    Ferritin with high and low iron content, 2000 and 790 iron atoms/molecule, was isolated from the spleens of copper-poisoned and control lambs, respectively. Differences in the iron content in vivo were reflected in the properties of the apoferritin protein shells, since the apoprotein from the low iron ferritin took up iron relatively more slowly (0.52 +/- 0.09) and released it more rapidly (1.68 +/- 0.06) in vitro. Although the two types of apoferritin were indistinguishable in terms of surface charge (pI range 4.98-5.43) and in consisting of both heavy and light subunits, the subunit interactions differed markedly; 40-50% of the subunits of low iron ferritin were in dimers stable to reduction and carboxylmethylation, 4% mercaptoethanol, 8% sodium dodecyl sulfate, and 100 degrees C for 30 min, 70% formic acid, and 30% methanol. Subunit dimers were also observed in liver ferritin from mouse and neonatal pig and were enriched in a low iron fraction of horse spleen ferritin. Based on cyanogen bromide fragmentation and NH2-terminal analysis, the natural and chemically cross-linked subunit dimers had two peptides in common; natural subunit dimers also appeared to have a second region cross-linked, suggesting the possibility of both intra- and intersubunit links in the natural dimers. In sheep spleen ferritin, both heavy and light subunits appeared to participate in subunit dimerization. Natural subunit dimers were enriched in low iron ferritin fractions of all ferritin preparations tested (linear correlation = 0.94) and can explain, at least in part, the previously observed effects of iron core size on the apoferritin shell. Whether the subunit cross-links represent part of the subunit assembly process subsequently cleaved by iron (or copper) or whether the cross-links form after iron core formation in vivo has yet to determined. In either case, it is clear that such post-translational variations can affect iron uptake and release and emphasize the importance of the

  3. Exploring virulence and immunogenicity in the emerging pathogen Sporothrix brasiliensis.

    Science.gov (United States)

    Della Terra, Paula Portella; Rodrigues, Anderson Messias; Fernandes, Geisa Ferreira; Nishikaku, Angela Satie; Burger, Eva; de Camargo, Zoilo Pires

    2017-08-01

    Sporotrichosis is a polymorphic chronic infection of humans and animals classically acquired after traumatic inoculation with soil and plant material contaminated with Sporothrix spp. propagules. An alternative and successful route of transmission is bites and scratches from diseased cats, through which Sporothrix yeasts are inoculated into mammalian tissue. The development of a murine model of subcutaneous sporotrichosis mimicking the alternative route of transmission is essential to understanding disease pathogenesis and the development of novel therapeutic strategies. To explore the impact of horizontal transmission in animals (e.g., cat-cat) and zoonotic transmission on Sporothrix fitness, the left hind footpads of BALB/c mice were inoculated with 5×106 yeasts (n = 11 S. brasiliensis, n = 2 S. schenckii, or n = 1 S. globosa). Twenty days post-infection, our model reproduced both the pathophysiology and symptomology of sporotrichosis with suppurating subcutaneous nodules that progressed proximally along lymphatic channels. Across the main pathogenic members of the S. schenckii clade, S. brasiliensis was usually more virulent than S. schenckii and S. globosa. However, the virulence in S. brasiliensis was strain-dependent, and we demonstrated that highly virulent isolates disseminate from the left hind footpad to the liver, spleen, kidneys, lungs, heart, and brain of infected animals, inducing significant and chronic weight loss (losing up to 15% of their body weight). The weight loss correlated with host death between 2 and 16 weeks post-infection. Histopathological features included necrosis, suppurative inflammation, and polymorphonuclear and mononuclear inflammatory infiltrates. Immunoblot using specific antisera and homologous exoantigen investigated the humoral response. Antigenic profiles were isolate-specific, supporting the hypothesis that different Sporothrix species can elicit a heterogeneous humoral response over time, but cross reaction was observed

  4. Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis

    DEFF Research Database (Denmark)

    Shaw, A C; Christiansen, G; Roepstorff, P

    2000-01-01

    (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies...... and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses......-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the Trp...

  5. Stronger T Cell Immunogenicity of Ovalbumin Expressed Intracellularly in Gram-Negative than in Gram-Positive Bacteria

    Science.gov (United States)

    Martner, Anna; Östman, Sofia; Lundin, Samuel; Rask, Carola; Björnsson, Viktor; Telemo, Esbjörn; Collins, L. Vincent; Axelsson, Lars; Wold, Agnes E.

    2013-01-01

    This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G−) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4+ T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G− bacteria and may be relevant for the use of bacterial carriers in vaccine development. PMID:23741469

  6. A novel peptide-based pan-influenza A vaccine: a double blind, randomised clinical trial of immunogenicity and safety.

    Science.gov (United States)

    Francis, James N; Bunce, Campbell J; Horlock, Claire; Watson, Jeannette M; Warrington, Steven J; Georges, Bertrand; Brown, Carlton B

    2015-01-03

    FP-01.1 is a novel synthetic influenza A vaccine consisting of six fluorocarbon-modified 35-mer peptides that encapsulate multiple CD4+ and CD8+ T-cell epitopes and is designed to induce an immune response across a broad population. FP-01.1 was evaluated for safety and immunogenicity in a randomised, double-blind, placebo-controlled, dose-escalation, phase I clinical study in healthy adult volunteers (n=49). IFNγ ELISpot assays and multicolour flow cytometry were used to characterise the immune response. FP-01.1 was safe and well tolerated at all doses tested with a similar adverse event profile in actively vaccinated subjects compared with controls. Maximum immunogenicity was in the 150 μg/peptide dose group where a robust response (243 spots/million PBMC) was demonstrated in 75% subjects compared with 0% in placebo controls. All six peptides were immunogenic. FP-01.1 induced dual CD4+ and CD8+ T cell responses and vaccine-specific T cells cross-recognise divergent influenza strains. This first-in-human study showed that FP-01.1 has an acceptable safety and tolerability profile and generated robust anti-viral T cell responses in a high proportion of subjects tested. The results support the further clinical testing of FP-01.1 prior to clinical, proof-of-concept, live viral challenge studies. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Homology analysis and cross-immunogenicity of OmpA from pathogenic Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis.

    Science.gov (United States)

    Chen, Yuhuang; Duan, Ran; Li, Xu; Li, Kewei; Liang, Junrong; Liu, Chang; Qiu, Haiyan; Xiao, Yuchun; Jing, Huaiqi; Wang, Xin

    2015-12-01

    The outer membrane protein A (OmpA) is one of the intra-species conserved proteins with immunogenicity widely found in the family of Enterobacteriaceae. Here we first confirmed OmpA is conserved in the three pathogenic Yersinia: Yersinia pestis, Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica, with high homology at the nucleotide level and at the amino acid sequence level. The identity of ompA sequences for 262 Y. pestis strains, 134 Y. pseudotuberculosis strains and 219 pathogenic Y. enterocolitica strains are 100%, 98.8% and 97.7% similar. The main pattern of OmpA of pathogenic Yersinia are 86.2% and 88.8% identical at the nucleotide and amino acid sequence levels, respectively. Immunological analysis showed the immunogenicity of each OmpA and cross-immunogenicity of OmpA for pathogenic Yersinia where OmpA may be a vaccine candidate for Y. pestis and other pathogenic Yersinia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Cleft Lip Repair: The Hybrid Subunit Method

    OpenAIRE

    Tollefson, TT

    2016-01-01

    Copyright © 2016 by Thieme Medical Publishers, Inc. The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the ...

  9. Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death.

    Science.gov (United States)

    Hodge, James W; Garnett, Charlie T; Farsaci, Benedetto; Palena, Claudia; Tsang, Kwong-Yok; Ferrone, Soldano; Gameiro, Sofia R

    2013-08-01

    Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high-mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC-1, or PSA-specific CD8(+) CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1(+), CD133(+)) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of "immunogenic modulation," where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy. Copyright © 2013 UICC.

  10. YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

    Science.gov (United States)

    Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

    2014-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

  11. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, R.M.B. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia]|[Universidade Nova de Lisboa, Oeiras (Portugal). Instituto de Tecnologia Quimica e Biologica; Franco, E.; Teixeira, A.R.N. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of {sup 35}S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author).

  12. STUDIES ON THE BIOCHEMICAL, BIOPHYSICAL, AND IMMUNOGENIC PROPERTIES OF JAPANESE B TYPE ENCEPHALITIS VIRUS AND VACCINES.

    Science.gov (United States)

    Duffy, C E; Stanley, W M

    1945-11-30

    Studies on the biochemical, biophysical, and immunogenic properties of Japanese B type encephalitis virus and vaccines have been made in order to determine whether a purified vaccine suitable for human use could be obtained by means of differential centrifugation of extracts of infected mouse brains. Studies were also made on extracts of normal mouse brains, it was found that extracts of normal as well as of infected mouse brains contained fairly large amounts of several components of high molecular weight. Components having sedimentation constants near 5 and 40 Svedberg units were found in extracts of infected brains. However the rates of sedimentation of the different components were so similar that it was found impossible, from a practical standpoint, to secure a vaccine consisting largely of virus by means of differential centrifugation. It was also found that a considerable portion of virus was lost or destroyed in the centrifugation process so that it was impossible to secure an effective degree of concentration of immunogenic potency. Although vaccines possessing about twice the immunogenic potency of the starting material were obtained, it was concluded that it was not practical to purify and concentrate Japanese B type encephalitis virus in infected mouse brain extracts by means of differential centrifugation for the production of a vaccine on a large scale. The optimum pH stability range of Japanese B type encephalitis virus activity was found to be near pH 8.5. The virus is inactivated fairly rapidly at pH 7 and very rapidly at more acid reactions. The virus is inactivated rapidly near pH 10. Extracts of infected mouse brains with buffers near pH 8 containing disodium phosphate were found to possess slightly higher titers than saline extracts near pH 7. However vaccines prepared from such extracts were found to possess essentially the same immunogenic potency, hence, although extraction at the more alkaline reaction may perhaps remove more active virus

  13. Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits

    DEFF Research Database (Denmark)

    Blumenthal, T; Young, R A; Brown, S

    1976-01-01

    Qbeta replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein S1 and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The affinity of the two subcomplexes...... alters its quaternary structure: the EF-Tu-Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA. This conformational change is not influenced by ionic strength. The addition of Qbeta RNA to the enzyme, does not result in the release...... of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP...

  14. Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F of Flavobacterium meningosepticum.

    Directory of Open Access Journals (Sweden)

    Tarlan Mamedov

    Full Text Available Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83 is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83 was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may

  15. Immunogenicity of protein aggregates of a monoclonal antibody generated by forced shaking stress with siliconized and nonsiliconized syringes in BALB/c mice.

    Science.gov (United States)

    Uchino, Tomonobu; Miyazaki, Yasunori; Yamazaki, Takuto; Kagawa, Yoshiyuki

    2017-10-01

    In this study, we aimed to investigate the immunogenicity of protein aggregates of monoclonal antibodies (mAbs), generated by forced shaking stress with siliconized and nonsiliconized syringes in a mouse model. Samples were filled in siliconized and nonsiliconized syringes with shaking and headspace air. Characterization studies were performed using high-performance size-exclusion chromatography, nanoparticle tracking analysis, flow cytometry, micro-flow imaging and resonant mass measurement. The samples (10 or 100 μg) were subcutaneously injected into BALB/c mice for 21 days, and the anti-drug antibody (ADA) concentrations were monitored. In samples shaken with siliconized syringes [SO (+)], large amounts of submicron and subvisible protein aggregates were formed by interactions with silicone oil droplets. The characteristics of protein aggregates differed between the mAb solution and shaken samples, which strongly indicates that silicone oil accelerates protein aggregation. When administered at low doses, the ADA concentration in all samples increased with repeated injections, and SO (+) induced the highest immunogenicity. However, when administered at high doses, ADA concentration decreased following prolonged repeated administration for tolerance. These results indicated that mAb protein aggregation induced immunogenicity in mice, and SO (+) induced higher immunogenicity than samples shaken with nonsiliconized syringe. © 2017 Royal Pharmaceutical Society.

  16. Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks.

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    Krystal Teasley Hamorsky

    Full Text Available INTRODUCTION: Cholera toxin B subunit (CTB is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants. METHODOLOGY/PRINCIPAL FINDINGS: In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major

  17. Preclinical immunogenicity and safety of a Group A streptococcal M protein-based vaccine candidate.

    Science.gov (United States)

    Batzloff, Michael R; Fane, Anne; Gorton, Davina; Pandey, Manisha; Rivera-Hernandez, Tania; Calcutt, Ainslie; Yeung, Grace; Hartas, Jon; Johnson, Linda; Rush, Catherine M; McCarthy, James; Ketheesan, Natkunam; Good, Michael F

    2016-12-01

    Streptococcus pyogenes (group A streptococcus, GAS) causes a wide range of clinical manifestations ranging from mild self-limiting pyoderma to invasive diseases such as sepsis. Also of concern are the post-infectious immune-mediated diseases including rheumatic heart disease. The development of a vaccine against GAS would have a large health impact on populations at risk of these diseases. However, there is a lack of suitable models for the safety evaluation of vaccines with respect to post-infectious complications. We have utilized the Lewis Rat model for cardiac valvulitis to evaluate the safety of the J8-DT vaccine formulation in parallel with a rabbit toxicology study. These studies demonstrated that the vaccine did not induce abnormal pathology. We also show that in mice the vaccine is highly immunogenic but that 3 doses are required to induce protection from a GAS skin challenge even though 2 doses are sufficient to induce a high antibody titer.

  18. [Immunogenicity and safety of the influenza vaccine, in a population older than 55-years in Mexico].

    Science.gov (United States)

    Ayala-Montiel, Octavio; Mascareñas, César; García-Hernández, Delfino; Rendón-Muñiz, Jorge; López, Irma; Felipe Montaño, Luis; Zenteno, I; Franco-Paredes, C

    2005-01-01

    To confirm the immunogenicity and tolerance of the inactivated, fractionated, and purified influenza vaccine, in a Mexican adult population aged 55 and older, medically served at a Petróleos Mexicanos Hospital (Pemex, Mexican Oil Company). The study was conducted between November and December, 2000, among ninety adult subjects aged 55 years and older who were seen at the Hospital Central Sur Pemex. The primary endpoints regarding immunogenicity were the percentage of individuals with protective antibodies targeting hemagglutinins higher than or equal to 1:40, and the percentage of subjects who seroconverted as measured by a four-fold increase in protective antibody production. Secondary endpoints included the frequency of local and systemic reactions to the vaccine. An additional criterion that was evaluated included antigen-antibody affinity assays to measure the polyclonal antibody response to the vaccine and the specific generation of high-affinity antibodies to viral proteins, before and after vaccination. The antibody protection rate was 95.6% against the HINI strain, 98.9% against the H3N2 strain, and a 100% against the B/Yamanashi strain. Seroconversion to the HINI strain was elicited in 74.4% of subjects, to the H3N2 strain in 88.9%, and to the B/Yamanashi strain in 82.2%. Eighteen (20%) subjects developed local reactions; 17 (18.8%) developed a systemic reaction post vaccination at day 5 and nine subjects (10%) at day 28. Local reactions consisted of pain in 10 (11.1%) subjects, redness in 8 (8.8%), and induration in 6 (6.6%). General malaise, headache, and fever were identified in 10, 8.8, and 0% of subjects, respectively, at day 5, and in 4.4, 6.6, and 0%, respectively, at day 28. Influenza vaccine was highly immunogenic in a healthy Mexican adult population aged 55 years and older. The generation of high-affinity antibodies to the virus after vaccination was also demonstrated. Local and systemic adverse reactions to the vaccine identified in our study

  19. Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

    Directory of Open Access Journals (Sweden)

    Widiastuti R

    2013-06-01

    Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

  20. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

    Directory of Open Access Journals (Sweden)

    Amanda R Bitencourt

    Full Text Available A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3 as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2% and at least 1 recombinant protein representing PvMSP-3β (79.1%. In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin. Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.

  1. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

    Science.gov (United States)

    Roier, Sandro; Fenninger, Judith C.; Leitner, Deborah R.; Rechberger, Gerald N.; Reidl, Joachim; Schild, Stefan

    2013-01-01

    Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica. PMID:23731905

  2. Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination.

    Science.gov (United States)

    Sanchez, J; Johansson, S; Löwenadler, B; Svennerholm, A M; Holmgren, J

    1990-01-01

    The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (non-cholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNA-polymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coli enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 micrograms/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a near-native conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. The effect of gamma-irradiation conditions on the immunogenicity of whole-inactivated Influenza A virus vaccine.

    Science.gov (United States)

    David, Shannon C; Lau, Josyane; Singleton, Eve V; Babb, Rachelle; Davies, Justin; Hirst, Timothy R; McColl, Shaun R; Paton, James C; Alsharifi, Mohammed

    2017-02-15

    Gamma-irradiation, particularly an irradiation dose of 50kGy, has been utilised widely to sterilise highly pathogenic agents such as Ebola, Marburg Virus, and Avian Influenza H5N1. We have reported previously that intranasal vaccination with a gamma-irradiated Influenza A virus vaccine (γ-Flu) results in cross-protective immunity. Considering the possible inclusion of highly pathogenic Influenza strains in future clinical development of γ-Flu, an irradiation dose of 50kGy may be used to enhance vaccine safety beyond the internationally accepted Sterility Assurance Level (SAL). Thus, we investigated the effect of irradiation conditions, including high irradiation doses, on the immunogenicity of γ-Flu. Our data confirm that irradiation at low temperatures (using dry-ice) is associated with reduced damage to viral structure compared with irradiation at room temperature. In addition, a single intranasal vaccination with γ-Flu irradiated on dry-ice with either 25 or 50kGy induced seroconversion and provided complete protection against lethal Influenza A challenge. Considering that low temperature is expected to reduce the protein damage associated with exposure to high irradiation doses, we titrated the vaccine dose to verify the efficacy of 50kGy γ-Flu. Our data demonstrate that exposure to 50kGy on dry-ice is associated with limited effect on vaccine immunogenicity, apparent only when using very low vaccine doses. Overall, our data highlight the immunogenicity of influenza virus irradiated at 50kGy for induction of high titre antibody and cytotoxic T-cell responses. This suggests these conditions are suitable for development of γ-Flu vaccines based on highly pathogenic Influenza A viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    Directory of Open Access Journals (Sweden)

    Sasaki Yukako

    2008-10-01

    Full Text Available Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit. Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas. The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98 ; L: 98–100%. The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

  5. Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family.

    Science.gov (United States)

    Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S

    1998-02-01

    RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

  6. Immunogenic Targets for Specific Immunotherapy in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2012-01-01

    Full Text Available Multiple myeloma remains an incurable disease although the prognosis has been improved by novel therapeutics and agents recently. Relapse occurs in the majority of patients and becomes fatal finally. Immunotherapy might be a powerful intervention to maintain a long-lasting control of minimal residual disease or to even eradicate disseminated tumor cells. Several tumor-associated antigens have been identified in patients with multiple myeloma. These antigens are expressed in a tumor-specific or tumor-restricted pattern, are able to elicit immune response, and thus could serve as targets for immunotherapy. This review discusses immunogenic antigens with therapeutic potential for multiple myeloma.

  7. Safety, immunogenicity and infectivity of new live attenuated influenza vaccines.

    Science.gov (United States)

    Isakova-Sivak, Irina; Rudenko, Larisa

    2015-01-01

    Live attenuated influenza vaccines (LAIVs) are believed to be immunologically superior to inactivated influenza vaccines, because they can induce a variety of adaptive immune responses, including serum antibodies, mucosal and cell-mediated immunity. In addition to the licensed cold-adapted LAIV backbones, a number of alternative LAIV approaches are currently being developed and evaluated in preclinical and clinical studies. This review summarizes recent progress in the development and evaluation of LAIVs, with special attention to their safety, immunogenicity and infectivity for humans, and discusses their perspectives for the future.

  8. Non-immunogenicity of overlapping gag peptides pulsed on autologous cells after vaccination of HIV infected individuals.

    Directory of Open Access Journals (Sweden)

    Henrik N Kløverpris

    several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.ClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search.

  9. Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

    Science.gov (United States)

    Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

    2016-01-01

    The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Transcriptomes and pathways associated with infectivity, survival and immunogenicity in Brugia malayi L3

    Directory of Open Access Journals (Sweden)

    Spiro David

    2009-06-01

    Full Text Available Abstract Background Filarial nematode parasites cause serious diseases such as elephantiasis and river blindness in humans, and heartworm infections in dogs. Third stage filarial larvae (L3 are a critical stage in the life cycle of filarial parasites, because this is the stage that is transmitted by arthropod vectors to initiate infections in mammals. Improved understanding of molecular mechanisms associated with this transition may provide important leads for development of new therapies and vaccines to prevent filarial infections. This study explores changes in gene expression associated with the transition of Brugia malayi third stage larvae (BmL3 from mosquitoes into mammalian hosts and how these changes are affected by radiation. Radiation effects are especially interesting because irradiated L3 induce partial immunity to filarial infections. The underlying molecular mechanisms responsible for the efficacy of such vaccines are unkown. Results Expression profiles were obtained using a new filarial microarray with 18, 104 64-mer elements. 771 genes were identified as differentially expressed in two-way comparative analyses of the three L3 types. 353 genes were up-regulated in mosquito L3 (L3i relative to cultured L3 (L3c. These genes are important for establishment of filarial infections in mammalian hosts. Other genes were up-regulated in L3c relative to L3i (234 or irradiated L3 (L3ir (22. These culture-induced transcripts include key molecules required for growth and development. 165 genes were up-regulated in L3ir relative to L3c; these genes encode highly immunogenic proteins and proteins involved in radiation repair. L3ir and L3i have similar transcription profiles for genes that encode highly immunogenic proteins, antioxidants and cuticle components. Conclusion Changes in gene expression that normally occur during culture under conditions that support L3 development and molting are prevented or delayed by radiation. This may explain

  11. EpiSweep: Computationally Driven Reengineering of Therapeutic Proteins to Reduce Immunogenicity While Maintaining Function.

    Science.gov (United States)

    Choi, Yoonjoo; Verma, Deeptak; Griswold, Karl E; Bailey-Kellogg, Chris

    2017-01-01

    Therapeutic proteins are yielding ever more advanced and efficacious new drugs, but the biological origins of these highly effective therapeutics render them subject to immune surveillance within the patient's body. When recognized by the immune system as a foreign agent, protein drugs elicit a coordinated response that can manifest a range of clinical complications including rapid drug clearance, loss of functionality and efficacy, delayed infusion-like allergic reactions, more serious anaphylactic shock, and even induced auto-immunity. It is thus often necessary to deimmunize an exogenous protein in order to enable its clinical application; critically, the deimmunization process must also maintain the desired therapeutic activity.To meet the growing need for effective, efficient, and broadly applicable protein deimmunization technologies, we have developed the EpiSweep suite of protein design algorithms. EpiSweep seamlessly integrates computational prediction of immunogenic T cell epitopes with sequence- or structure-based assessment of the impacts of mutations on protein stability and function, in order to select combinations of mutations that make Pareto optimal trade-offs between the competing goals of low immunogenicity and high-level function. The methods are applicable both to the design of individual functionally deimmunized variants as well as the design of combinatorial libraries enriched in functionally deimmunized variants. After validating EpiSweep in a series of retrospective case studies providing comparisons to conventional approaches to T cell epitope deletion, we have experimentally demonstrated it to be highly effective in prospective application to deimmunization of a number of different therapeutic candidates. We conclude that our broadly applicable computational protein design algorithms guide the engineer towards the most promising deimmunized therapeutic candidates, and thereby have the potential to accelerate development of new protein

  12. Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta.

    Science.gov (United States)

    Walker, J; Hoekstra, R; Roos, M H; Wiley, L J; Weiss, A S; Sangster, N C; Tait, A

    2001-06-28

    Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.

  13. Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity

    DEFF Research Database (Denmark)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Tesser, G.I.

    1997-01-01

    Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attem...

  14. Synthetic peptide vaccines: palmitoylation of peptide antigens by an thioester bond increases immunogenicity

    NARCIS (Netherlands)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Tesser, G.I.; Dalsgaard, K.; Langeveld, J.P.M.; Boshuizen, R.S.; Meloen, R.H.

    1997-01-01

    Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts

  15. Immunogenicity and diagnostic potential of synthetic antigenic cell surface glycans of Leishmania.

    Science.gov (United States)

    Anish, Chakkumkal; Martin, Christopher E; Wahlbrink, Annette; Bogdan, Christian; Ntais, Pantelis; Antoniou, Maria; Seeberger, Peter H

    2013-11-15

    Detection and quantification of pathogen-derived antigenic structures is a key method for the initial diagnosis and follow-up of various infectious diseases. Complex parasitic diseases such as leishmaniasis require highly sensitive and specific tests prior to treatment with potentially toxic drugs. To investigate the diagnostic potential of cell surface glycans found on Leishmania parasites, we identified diagnostically relevant glycan epitopes and used synthetic glycan microarrays to screen sera from infected humans and dogs. On the basis of the screening results, we selected a tetrasaccharide to generate anti-glycan antibodies. The corresponding tetrasaccharide-carrier protein conjugate was immunogenic in mice, and sera obtained from immunized mice specifically detected the Leishmania parasite. These results demonstrate how synthetic glycan arrays, in combination with immunological methods, help to identify promising carbohydrate antigens for pathogen detection.

  16. A review of immunogenicity and tolerability of live attenuated Hepatitis A vaccine in children.

    Science.gov (United States)

    Rao, Sameer; Mao, J S; Motlekar, Salman; Fangcheng, Zhuang; Kadhe, Ganesh

    2016-12-01

    Changing epidemiology of Hepatitis A virus (HAV) has led to an increased susceptibility of adolescents and adults to the infection. Vaccination can remarkably reduce the incidence and associated morbidity of HAV infection. This review is focused on the safety and efficacy of H2 strain derived live attenuated Hepatitis A vaccine. We found the vaccine to be highly immunogenic with minimal or negligible safety issues. Moreover, a single dose of live attenuated vaccine persists a long term immune response and can be a preferred option for developing countries. In 2014, Indian Academy of Paediatrics (IAP) also updated their recommendations for H2 vaccine as a single dose as against the previous 2 dose schedule. A focused approach to include the vaccine in national immunization program should be explored.

  17. Preclinical evaluation of the immunogenicity and safety of an inactivated enterovirus 71 candidate vaccine.

    Directory of Open Access Journals (Sweden)

    Shi-Hsia Hwa

    2013-11-01

    Full Text Available Human enterovirus 71 (EV71 is a significant cause of morbidity and mortality from Hand, Foot and Mouth Disease (HFMD and neurological complications, particularly in young children in the Asia-Pacific region. There are no vaccines or antiviral therapies currently available for prevention or treatment of HFMD caused by EV71. Therefore, the development of therapeutic and preventive strategies against HFMD is of growing importance. We report the immunogenic and safety profile of inactivated, purified EV71 preparations formulated with aluminum hydroxide adjuvant in preclinical studies in mice and rabbits. In mice, the candidate vaccine formulations elicited high neutralizing antibody responses. A toxicology study of the vaccine formulations planned for human use performed in rabbits showed no vaccine-related pathological changes and all animals remained healthy. Based on these preclinical studies, Phase 1 clinical testing of the EV71 inactivated vaccine was initiated.

  18. Novel Vaccine Against Mycoplasma Hyosynoviae: The Immunogenic Effect of Iscom-Based Vaccines in Swine

    DEFF Research Database (Denmark)

    Lauritsen, Klara Tølbøll; Vinther Heydenreich, Annette; Riber, Ulla

    Arthritis in swine is frequently caused by Mycoplasma hyosynoviae (Mhs). For the development of an effective vaccine we investigated the immunogenic effect of three vaccine preparations with the ISCOM adjuvant Posintro™ from Nordic Vaccine. A: formalin fixed whole-cells Mhs (300 µg/dose) mixed...... with Posintro, B: Deoxycholate extracted lipoproteins from Mhs organisms (DOC-antigen, 300 μg/dose) in Posintro and C: DOC-antigen (50 μg/dose) in Posintro. Each vaccine-group contained three pigs. Vaccinations (i.m.) were performed at 12 and 15 weeks of age. The development of specific IgG and secretion...... of IFNγ were measured. Three weeks after the second vaccination, pigs were euthanised and autopsied. Vaccine B induced a high level of specific serum IgG in all pigs a week after boost. Vaccine C gave a variable response after boost, with two pigs seroconverting, while no response was seen by vaccine A...

  19. Safety, Tolerability, and Immunogenicity of Interferons

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    Michael G. Tovey

    2010-04-01

    Full Text Available Interferons (IFNs are class II cytokines that are key components of the innate immune response to virus infection. Three IFN sub-families, type I, II, and III IFNs have been identified in man, Recombinant analogues of type I IFNs, in particular IFNα2 and IFNβ1, have found wide application for the treatment of chronic viral hepatitis and remitting relapsing multiple sclerosis respectively. Type II IFN, or IFN gamma, is used principally for the treatment of chronic granulomatous disease, while the recently discovered type III IFNs, also known as IFN lambda or IL-28/29, are currently being evaluated for the treatment of chronic viral hepatitis. IFNs are in general well tolerated and the most common adverse events observed with IFNα or IFNβ therapy are “flu-like” symptoms such as fever, headache, chills, and myalgia. Prolonged treatment is associated with more serious adverse events including leucopenia, thrombocytopenia, increased hepatic transaminases, and neuropsychiatric effects. Type I IFNs bind to high-affinity cell surface receptors, composed of two transmembrane polypeptides IFNAR1 and IFNAR2, resulting in activation of the Janus kinases Jak1 and Tyk2, phosphorylation and activation of the latent cytoplasmic signal transducers and activators of transcription (STAT1 and STAT2, formation of a transcription complex together with IRF9, and activation of a specific set of genes that encode the effector molecules responsible for mediating the biological activities of type I IFNs. Systemic administration of type I IFN results in activation of IFN receptors present on essentially all types of nucleated cells, including neurons and hematopoietic stem cells, in addition to target cells. This may well explain the wide spectrum of IFN associated toxicities. Recent reports suggest that certain polymorphisms in type I IFN signaling molecules are associated with IFN-induced neutropenia and thrombocytopenia in patients with chronic hepatitis C. IFN

  20. Immunogenicity and ecotoxicity of engineered nanoparticles

    Science.gov (United States)

    Maurer-Jones, Melissa Ann

    -induced oxidative stress. The generalizability of the mechanism of TiO2 toxicity, as detailed in Chapter Two and Three, is explored in Chapter Four in a bacteria model, Shewanella oneidensis, studying the functions of biofilm formation using a quartz crystal microbalance (QCM) and flavin secretion using high performance liquid chromatography (HPLC). This study revealed that the proximity of the TiO 2 nanoparticles to S. oneidensis caused changes in gene expression resulting in an observed delay in biofilm growth and increase in riboflavin secretion. Chapter Five works to develop an in situ Ag nanoparticle characterization tool using fluorous-phase ion selective electrodes to measure dissolved Ag+, with preliminary investigation into the toxicity of Ag nanoparticles and Ag+ ions to S. oneidensis, resulting in one of the first in situ characterization tools for nanoparticles during toxicity assessments. Moving beyond laboratory work, Chapter Six examines bench scientists' perspective on the regulation of nanotherapies moving from pre-clinical to first-in-human trials and the ethical considerations for the implementation of nanotechnology. Finally, Chapter Seven details the development of a 3-day nanotoxicity laboratory for introductory chemistry classes to introduce students to interdisciplinary science and the cutting edge research field of nanotoxicology. In total, my project has considered the scientific, ethical, and educational implications for nanotoxicology and has ultimately contributed to a better understanding of the nanoparticle-cell interaction.

  1. Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals

    DEFF Research Database (Denmark)

    Rup, B; Pallardy, M; Sikkema, D

    2015-01-01

    immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic...... scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities...... is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti-Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce...

  2. Proteomics, peptidomics, and immunogenic potential of wheat beer (Weissbier).

    Science.gov (United States)

    Picariello, Gianluca; Mamone, Gianfranco; Cutignano, Adele; Fontana, Angelo; Zurlo, Lucia; Addeo, Francesco; Ferranti, Pasquale

    2015-04-08

    Wheat beer is a traditional light-colored top-fermenting beer brewed with at least 50% malted (e.g., German Weissbier) or unmalted (e.g., Belgian Witbier) wheat (Triticum aestivum) as an adjunct to barley (Hordeum vulgare) malt. For the first time, we explored the proteome of three Weissbier samples, using both 2D electrophoresis (2DE)-based and 2DE-free strategies. Overall, 58 different gene products arising from barley, wheat, and yeast (Saccharomyces spp.) were identified in the protein fraction of a representative Weissbier sample analyzed in detail. Analogous to all-barley-malt beers (BMB), barley and wheat Z-type serpins and nonspecific lipid transfer proteins dominated the proteome of Weissbier. Several α-amylase/trypsin inhibitors also survived the harsh brewing conditions. During brewing, hundreds of peptides are released into beer. By liquid chromatography-electrospray tandem mass spectrometry (LC-ESI MS/MS) analysis, we characterized 167 peptides belonging to 44 proteins, including gliadins, hordeins, and high- and low-molecular-weight glutenin subunits. Because of the interference from the overabundant yeast-derived peptides, we identified only a limited number of epitopes potentially triggering celiac disease. However, Weissbier samples contained 374, 372, and 382 ppm gliadin-equivalent peptides, as determined with the competitive G12 ELISA, which is roughly 10-fold higher than a lager BMB (41 ppm), thereby confirming that Weissbier is unsuited for celiacs. Western blot analysis demonstrated that Weissbier also contained large-sized prolamins immunoresponsive to antigliadin IgA antibodies from the pooled sera of celiac patients (n = 4).

  3. The calcium channel β2 (CACNB2 subunit repertoire in teleosts

    Directory of Open Access Journals (Sweden)

    Mueller Rachel

    2008-04-01

    Full Text Available Abstract Background Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Results Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2 indicated they are membrane-associated guanylate kinase (MAGUK-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and

  4. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    DEFF Research Database (Denmark)

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens O L

    2010-01-01

    In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit.......In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit....

  5. Immunogenicity to Biotherapeutics – The Role of Anti-drug Immune Complexes

    Science.gov (United States)

    Krishna, Murli; Nadler, Steven G.

    2016-01-01

    Biological molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However, by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. In the context of immune-mediated adverse effects that have been documented to biological drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biological drugs in instances where there have been immune-mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs) elicited in vivo to a biotherapeutic; alongside with the structural, biophysical, and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However, many of the immune-mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex (IC) intermediate that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing ICs, risk factors that are intrinsic either to the therapeutic molecule or to the host that might predispose to IC-mediated effects, and review the recent literature on

  6. Immunogenicity to Biotherapeutics – the role of Anti-drug Immune complexes

    Directory of Open Access Journals (Sweden)

    Murli eKrishna

    2016-02-01

    Full Text Available AbstractBiologic molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. Given the range of immune mediated adverse effects that have been documented to biologic drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biologic drugs in instances where there have been immune mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs elicited in vivo to a biotherapeutic; alongside with the structural, biophysical and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However many of the immune mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex intermediate (ICs that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing-ICs, risk factors that are either intrinsic to the therapeutic molecule or to the host which might predispose to IC mediated effects, and review the recent

  7. Size, Shape, and Sequence-Dependent Immunogenicity of RNA Nanoparticles

    Directory of Open Access Journals (Sweden)

    Sijin Guo

    2017-12-01

    Full Text Available RNA molecules have emerged as promising therapeutics. Like all other drugs, the safety profile and immune response are important criteria for drug evaluation. However, the literature on RNA immunogenicity has been controversial. Here, we used the approach of RNA nanotechnology to demonstrate that the immune response of RNA nanoparticles is size, shape, and sequence dependent. RNA triangle, square, pentagon, and tetrahedron with same shape but different sizes, or same size but different shapes were used as models to investigate the immune response. The levels of pro-inflammatory cytokines induced by these RNA nanoarchitectures were assessed in macrophage-like cells and animals. It was found that RNA polygons without extension at the vertexes were immune inert. However, when single-stranded RNA with a specific sequence was extended from the vertexes of RNA polygons, strong immune responses were detected. These immunostimulations are sequence specific, because some other extended sequences induced little or no immune response. Additionally, larger-size RNA square induced stronger cytokine secretion. 3D RNA tetrahedron showed stronger immunostimulation than planar RNA triangle. These results suggest that the immunogenicity of RNA nanoparticles is tunable to produce either a minimal immune response that can serve as safe therapeutic vectors, or a strong immune response for cancer immunotherapy or vaccine adjuvants.

  8. Cytokines in immunogenic cell death: Applications for cancer immunotherapy.

    Science.gov (United States)

    Showalter, Anne; Limaye, Arati; Oyer, Jeremiah L; Igarashi, Robert; Kittipatarin, Christina; Copik, Alicja J; Khaled, Annette R

    2017-09-01

    Despite advances in treatments like chemotherapy and radiotherapy, metastatic cancer remains a leading cause of death for cancer patients. While many chemotherapeutic agents can efficiently eliminate cancer cells, long-term protection against cancer is not achieved and many patients experience cancer recurrence. Mobilizing and stimulating the immune system against tumor cells is one of the most effective ways to protect against cancers that recur and/or metastasize. Activated tumor specific cytotoxic T lymphocytes (CTLs) can seek out and destroy metastatic tumor cells and reduce tumor lesions. Natural Killer (NK) cells are a front-line defense against drug-resistant tumors and can provide tumoricidal activity to enhance tumor immune surveillance. Cytokines like IFN-γ or TNF play a crucial role in creating an immunogenic microenvironment and therefore are key players in the fight against metastatic cancer. To this end, a group of anthracyclines or treatments like photodynamic therapy (PDT) exert their effects on cancer cells in a manner that activates the immune system. This process, known as immunogenic cell death (ICD), is characterized by the release of membrane-bound and soluble factors that boost the function of immune cells. This review will explore different types of ICD inducers, some in clinical trials, to demonstrate that optimizing the cytokine response brought about by treatments with ICD-inducing agents is central to promoting anti-cancer immunity that provides long-lasting protection against disease recurrence and metastasis. Copyright © 2017. Published by Elsevier Ltd.

  9. Influences of obesity on the immunogenicity of Hepatitis B vaccine.

    Science.gov (United States)

    Liu, Fang; Guo, Zhirong; Dong, Chen

    2017-05-04

    Hepatitis B vaccine is regarded as the most effective method for the prevention of hepatitis B virus (HBV) infection. However, several factors such as age, body mass index and immunocompetent state have been reported to be associated with reduced immunization responses. The present commentary was aimed to discuss the influences of obesity on the immunogenicity of hepatitis B vaccines. Available peer-reviewed literatures, practice guidelines, and statistics published on hepatitis B vaccine in obesity between 1973 and 2015. Obesity was significantly associated with non-response to hepatitis B vaccine immunization. The risk of nonresponsiveness of hepatitis B vaccine among obese people increased with BMI. Moreover, the obesity might lead to an increased risk of HBV vaccine-escape mutations. The mechanism responsible for decreased immunization responses in obesity included leptin-induced systemic and B cell intrinsic inflammation, impaired T cell responses and lymphocyte division and proliferation. Therefore, more studies should be performed to analyze the influences of obesity on the immunogenicity of hepatitis B vaccines to improve the immunoprotecive effect of hepatitis B vaccines in future.

  10. Epitope determination of immunogenic proteins of Neisseria gonorrhoeae.

    Science.gov (United States)

    Connor, Daniel O; Danckert, Lena; Hoppe, Sebastian; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2017-01-01

    Neisseria gonorrhoeae is the causative organism of gonorrhoea, a sexually transmitted disease that globally accounts for an estimated 80 to 100 million new infections per year. Increasing resistances to all common antibiotics used for N. gonorrhoeae treatment pose the risk of an untreatable disease. Further knowledge of ways of infection and host immune response are needed to understand the pathogen-host interaction and to discover new treatment alternatives against this disease. Therefore, detailed information about immunogenic proteins and their properties like epitope sites could advance further research in this area. In this work, we investigated immunogenic proteins of N. gonorrhoeae for linear epitopes by microarrays. Dominant linear epitopes were identified for eleven of the nineteen investigated proteins with three polyclonal rabbit antibodies from different immunisations. Identified linear epitopes were further examined for non-specific binding with antibodies to Escherichia coli and the closely related pathogen Neisseria meningitidis. On top of that, amino acids crucial for the antibody epitope binding were detected by microarray based alanine scans.

  11. Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein.

    Science.gov (United States)

    Céspedes, Nora; Arévalo-Herrera, Myriam; Felger, Ingrid; Reed, Steve; Kajava, Andrey V; Corradin, Giampietro; Herrera, Sócrates

    2013-10-01

    Plasmodium vivax circumsporozoite (PvCS) protein is a major sporozoite surface antigen involved in parasite invasion of hepatocytes and is currently being considered as vaccine candidate. PvCS contains a dimorphic central repetitive fragment flanked by conserved regions that contain functional domains. We have developed a chimeric 137-mer synthetic polypeptide (PvCS-NRC) that includes the conserved region I and region II-plus and the two natural repeat variants known as VK210 and VK247. The antigenicity of PvCS-NRC was tested using human sera from PNG and Colombia endemic areas and its immunogenicity was confirmed in mice with different genetic backgrounds, the polypeptide formulated either in Alum or GLA-SE adjuvants was assessed in inbred C3H, CB6F1 and outbred ICR mice, whereas a formulation in Montanide ISA51 was tested in C3H mice. Antigenicity studies indicated that the chimeric peptide is recognized by a high proportion (60-70%) of residents of malaria-endemic areas. Peptides formulated with either GLA-SE or Montanide ISA51 adjuvants induced stronger antibody responses as compared with the Alum formulation. Sera from immunized mice as well as antigen-specific affinity purified human IgG antibodies reacted with sporozoite preparations in immunofluorescence and Western blot assays, and displayed strong in vitro inhibition of sporozoite invasion (ISI) into hepatoma cells. The polypeptide was recognized at high prevalence when tested against naturally induced human antibodies and was able to induce significant immunogenicity in mice. Additionally, specific antibodies were able to recognize sporozoites and were able to block sporozoite invasion in vitro. Further evaluation of this chimeric protein construct in preclinical phase e.g. in Aotus monkeys in order to assess the humoral and cellular immune responses as well as protective efficacy against parasite challenge of the vaccine candidate must be conducted. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Comparative immunogenicity of HIV-1 clade C envelope proteins for prime/boost studies.

    Directory of Open Access Journals (Sweden)

    Douglas H Smith

    2010-08-01

    Full Text Available Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120 as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses.A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested.While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies.

  13. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals.

    Science.gov (United States)

    Dao, E Han; Sierra, Raymond G; Laksmono, Hartawan; Lemke, Henrik T; Alonso-Mori, Roberto; Coey, Aaron; Larsen, Kevin; Baxter, Elizabeth L; Cohen, Aina E; Soltis, S Michael; DeMirci, Hasan

    2015-07-01

    In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

  14. Evaluation of peptide designing strategy against subunit reassociation in mucin 1: A steered molecular dynamics approach.

    Directory of Open Access Journals (Sweden)

    J Lesitha Jeeva Kumari

    Full Text Available Subunit reassociation in mucin 1, a breast cancer tumor marker, is reported as one of the critical factors for its cytoplasmic activation. Inhibition of its heterodimeric association would therefore result in loss of its function and alter disease progression. The present study aimed at evaluating peptide inhibitor designing strategies that may serve as antagonist against this receptor-ligand alliance. Several peptides and their derivatives were designed based on native residues, subunit interface, hydrogen bonding and secondary structure. Docking studies with the peptides were carried on the receptor subunit and their binding affinities were evaluated using steered molecular dynamics simulation and umbrella sampling. Our results showed that among all the different classes of peptides evaluated, the receptor based peptide showed the highest binding affinity. This result was concurrent with the experimental observation that the receptor-ligand alliance in mucin 1 is highly specific. Our results also show that peptide ligand against this subunit association is only stabilized through native residue inter-protein interaction irrespective of the peptide structure, peptide length and number of hydrogen bonds. Consistency in binding affinity, pull force and free energy barrier was observed with only the receptor derived peptides which resulted in favorable interprotein interactions at the interface. Several observations were made and discussed which will eventually lead to designing efficient peptide inhibitors against mucin 1 heterodimeric subunit reassociation.

  15. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

    Directory of Open Access Journals (Sweden)

    E. Han Dao

    2015-07-01

    Full Text Available In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

  16. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface

    Directory of Open Access Journals (Sweden)

    Benoît Bestgen

    2017-02-01

    Full Text Available Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α’ subunits and two regulatory (β subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein–protein interaction, we previously designed an active cyclic peptide (Pc derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way.

  17. B epitope multiplicity and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines.

    Science.gov (United States)

    Blanco, Esther; Cubillos, Carolina; Moreno, Noelia; Bárcena, Juan; de la Torre, Beatriz G; Andreu, David; Sobrino, Francisco

    2013-01-01

    Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD) vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154) linked to a T-cell epitope (3A 21 to 35) of FMD virus (FMDV) elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T) or four (B4T) copies of the B-cell epitope from type O FMDV (a widespread circulating serotype) were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFN γ . Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

  18. B Epitope Multiplicity and B/T Epitope Orientation Influence Immunogenicity of Foot-and-Mouth Disease Peptide Vaccines

    Directory of Open Access Journals (Sweden)

    Esther Blanco

    2013-01-01

    Full Text Available Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154 linked to a T-cell epitope (3A 21 to 35 of FMD virus (FMDV elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T or four (B4T copies of the B-cell epitope from type O FMDV (a widespread circulating serotype were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFNγ. Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

  19. A PILOT AND FEASIBILITY CLINICAL TRIAL EVALUATING IMMUNO-GENE THERAPY OF MALIGNANT PLEURAL MESOTHELIOMA (MPM) USING INTRAPLEURAL DELIVERY OF ADENOVIRUS- INTERFERON-ALPHA (Ad.hIFN-α2b) IN COMBINATION WITH HIGH-DOSE CELECOXIB AND SYSTEMIC CHEMOTHERAPY

    Science.gov (United States)

    Sterman, Daniel H; Alley, Evan; Stevenson, James; Friedberg, Joseph; Metzger, Susan; Recio, Adri; Moon, Edmund; Haas, Andrew R; Vachani, Anil; Katz, Sharyn I; Sun, Jing; Heitjan, Daniel F; Hwang, Wei-Ting; Litzky, Leslie; Yearley, Jennifer H; Tan, Kay See; Papasavvas, Emmanouil; Kennedy, Paul; Montaner, Luis J.; Cengel, Keith; Simone, Charles B; Culligan, Melissa; Langer, Corey J; Albelda, Steven M

    2016-01-01

    Purpose “In situ vaccination” using immuno-gene therapy has the ability to induce polyclonal anti-tumor responses directed by the patient’s immune system. Experimental Design Patients with unresectable MPM received two intrapleural doses of a replication-defective adenoviral vector containing the human interferon-alpha2b gene (Ad.IFN) concomitant with a 14-day course of celecoxib followed by chemotherapy. Primary outcomes were safety, toxicity, and objective response rate; secondary outcomes included progression-free and overall survival. Bio-correlates on blood and tumor were measured. Results Forty subjects were treated: 18 received first-line pemetrexed-based chemotherapy, 22 received second-line chemotherapy with pemetrexed (n=7) or gemcitabine (n=15). Treatment was generally well tolerated. The overall response rate was 25% and the disease control rate was 88%. Median overall survival (MOS) for all patients with epithelial histology was 21 months versus 7 months for patients with non-epithelial histology. MOS in the first-line cohort was 12.5 months, while MOS for the second-line cohort was 21.5 months, with 32% of patients alive at 2 years. No biologic parameters were found to correlate with response, including numbers of activated blood T cells or NK cells, regulatory T cells in blood, peak levels of interferon-α in blood or pleural fluid, induction of anti-tumor antibodies, nor an immune-gene signature in pretreatment biopsies. Conclusions The combination of intrapleural Ad.IFN, celecoxib, and chemotherapy proved safe in patients with MPM. Overall survival rate was significantly higher than historical controls in the second-line group. Results of this study support proceeding with a multi-center randomized clinical trial of chemo-immunogene therapy versus standard chemotherapy alone. PMID:26968202

  20. Diet-induced bacterial immunogens in the gastrointestinal tract of dairy cows: Impacts on immunity and metabolism

    Directory of Open Access Journals (Sweden)

    Zhou Jun

    2011-08-01

    Full Text Available Abstract Dairy cows are often fed high grain diets to meet the energy demand for high milk production or simply due to a lack of forages at times. As a result, ruminal acidosis, especially subacute ruminal acidosis (SARA, occurs frequently in practical dairy production. When SARA occurs, bacterial endotoxin (or lipopolysaccharide, LPS is released in the rumen and the large intestine in a large amount. Many other bacterial immunogens may also be released in the digestive tract following feeding dairy cows diets containing high proportions of grain. LPS can be translocated into the bloodstream across the epithelium of the digestive tract, especially the lower tract, due to possible alterations of permeability and injuries of the epithelial tissue. As a result, the concentration of blood LPS increases. Immune responses are subsequently caused by circulating LPS, and the systemic effects include increases in concentrations of neutrophils and the acute phase proteins such as serum amyloid-A (SAA, haptoglobin (Hp, LPS binding protein (LBP, and C-reactive protein (CRP in blood. Entry of LPS into blood can also result in metabolic alterations. Blood glucose and nonesterified fatty acid concentrations are enhanced accompanying an increase of blood LPS after increasing the amount of grain in the diet, which adversely affects feed intake of dairy cows. As the proportions of grain in the diet increase, patterns of plasma β-hydoxybutyric acid, cholesterol, and minerals (Ca, Fe, and Zn are also perturbed. The bacterial immunogens can also lead to reduced supply of nutrients for synthesis of milk components and depressed functions of the epithelial cells in the mammary gland. The immune responses and metabolic alterations caused by circulating bacterial immunogens will exert an effect on milk production. It has been demonstrated that increases in concentrations of ruminal LPS and plasma acute phase proteins (CRP, SAA, and LBP are associated with declines in

  1. A single or multistage mycobacterium avium subsp. paratuberculosis subunit vaccine

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention provides one or more immunogenic polypeptides for use in a preventive or therapeutic vaccine against latent or active infection in a human or animal caused by a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Furthermore a single or multi-phase vaccine...... comprising the one or more immunogenic polypeptides is provided for administration for the prevention or treatment of infection with a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Additionally, nucleic acid vaccines, capable of in vivo expression of the multi-phase vaccine...

  2. Assembly of catalytic subunits of aspartate transcarbamoylase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Burns, D.L.; Schachman, H.K.

    1980-10-01

    Although extensive studies have been conducted on the assembly of the allosteric enzyme, aspartate transcarbamoylase (ATCase) from isolate, intact catalytic (C) and regulatory (R) subunits, there has been little research on the formation of these subunits from individual catalytic (c) and regulatory (r) polypeptide chains. Such studies would be useful for evaluating the strengths of the interchain bonding domains within the subunits just as earlier experiments provided valuable data regarding interactions between the subunits in ATCase. The intact enzyme comprising two C trimers and three R dimers is designated as C/sub 2/R/sub 3/ or c/sub 6/r/sub 6/.

  3. The priming effect of previous natural pandemic H1N1 infection on the immunogenicity to subsequent 2010-2011 influenza vaccination in children: a prospective cohort study.

    Science.gov (United States)

    Kang, Eun Kyeong; Eun, Byung Wook; Kim, Nam Hee; Lim, Jung Sub; Lee, Jun Ah; Kim, Dong Ho

    2016-08-22

    The effect of previous natural pandemic H1N1 (H1N1 pdm09) influenza infection on the immunogenicity to subsequent inactivated influenza vaccination in children has not been well studied. We aimed to evaluate the effect of H1N1 pdm09 natural infection and vaccination on the immunogenicity to subsequent 2010-2011 seasonal inactivated influenza vaccination in children. From October 2010 to May 2011, we conducted an open-label, multi-center study in children aged 6 months -18 years in Korea. We measured antibody titers with a hemagglutination-inhibition (HI) assay at baseline, 1 month, and 6 months after vaccination with trivalent split or subunit vaccines containing H1N1 pdm, A/H3N2, and B. The subjects were classified into 4 groups depending on the presence of laboratory-confirmed H1N1 pdm09 infection and/or vaccination in the 2009-2010 season; Group I: vaccination (-)/infection(-), Group II: vaccination (-)/infection(+), Group III: vaccination (+)/infection(-), Group IV: vaccination (+)/infection(+). Among the subjects in group I, 47 subjects who had a baseline titer >1:10 were considered to have an asymptomatic infection. They were included into the final group II (n = 80). We defined the new group II as the infection-primed (IP) group and group III as the vaccine-primed (VP) group. Seroconversion rate (57.5 % vs 35.9 %, p = 0.001), seroprotection rate at 6 months after vaccination (70.8 % vs 61.8 %, p = 0.032), and GMT at 1 month after vaccination (129.9 vs 66.5, p = 0.002) were significantly higher in the IP group than in the VP group. In the 9-18 year-old group, seroconversion rate and immunogenicity at 1 and 6 months were significantly higher in the IP group than in the VP group. However in the 1-7 year-old age group, there was no significant difference between the two groups. Previous H1N1 pdm09 infection appears to have positive effects on immunogenicity of subsequent inactivated influenza vaccines against H1N1 pdm09 in older

  4. [Immunogenicity and safety of DTaP-IPV//PRP-T combined vaccine in infants in China].

    Science.gov (United States)

    Li, Yan-ping; Li, Feng-xiang; Hou, Qi-ming; Li, Chang-gui; Li, Ya-nan; Chen, Fu-sheng; Hu, Xue-zhong; Su, Wen-bin; Zhang, Shu-min; Fang, Han-hua; Ye, Qiang; Zeng, Tian-de; Liu, Tao-xuan; Li, Xiu-bi; Huang, Yun-neng; Deng, Man-ling; Li, Rong-cheng; Zhang, Yan-ping; Esteban, Ortiz

    2011-08-01

    The aim of this study was to demonstrate the immunogenicity and safety of diphtheria, tetanus, pertussis (acellular, component), poliomyelitis (inactivated) vaccine (adsorbed) and Haemophilus influenzae type b conjugate vaccine (DTaP-IPV//PRP-T) combined vaccine compared with commercially available DTaP (diphtheria, tetanus and pertussis), Haemophilus influenzae type b (Hib), tetanus conjugate and IPV monovalent vaccine. Subjects were randomly divided into three groups, Group A and Group B were DTaP-IPV//PRP-T combined vaccine (PENTAXIM(TM)) vaccinated at 2, 3, 4 months of age or 3, 4, 5 months of age respectively; Group C was commercially available DTaP. Hib tetanus conjugate (Act-HIB(TM)) and IPV (IMOVAX PolioTM(TM)) vaccines vaccinated at 3, 4, 5 months of age. All groups received booster dose at 18 to 20 months of age, with antibody titers tested. Non-inferiority analysis was demonstrated in terms of seroprotection/seroconversion rates between Group A, Group B respectively and Group C. Safety information was collected after each vaccination to assess the safety of investigational vaccines. The non-inferiority of DTaP-IPV//PRP-T combined vaccine vaccinated at 2, 3, 4 or 3, 4, 5 months of age versus DTaP, Hib tetanus conjugate and IPV vaccine was demonstrated for all vaccine antigens in both primary and booster phases in terms of seroprotection/seroconversion rates. DTaP-IPV//PRP-T combined vaccine was well tolerated. The rate of solicited/unsolicited severe adverse reactions was very low and similar to the control vaccines. DTaP-IPV//PRP-T combined vaccine was highly immunogenic with good safety profile in Chinese infants, which was comparable to the commercially available control vaccines.

  5. The safety and immunogenicity of Quadrivalent HPV (qHPV) vaccine in systemic lupus erythematosus.

    Science.gov (United States)

    Dhar, J Patricia; Essenmacher, Lynnette; Dhar, Renee; Magee, Ardella; Ager, Joel; Sokol, Robert J

    2017-05-09

    This study evaluated the safety and immunogenicity of qHPV vaccine in SLE. Subjects: 34 women ages 19-50years (yrs.) with mild to moderate SLE & minimally active or inactive SLE received qHPV vaccine at the standard dosing schedule. active SLE disease (SELENA-SLEDAI>2), history of severe SLE disease, deep venous thrombosis, on >400mg/day of hydroxychloroquine, on >15mg/day of prednisone, or active infections. Patients were monitored for adverse events (AE), SLE flare, generation of thrombogenic antibodies and thrombosis. Antibody (Ab) levels to HPV 6, 11, 16 & 18 were measured by HPV competitive Luminex Immunoassay and Geometric Mean Titers (GMTs) were calculated for each HPV type. Seroconversion was assessed for those seronegative at baseline. The women in the study: African-American (79%), mean age=38.1years, mean age at diagnosis of SLE=28.6years, 35.3% had a history of smoking, 91% had 4 or more sexual partners, 50% had a history of sexually transmitted diseases, and 27.3% used condoms on a regular basis. Vaccine site reactions (VSRs) occurred in 62%, all mild. Ninety-seven percent experienced at least 1 non vaccine adverse event (nvAE) with a total of 493 nvAEs in 33 patients, of which 90% were mild and none were related to vaccine or SLE. There were 9 serious AEs, none were related to vaccine or SLE, all resolved. No patient experienced an SLE flare, thrombosis, or generation of thrombogenic antibodies. Seroconversion rate was 100% with mean GMTs comparable to Gardasil® package insert data. In this SLE vaccine study, qHPV vaccine was generally safe, well tolerated, and highly immunogenic. This clinical trial is registered on Clinical Trials.gov under number, NCT01741012 and was conducted under the FDA IND BB14113. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Immunogenicity of hydrolysate formulas in children (part 1). Analysis of 202 reactions.

    Science.gov (United States)

    Cantani, A; Micera, M

    2000-01-01

    Cow's milk protein hydrolyzed formulas appeared in the 1940s with the aim of decreasing or eliminating the allergenicity of cow's milk proteins, in addition to reducing the risk of sensitization. In recent years, the so-called "hypoallergenic" formulas have been developed. The use of such hydrolyzed formulas is based on the premise that predigested proteins, when fed as amino acids and peptides, provide nutrients in a nonantigenic form. Thus, protein hydrolyzed formulas have been classified as hypoallergenic. These formulas are processed by heat and enzymatic hydrolysis, and the conformational and sequential structures are more or less changed. The formulas contain peptides of lower molecular weight than the native protein source, which are thought to be less immunogenic. Hydrolyzed formulas appear to be nutritionally adequate and infants generally gain weight until they refuse the formula because of its bad taste. However, caution should be taken when such formulas are given for prolonged periods since no data are available on nutritional assessment of infants exclusively fed hydrolyzed formulas for several months. In this paper we report and discuss more than 202 reactions to different hydrolyzed formulas, including cases of anaphylactic shock and apparent life-threatening events. The cross-reactivity between different hydrolyzed formulas and cow's milk proteins, and the potential immunogenicity of such formulas are discussed. We conclude that none of the hydrolyzed formulas are nonallergenic, both for allergic children and for high-risk babies. Moreover, we suggest that double-blind placebo-controlled food challenge studies in larger cohorts of babies evaluated with well-defined and well-validated diagnostic methods may establish a more reliable prevalence of allergy to hydrolyzed formulas.

  7. Characterization of BIP protein of G. lamblia as a potential immunogen in a mouse infection model.

    Science.gov (United States)

    Lopez-Romero, Gloria; Garzon, Thania; Rascon, Raul; Valdez, Alejandra; Quintero, Jael; Arvizu-Flores, Aldo A; Garibay-Escobar, Adriana; Rascon, Lucila; Astiazarán-García, Humberto; Velazquez, Carlos

    2017-08-01

    Giardia lamblia is a protozoan parasite that causes one of the most common gastrointestinal diseases worldwide. To eliminate the parasite from the host intestine, it is necessary the activation of B-cell and T-cell dependent mechanisms. The knowledge about Giardia antigens that can stimulate the host immune response is limited. Recently, it has been described the Binding Immunoglobulin Protein (BIP) of G. lamblia (71kDa) as a potential immunogen. Additionally, our group has identified a highly immunogenic antigen (5G8 protein) of G. lamblia with a relative molecular mass of approximately 70kDa. There is some evidence suggesting that the 5G8 protein may activate both humoral and cellular immune responses. Based on these observations and preliminary mass spectrometry analyses, we hypothesized that the antigen 5G8 could be the BIP protein. In the present study, we characterize immunochemically the BIP protein of Giardia. Flow cytometric assays and western blotting were used to determine the expression profile of BIP and 5G8 antigens in Giardia trophozoites. The differences in expression profile indicated that BIP and 5G8 are not the same molecule. ELISA and Western blotting assays revealed that BIP protein was recognized by antibodies produced during G. lamblia infection in C3H/HeN mice. MTT assays did not reveal the activation of cellular immune response induced by BIP protein in vitro. In addition, we identified the potential B-cell and T-cell epitopes of G. lamblia BIP protein. This molecule is a conserved protein among Giardia strains and other pathogens. The complete immunological characterization of this antigen will contribute to a better understanding of the host-parasite interactions in Giardia infection. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Accelerated evolution and coevolution drove the evolutionary history of AGPase sub-units during angiosperm radiation.

    Science.gov (United States)

    Corbi, Jonathan; Dutheil, Julien Y; Damerval, Catherine; Tenaillon, Maud I; Manicacci, Domenica

    2012-03-01

    ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch biosynthesis. In the green plant lineage, it is composed of two large (LSU) and two small (SSU) sub-units encoded by paralogous genes, as a consequence of several rounds of duplication. First, our aim was to detect specific patterns of molecular evolution following duplication events and the divergence between monocotyledons and dicotyledons. Secondly, we investigated coevolution between amino acids both within and between sub-units. A phylogeny of each AGPase sub-unit was built using all gymnosperm and angiosperm sequences available in databases. Accelerated evolution along specific branches was tested using the ratio of the non-synonymous to the synonymous substitution rate. Coevolution between amino acids was investigated taking into account compensatory changes between co-substitutions. We showed that SSU paralogues evolved under high functional constraints during angiosperm radiation, with a significant level of coevolution between amino acids that participate in SSU major functions. In contrast, in the LSU paralogues, we identified residues under positive selection (1) following the first LSU duplication that gave rise to two paralogues mainly expressed in angiosperm source and sink tissues, respectively; and (2) following the emergence of grass-specific paralogues expressed in the endosperm. Finally, we found coevolution between residues that belong to the interaction domains of both sub-units. Our results support the view that coevolution among amino acid residues, especially those lying in the interaction domain of each sub-unit, played an important role in AGPase evolution. First, within SSU, coevolution allowed compensating mutations in a highly constrained context. Secondly, the LSU paralogues probably acquired tissue-specific expression and regulatory properties via the coevolution between sub-unit interacting domains. Finally, the pattern we observed during LSU evolution is consistent

  9. Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions.

    Directory of Open Access Journals (Sweden)

    Daniela Almeida

    Full Text Available Mitochondrial protein-coding genes (mt genes encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS. Despite the vital role of the mitochondrial genome (mt genome in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments ranging from the intertidal to the deep sea, having distinct metabolic requirements, varied body shapes and highly advanced visual and nervous systems that make them highly competitive and successful worldwide predators. Thus, cephalopods are valuable models for testing natural selection acting on their mitochondrial subunits (mt subunits. Here, we used concatenated mt genes from 17 fully sequenced mt genomes of diverse cephalopod species to generate a robust mitochondrial phylogeny for the Class Cephalopoda. We followed an integrative approach considering several branches of interest-covering cephalopods with distinct morphologies, metabolic rates and habitats-to identify sites under positive selection and localize them in the respective protein alignment and/or tridimensional structure of the mt subunits. Our results revealed significant adaptive variation in several mt subunits involved in the energy production pathway of cephalopods: ND5 and ND6 from Complex I, CYTB from Complex III, COX2 and COX3 from Complex IV, and in ATP8 from Complex V. Furthermore, we identified relevant sites involved in protein-interactions, lining proton translocation channels, as well as disease/deficiencies related sites in the aforementioned complexes. A particular case, revealed by this study, is the involvement of some positively selected sites, found in Octopoda lineage in lining proton translocation channels (site 74 from ND5 and in interactions between subunits (site 507 from ND

  10. A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats.

    Science.gov (United States)

    Rostad, Christina A; Stobart, Christopher C; Gilbert, Brian E; Pickles, Ray J; Hotard, Anne L; Meng, Jia; Blanco, Jorge C G; Moin, Syed M; Graham, Barney S; Piedra, Pedro A; Moore, Martin L

    2016-08-15

    Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by

  11. The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae.

    Science.gov (United States)

    Perez, Emilie; Lapaille, Marie; Degand, Hervé; Cilibrasi, Laura; Villavicencio-Queijeiro, Alexa; Morsomme, Pierre; González-Halphen, Diego; Field, Mark C; Remacle, Claire; Baurain, Denis; Cardol, Pierre

    2014-11-01

    The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologues to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE both to confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance

    Directory of Open Access Journals (Sweden)

    Boris C. Dunkov

    2002-04-01

    Full Text Available Secreted ferritin in the mosquito, Aedes aegypti, has several subunits that are the products of at least two genes, one encoding a homologue of the vertebrate heavy chain (HCH and the other the light chain homologue (LCH. Here we report the developmental and organ specific pattern of expression of the ferritin HCH messages and of both subunit types in control sugar-fed mosquitoes, in those exposed to high levels of dietary iron, and after blood feeding.

  13. Reconstruction of the nasal soft triangle subunit.

    Science.gov (United States)

    Constantine, Fadi C; Lee, Michael R; Sinno, Sammy; Thornton, James F

    2013-05-01

    Of all nine subunits, the soft triangle is perhaps the most challenging to recreate. The complexity of soft triangle reconstruction resides in its proximity to such important structures as the nasal tip, nasal ala, and distal columella. If the soft triangle is not properly reconstructed, problems with nasal function and aesthetics often arise. Anatomical asymmetries in the lower third and abnormal shadowing can occur following insufficient restoration. A retrospective review was completed of all patients undergoing reconstruction of the nasal soft triangle subunit at the University of Texas Southwestern Medical Center in Dallas, Texas, from 1995 to 2010. Defects with only external skin intact were classified as type I. Defects involving both skin and underlying soft tissue with intact mucosa were classified as type II. Finally, transmural defects with violated mucosa were classified as type III. Surgical outcomes were graded on a scale of I to IV. Grades given were based on the complexity of the existing defect and restoration of the soft triangle, with higher grades given when adjacent structures were not distorted. Of the 14 cases reviewed, two (14 percent) were type I defects, nine (64 percent) were type II defects, and three (21 percent) were type III defects. Three patients (21 percent) required revision with subsequent resurfacing and two (14 percent) required resurfacing alone. All but one patient (93 percent) had a grade of 2.0 or better, with the one patient opting not to undergo revision. The authors believe their method of soft triangle reconstruction using the proposed algorithm is an easy approach to soft triangle reconstruction that will yield consistent surgical and clinical success from aesthetic and functional perspectives. Furthermore, the authors were able to achieve excellent aesthetic outcomes without compromise or facing any structural complications. Therapeutic, IV.

  14. Interactions between subunits in heterodimeric Ncd molecules.

    Science.gov (United States)

    Kocik, Elzbieta; Skowronek, Krzysztof J; Kasprzak, Andrzej A

    2009-12-18

    The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

  15. Induction of Immunogenic Cell Death with Non-Thermal Plasma for Cancer Immunotherapy

    Science.gov (United States)

    Lin, Abraham G.

    treatment, ROS immediately increased. When chemical attenuators of ROS were used, intracellular ROS was abrogated and emission of ICD markers were attenuated. This strongly suggests that plasma-induced ICD is associated with increased intracellular ROS. The gold-standard approach to evaluating whether a stimulus can elicit genuine ICD relies on a vaccination assay. CT26 colorectal cancer cells were treated at ICD-inducing regimes of plasma and injected into syngeneic Balb/c mice. One week later, mice were challenged with live CT26 cancer cells. Tumor progression was moderated in animals immunized with plasma-treated CT26 cells. Altogether, these provide strong evidence that plasma regimes can be adapted for a new application: ICD induction. Next, a study was conducted to test the potential of plasma to induce ICD in tumors in animals. Plasma treatment of subcutaneous tumors in mice elicited the emission of ecto-CRT and high mobility group box 1 (HMGB1), another marker of ICD, in the tumor and also recruited CD11c+ and CD45+ immune cells locally. This was followed by development of cancer-specific splenic T cells, indicating that a systemic anti-tumor response was elicited from localized plasma treatment of the tumor. Overall, this work demonstrates the development of non-thermal plasma as a novel method of inducing immunogenic cell death for cancer immunotherapy. The obtained results further our understanding of plasma-cellular interaction mechanisms and highlight the potential for clinical translation.

  16. Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

    Directory of Open Access Journals (Sweden)

    Qiong Liu

    2016-03-01

    Full Text Available Lipopolysaccharide (LPS is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium. Outer membrane proteins (OMPs from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46 induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa.

  17. The interleukin 2 receptor (IL-2R): the IL-2R alpha subunit alters the function of the IL-2R beta subunit to enhance IL-2 binding and signaling by mechanisms that do not require binding of IL-2 to IL-2R alpha subunit.

    OpenAIRE

    Grant, A J; Roessler, E; Ju, G; Tsudo, M; Sugamura, K; Waldmann, T A

    1992-01-01

    Interleukin 2 (IL-2)-mediated signaling through its high-affinity receptor involves a complex interrelationship between IL-2 and two IL-2-binding chains, IL-2R alpha and beta chains. Previously with the reagents available it was difficult to define functional interactions between these two IL-2R subunits involved in IL-2 binding and signal transduction. To extend our understanding of the interplay between the two binding subunits we have done studies with the monoclonal antibody HIEI, which i...

  18. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James

    2004-01-01

    recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... on GPCR-to-effector specificity. Dually modified Galpha proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves...

  19. The cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly

    Science.gov (United States)

    Razi, Aida; Guarné, Alba; Ortega, Joaquin

    2017-01-01

    Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit. PMID:28396444

  20. Polyphenol Interactions Mitigate the Immunogenicity and Allergenicity of Gliadins.

    Science.gov (United States)

    Pérot, Maxime; Lupi, Roberta; Guyot, Sylvain; Delayre-Orthez, Carine; Gadonna-Widehem, Pascale; Thébaudin, Jean-Yves; Bodinier, Marie; Larré, Colette

    2017-08-09

    Wheat allergy is an IgE-mediated disorder. Polyphenols, which are known to interact with certain proteins, could be used to reduce allergic reactions. This study screened several polyphenol sources for their ability to interact with gliadins, mask epitopes, and affect basophil degranulation. Polyphenol extracts from artichoke leaves, cranberries, apples, and green tea leaves were examined. Of these extracts, the first three formed insoluble complexes with gliadins. Only the cranberry and apple extracts masked epitopes in dot blot assays using anti-gliadin IgG and IgE antibodies from patients with wheat allergies. The cranberry and artichoke extracts limited cellular degranulation by reducing mouse anti-gliadin IgE recognition. In conclusion, the cranberry extract is the most effective polyphenol source at reducing the immunogenicity and allergenicity of wheat gliadins.

  1. Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Reimann, Ilona

    2007-01-01

    A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination–challenge experiment in pigs. The chimeric virus proved...... to be avirulent and neither chimeric virus nor viral RNA was detected in serum after vaccination. The safety of the vaccine was tested by horizontal transmission to sentinel pigs, which remained uninfected. The vaccine efficacy was examined by challenge infection with classical swine fever virus (CSFV) Eystrup....... In ‘challenge controls’, the viral load of CSFV coincided with the development of pronounced clinical symptoms. In contrast, the vaccinated pigs showed transient and weak clinical signs. Analysis of the viral load in these pigs showed 1000-fold lower viral RNA levels compared to ‘challenge controls...

  2. Alcohol- and alcohol antagonist-sensitive human GABAA receptors: tracking δ subunit incorporation into functional receptors.

    Science.gov (United States)

    Meera, Pratap; Olsen, Richard W; Otis, Thomas S; Wallner, Martin

    2010-11-01

    GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs.

  3. Characterization of Autoantibodies against the E1 Subunit of Branched-Chain 2-Oxoacid Dehydrogenase in Patients with Primary Biliary Cirrhosis

    Directory of Open Access Journals (Sweden)

    Tsutomu Mori

    2012-01-01

    Full Text Available Primary biliary cirrhosis (PBC is characterized by antimitochondrial antibodies (AMAs that react with the lipoyl-containing E2 subunits of 2-oxoacid dehydrogenase complexes such as BCOADC and PDC. The lipoyl domains of E2 contain the major epitopes essential for immunopathology. However, the non-lipoyl-containing E1 subunits are also frequently targeted. Since anti-E1 antibodies always appear in combination with anti-E2 antibodies, the mechanisms underlying the autoimmunity against E1 may be linked to, but distinct from, those against E2. Here, we demonstrate that intermolecular and intramolecular determinant spreading underlies the autoimmunity against E1. We performed characterizations and epitope mapping for anti-BCOADC-E1 antibodies from both the intermolecular and intramolecular points of view. The antibody reactivities form a cluster against the BCOADC complex that is distinct from that against the PDC complex, and the anti-BCOADC-E1 antibodies arise as part of the cluster against the BCOADC complex. Multiple epitopes are present on the surface of the BCOADC-E1 molecule, and the major epitope overlaps with the active center. Sera with anti-BCOADC-E1 antibodies strongly inhibited the enzyme activity. These findings suggest that the E1 subunit as part of the native BCOADC complex is an immunogen, and that determinant spreading is involved in the pathogenesis of AMA production.

  4. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development.

    Science.gov (United States)

    Kaeser, Gwendolyn E; Rabe, Brian A; Saha, Margaret S

    2011-04-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA(A) and metabotropic GABA(B) receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA(A) and GABA(B) receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABA(A) α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Copyright © 2011 Wiley-Liss, Inc.

  5. A Cooperative Escherichia coli Aspartate Transcarbamoylase without Regulatory Subunits

    Energy Technology Data Exchange (ETDEWEB)

    Mendes, K.; Kantrowitz, E

    2010-01-01

    Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative Escherichia coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c{sub 6}r{sub 6}. Dissociation of the native holoenzyme produces catalytically active trimers, c{sub 3}, and nucleotide-binding regulatory dimers, r{sub 2}. By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c{sub 6}, was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c{sub 6} species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c3. The c{sub 6} ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 {angstrom} resolution. The structure of c{sub 6} ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240s loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c{sub 6} enzyme, enhancing its activity over the wild-type holoenzyme (c{sub 6}r{sub 6}) and c{sub 3}. The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction

  6. Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

    Science.gov (United States)

    Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

    2017-05-15

    The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation.IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

  7. Expression and purification of the central stalk subunits of Na + ...

    African Journals Online (AJOL)

    , NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na+-ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, ...

  8. Safety and immunogenicity of indigenous recombinant hepatitis B vaccine (Shanvac-B) in comparison with commercially available vaccine.

    Science.gov (United States)

    Joshi, N; Kumar, A; Sreenivas, D V; Palan, S; Nagarjuna Kumar, Y R

    2000-01-01

    To assess the clinical safety, reactogenicity and immunogenicity of an indigenously developed recombinant hepatitis B vaccine (Shanvac-B; Shantha Biotechnics) and to compare it with another commercially available vaccine (Engerix-B, SmithKline Beecham) in healthy adults. 120 healthy adults randomLy received 20 micrograms of either Engerix-B (Group A; n = 61) or Shanvac-B (Group B; n = 59) in 0, 1, 2 months schedule. Anti HBs was assessed using commercially available AUSAB kits (Abbott Laboratories) one month after each dose. Protective seroconversion rates after first, second and third dose were 10%, 62.7% and 91.4%, respectively in Group A and 22.4%, 68.9% and 96.4% in Group B, respectively. The geometric mean titer (GMT) after the third dose was significantly high in Group B (419 mIU/mL) than in Group A (140 mIU/mL; p < 0.001). The GMT was significantly higher in women in both the groups. The indigenous vaccine was found to be clinically safe and well tolerated without significant side effects. The recombinant hepatitis B vaccine (Shanvac-B) developed in India is safe, well tolerated, and highly immunogenic, with high seroconversion and GMT response.

  9. In silico analysis to identify vaccine candidates common to multiple serotypes of Shigella and evaluation of their immunogenicity

    KAUST Repository

    Pahil, Sapna

    2017-08-02

    Shigellosis or bacillary dysentery is an important cause of diarrhea, with the majority of the cases occurring in developing countries. Considering the high disease burden, increasing antibiotic resistance, serotype-specific immunity and the post-infectious sequelae associated with shigellosis, there is a pressing need of an effective vaccine against multiple serotypes of the pathogen. In the present study, we used bio-informatics approach to identify antigens shared among multiple serotypes of Shigella spp. This approach led to the identification of many immunogenic peptides. The five most promising peptides based on MHC binding efficiency were a putative lipoprotein (EL PGI I), a putative heat shock protein (EL PGI II), Spa32 (EL PGI III), IcsB (EL PGI IV) and a hypothetical protein (EL PGI V). These peptides were synthesized and the immunogenicity was evaluated in BALB/c mice by ELISA and cytokine assays. The putative heat shock protein (HSP) and the hypothetical protein elicited good humoral response, whereas putative lipoprotein, Spa32 and IcsB elicited good T-cell response as revealed by increased IFN-γ and TNF-α cytokine levels. The patient sera from confirmed cases of shigellosis were also evaluated for the presence of peptide specific antibodies with significant IgG and IgA antibodies against the HSP and the hypothetical protein, bestowing them as potential future vaccine candidates. The antigens reported in this study are novel and have not been tested as vaccine candidates against Shigella. This study offers time and cost-effective way of identifying unprecedented immunogenic antigens to be used as potential vaccine candidates. Moreover, this approach should easily be extendable to find new potential vaccine candidates for other pathogenic bacteria.

  10. In silico analysis to identify vaccine candidates common to multiple serotypes of Shigella and evaluation of their immunogenicity.

    Science.gov (United States)

    Pahil, Sapna; Taneja, Neelam; Ansari, Hifzur Rahman; Raghava, G P S

    2017-01-01

    Shigellosis or bacillary dysentery is an important cause of diarrhea, with the majority of the cases occurring in developing countries. Considering the high disease burden, increasing antibiotic resistance, serotype-specific immunity and the post-infectious sequelae associated with shigellosis, there is a pressing need of an effective vaccine against multiple serotypes of the pathogen. In the present study, we used bio-informatics approach to identify antigens shared among multiple serotypes of Shigella spp. This approach led to the identification of many immunogenic peptides. The five most promising peptides based on MHC binding efficiency were a putative lipoprotein (EL PGI I), a putative heat shock protein (EL PGI II), Spa32 (EL PGI III), IcsB (EL PGI IV) and a hypothetical protein (EL PGI V). These peptides were synthesized and the immunogenicity was evaluated in BALB/c mice by ELISA and cytokine assays. The putative heat shock protein (HSP) and the hypothetical protein elicited good humoral response, whereas putative lipoprotein, Spa32 and IcsB elicited good T-cell response as revealed by increased IFN-γ and TNF-α cytokine levels. The patient sera from confirmed cases of shigellosis were also evaluated for the presence of peptide specific antibodies with significant IgG and IgA antibodies against the HSP and the hypothetical protein, bestowing them as potential future vaccine candidates. The antigens reported in this study are novel and have not been tested as vaccine candidates against Shigella. This study offers time and cost-effective way of identifying unprecedented immunogenic antigens to be used as potential vaccine candidates. Moreover, this approach should easily be extendable to find new potential vaccine candidates for other pathogenic bacteria.

  11. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

    2016-06-13

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

  12. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  13. [Penicillin acylase from Escherichia coli: catalytically active subunits].

    Science.gov (United States)

    Kabakov, V E; Kliachko, N L; Levashov, A V

    1995-05-01

    Gel filtration under denaturing conditions was used to isolate the alpha- and beta-subunits of penicillin acylase (PA). Refolded subunits were obtained through removing urea by dialysis. Both renatured subunits were catalytically active during hydrolysis of phenylacetic acid p-nitroanilide; this activity decreased after addition of a serine-specific inhibitor--phenylmethanesulfonyl fluoride. The subunits were also active in reversed micelles of Aerosol OT (AOT) in octane, the optimum hydration degree being 11.9 and 17.5 for the light (alpha) and heavy (beta) subunits, respectively. The positions of the maxima were consistent with both theoretically calculated optimum hydration degrees and the earlier reported profile of enzymatic activity for native PA in reversed micelles.

  14. Genetic diversity of levamisole receptor subunits in parasitic nematode species and abbreviated transcripts associated with resistance.

    Science.gov (United States)

    Neveu, Cédric; Charvet, Claude L; Fauvin, Aymeric; Cortet, Jacques; Beech, Robin N; Cabaret, Jacques

    2010-07-01

    and T. colubriformis were essentially unchanged, but abbreviated transcripts of the unc-63 subunit were specifically expressed in resistant isolates of all three species. The candidate gene strategy developed in this study revealed an unexpectedly high diversity of L-AChR subunits specific to the trichostrongylid parasites that are a principal target for the drug LEV. Abbreviated variants, predicted to produce nonfunctional unc-63, were associated with LEV resistance. This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.

  15. Cauliflower Mosaic Virus: A 420 Subunit (T = 7), Multilayer Structure

    Science.gov (United States)

    Cheng, R. H.; Olson, N. H.

    2014-01-01

    The structures of the Cabb-B and CM 1841 strains of cauliflower mosaic virus (CaMV) have been solved to about 3 nm resolution from unstained, frozen-hydrated samples that were examined with low-irradiation cryo-electron microscopy and three-dimensional image reconstruction procedures. CaMV is highly susceptible to distortions. Spherical particles, with a maximum diameter of 53.8 nm, are composed of three concentric layers (I–III) of solvent-excluded density that surround a large, solvent-filled cavity (∼27 nm dia.). The outermost layer (I) contains 72 capsomeric morphological units, with 12 pentavalent pentamers and 60 hexavalent hexamers for a total of 420 subunits (37–42 kDa each) arranged with T = 7 icosahedral symmetry. CaMV is the first example of a T = 7 virus that obeys the rules of stoichiometry proposed for isometric viruses by Caspar and Klug (1962, Cold Spring Harb. Symp. Quant. Biol. 27, 1–24), although the hexameric capsomers exhibit marked departure from the regular sixfold symmetry expected for a structure in which the capsid protein subunits are quasi-equivalently related. The double-stranded DNA genome is distributed in layers II and III along with a portion of the viral protein. The CaMV reconstructions are consistent with the model based on neutron diffraction studies (Kruse et al., 1987, Virology 159, 166–168) and, together, these structural models are discussed in relation to a replication-assembly model (Hull et al., 1987, J. Cell Sci. (Suppl.) 7, 213–229). Remarkable agreement between the reconstructions of CaMV Cabb-B and CM1841 suggests that other strains of CaMV adopt the Same basic Structure. PMID:1733107

  16. Immunogenicity and performance of an enterovirus 71 virus-like-particle vaccine in nonhuman primates.

    Science.gov (United States)

    Lim, Pei-Yin; Hickey, Andrew C; Jamiluddin, Mohamad F; Hamid, Sharifah; Kramer, Joshua; Santos, Rosemary; Bossart, Katharine N; Cardosa, M Jane

    2015-11-04

    A vaccine against human enterovirus 71 (EV-A71) is urgently needed to combat outbreaks of EV-A71 and in particular, the serious neurological complications that manifest during these outbreaks. In this study, an EV-A71 virus-like-particle (VLP) based on a B5 subgenogroup (EV-A71-B5 VLP) was generated using an insect cell/baculovirus platform. Biochemical analysis demonstrated that the purified VLP had a highly native procapsid structure and initial studies in vivo demonstrated that the VLPs were immunogenic in mice. The impact of VLP immunization on infection was examined in non-human primates using a VLP prime-boost strategy prior to EV-A71 challenge. Rhesus macaques were immunized on day 0 and day 21 with VLPs (100 μg/dose) containing adjuvant or with adjuvant alone (controls), and were challenged with EV-A71 on day 42. Complete blood counts, serum chemistry, magnetic resonance imaging (MRI) scans, and histopathology results were mostly normal in vaccinated and control animals after virus challenge demonstrating that the fatal EV-A71-B3 clinical isolate used in this study was not highly virulent in rhesus macaques. Viral genome and/or infectious virus were detected in blood, spleen or brain of two of three control animals, but not in any specimens from the vaccinated animals, indicating that VLP immunization prevented systemic spread of EV-A71 in rhesus macaques. High levels of IgM and IgG were detected in VLP-vaccinated animals and these responses were highly specific for EV-A71 particles and capsid proteins. Serum from vaccinated animals also exhibited similar neutralizing activity against different subgenogroups of EV-A71 demonstrating that the VLPs induced cross-neutralizing antibodies. In conclusion, our EV-A71-B5 VLP is safe, highly immunogenic, and prevents systemic EV-A71-B3 infection in nonhuman primates making it a viable attractive vaccine candidate for EV-A71. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Computing the Diamagnetic Susceptibility and Diamagnetic Anisotropy of Membrane Proteins from Structural Subunits.

    Science.gov (United States)

    Babaei, Mahnoush; Jones, Isaac C; Dayal, Kaushik; Mauter, Meagan S

    2017-06-13

    The behavior of large, complex molecules in the presence of magnetic fields is experimentally challenging to measure and computationally intensive to predict. This work proposes a novel, mixed-methods approach for efficiently computing the principal magnetic susceptibilities and diamagnetic anisotropy of membrane proteins. The hierarchical primary (amino acid), secondary (α helical and β sheet), and tertiary (α helix and β barrel) structure of transmembrane proteins enables analysis of a complex molecule using discrete subunits of varying size and resolution. The proposed method converts the magnetic susceptibility tensor for all protein subunits to a unit coordinate system and sums them to build the magnetic susceptibility tensor for the membrane protein. Using this approach, we calculate the diamagnetic anisotropy for all transmembrane proteins of known structure and investigate the effect of different subunit resolutions on the resulting predictions of diamagnetic anisotropy. We demonstrate that amino acid residues with aromatic side groups exhibit higher diamagnetic anisotropies. On average, high percentages of aromatic amino acid subunits, a β barrel tertiary structure, and a small volume are correlated with high volumetric diamagnetic anisotropy. Finally, we demonstrate that accounting for the spatial position of the residues with respect to one another is critical to accurately computing the magnetic properties of the complex protein molecule.

  18. Unassigned MURF1 of kinetoplastids codes for NADH dehydrogenase subunit 2

    Directory of Open Access Journals (Sweden)

    Burger Gertraud

    2008-10-01

    Full Text Available Abstract Background In a previous study, we conducted a large-scale similarity-free function prediction of mitochondrion-encoded hypothetical proteins, by which the hypothetical gene murf1 (maxicircle unidentified reading frame 1 was assigned as nad2, encoding subunit 2 of NADH dehydrogenase (Complex I of the respiratory chain. This hypothetical gene occurs in the mitochondrial genome of kinetoplastids, a group of unicellular eukaryotes including the causative agents of African sleeping sickness and leishmaniasis. In the present study, we test this assignment by using bioinformatics methods that are highly sensitive in identifying remote homologs and confront the prediction with available biological knowledge. Results Comparison of MURF1 profile Hidden Markov Model (HMM against function-known profile HMMs in Pfam, Panther and TIGR shows that MURF1 is a Complex I protein, but without specifying the exact subunit. Therefore, we constructed profile HMMs for each individual subunit, using all available sequences clustered at various identity thresholds. HMM-HMM comparison of these individual NADH subunits against MURF1 clearly identifies this hypothetical protein as NAD2. Further, we collected the relevant experimental information about kinetoplastids, which provides additional evidence in support of this prediction. Conclusion Our in silico analyses provide convincing evidence for MURF1 being a highly divergent member of NAD2.

  19. Immunogenicity and safety of a quadrivalent meningococcal polysaccharide CRM conjugate vaccine in infants and toddlers

    Directory of Open Access Journals (Sweden)

    Miguel Tregnaghi

    2014-09-01

    Conclusions: MenACWY-CRM vaccination regimens in infants and toddlers were immunogenic and well tolerated. No clinically meaningful effects of concomitant administration with routine infant and toddler vaccines were observed.

  20. How real is the long-lasting effect of tumor necrosis factor α inhibitors? Focus on immunogenicity

    Directory of Open Access Journals (Sweden)

    D.E. Karateev

    2014-05-01

    Full Text Available Tumor necrosis factor (TNF α inhibitors are the most commonly used agents to treat rheumatoid arthritis (RA and other inflammatory arthropathies. Five drugs belonging to the family of TNFα inhibitors have been certified in Russia for treating RA: infliximab (INF, adali- mumab (ADA, golimumab, certolizumab pegol, and etanercept (ETN. These drugs have different compositions. ETN does not belong to the family of monoclonal antibodies (mAbs and has a different mechanism of action. It is a dimeric molecule of synthetic fusion protein contain- ing TNF receptor and bound to the Fc-fragment of human Ig1. ETN can inhibit both TNFα and lymphotoxin α. ETN contains only the pro- tein identical to human protein. All TNFα inhibitors exhibit a virtually identical anti-inflammatory activity. The data from the registries show that the risk of discontinuation of therapy with TNFα during the first 2–3 years is appreciably high; there is a trend toward increased frequency of therapy discontinuation because of loss of effectiveness. It was found that the risk of therapy discontinuation because of insufficient effectiveness and adverse events (AEs is minimal for ETN and maximal for INF. The structure of biological drugs (which also affects their immunogenicity has the key neg- ative effect on maintaining the response to therapy and frequency of AEs. However, since ETN is a fusion molecule and contains less poten- tially immunogenic epitopes compared to mAbs, the frequency of detecting anti-drug antibodies (ADAbs is appreciably lower. The fact that ETN has a lower immunogenicity can be used to explain the significantly lower probability of discontinuing therapy using this drug as compared to INF and ADA. The risk that the need to increase the dose because of gradual loss of effectiveness of therapy with ADA and INF, was 4.9- and 28-fold higher, respectively, as compared to ETN. Therapeutic algorithms make it possible to control therapy with TGFα inhibitors

  1. How real is the long-lasting effect of tumor necrosis factor α inhibitors? Focus on immunogenicity

    Directory of Open Access Journals (Sweden)

    D.E. Karateev

    2014-01-01

    Full Text Available Tumor necrosis factor (TNF α inhibitors are the most commonly used agents to treat rheumatoid arthritis (RA and other inflammatory arthropathies. Five drugs belonging to the family of TNFα inhibitors have been certified in Russia for treating RA: infliximab (INF, adali- mumab (ADA, golimumab, certolizumab pegol, and etanercept (ETN. These drugs have different compositions. ETN does not belong to the family of monoclonal antibodies (mAbs and has a different mechanism of action. It is a dimeric molecule of synthetic fusion protein contain- ing TNF receptor and bound to the Fc-fragment of human Ig1. ETN can inhibit both TNFα and lymphotoxin α. ETN contains only the pro- tein identical to human protein. All TNFα inhibitors exhibit a virtually identical anti-inflammatory activity. The data from the registries show that the risk of discontinuation of therapy with TNFα during the first 2–3 years is appreciably high; there is a trend toward increased frequency of therapy discontinuation because of loss of effectiveness. It was found that the risk of therapy discontinuation because of insufficient effectiveness and adverse events (AEs is minimal for ETN and maximal for INF. The structure of biological drugs (which also affects their immunogenicity has the key neg- ative effect on maintaining the response to therapy and frequency of AEs. However, since ETN is a fusion molecule and contains less poten- tially immunogenic epitopes compared to mAbs, the frequency of detecting anti-drug antibodies (ADAbs is appreciably lower. The fact that ETN has a lower immunogenicity can be used to explain the significantly lower probability of discontinuing therapy using this drug as compared to INF and ADA. The risk that the need to increase the dose because of gradual loss of effectiveness of therapy with ADA and INF, was 4.9- and 28-fold higher, respectively, as compared to ETN. Therapeutic algorithms make it possible to control therapy with TGFα inhibitors

  2. Impact of baseline covariates on the immunogenicity of the 9-valent HPV vaccine – A combined analysis of five phase III clinical trials

    Directory of Open Access Journals (Sweden)

    Lone K. Petersen

    2017-06-01

    Full Text Available Background: The immunogenicity profile of the 9-valent HPV (9vHPV vaccine was evaluated across five phase III clinical studies conducted in girls and boys 9–15 years of age and young women 16–26 years of age. The effect of baseline characteristics of subjects on vaccine-induced HPV antibody responses was assessed. Methods: Immunogenicity data from 11,304 subjects who received ≥1 dose of 9vHPV vaccine in five Phase III studies were analyzed. Vaccine was administered as a 3-dose regimen. HPV antibody titers were assessed 1 month after dose 3 using a competitive Luminex immunoassay and summarized as geometric mean titers (GMTs. Covariates examined were age, gender, race, region of residence, and HPV serostatus and PCR status at day 1. Results: GMTs to all 9 vaccine HPV types decreased with age at vaccination initiation, and were otherwise generally similar among the demographic subgroups defined by gender, race and region of residence. For all subgroups defined by race or region of residence, GMTs were higher in girls and boys than in young women. Vaccination of subjects who were seropositive at day 1 to a vaccine HPV type resulted in higher GMTs to that type, compared with those in subjects who were seronegative for that type at day 1. Conclusions: 9vHPV vaccine immunogenicity was robust among subjects with differing baseline characteristics. It was generally comparable across subjects of different races and from different regions. Greater immunogenicity in girls and boys versus young women (the population used to establish 9vHPV vaccine efficacy in clinical studies indicates that the anti-HPV responses generated by the vaccine in adolescents from all races or regions were sufficient to induce high-level protective efficacy. This immunogenicity profile supports a widespread 9vHPV vaccination program and early vaccination. Studies in the meta-analysis: V503-001, V503-002, V503-005, V503-007, V503-009/GDS01C, Clinical trials

  3. Isolation, characterization, virulence and immunogenicity testing of field isolates of Pasteurella multocida, Staphylococcus aureus, and Streptococcus agalactiae in laboratory settings.

    Science.gov (United States)

    Qudratullah; Muhammad, G; Saqib, M; Bilal, M Qamar

    2017-08-01

    The present study was designed to investigate isolation, characterization, virulence and immunogenicity testing of field isolates of Pasteurella multocida, Staphylococcus aureus, and Streptococcus agalactiae in rabbits and mice. Isolates of P. multocida, S. aureus and Str. agalactiae recovered from field cases of Hemorragic septicemia and mastitis were scrutinized for virulence/pathogenicity and immunogenicity. Mouse LD50 of P. multocida showed that P. multocida isolate No.1 was more virulent than isolates No. 2 and 3. Virulence of isolate No.1S. aureus and Str. agalactiae revealed that 100, 80% rabbits died within 18h of inoculation. Seven-digit numerical profiles of these 4 isolates with API® Staph test strips isolates, No.1 (6736153) showed good identification (S. aureus id=90.3%). Indirect ELISA-based serum antibody titers to P. multocida isolate No.1, S. aureus No.1, Str. agalactiae, isolate No.1 elicited high antibody titers 1.9, 1.23, 1.12 respectively. All the pathogens of Isolate No. 1 (P. multocida, S. aureus Str. agalactiae), were high antibody than others isolates. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Immunogenicity of HPV prophylactic vaccines: Serology assays and their use in HPV vaccine evaluation and development.

    Science.gov (United States)

    Pinto, Ligia A; Dillner, Joakim; Beddows, Simon; Unger, Elizabeth R

    2018-01-17

    When administered as standard three-dose schedules, the licensed HPV prophylactic vaccines have demonstrated extraordinary immunogenicity and efficacy. We summarize the immunogenicity of these licensed vaccines and the most commonly used serology assays, with a focus on key considerations for one-dose vaccine schedules. Although immune correlates of protection against infection are not entirely clear, both preclinical and clinical evidence point to neutralizing antibodies as the principal mechanism of protection. Thus, immunogenicity assessments in vaccine trials have focused on measurements of antibody responses to the vaccine. Non-inferiority of antibody responses after two doses of HPV vaccines separated by 6 months has been demonstrated and this evidence supported the recent WHO recommendations for two-dose vaccination schedules in both boys and girls 9-14 years of age. There is also some evidence suggesting that one dose of HPV vaccines may provide protection similar to the currently recommended two-dose regimens but robust data on efficacy and immunogenicity of one-dose vaccine schedules are lacking. In addition, immunogenicity has been assessed and reported using different methods, precluding direct comparison of results between different studies and vaccines. New head-to-head vaccine trials evaluating one-dose immunogenicity and efficacy have been initiated and an increase in the number of trials relying on immunobridging is anticipated. Therefore, standardized measurement and reporting of immunogenicity for the up to nine HPV types targeted by the current vaccines is now critical. Building on previous HPV serology assay standardization and harmonization efforts initiated by the WHO HPV LabNet in 2006, new secondary standards, critical reference reagents and testing guidelines will be generated as part of a new partnership to facilitate harmonization of the immunogenicity testing in new HPV vaccine trials. Copyright © 2018 Elsevier Ltd. All rights

  5. Monoclonal antibody aggregates: physicochemical characteristics, stability in biological fluids and immunogenicity

    OpenAIRE

    Filipe, V.L.S.

    2012-01-01

    Therapeutic proteins have become invaluable in treating a wide range of serious and life-threatening diseases. However, repeated administration of these drugs to patients often induces the formation of undesirable anti-drug antibodies, also known as immunogenicity. Among the factors that are known to play a role in immunogenicity of therapeutic proteins, the presence of protein aggregates has been indicated as one of the main product-related risk factors. Even though several studies have show...

  6. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Rebecca Dovega

    Full Text Available Protein Phosphatase 2A (PP2A is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A subunit, a catalytic (C subunit and various regulatory (B subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  7. Safety and immunogenicity of two live attenuated human rotavirus vaccine candidates, 116E and I321, in infants: results of a randomised controlled trial.

    Science.gov (United States)

    Bhandari, Nita; Sharma, Pooja; Glass, Roger I; Ray, Pratima; Greenberg, Harry; Taneja, Sunita; Saksena, Manju; Rao, C Durga; Gentsch, Jon R; Parashar, Umesh; Maldonado, Yvonne; Ward, Richard L; Bhan, M K

    2006-07-26

    We evaluated safety and immunogenicity of two orally administered human rotavirus vaccine candidates 116E and I321. Ninety healthy infants aged 8 weeks received a single dose of 116E (10(5)FFu (florescence focus units)), I321 (10(5)FFu) or placebo. There were no significant differences in the number of adverse events. Fever was reported by 6/30, 1/30 and 5/30 in the 116E, I321 and placebo groups; the corresponding figures for diarrhoea were 5/30, 8/29 and 3/30. Serum IgA seroconversion rates were 73%, 39% and 20% in the 116E, I321 and placebo groups, respectively. Vaccine virus was shed on days 3, 7 or 28 in 11/30 infants of the 116E and none in the other two groups. The 116E strain is attenuated, clinically safe and highly immunogenic with a single dose.

  8. Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

    Directory of Open Access Journals (Sweden)

    Janssens Veerle

    2008-08-01

    Full Text Available Abstract Background Protein phosphatase 2A (PP2A is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

  9. Rice heterotrimeric G-protein gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress.

    Science.gov (United States)

    Yadav, Dinesh Kumar; Islam, S M Shahinul; Tuteja, Narendra

    2012-07-01

    Heterotrimeric G-proteins (α, β and γ subunits) are primarily involved in diverse signaling processes by transducing signals from an activated transmembrane G-protein coupled receptor (GPCR) to appropriate downstream effectors within cells. The role of α and β G-protein subunits in salinity and heat stress has been reported but the regulation of γ subunit of plant G-proteins in response to abiotic stress has not heretofore been described. In the present study we report the isolation of full-length cDNAs of two isoforms of Gγ [RGG1(I), 282 bp and RGG2(I), 453 bp] from rice (Oryza sativa cv Indica group Swarna) and described their transcript regulation in response to abiotic stresses. Protein sequence alignment and pairwise comparison of γ subunits of Indica rice [RGG(I)] with other known plant G-protein γ subunits demonstrated high homology to barley (HvGs) while soybean (GmG2) and Arabidopsis (AGG1) were least related. The numbers of the exons and introns were found to be similar between RGG1(I) and RGG2(I), but their sizes were different. Analyses of promoter sequences of RGG(I) confirmed the presence of stress-related cis-regulatory signature motifs suggesting their active and possible independent roles in abiotic stress signaling. The transcript levels of RGG1(I) and RGG2(I) were upregulated following NaCl, cold, heat and ABA treatments. However, in drought stress only RGG1(I) was upregulated. Strong support by transcript profiling suggests that γ subunits play a critical role via cross talk in signaling pathways. These findings provide first direct evidence for roles of Gγ subunits of rice G-proteins in regulation of abiotic stresses. These findings suggest the possible exploitation of γ subunits of G-protein machinery for promoting stress tolerance in plants.

  10. Effects of pyruvate dehydrogenase subunits overexpression on the α-ketoglutarate production in Yarrowia lipolytica WSH-Z06.

    Science.gov (United States)

    Guo, Hongwei; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-08-01

    Yarrowia lipolytica WSH-Z06 harbours a promising capability to oversynthesize α-ketoglutarate (α-KG). Its wide utilization is hampered by the formation of high concentrations of pyruvate. In this study, a metabolic strategy for the overexpression of the α and β subunits of pyruvate dehydrogenase E1, E2 and E3 components was designed to reduce the accumulation of pyruvate. Elevated expression level of α subunit of E1 component improved the α-KG production and reduced the pyruvate accumulation. Due to a reduction in the acetyl-CoA supply, neither the growth of cells nor the synthesis of α-KG was restrained by the overexpression of β subunit of E1, E2 and E3 components. Furthermore, via the overexpression of these thiamine pyrophosphate (TPP)-binding subunits, the dependency of pyruvate dehydrogenase on thiamine was diminished in strains T1 and T2, in which α and β subunits of E1 component were separately overexpressed. In these two recombinant strains, the accumulation of pyruvate was insensitive to variations in exogenous thiamine. The results suggest that α-KG production can be enhanced by altering the dependence on TPP of pyruvate dehydrogenase and that the competition for the cofactor can be switched to ketoglutarate dehydrogenase via separate overexpression of the TPP-binding subunits of pyruvate dehydrogenase. The results presented here provided new clue to improve α-KG production.

  11. Conjugation of the CRM197-inulin conjugate significantly increases the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein.

    Science.gov (United States)

    Hu, Shun; Yu, Weili; Hu, Chunyang; Wei, Dong; Shen, Lijuan; Hu, Tao; Yi, Youjin

    2017-11-01

    Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen that causes tuberculosis (TB). Effective vaccination is urgently needed to deal with the serious threat from TB. Mtb-secreted protein antigens are important virulence determinants of Mtb with poor immunogenicity. Adjuvants and antigen delivery systems are thus highly desired to improve the immunogenicity of protein antigens. Inulin is a biocompatible polysaccharide (PS) adjuvant that can stimulate a strong cellular and humoral immunity. Bacterial capsular PS and haptens have been conjugated with cross-reacting material 197 (CRM 197 ) to improve their immunogenicity. CFP10 and TB10.4 were two Mtb-secreted immunodominant protein antigens. A CFP10-TB10.4 fusion protein (CT) was used as the antigen for covalent conjugation with the CRM 197 -inulin conjugate (CRM-inu). The resultant conjugate (CT-CRM-inu) elicited high CT-specific IgG titers, stimulated splenocyte proliferation and provoked the secretion of Th1-type and Th2-type cytokines. Conjugation with CRM-inu significantly prolonged the systemic circulation of CT and exposure to the immune system. Moreover, CT-CRM-inu showed no apparent toxicity to cardiac, hepatic and renal functions. Thus, conjugation of CT with CRM-inu provided an effective strategy for development of protein-based vaccines against Mtb infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Immunogenic Display of Purified Chemically Cross-Linked HIV-1 Spikes

    Science.gov (United States)

    Leaman, Daniel P.; Lee, Jeong Hyun; Ward, Andrew B.

    2015-01-01

    -neutralizing antibodies. Fixed native spikes were recognized by all classes of known broadly neutralizing antibodies but not by non-neutralizing antibodies and displayed on nanoparticles in high copy number. An immunization experiment in rabbits revealed that cross-linking Env reduced its overall immunogenicity; however, high-copy display on nanoparticles enabled boosting of antibodies that sporadically neutralized some relatively resistant HIV-1 isolates, albeit at a low titer. This study describes the purification of stable and antigenically correct Env spikes from virions that can be used as immunogens. PMID:25878116

  13. Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum).

    Science.gov (United States)

    He, Dong-Mei; Qian, Kai-Xian; Shen, Gui-Fang; Li, Yi-Nü; Zhang, Zhi-Fang; Su, Zhong-Liang; Shao, Hong-Bo

    2007-04-01

    The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity.

  14. Construction and Characterization of Human Rotavirus Recombinant VP8* Subunit Parenteral Vaccine Candidates

    OpenAIRE

    Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W.; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

    2012-01-01

    Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effec...

  15. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    Science.gov (United States)

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  16. Protective Efficacy and Immunogenicity of an Adenoviral Vector Vaccine Encoding the Codon-Optimized F Protein of Respiratory Syncytial Virus▿

    Science.gov (United States)

    Kohlmann, Rebekka; Schwannecke, Sarah; Tippler, Bettina; Ternette, Nicola; Temchura, Vladimir V.; Tenbusch, Matthias; Überla, Klaus; Grunwald, Thomas

    2009-01-01

    Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans. PMID:19776123

  17. Protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized F protein of respiratory syncytial virus.

    Science.gov (United States)

    Kohlmann, Rebekka; Schwannecke, Sarah; Tippler, Bettina; Ternette, Nicola; Temchura, Vladimir V; Tenbusch, Matthias; Uberla, Klaus; Grunwald, Thomas

    2009-12-01

    Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.

  18. The immunogenicity of thin-film freeze-dried, aluminum salt-adjuvanted vaccine when exposed to different temperatures.

    Science.gov (United States)

    Thakkar, Sachin G; Ruwona, Tinashe B; Williams, Robert O; Cui, Zhengrong

    2017-04-03

    Insoluble aluminum salts such as aluminum oxyhydroxide have been used for decades as adjuvants in human vaccines, and many vaccines contain aluminum salts as adjuvants. Aluminum salt-adjuvanted vaccines must be managed in cold-chain (2-8° C) during transport and storage, as vaccine antigens in general are too fragile to be stable in ambient temperatures, and unintentional slowing freezing causes irreversible aggregation and permanent damage to the vaccines. Previously, we reported that thin-film freeze-drying can be used to convert vaccines adjuvanted with an aluminum salt from liquid suspension into dry powder without causing particle aggregation or decreasing in immunogenicity following reconstitution. In the present study, using ovalbumin (OVA)-adsorbed Alhydrogel® (i.e. aluminum oxyhydroxide, 2% w/v) as a model vaccine, we showed that the immunogenicity of thin-film freeze-dried OVA-adsorbed Alhydrogel® vaccine powder was not significantly changed after it was exposed for an extended period of time in temperatures as high as 40° C or subjected to repeated slow freezing-and-thawing. It is expected that immunization programs can potentially benefit by integrating thin-film freeze-drying into vaccine preparations.

  19. Regulatory properties of statins and rho gtpases prenylation inhibitiors to stimulate melanoma immunogenicity and promote anti-melanoma immune response.

    Science.gov (United States)

    Sarrabayrouse, Guillaume; Pich, Christine; Teiti, Iotefa; Tilkin-Mariame, Anne Françoise

    2017-02-15

    Melanoma is a highly lethal cutaneous tumor, killing affected patients through development of multiple poorly immunogenic metastases. Suboptimal activation of immune system by melanoma cells is often due to molecular modifications occurring during tumor progression that prevent efficient recognition of melanoma cells by immune effectors. Statins are HMG-CoA reductase inhibitors, which block the mevalonate synthesis pathway, used by millions of people as hypocholesterolemic agents in cardiovascular and cerebrovascular diseases. They are also known to inhibit Rho GTPase activation and Rho dependent signaling pathways. Rho GTPases are regarded as molecular switches that regulate a wide spectrum of cellular functions and their dysfunction has been characterized in various oncogenic process notably in melanoma progression. Moreover, these molecules can modulate the immune response. Since 10 years we have demonstrated that Statins and other Rho GTPases inhibitors are critical regulators of molecules involved in adaptive and innate anti-melanoma immune response. In this review we summarize our major observations demonstrating that these pharmacological agents stimulate melanoma immunogenicity and suggest a potential use of these molecules to promote anti-melanoma immune response. © 2016 UICC.

  20. Protectio