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Sample records for antigens recombinant c100-3

  1. Microbial antigenic variation mediated by homologous DNA recombination.

    Science.gov (United States)

    Vink, Cornelis; Rudenko, Gloria; Seifert, H Steven

    2012-09-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to the modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in the expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and that are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum, and Mycoplasma spp.), fungi (such as Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved. PMID:22212019

  2. Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91.

    Science.gov (United States)

    Willadsen, P; Smith, D; Cobon, G; McKenna, R V

    1996-05-01

    Cattle were vaccinated either with a single recombinant tick antigen, Bm86 or with a combination of two recombinant antigens, Bm86 and Bm91 from the tick Boophilus microplus. In three experiments, the responses of cattle to subsequent challenge with the tick were assessed. The addition of the Bm91 antigen enhanced the efficacy of the vaccination over that with Bm86 alone to a statistically significant degree. Moreover, co-vaccination with two antigens did not impair the response of cattle to the Bm86 antigen. Finally, responses of individual cattle to the two antigens were independent. All of these results may be relevant to the increase in efficacy expected from a dual antigen vaccine. PMID:9229376

  3. Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

    OpenAIRE

    Cho, Jung-Hwa; Chung, Woo-Suk; Song, Kyoung-Ju; Na, Byoung-kuk; Kang, Seung-Won; Song, Chul-Yong; Kim, Tong-Soo

    2005-01-01

    Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites ...

  4. Use of recombinant chimeric antigens for the serodiagnosis of Mycoplasma pneumoniae infection.

    Science.gov (United States)

    Montagnani, F; De Paolis, F; Beghetto, E; Gargano, N

    2010-11-01

    In this paper, we have evaluated the diagnostic utility of three antigenic regions of the Mycoplasma pneumoniae P1, P30, and MPN456 gene products in order to replace the soluble, whole-cell bacterial extract in serological assays. Antigenic regions, being previously identified as B-cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering. Paired serum samples from 47 patients with M. pneumoniae infection and from 39 subjects with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection were included. Immunoglobulin G (IgG) antibodies against epitopes carried by recombinant antigens were measured by performing recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Rec-ELISA results were compared to those obtained by a commercial assay using the whole-cell Mycoplasma antigen. Our study demonstrates that all IgG Rec-ELISAs using recombinant antigens have better sensitivity with respect to the commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays. The use of recombinant antigens is effective in distinguishing M. pneumoniae-infected patients from uninfected individuals and shows that immunoassays based on recombinant antigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases. PMID:20632053

  5. SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

    OpenAIRE

    Magnarelli, Louis A.; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37...

  6. Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays

    OpenAIRE

    LaBrecque, F. D.; LaBrecque, D. R.; Klinzman, D.; Perlman, S; Cederna, J. B.; Winokur, P. L.; Han, J.-Q.; Stapleton, J. T.

    1998-01-01

    Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies whi...

  7. Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool

    OpenAIRE

    Alleman, A. Rick; McSherry, Leo J.; Barbet, Anthony F.; Breitschwerdt, Edward B.; Sorenson, Heather L.; Bowie, Michael V.; Bélanger, Myriam

    2001-01-01

    The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum ...

  8. Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Wu, Anna M. [David Geffen School of Medicine at the University of California Los Angeles, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Los Angeles, CA (United States); Braun, Jonathan [David Geffen School of Medicine at the University of California Los Angeles, Department of Pathology and Laboratory Medicine, Los Angeles, CA (United States)

    2009-01-15

    Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human Fc{gamma}RIIb receptor. The NA3-Fc{gamma}RIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Flow cytometry identified Jurkat clones stably expressing NA3-Fc{gamma}RIIb at low, medium, and high levels. High and medium NA3-Fc{gamma}RIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA {sup 124}I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 {+-} 0.2, 15.2 {+-} 1.3, and 4.6 {+-} 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 {+-} 0.8%ID/g. The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. (orig.)

  9. Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

    OpenAIRE

    Ortiz, B L; Garcia, A. M.; A. Restrepo; McEwen, J. G.

    1996-01-01

    We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.

  10. Production of schistosome antigens for immunodiagnosis and vaccines: the role of recombinant DNA technology

    International Nuclear Information System (INIS)

    A major problem to confront biochemists studying the immunology of parasitic infection is a paucity of the organisms themselves. Conventional biochemical techniques for the isolation and purification of individual antigens are inappropriate. This problem has been alleviated by the application of recombinant DNA technology. It is now possible to produce large quantities of individual antigens by cloning the corresponding genes into plasmids (or other vectors) and subsequent expression in bacteria. Antigens produced in this way may provide the basis of a specific diagnostic test and vaccines. This paper describes the identification of cDNA clones of Schistosoma mansoni which encode a major egg antigen and schistosomula surface antigens. These antigens are thought to be species specific and may form the basis of a diagnostic test. The schistosomula antigens are also possible candidates for inclusion in an experimental vaccine against infection with S. mansoni. (author)

  11. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  12. Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine

    OpenAIRE

    Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming

    2015-01-01

    The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the “next-generation” recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA...

  13. Expression of hepatitis A virus cDNA in Escherichia coli: antigenic VP1 recombinant protein.

    OpenAIRE

    Ostermayr, R; von der Helm, K; Gauss-Müller, V; Winnacker, E L; Deinhardt, F.

    1987-01-01

    The genome of hepatitis A virus (HAV) was reverse transcribed into cDNA and molecularly cloned. cDNA clones coding for the capsid protein VP1 that carries the major HAV antigen were cloned into the expression vector pUR290 and expressed in Escherichia coli. The recombinant fusion protein reacted in an immunoblot with rabbit anti-HAV serum, suggesting that it possesses HAV antigenicity.

  14. Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen.

    OpenAIRE

    Lagranderie, M; Lo-Man, R; Dériaud, E; Gicquel, B; Gheorghiu, M; Leclerc, C

    1997-01-01

    Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various...

  15. Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes

    OpenAIRE

    Sadoogh Abbasian, Shabnam; Ghaznavi Rad, Ehsanollah; Akbari, Neda; Zolfaghari, Mohammad Reza; Pakzad, Iraj; ABTAHI, Hamid

    2014-01-01

    Background: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. Objectives: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activ...

  16. Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

    Directory of Open Access Journals (Sweden)

    Steindel Mario

    2009-01-01

    Full Text Available Abstract Background Antigen specificity and IgG subclass could be significant in the natural history of Chagas' disease. The relationship between the different stages of human Chagas' disease and the profiles of total IgG and its subclasses were thus analysed here; they were directed against a crude T. cruzi extract and three recombinant antigens: the T. cruzi kinetoplastid membrane protein-11 (rKMP-11, an internal fragment of the T. cruzi HSP-70 protein192-433, and the entire Trypanosoma rangeli HSP-70 protein. Methods Seventeen Brazilian acute chagasic patients, 50 Colombian chronic chagasic patients (21 indeterminate and 29 cardiopathic patients and 30 healthy individuals were included. Total IgG and its subtypes directed against the above-mentioned recombinant antigens were determined by ELISA tests. Results The T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins were able to distinguish both acute from chronic chagasic patients and infected people from healthy individuals. Specific antibodies to T. cruzi crude antigen in acute patients came from IgG3 and IgG4 subclasses whereas IgG1 and IgG3 were the prevalent isotypes in indeterminate and chronic chagasic patients. By contrast, the specific prominent antibodies in all disease stages against T. cruzi KMP-11 and T. rangeli HSP-70 recombinant antigens were the IgG1 subclass. Conclusion T. cruzi KMP-11 and the T. rangeli HSP-70 recombinant proteins may be explored together in the immunodiagnosis of Chagas' disease. Polarising the IgG1 subclass of the IgG response to T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins could have important biological effects, taking into account that this is a complement fixing antibody.

  17. Construction of Recombinant Modified Vaccinia Ankara (MVA) Expressing Hepatitis B Virus Surface Antigen

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections.Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system.Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

  18. Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits.

    Science.gov (United States)

    Magnarelli, Louis A; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections. PMID:22247369

  19. Use of the Recombinant Particles of Potato Virus a (PVA) for Presentation of Foreign Antigens

    Czech Academy of Sciences Publication Activity Database

    Moravec, Tomáš; Čeřovská, Noemi; Velemínský, Jiří

    Aschersleben : International Society of Plant Pathology, 2002, s. 103. [International Plant Virus Epidemiology Symposium /8./.. Aschersleben (DE), 12.03.2002-17.03.2002] Institutional research plan: CEZ:AV0Z5038910 Keywords : recombinant particles * potato virus (PVA) * antigens Subject RIV: EB - Genetics ; Molecular Biology

  20. Antigenic profile of human recombinant PrP: generation and chracterization of a versatile polyclonal antiserum

    NARCIS (Netherlands)

    Sachsamanoglou, M.; Paspaltzis, I.; Petrakis, S.; Verghese-Nikolakaki, S.; Panagiotidis, C.H.; Voitlander, T.; Budka, H.; Langeveld, J.P.M.; Sklaviadis, T.

    2004-01-01

    We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antise

  1. Evaluating Recombinant Antigen ROP1 Efficacy in Diagnosis of Toxoplasma Gondii Infection

    Directory of Open Access Journals (Sweden)

    F Keshavarzi

    2015-07-01

    Full Text Available Introduction:Toxoplasma gondii is a ubiquitous obligate intracellular parasite with a relatively broad host range infecting both mammals and birds. Toxoplasma proteins are strong antigens that can begin strong immune reactions, among which Rhoptry protein 1 (ROP1 can be named discharging from rhoptry cell-organ. ROP1 is regarded as a competitor for recombinant vaccines against toxoplasmosis. Therefore, the main objective of the current study was to evaluate the cloning and expression of ROP1 Toxoplasma gondii in a cloning vector as well as to create this recombinant antigen in order to be applied for later uses. Methods:Genomic DNA of Toxoplasma gondii was removed and reproduced by PCR, then the PCR product was cloned into the EcoR1 and BamH1 sites of cloning vector, pUET1, and transformed into Escherichia coli BL21 plysS strain. Moreover, pcROP1 was sub-cloned into the HindIII and EcoRI sites of the pcDNA3 in order to produce recombining eukaryotic declaration vector. The cloned ROP1 was verified by PCR, limitation enzymes (HindIII and BglΙ digestion and nucleotide sequencing. Then, this recombinant antigen was covered applying IgM and ELISAIgG. Results:The study results demonstrated that a fragment of 757 bp was separated. In addition, nucleotide sequence analysis of the ROP1 cloned in pUET1vector revealed high homology (96% with RH strain Gene Bank Accession (No. M71274. Conclusion:The recombinant ROP1 antigen in an IgM Rec-ELISA test can be replaced with the tachyzoite antigen in IgG and IgM serologic tests.

  2. Production of Recombinant Antigens of Ureaplasma parvum Serotypes 3 and 6 for Development of a Serological Assay▿

    OpenAIRE

    Vancutsem, E.; Echahidi, F.; Van Geel, K.; Muyldermans, G.; Soetens, O; Naessens, A.

    2007-01-01

    Recombinant antigens of Ureaplasma parvum serotypes 3 and 6 were produced in order to develop a serological assay for Ureaplasma antibody detection. The genes of the multiple banded antigen (MBA) were amplified by PCR and cloned in a pTrcHis TOPO plasmid. Purified recombinant proteins were evaluated in Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and human sera. Our approach was successful in the production of the recombinant MBAs (rMBAs) for serot...

  3. Induction of HLA-DR antigen on human squamous carcinoma by recombinant interferon gamma.

    Science.gov (United States)

    Koch, W M; Dugan, E; Diaz, L A; Richtsmeier, W J

    1988-05-01

    The antigen recognition system which plays the major role in immunologic attraction mechanisms, including graft rejection, is the class II major histocompatibility complex containing the HLA-DR locus. Few types of cells constitutively express this antigen, as it is a potent immunological activating signal usually confined to antigen processing cells, activated lymphocytes, and endothelium. Using indirect immunofluorescence, we have observed induction of the HLA-DR glycoprotein in selected head and neck squamous cell carcinoma tissue cultures treated with recombinant interferon gamma. This occurs in concert with growth arrest and morphological changes after rHuIFN-gamma treatment. This report describes the induction of a surface antigen that may have profound prognostic significance. Understanding the kinetics of HLA-DR induction will aid in the design and assessment of adoptive immunotherapy with rHuIFN-gamma. PMID:3129628

  4. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  5. Vaccination with a cocktail of Ancylostoma ceylanicum recombinant antigens leads to worm burden reduction in hamsters.

    Science.gov (United States)

    Wiśniewski, Marcin; Łapiński, Maciej; Daniłowicz-Luebert, Emilia; Jaros, Sławomir; Długosz, Ewa; Wędrychowicz, Halina

    2016-09-01

    Hookworms, a group to which Ancylostoma ceylanicum belongs, are gastrointestinal nematodes that infect more than 700 million people around the world. They are a leading cause of anemia in developing countries. In order to effectively prevent hookworm infections research is conducted to develop an effective vaccine using recombinant antigens of the parasite. The aim of this study was to examine the influence of the hosts' on protection against ancylostomiasis and the shaping of the humoral immune response among Syrian hamsters after immunization with a cocktail of five A. ceylanicum recombinant antigens. Ace-ASP-3, Ace-ASP-4, Ace-APR-1, Ace-MEP-6 and Ace-MEP-7 were obtained in the pET expression system. Immunization with a vaccine cocktail resulted in a 33.5% worm burden reduction. The immunogenicity of the recombinant proteins were determined using ELISA. Statistical analysis showed that vaccinated hamsters developed stronger humoral responses to four of five recombinant antigens (the exception being Ace-ASP-3) compared to hamsters from the control group. PMID:27447220

  6. High expression of water-soluble recombinant antigenic domains of Toxoplasma gondii secretory organelles.

    Science.gov (United States)

    Yang, Zhaoshou; Ahn, Hye-Jin; Nam, Ho-Woo

    2014-08-01

    Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1. PMID:25246715

  7. Isolation, Cloning, Expression and Purification of Recombinant RhD Antigen from Cord Blood

    OpenAIRE

    Habibi Roudkenar, M; A Oodi; Halabian, R.; M Mohammadipour; N Amirizadeh; N Massrori; P Mozafari; Kamali, E; A. Mohammadi Roushandeh; H Rezvan

    2008-01-01

    "nBackground: Rh (Rhesus) is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blod, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study."nMethods: Total RNAs were extracted from cord blood (O+).  The quality of RNA was determined by electrophoresis. In or­der to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT...

  8. Mapping of T cell epitopes using recombinant antigens and synthetic peptides.

    OpenAIRE

    Lamb, J R; Ivanyi, J.; Rees, A D; Rothbard, J B; Howland, K; Young, R. A.; Young, D B

    1987-01-01

    Two complementary approaches were used to determine the epitope specificity of clonal and polyclonal human T lymphocytes reactive with the 65-kd antigen of Mycobacterium leprae. A recombinant DNA sublibrary constructed from portions of the 65-kd gene was used to map T cell determinants within amino acid sequences 101-146 and 409-526. Independently, potential T cell epitopes within the protein were predicted based on an empirical analysis of specific patterns in the amino acid sequence. Of six...

  9. Decreased Serum Antibody Responses to Recombinant Pneumocystis Antigens in HIV-Infected and Uninfected Current Smokers▿

    OpenAIRE

    Crothers, Kristina; Daly, Kieran R.; Rimland, David; Goetz, Matthew Bidwell; Gibert, Cynthia L.; Butt, Adeel A.; Justice, Amy C.; Djawe, Kpandja; Levin, Linda; Walzer, Peter D.

    2010-01-01

    Serologic studies can provide important insights into the epidemiology and transmission of Pneumocystis jirovecii. Exposure to P. jirovecii can be assessed by serum antibody responses to recombinant antigens from the major surface glycoprotein (MsgC), although factors that influence the magnitude of the antibody response are incompletely understood. We determined the magnitudes of antibody responses to P. jirovecii in comparison to adenovirus and respiratory syncytial virus (RSV) in HIV-infec...

  10. Isolation, Cloning, Expression and Purification of Recombinant RhD Antigen from Cord Blood

    Directory of Open Access Journals (Sweden)

    M Habibi Roudkenar

    2008-09-01

    Full Text Available "nBackground: Rh (Rhesus is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blod, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study."nMethods: Total RNAs were extracted from cord blood (O+.  The quality of RNA was determined by electrophoresis. In or­der to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The iso­lated RhD gene was   cloned to pUC18 vector and transformed to DH5α. The confirmed construct was sub cloned into expres­sion vector, pBADgIII/A, and expressed in Top10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available hu­man anti-RhD polyclonal   antibody with   peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. "nRe­sults: RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane."nConclusion: The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applica­ble for production anti- D antibody in an animal model.

  11. Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery▿

    OpenAIRE

    Li, Lingling; Munir, Shirin; Bannantine, John P.; Sreevatsan, Srinand; Kanjilal, Sagarika; Kapur, Vivek

    2006-01-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed...

  12. MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

    Directory of Open Access Journals (Sweden)

    Cunha-Neto E.

    1999-01-01

    Full Text Available The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

  13. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    Science.gov (United States)

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  14. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    Directory of Open Access Journals (Sweden)

    Ryuichi Miura

    Full Text Available Canine distemper virus (CDV vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively. Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

  15. Role of recombination activating genes in the generation of antigen receptor diversity and beyond.

    Science.gov (United States)

    Nishana, Mayilaadumveettil; Raghavan, Sathees C

    2012-12-01

    V(D)J recombination is the process by which antibody and T-cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non-homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence-specific nuclease and its pathological role as a structure-specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence-specific and structure-specific nuclease. It also deals with the off-target cleavage of RAGs and its implications in genomic instability. PMID:23039142

  16. Production of Recombinant Echinococcus granulosus Antigen B Subunits, In Order to Using Of Them in Serodiagnostic Tests of Hydatidosis

    Directory of Open Access Journals (Sweden)

    R Pazoki

    2007-06-01

    Full Text Available Background: Hydatidosis is one of the most important helminthiasis, and is a public health problem in many regions of the world. Methods: With the aim of production of recombinant subunits of antigen B, two different sequences of Echinococcus granulosus Antigen B, acquired from Gene Bank and amplified with specific primers via RT-PCR reaction. The amplified fragments (HI, HII cloned into pTZ57R T.vector, and then subcloned into pGEMEX-1 expression vector. Resaults: The SDS-PAGE performed after induction of cloned genes, and production of about 35 K.Da recombinant fusion proteins were confirmed for either two cloned genes. The immunogenicity of the recombinant fusion proteins were tested using double diffusion and immunoblotting. Both recombinant fusion proteins derived from lysate of transformed bacteria, were reactive for antibodies in serum of cystic hydatid patient. Conclusion: The produced recombinant antigen B subunits can be use in seroldiagnostic tests of hydatidosis, after purification.

  17. Soluble recombinant merozoite surface antigen-142kDa of Plasmodium vivax: An improved diagnostic antigen for vivax malaria.

    Science.gov (United States)

    Mirahmadi, Hadi; Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad

    2016-04-01

    Enzyme Linked Immunosorbent Assay (ELISA), as a serological test, can be a beneficial tool for epidemiological studies by screening blood donors and diagnosis of specific antibodies from Plasmodium vivax (P. vivax) infected cases. Since P. vivax cannot easily be acquired in vitro, ELISA assays using total or semi-purified antigens are seldom used. On the basis of this restriction, we examined whether recombinant protein 42 kDa related to C-terminal region of the merozoite surface antigen-1 of P. vivax (MSA-1(42)) could be suitable for serological detection of vivax malaria infection. Purified recombinant protein produced in Escherichia coli (E. coli) (GST-MSA-1(42)) was examined for its ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 262 serum samples collected from individuals living in the south and southeastern regions of Iran where malaria is endemic. Samples exposed to Plasmodium falciparum (P. falciparum) infection and patients with other infectious disease (toxoplasmosis, Leishmania infantum infection, echinococcosis and FUO (fever with unknown origin)) except for P. falciparum were residing in non- malaria endemic areas in Iran. Generally, the sensitivity of ELISA evaluated with sera from naturally infected individuals was 86.9%. The specificity value of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases was 94.05%. The positive predictive value (PPV), negative predictive value (NPV) provided, and the diagnostic efficiency of anti-rPvMSA-1(42) antibody using indirect ELISA were determined 93.58, 87.77 and 91.06% respectively. Our study demonstrated that, because MSA-1(42) kDa contains both the 33 and 19 kDa fragments in its structure, it can serve as the basis for the development of a sensitive serological test which can be used for epidemiological studies, screening blood donors and diagnosis of P. vivax malaria. PMID:26851675

  18. The expression and antigenicity identification of recombinant rat TGF-β1 n bacteria

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study structure-function details of TGF-β1,the recombinant mature form of rat TGF-β1 was expressed in bacteria.Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-β1(amino acid 279390)was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase.This system allowed an active and selective synthesis of recombinant TGF-β1.The molecular weight of expressed TGF-β1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD.Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity.Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data.In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-β1 antibody.The mature recombinant rat TGF-β1 expressed in this study provides a useful tool for future detailed structural and functional studies.

  19. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate

    Science.gov (United States)

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen. PMID:25891359

  20. Mycobacterium bovis BCG priming induces a strong potentiation of the antibody response induced by recombinant BCG expressing a foreign antigen.

    OpenAIRE

    Gheorghiu, M; Lagranderie, M R; Gicquel, B M; Leclerc, C D

    1994-01-01

    Several recent studies have demonstrated that strong cellular or humoral immune responses can be induced against foreign antigens expressed by recombinant Mycobacterium bovis BCG. It has therefore been suggested that BCG could represent one of the best candidate vectors for live recombinant vaccines. However, a large percentage of the human population has been immunized by BCG, and this priming could modify the immune response to future recombinant BCG vaccines. In the present study, we have ...

  1. TH1 and TH2 responses are influenced by HLA antigens in healthy neonates vaccinated with recombinant hepatitis B vaccine.

    OpenAIRE

    Abdollah Jafarzadeh; Fazel Shokri

    2012-01-01

    The immune response to hepatitis B surface antigen (HBsAg) is influenced by several factors, of which HLA antigens and balanced secretion of Th1/Th2 cytokines play important roles. The aim of this study was to evaluate the influence of HLA antigens on cytokine secretion by HBsAg-stimulated peripheral blood mononuclear cells (PBMC) from healthy neonates vaccinated with recombinant HBsAg. PBMCs were isolated from 48 Iranian neonates vaccinated with a recombinant HBV vaccine. The cells were stim...

  2. Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

    Science.gov (United States)

    Rocha, T S; Tramuta, C; Catania, S; Matucci, A; Giuffrida, M G; Baro, C; Profiti, M; Bertolotti, L; Rosati, S

    2015-04-01

    Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications. PMID:25667423

  3. Characterisation of recombinant immunoreactive antigens of the scab mite Sarcoptes scabiei.

    Science.gov (United States)

    Kuhn, C; Lucius, R; Matthes, H F; Meusel, G; Reich, B; Kalinna, B H

    2008-05-31

    Sarcoptic mange (or scabies) is an important skin disease which can affect a variety of species including humans, cattle, goats, sheep, horses, pigs, rabbits, and dogs. Approximately 300 million people are affected worldwide and in lifestock animals the infestation may lead to substantial economic losses caused by depression in growth and feed conversion rates. Diagnosis of Sarcoptes infestation is difficult and only a few serological tests have been developed using whole mite antigen for diagnosis of mange in animals. Here we describe the isolation and characterisation of cDNAs of several immunoreactive clones and their recombinant expression in Escherichia coli. Three of the proteins contain repetitive sequences which suggests that they might be involved in immune evasion. The application of these antigens in serodiagnosis and the suitability for diagnosis is discussed. PMID:18359167

  4. Bovine immunoprotection against Rhipicephalus (Boophilus) microplus with recombinant Bm86-Campo Grande antigen.

    Science.gov (United States)

    Cunha, Rodrigo Casquero; Pérez de León, Adalberto Angel; Leite, Fábio Pereira Leivas; Pinto, Luciano da Silva; Dos Santos Júnior, Alceu Gonçalves; Andreotti, Renato

    2012-01-01

    The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul. PMID:23070436

  5. ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

    OpenAIRE

    SATO Neuza Satomi; HIRATA Mário H.; HIRATA Rosário D.C.; Lia Carmen M.S. ZERBINI; Edilene P.R. SILVEIRA; MELO Carmem Silvia de; Ueda, Mirthes

    1999-01-01

    Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rT...

  6. Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed Pichia pastoris cells.

    Science.gov (United States)

    Montesino, R; Cremata, J; Rodríguez, M; Besada, V; Falcón, V; de la Fuente, J

    1996-02-01

    In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris. The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm. The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein. PMID:8867893

  7. Recombinant Virus Vaccination against “Self” Antigens Using Anchor-fixed Immunogens

    OpenAIRE

    Irvine, Kari R.; Parkhurst, Maria R.; Shulman, Eliza P.; Tupesis, Janis P; Custer, Mary; Touloukian, Christopher E; Robbins, Paul F.; Yafal, Alicia Gómez; Greenhalgh, Patricia; Sutmuller, Roger P.M.; Offringa, Rienk; Rosenberg, Steven A.; Restifo, Nicholas P

    1999-01-01

    To study the induction of anti-“self” CD8+ T-cell reactivity against the tumor antigen gp100, we used a mouse transgenic for a chimeric HLA-A*0201/H-2 Kb molecule (A2/Kb). We immunized the mice with a recombinant vaccinia virus encoding a form of gp100 that had been modified at position 210 (from a threonine to a methionine) to increase epitope binding to the restricting class I molecule. Immunogens containing the “anchor-fixed” modification elicited anti-self CD8+ T cells specific for the wi...

  8. Effect of particulation on the immunogenic and protective properties of the recombinant Bm86 antigen expressed in Pichia pastoris.

    Science.gov (United States)

    García-García, J C; Montero, C; Rodríguez, M; Soto, A; Redondo, M; Valdés, M; Méndez, L; de la Fuente, J

    1998-02-01

    The recombinant Bm86 tick antigen expressed in Pichia pastoris is obtained in a highly particulated form, as a distinguish feature of this expression system. This particulated protein, the active principle of the recombinant vaccine Gavac against the cattle tick, have shown high immunogenic and protective properties, probably associated with its own characteristics. To evaluate the effects of particulation on the properties of Bm86, three groups of calves were immunized with particulated or non-particulated recombinant Bm86 and the anti-Bm86 antibody response determined. Animals were challenged with a controlled tick infestation and the protective capacities of both proteins assessed. Humoral immune response and protection in cattle vaccinated with the particulated antigen were higher. These experiments suggested that particulation of the Bm86 expressed in P. pastoris is an important feature for the protective properties of the antigen in vaccine preparations. PMID:9607058

  9. Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector.

    Directory of Open Access Journals (Sweden)

    Jan Drahota

    Full Text Available BACKGROUND: Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. METHODOLOGY/PRINCIPAL FINDINGS: The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. CONCLUSIONS/SIGNIFICANCE: Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral

  10. Enzyme-Linked Immunosorbent Assay Employing a Recombinant Antigen for Detection of Protective Antibody against Swine Erysipelas

    OpenAIRE

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-01-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher y...

  11. Chromatin architecture, CTCF and V(D)J recombination: managing long-distance relationships at antigen receptor loci1

    OpenAIRE

    Shih, Han-Yu; Krangel, Michael S.

    2013-01-01

    The rearrangement of T and B lymphocyte antigen receptor loci occurs within a highly complex chromosomal environment and is orchestrated through complex mechanisms. Over the past decade, a large body of literature has highlighted the significance of chromatin architecture at antigen receptor loci in supporting the genomic assembly process: in preparation for recombination, these loci tend to contract and form multiple loops that shorten the distances between gene segments and facilitate recom...

  12. Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

    OpenAIRE

    Kröncke, K D; Boulnois, G; Roberts, I.; Bitter-Suermann, D; Golecki, J R; Jann, B; Jann, K

    1990-01-01

    The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or r...

  13. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    Science.gov (United States)

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  14. Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

    Science.gov (United States)

    Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei

    2015-05-01

    The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design. PMID:25787135

  15. Control of Boophilus microplus populations in grazing cattle vaccinated with a recombinant Bm86 antigen preparation.

    Science.gov (United States)

    Rodríguez, M; Penichet, M L; Mouris, A E; Labarta, V; Luaces, L L; Rubiera, R; Cordovés, C; Sánchez, P A; Ramos, E; Soto, A

    1995-04-01

    Current methods for the control of cattle tick Boophilus microplus infestations are not effective and the parasite remains a serious problem for the cattle industry in tropical and subtropical areas. Recently, we developed a vaccine against B. microplus employing a recombinant Bm86 (rBm86) antigen preparation (Gavac, Heber Biotec) and it was shown to induce a protective response in vaccinated animals under controlled conditions. Here we show that, under field conditions in grazing cattle, the vaccine is able to control B. microplus populations. Two parasite-free farms were employed for the study. In the first farm, animals were vaccinated with the recombinant vaccine, while, in the second, animals received a saline injection in adjuvant. After immunization, animals were artificially infected and the infestation rate was recorded. Over the 33 weeks of the experiment, the infestation rate was lower in the vaccinated group compared with the control group. At the end of the experiment it was necessary to use chemicals in the control farm after serious losses in production and animals. PMID:7660571

  16. Influence of culture medium on the production of eif antigen from Leishmania chagasi in recombinant Escherichia coli

    Directory of Open Access Journals (Sweden)

    Michelle Rossana Ferreira Vaz

    2011-12-01

    Full Text Available With the advent of recombinant DNA technology, recombinant protein expression has become an important tool in the study of the structure, function and identification of new proteins, especially those with therapeutic functions. Escherichia coli has been the predominant prokaryote used in genetic engineering studies due to the abundance of information about its metabolism. Despite significant advances in molecular biology and immunology of infections, there are as yet no prophylactic drugs capable of preventing visceral leishmaniasis. It is therefore important to identify specific antigens in order to develop vaccines and diagnostic kits against this disease. The objective of this study was to evaluate the influence of culture medium on the production of eIF antigen from Leishmania chagasi in recombinant Escherichia coli. An induction procedure using IPTG was carried out in a series of trials, to observe the influence of culture medium (2xTY, TB under expression of the recombinant eIF protein. Results showed that recombinant protein expression was associated to growth and that the highest eIF antigen expression was obtained in the 2xTY medium.

  17. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

    Directory of Open Access Journals (Sweden)

    Michal Sima

    2016-03-01

    Full Text Available Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs using salivary gland homogenates (SGHs as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species.Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera.Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

  18. Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens

    Directory of Open Access Journals (Sweden)

    María Susana Imaz

    2008-06-01

    Full Text Available The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B, singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50, smear-negative pulmonary TB (pTBsm- (60 and extrapulmonary TB (51 and 214 control patients (mycobacteriosis (14, mycoses(14, leprosy(4, other underlying diseases (82 and healthy people (100]. The individual antigens ranged from 25% to 42% in sensitivity and from 93% to 96% in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55%. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76% sensitivity and 83% specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63% but with a good specificity (95%, even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70% of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.

  19. Antigen

    Science.gov (United States)

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  20. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    Science.gov (United States)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  1. Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis.

    Science.gov (United States)

    Flannery, B; Costa, D; Carvalho, F P; Guerreiro, H; Matsunaga, J; Da Silva, E D; Ferreira, A G; Riley, L W; Reis, M G; Haake, D A; Ko, A I

    2001-09-01

    There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis. PMID:11526167

  2. Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

    Directory of Open Access Journals (Sweden)

    Eliza Yoshie do Rosário

    2005-04-01

    Full Text Available The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2% were positive in parasitological exams (skin and/or spleen. These animals were compared to healthy animals (n = 25, negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%. Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%. Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00. High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97. Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

  3. Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

    OpenAIRE

    Fernandes, Ana Paula; Elizabeth Cortez HERRERA; Wilson MAYRINK; Gazzinelli, Ricardo T.; LIU Wen Yu; Carlos Alberto da COSTA; Tavares, Carlos Alberto Pereira; Melo, Maria Norma; Michalick, Marilene Susan Marques; Gentz, Reiner; NASCIMENTO Evaldo

    1997-01-01

    In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting ana...

  4. Advax-Adjuvanted Recombinant Protective Antigen Provides Protection against Inhalational Anthrax That Is Further Enhanced by Addition of Murabutide Adjuvant

    OpenAIRE

    Feinen, Brandon; Petrovsky, Nikolai; Verma, Anita; Tod J Merkel

    2014-01-01

    Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax a...

  5. Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

    OpenAIRE

    Chun, Jeong-Hoon; Choi, On-Jee; Cho, Min-Hee; Hong, Kee-Jong; Seong, Won Keun; Oh, Hee-Bok; Rhie, Gi-eun

    2012-01-01

    Objective Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. Methods Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (inte...

  6. A multicenter evaluation of a new antibody test kit for lymphatic filariasis employing recombinant Brugia malayi antigen Bm-14

    OpenAIRE

    Weil, Gary J; Curtis, Kurt C.; Fischer, Peter U.; Kimberly Y Won; Lammie, Patrick J; Joseph, Hayley; Melrose, Wayne D; Brattig, Norbert W.

    2010-01-01

    Antibody tests are useful for mapping the distribution of lymphatic filariasis (LF) in countries and regions and for monitoring progress in elimination programs based on mass drug administration (MDA). Prior antibody tests have suffered from poor sensitivity and/or specificity or from a lack of standardization. We conducted a multicenter evaluation of a new commercial ELISA that detects IgG4 antibodies to the recombinant filarial antigen Bm14. Four laboratories tested a shared panel of coded ...

  7. The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen.

    Directory of Open Access Journals (Sweden)

    Rosamund Chapman

    Full Text Available Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP. Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206] were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]. The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]. Mice primed with 10(7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10(6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.

  8. The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen.

    Science.gov (United States)

    Chapman, Rosamund; Bourn, William R; Shephard, Enid; Stutz, Helen; Douglass, Nicola; Mgwebi, Thandi; Meyers, Ann; Chin'ombe, Nyasha; Williamson, Anna-Lise

    2014-01-01

    Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10(7) CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10(6) splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge. PMID:25061753

  9. Establishment of an in vivo potency assay for the recombinant hepatit is B surface antigen in monovalent and combined vaccines

    OpenAIRE

    Mabel Izquierdo-López; Karelia Cosme-Diaz; Gerardo García-Illera; Zoe Núñez-Lamotte; Yamila Martínez- Cuéllar; Maribel Vega-Simón; Lourdes Costa-Anguiano; Marisel Quintana-Esquivel; Ileana Rosales-Torres; Omar Mosqueda-Lobaina

    2014-01-01

    In this paper the development of potency assay in animals (mice) was made, with the objective of demonstrating the immunogenic power of the recombinant Hepatitis B surface antigen in monovalent and combined vaccines, produced at the Center of Genetic Engineering and Biotechnology. The potency test is a parameter in quality control and it is also a tool to demonstrate the consistency of the production process. Parameters such as duration of the test, number of animals in the test, ...

  10. A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

    Science.gov (United States)

    Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  11. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague.

    Science.gov (United States)

    Rocke, Tonie E; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

  12. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague

    Directory of Open Access Journals (Sweden)

    Tonie E. Rocke

    2014-10-01

    Full Text Available In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  13. TH1 and TH2 responses are influenced by HLA antigens in healthy neonates vaccinated with recombinant hepatitis B vaccine.

    Directory of Open Access Journals (Sweden)

    Abdollah Jafarzadeh

    2012-12-01

    Full Text Available The immune response to hepatitis B surface antigen (HBsAg is influenced by several factors, of which HLA antigens and balanced secretion of Th1/Th2 cytokines play important roles. The aim of this study was to evaluate the influence of HLA antigens on cytokine secretion by HBsAg-stimulated peripheral blood mononuclear cells (PBMC from healthy neonates vaccinated with recombinant HBsAg. PBMCs were isolated from 48 Iranian neonates vaccinated with a recombinant HBV vaccine. The cells were stimulated in vitro with rHBsAg and the concentration of IL-4, IL-10, IL-12 and IFN-γ were quantitated in culture supernatant by sandwich ELISA. HLA typing was performed by microlymphocytotoxicity method. Significant diminished secretion of both Th1 (IFN-γ and Th2 (IL-4, IL-10 cytokines was observed in HBsAg-stimulated PBMC from vaccinees expressing the HLA-DR7 compared to DR7 negative vaccinees. Similarly, lower production of these cytokines was also observed in vaccinees with DR7-DR53-DQ2, B7-DR7-DR53-DQ2 and A2-DR7-DR53-DQ2 haplotypes (p<0.05, p <0.005. While HBsAg-stimulated PBMC of DR13+ subjects produced lower levels of Th2-type cytokines (IL-4 and IL-10, those of HLA-B8+ or HLA-A9+ subjects produced higher levels of Th2-type cytokines. Cytokine secretion in response to PHA mitogen was not associated with a given HLA antigen or haplotype and was similarly represented in all groups of subjects irrespective of their HLA complex. These results indicate that HLA antigens may differentially influence cytokine secretion by HBsAg-specific T-cells of healthy neonates vaccinated with recombinant HB vaccine. This phenomenon may have an important implication for control of the immune response to HBsAg vaccine.

  14. Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D.

    Science.gov (United States)

    Radolf, J D; Lernhardt, E B; Fehniger, T E; Lovett, M A

    1986-06-01

    An enzyme-linked immunosorbent assay (ELISA) for syphilis has been developed that detects IgG antibody to purified recombinant Treponema pallidum surface antigen 4D. The 4D ELISA was capable of detecting 25 ng of 4D antigen-specific antibody. Neither 172 nonsyphilitic sera nor 20 false-positive sera in the Venereal Disease Research Laboratory test reacted in the 4D ELISA. The sensitivity of the 4D ELISA was comparable to that of the adsorbed fluorescent treponemal antibody test in primary, secondary, and latent disease. Most sera from patients with yaws or pinta were also reactive, a result indicating that a 4D antigen-like molecule also exists in the closely related pathogenic treponemes Treponema pertenue and Treponema carateum. PMID:3517186

  15. Temperature-mediated recombinant anthrax protective antigen aggregate development: Implications for toxin formation and immunogenicity.

    Science.gov (United States)

    Amador-Molina, Juan C; Valerdi-Madrigal, Esther D; Domínguez-Castillo, Rocío I; Sirota, Lev A; Arciniega, Juan L

    2016-07-29

    Anthrax vaccines containing recombinant PA (rPA) as the only antigen face a stability issue: rPA forms aggregates in solution after exposure to temperatures ⩾40°C, thus losing its ability to form lethal toxin (LeTx) with Lethal Factor. To study rPA aggregation's impact on immune response, we subjected rPA to several time and temperature combinations. rPA treated at 50°C for 30min formed high mass aggregates when analyzed by gel electrophoresis and failed to form LeTx as measured by a macrophage lysis assay (MLA). Aggregated rPA-formed LeTx was about 30 times less active than LeTx containing native rPA. Mice immunized with heat-treated rPA combined with Al(OH)3 developed antibody titers about 49 times lower than mice immunized with native rPA, as measured by a Toxicity Neutralization Assay (TNA). Enzyme Linked Immunosorbent Assay (ELISA) of the same immune sera showed anti-rPA titers only 2-7 times lower than titers elicited by native rPA. Thus, rPA's ability to form LeTx correlates with its production of neutralizing antibodies, and aggregation significantly impairs the protein's antibody response. However, while these findings suggest MLA has some value as an in-process quality test for rPA in new anthrax vaccines, they also confirm the superiority of TNA for use in vaccine potency. PMID:27364097

  16. Mitotic evolution of Plasmodium falciparum shows a stable core genome but recombination in antigen families.

    Directory of Open Access Journals (Sweden)

    Selina E R Bopp

    Full Text Available Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone. In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1 on chromosome 1. We observed 18 large-scale (>1 kb on average deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10(-6 structural variants per base pair per generation. Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0-9.7×10(-9 mutations per base pair per generation, we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum

  17. Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

    Institute of Scientific and Technical Information of China (English)

    Azam Roohi; Yaghoub Yazdani; Jalal Khoshnoodi; Seyed Mohammad Jazayeri; William F Carman; Mahmood Chamankhah; Manley Rashedan; Fazel Shokri

    2006-01-01

    AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the "a" region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the "a"determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

  18. Cytokine Switch and Bystander Suppression of Autoimmune Responses to Multiple Antigens in Experimental Autoimmune Encephalomyelitis by a Single Recombinant T-Cell Receptor Ligand

    OpenAIRE

    Sinha, Sushmita; Subramanian, Sandhya; Miller, Lisa; Proctor, Thomas M.; Roberts, Chris; Burrows, Gregory G.; Vandenbark, Arthur A.; Offner, Halina

    2009-01-01

    Recombinant T-cell receptor ligands (RTLs) can reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner, and are currently in clinical trials for treatment of subjects with multiple sclerosis (MS). Antigen specificity of RTL raises the question as to whether this treatment would be successful in MS patients where target antigens are unknown. Using spinal cord homogenate or combinations of two different peptides to induce disease,...

  19. Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction

    DEFF Research Database (Denmark)

    Kolesar, Peter; Altmannova, Veronika; Silva, Sonia;

    2016-01-01

    -interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation of...

  20. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

    Science.gov (United States)

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Lülf, Anna; Marr, Lisa; Jany, Sylvia; Deeg, Cornelia A; Pijlman, Gorben P; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E; Sutter, Gerd

    2016-04-01

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans. PMID:26939903

  1. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    Science.gov (United States)

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins. PMID:26777581

  2. Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wolff Jose LC

    2011-05-01

    Full Text Available Abstract Background Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS. Methods In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

  3. Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.

    OpenAIRE

    Gurramkonda, Chandrasekhar; Zahid, Maria; Nemani, Satish Kumar; Adnan, Ahmad; Gudi, Satheesh Kumar; Khanna, Navin; Ebensen, Thomas; Lünsdorf, Heinrich; Guzmán, Carlos A.; Rinas, Ursula

    2013-01-01

    Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg....

  4. Recombinant Heat Shock Protein 70 in Combination with Radiotherapy as a Source of Tumor Antigens to Improve Dendritic Cell Immunotherapy

    OpenAIRE

    Kwan-HwaChi; Shih-JenLiu

    2012-01-01

    Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C'-PSA) and alpha-fetoprotein (rHSP70C'-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumo...

  5. Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy

    OpenAIRE

    Wang, Yu-Shan; Liu, Shih-Jen; Huang, Su-Chen; Chang, Chao-Chun; Huang, Yi-Chun; Fong, Weng-Lam; Chi, Mau-Shin; Chi, Kwan-Hwa

    2012-01-01

    Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C′-PSA) and α-fetoprotein (rHSP70C′-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor im...

  6. Immunogenicity and Protective Efficacy of a Novel Recombinant BCG Strain Overexpressing Antigens Ag85A and Ag85B

    OpenAIRE

    Chun Wang; Ruiling Fu; Xionglin Fan; Chunwei Shi; Jia Lu; Xindong Teng; Lingxia Chen; Kun Tan; Zhenhua Chen

    2012-01-01

    Recombinant Bacillus Calmette-Guérin (rBCG) strain is the promising vaccine candidate for tuberculosis (TB) prevention, which aims at providing more enduring and enhanced protection than the parental BCG vaccine. In this study, three rBCG strains overexpressing immunodominant antigens Ag85B (rBCG::85B), Ag85A (rBCG::85A), or both (rBCG::AB) of Mycobacterium tuberculosis were constructed, respectively. rBCG strains showed higher level of overexpression of Ag85A and/or Ag85B proteins than BCG c...

  7. Protective Effect of Vaccination with a Combination of Recombinant Surface Antigen 1 and Interleukin-12 against Toxoplasmosis in Mice

    OpenAIRE

    Letscher-Bru, Valerie; Villard, Odile; Risse, Bernhard; Zauke, Michael; Klein, Jean-Paul; Kien, Truong T.

    1998-01-01

    We studied the immune response induced in mice by recombinant Toxoplasma gondii surface antigen 1 (rSAG1) protein, alone or combined with interleukin-12 (IL-12) as an adjuvant, and the protective effect against toxoplasmosis. Immunization with rSAG1 alone induced a specific humoral type 2 immunity and did not protect the animals from infection. In contrast, immunization with rSAG1 plus IL-12 redirected humoral and cellular immunity toward a type 1 pattern and reduced the brain parasite load b...

  8. Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast.

    Science.gov (United States)

    Monsalve, R I; Lu, G; King, T P

    1999-08-01

    Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast alpha signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis). PMID:10425162

  9. The physical stability of the recombinant tuberculosis fusion antigens h1 and h56

    DEFF Research Database (Denmark)

    Hamborg, Mette; Kramer, Ryan; Schanté, Carole E; Agger, Else Marie; Christensen, Dennis; Jorgensen, Lene; Foged, Camilla; Middaugh, C Russell

    2013-01-01

    gel electrophoresis analyses demonstrated that both antigens exist as a distribution of multimeric states under nonstressed conditions. Their conformational stability was monitored as a function of pH and temperature and visualized in three-index empirical phase diagrams. Both antigens showed a...... it is therefore important to characterize their conformational stability in solution as well as upon interaction with adjuvants. In this study, the physical stability of the two antigens was characterized using a number of biophysical techniques. Dynamic light scattering and sodium dodecyl sulfate-polyacrylamide...

  10. Development of a sandwich Dot-ELISA for detecting bovine viral diarrhea virus antigen with E2 recombinant protein

    Institute of Scientific and Technical Information of China (English)

    Yuelan ZHAO; Yuzhu ZUO; Lei ZHANG; Jinghui FAN; Hanchun YANG; Jianhua QIN

    2009-01-01

    The IgG antibodies of rabbit anti-E2 protein of the bovine viral diarrhea virus were prepared by a general method from high efficiency serum immunized by E2 recombinant protein antigen expressed in E. coli prokaryotic expression system and were labeled to make enzymelabeled antibody with the method of NaIO4. A sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the detection of BVDV was developed. The optimal reaction conditions of Dot-ELISAwere determined. The results show that optimal coating antibody was 300 μg·mL-1, the working concentration of HRP-labeled antibody was 1:50. The optimal blocking reagent and time were 5% bovine serum and 45 rain. The minimum detection of the content of antigen reached 1.35μg·mL-1. Compared with the routine IDEXX ELISA test kit with the whole virus, its specificity, sensitivity and coincidence rate were 90.48%, 96.55% and 95.24%, respectively. Compared with the sandwich Dot-ELISA with the negative staining electron microscope and RT-PCR, the coincidence rates were 90.9% and 93.1%, respectively. In addition, Bovine viral diarrhea virus (BVDV) antigen of 178 samples collected from cow farms in the Hebei Province, China, were detected by the developed Dot-ELISA and the IDEXX BVDV antigen Test Kit simultaneously, BVDV antigen positive rate was 39.89%-41.01%. The result of detecting clinical samples demonstrated that the established method showed its specificity, sensitivity and repeatability, whereas the results were easily interpreted without an ELISA reader.

  11. Development of Recombinant Flagellar Antigens for Serological Detection of Salmonella enterica Serotypes Enteritidis, Hadar, Heidelberg, and Typhimurium in Poultry

    Directory of Open Access Journals (Sweden)

    Charles L. Hofacre

    2013-07-01

    Full Text Available Accurate and fast detection of harmful Salmonella is a major concern of food safety. Common Salmonella serotypes responsible for human associated foodborne outbreaks are S. Enteritidis, S. Hadar, S. Heidelberg, and S. Typhimurium are also commonly isolated from poultry. Serology is commonly used to monitor disease in poultry, therefore application of Salmonella serotype-specific test will have added value in Salmonella surveillance or monitoring vaccine efficacy. Recombinant flagellins were purified to be used as antigens in an ELISA. In this study, an ELISA was developed for the serological detection of S. Enteritidis. Once optimized, 500 ng of purified recombinant S. Enteritidis flagellin and a 1:64 dilution were determined to be optimal for testing sera. A negative baseline cutoff was calculated to be an optical density (OD of 0.35. All sera from birds with history of S. Enteritidis exposure tested positive and all sera from chickens with no exposure tested negative to this Salmonella serotype. Current ELISA for serological detection of Salmonella suffers from cross reactivity inherent in lipopolysaccharide (LPS or whole cell antigen based serological tests. This new ELISA eliminates common cross reactivity by focusing specifically on the flagellins of the Salmonella serotypes common in poultry and associated with foodborne outbreaks.

  12. Control of Boophilus microplus ticks in cattle calves by immunization with a recombinant Bm86 glucoprotein antigen preparation.

    Science.gov (United States)

    Khalaf-Allah, S S

    1999-06-01

    Forty Egyptian native cattle calves of 4-6 months old randomly allocated into two groups of twenty animals each were used to assess the effect of immunization of animals with a recombinant Bm86 antigen derived from Boophilus microplus ticks on induction of immunity that could protect calves during tick season. The immunization protocol involved two injections administered intramuscularly, the first was applied with complete Freund's adjuvant and the second was given with incomplete Freund's adjuvant two months later. Control calves were given saline plus adjuvant. Immunization reduced the number of adult ticks developing from a subsequent challenge infestation by 78% in immunized calves. Vaccination also, significantly reduced the weight of adult ticks in immunized calves (30.51%). The results of skin delayed hypersensitivity reaction revealed that the diameter of sites injected with the recombinant Bm86 antigen was significantly larger in immunized calves than those in controls. Analysis of the immune response indicated that there was a significant increase in the level of IgG and IgA antibodies in serum of immunized calves and protection from reinfestation was correlated with the levels of circulating antibodies. PMID:10422372

  13. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli : Its application to a 12 kDa antigen from Cryptosporidium parvum

    OpenAIRE

    Costa, Sofia Judite; Silva, P.; Almeida, André; Conceição, A.; Domingues, Lucília; Castro, António G.

    2013-01-01

    The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimise recombinant antigen product...

  14. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA Induce Protective Immune Responses in Dogs.

    Directory of Open Access Journals (Sweden)

    Elodie Petitdidier

    2016-05-01

    Full Text Available Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA, from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA or its carboxy terminal part LaPSA-12S (Cter-rPSA, combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

  15. Alanine scanning of the rabies virus glycoprotein antigenic site III using recombinant rabies virus: implication for post-exposure treatment.

    Science.gov (United States)

    Papaneri, Amy B; Wirblich, Christoph; Marissen, Wilfred E; Schnell, Matthias J

    2013-12-01

    The safety and availability of the human polyclonal sera that is currently utilized for post-exposure treatment (PET) of rabies virus (RABV) infection remain a concern. Recombinant monoclonal antibodies have been postulated as suitable alternatives by WHO. To this extent, CL184, the RABV human antibody combination comprising monoclonal antibodies (mAbs) CR57 and CR4098, has been developed and has delivered promising clinical data to support its use for RABV PET. For this fully human IgG1 cocktail, mAbs CR57 and CR4098 are produced in the PER.C6 human cell line and combined in equal amounts in the final product. During preclinical evaluation, CR57 was shown to bind to antigenic site I whereas CR4098 neutralization was influenced by a mutation of position 336 (N336) located within antigenic site III. Here, alanine scanning was used to analyze the influence of mutations within the potential binding site for CR4098, antigenic site III, in order to evaluate the possibility of mutated rabies viruses escaping neutralization. For this approach, twenty flanking amino acids (10 upstream and 10 downstream) of the RABV glycoprotein (G) asparagine (N336) were exchanged to alanine (or serine, if already alanine) by site-directed mutagenesis. Analysis of G expression revealed four of the twenty mutant Gs to be non-functional, as shown by their lack of cell surface expression, which is a requirement for the production of infectious RABV. Therefore, these mutants were excluded from further study. The remaining sixteen mutants were introduced in an infectious clone of RABV, and recombinant RABVs (rRABVs) were recovered and utilized for in vitro neutralization assays. All of the viruses were effectively neutralized by CR4098 as well as by CR57, indicating that single amino acid exchanges in this region does not affect the broad neutralizing capability of the CL184 mAb combination. PMID:24120673

  16. Vaccine potential of recombinant antigens of Theileria annulata and Hyalomma anatolicum anatolicum against vector and parasite.

    Science.gov (United States)

    Jeyabal, L; Kumar, Binod; Ray, Debdatta; Azahahianambi, Palavesam; Ghosh, Srikanta

    2012-09-10

    In an attempt to develop vaccine against Hyalomma anatolicum anatolicum and Theileria annulata, three antigens were expressed in prokaryotic expression system and protective potentiality of the antigens was evaluated in cross bred calves. Two groups (grs. 1 and 4) of male cross-bred (Bos indicus × Bos taurus) calves were immunized with rHaa86, a Bm86 ortholog of H. a. anatolicum, while one group of calves (gr. 2) were immunized with cocktails of two antigens viz., surface antigens of T. annulata (rSPAG1, rTaSP). One group each was kept as negative controls (grs. 3 and 5). The animals of groups 1, 2 and 3 were challenged with T. annulata infected H. a. anatolicum adults while the animals of groups 1, 3, 4 and 5 were challenged with uninfected adult ticks. A significantly high (p<0.05) antibody responses to all the three antigens were detected in immunized calves, but the immune response was comparatively higher with rHaa86 followed by rTaSP and rSPAG1. Upon challenge with T. annulata infected ticks, animals of all groups showed symptoms of the disease but there was 50% survival of calves of group 1 while all non immunized control calves (group 3) and rSPAG1+rTaSP immunized calves died. The rHaa86 antigen was found efficacious to protect calves against more than 71.4-75.5% of the challenge infestation. The experiment has given a significant clue towards the development of rHaa86 based vaccine against both H. a. anatolicum and T. annulata. PMID:22546546

  17. Differential Effects of Prior Exposure to Environmental Mycobacteria on Vaccination with Mycobacterium bovis BCG or a Recombinant BCG Strain Expressing RD1 Antigens

    OpenAIRE

    Demangel, Caroline; Garnier, Thierry; Rosenkrands, Ida; Cole, Stewart T.

    2005-01-01

    In silico analysis reveals that most protective antigens expressed by the antituberculous vaccine Mycobacterium bovis BCG (BCG) are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization with M. avium, M. scrofulaceum, or M. vaccae on the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of expe...

  18. Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

    OpenAIRE

    Xiaodong Weng; Youlin Kuang; Xiuheng Liu; Zhiyuan Chen; Hengcheng Zhu; Hui Chen; Botao Jiang; Hao Shen

    2011-01-01

    Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-interna...

  19. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    DEFF Research Database (Denmark)

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas Salhøj;

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium ...

  20. Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

    OpenAIRE

    Chen-Ji Huang; Hwei-Ling Peng; Chih-Yu Cheng

    2011-01-01

    In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M ure...

  1. Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection.

    Science.gov (United States)

    Kalenda, Yombo Dan Justin; Kato, Kentaro; Goto, Yasuyuki; Fujii, Yoshito; Hamano, Shinjiro

    2015-12-01

    The diagnosis of schistosome infection, followed by effective treatment and/or mass drug administration, is crucial to reduce the disease burden. Suitable diagnostic tests and field-applicable tools are required to sustain schistosomiasis control programs. We therefore assessed the potential of tandem repeat (TR) proteins for sero-diagnosis of Schistosoma mansoni infection using an experimental mouse model. TR genes in the genome of S. mansoni were searched in silico and 7 candidates, named SmTR1, 3, 8, 9, 10, 11 and 15, were selected. Total RNA was extracted from S. mansoni adult worms and eggs. Target TR genes were amplified, cloned, and the proteins were expressed in Escherichia coli competent cells. Female BALB/c mice were infected with 100 S. mansoni cercariae and sera were collected each week post-infection for 18 weeks. The levels of IgG antibodies to SmTR antigens were compared to those to soluble egg antigen (SEA) and to soluble worm antigen preparation (SWAP). Sera of infected mice reacted to all the antigens whereas those of naïve mice did not. IgG responses to SmTR1, 3, 9 and 10 were detected at the early stage of infection. Interestingly, antibodies reacting to SmTR3, 9, 10 and 15 dramatically decreased 4 weeks after treatment with praziquantel, while those against SEA and SWAP remained elevated. Our study suggests that TR proteins, especially SmTR10, may be suitable antigens for sero-diagnosis of infection by S. mansoni and are potential markers for monitoring and surveillance of schistosomiasis, including re-infection after treatment with praziquantel. PMID:26148816

  2. Use of novel recombinant antigens in the interferon gamma assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, C.; Nielsen, Søren Saxmose; Jungersen, Gregers

    2012-01-01

    of the study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-¿ assay and to assess the consistency of IFN-¿ responses with repeated samplings. The antigens used were 4 ESAT-6 family members, 4 latency proteins, 4 secreted proteins including Ag85B, 3...... were positive to PPDj were non-cases and one case was negative to PPDj but positive to all other tested antigens. Furthermore, PPDj produced elevated IFN-¿ responses in both the infected and non-infected herds and showed low consistency. Immunogenicity was highest for the group of latency proteins (0...

  3. Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague.

    Science.gov (United States)

    Sanapala, Shilpa; Rahav, Hannah; Patel, Hetal; Sun, Wei; Curtiss, Roy

    2016-05-01

    Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine. PMID:27060051

  4. Study of Immunization against Anthrax with the Purified Recombinant Protective Antigen of Bacillus anthracis

    OpenAIRE

    Singh,Yogendra; Ivins, Bruce E.; Leppla, Stephen H.

    1998-01-01

    Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. We previously described the biological activity of a nontoxic mutant of PA expressed in Bacillus subtilis. In ...

  5. Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

    Science.gov (United States)

    Su, Qiudong; Guo, Minzhuo; Jia, Zhiyuan; Qiu, Feng; Lu, Xuexin; Gao, Yan; Meng, Qingling; Tian, Ruiguang; Bi, Shengli; Yi, Yao

    2016-07-01

    Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine. PMID:26994964

  6. Vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens Cb15, Cb42 and Cb48

    Directory of Open Access Journals (Sweden)

    Sukarsih

    2000-10-01

    Full Text Available Recombinant forms of a number of peritrophic membrane proteins from the screwworm fly Chrysomya bezziana have been assessed in vitro and in vivo for their efficacy as antigens in vaccination against the tissue-invasive, larval form of the parasite. The proteins included Cb15 and Cb42 expressed in Escherichia coli and Cb48 expressed in both Escherichia coli and Pichia pastoris. In all cases, the in vitro assays of larval growth on serum from vaccinated sheep failed to show inhibition of larval weight gain or any detrimental effect on larval survival relative to controls. Chrysomya bezziana Cb48 has a significant degree of sequence identity with the antigen PM48 from Lucilia cuprina. Feeding Lucilia cuprina larvae on antisera to Cb48 induced a small but statistically significant reduction in weight gain, as does feeding on antisera to PM48. In vivo, larvae feeding on sheep vaccinated with Escherichia coli-expressed Cb15 and Cb42 and Pichia pastoris-expressed Cb48 showed marginally greater weight gain and survival which was equal to or greater than that on non-vaccinated sheep. The significance of these observations is discussed.

  7. Regeneration of Recombinant Antigen Microarrays for the Automated Monitoring of Antibodies against Zoonotic Pathogens in Swine Sera

    Directory of Open Access Journals (Sweden)

    Verena K. Meyer

    2015-01-01

    Full Text Available The ability to regenerate immobilized proteins like recombinant antigens (rAgs on surfaces is an unsolved problem for flow-based immunoassays on microarray analysis systems. The regeneration on microarray chip surfaces is achieved by changing the protein structures and desorption of antibodies. Afterwards, reactivation of immobilized protein antigens is necessary for reconstitution processes. Any backfolding should be managed in a way that antibodies are able to detect the protein antigens in the next measurement cycle. The regeneration of rAg microarrays was examined for the first time on the MCR3 flow-based chemiluminescence (CL microarray analysis platform. The aim was to reuse rAg microarray chips in order to reduce the screening effort and costs. An antibody capturing format was used to detect antibodies against zoonotic pathogens in sera of slaughtered pigs. Different denaturation and reactivation buffers were tested. Acidic glycine-SDS buffer (pH 2.5 and 8 M guanidinium hydrochloride showed the best results in respect of denaturation efficiencies. The highest CL signals after regeneration were achieved with a carbonate buffer containing 10 mM DTT and 0.1% BSA for reactivation. Antibodies against Yersinia spp. and hepatitis E virus (HEV were detected in swine sera on one immunochip over 4 days and 25 measurement cycles. Each cycle took 10 min for detection and regeneration. By using the rAg microarray chip, a fast and automated screening of antibodies against pathogens in sera of slaughtered pigs would be possible for zoonosis monitoring.

  8. Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis

    DEFF Research Database (Denmark)

    Pietkiewicz, H; Hiszczyńska-Sawicka, E; Kur, J;

    2007-01-01

    The precise diagnosis of an acute and recent Toxoplasma infection in pregnant women and the newborn child is important before treatment. This study describes a new Toxoplasma gondii IgG avidity test based on a combination of recombinant GRA1, GRA7 and SAG1 antigens and shows that this test is use...

  9. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  10. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    Science.gov (United States)

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. PMID:27030586

  11. Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Liu, Lifeng; Qian, Suhong; Chen, Huanchun; Li, Xiangmin

    2014-12-01

    Virus-like particles (VLPs) of chimeric porcine circovirus type 2 (PCV2) were generated by replacing the nuclear localization signal (NLS; at 1-39 aa) of PCV2 capsid protein (Cap) with classical swine fever virus (CSFV) T-cell epitope (1446-1460 aa), CSFV B-cell epitope (693-716 aa) and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine. PMID:25490764

  12. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

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    Tam Yew

    2012-10-01

    Full Text Available Abstract Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg from Pichia pastoris expression cells were optimized using response surface methodology (RSM based on the central composite design (CCD. The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

  13. CLINICAL EVALUATION OF FOUR RECOMBINANT TREPONEMA PALLIDUM ANTIGEN-BASED RAPID TESTS IN THE DIAGNOSIS OF SYPHILIS

    Institute of Scientific and Technical Information of China (English)

    Lin-na Wang; Lei Yang; He-yi Zheng

    2007-01-01

    To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis.Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. pallidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests.T. pallidum haemagglutination test (TPHA) was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed.Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test,VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98.9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9%; for whole blood were 74.1% and 99.5%, 87.9% and 99.4%,73.2% and 99.7%, 64.7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA ( P>0.05 ).Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.

  14. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    Science.gov (United States)

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples. PMID:26693887

  15. Dose of Incorporated Immunodominant Antigen in Recombinant BCG Impacts Modestly on Th1 Immune Response and Protective Efficiency against Mycobacterium tuberculosis in Mice

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    Hui Ma

    2014-01-01

    Full Text Available One approach for improving BCG efficacy is to utilize BCG as vehicle to develop recombinant BCG (rBCG strains overexpressing Mycobacterium tuberculosis (M. tb antigens. Also expression level of a candidate antigen should impact the final T cell responses conferred by rBCG. In this study, based on our previously constructed differential expression system, we developed two rBCG strains overexpressing M. tb chimeric antigen Ag856A2 (coding a recombinant ag85a with 2 copies of esat-6 inserted at Acc I site of ag85a at differential levels under the control of the subtly modified furA promoters. These two rBCG strains were used to vaccinate C57BL/6 mice and exploit dose of incorporated antigen in rBCG to optimize immune response and protective efficiency against M. tb challenge in mouse model. The results showed that rBCG strains overexpressing Ag856A2 at differential levels induced different antigen-specific IFN-γ production and comparable number of M. tb-specific CD4 T cells expressing IL-2. M. tb challenge experiment showed that rBCG strains afforded enhanced but comparable immune protection characterized by reduced bacillary load, lung pathology, and inflammation. These results suggested that the dose of antigens incorporated in rBCG can impact T cell immune responses but imposed no significantly differential protective efficacies.

  16. Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2.

    Science.gov (United States)

    Chang, J W; Mechling, D E; Bächinger, H P; Burrows, G G

    2001-06-29

    Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the

  17. Immune responses induced by a Leishmania (Leishmania amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

    Directory of Open Access Journals (Sweden)

    Ana Paula FERNANDES

    1997-03-01

    Full Text Available In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania amazonensis recombinant protein of 33 kD (Larp33 which is recognized by antibodies and peripheral blood leukocytes (PBL from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L. amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L. amazonensis and L. (Viannia braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10 against L. (L. amazonensis after vaccination using Bacille Calmette-Guerin (BCG as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasisA resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania amazonensis de 33 kD (Larp33 foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L. amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao ant

  18. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

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    Chin'ombe Nyasha

    2009-04-01

    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  19. Evaluation of host humoral antibody production against Plasmodium falciparum recombinant circumsporozoite antigen in Nigerian children

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    Olalubi Adewole Oluwasogo , Ogunlana Oluseyi Ebenezer & Anumudu Chiaka

    2012-09-01

    Full Text Available Background & objectives: The challenge of malaria and efforts targeted at developing malaria vaccines triggeredthis study on the reactivity of IgG and its subclasses in the test serum specific to CSP. This work was directed atassessing the influence of age and gender on host humoral antibody against Plasmodium falciparum recombinantcircumsporozoite antigen in Nigerian children.Methods: In all, 67 serum samples (>10,000 parasites/μl of blood collected from malaria-infected children atthe University College Hospital, Ibadan during the transmission season were analyzed by ELISA.Results: The mean absorbance values of IgG subclasses reactive against P. falciparum CSP appeared to be agedependent and ranged from 0.01 for IgG4 in younger children to 0.95 for IgG3 in older children. The sixty-sevensubjects investigated in this study had significantly higher mean IgG1 and IgG3 than the uninfected controls(p IgG1>IgG2>IgG4 which confirmed the prevalence of the cytophilicantibodies (IgG1 and IgG3 in 65% of the malaria infected children over the non-cytophilic subclasses (IgG2and IgG4. Similarly, there was low production of IgG4 and IgG2 levels in 35% of the subjects compared withcontrol. IgG was detected in the serum of North American Subjects (NAS which served as negative control forCSP-specific IgG subclasses. Although the NAS titre was lower than that of the malaria subjects in Nigeria, itsIgG2 was, however, higher (0.16 than that of other subclasses. The mean absorbance values of total serum IgGsubclass were higher than those of IgG subclasses specific to P. falciparum circumsporozoite antigen. The meanabsorbance values of the total serum IgG subclass follows the order IgG2>IgG1>IgG4>IgG3.Interpretation & conclusion: Age and gender-dependent correlations of results suggest that acquired immunitycould play a significant role in protection from malaria. Antibody levels are higher in male than female childrenof the same age group. Antibody levels also

  20. Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China.

    Science.gov (United States)

    Lu, Shuai; Chen, Yingzhu; Qin, Kun; Zhou, Jianfang; Lou, Yongliang; Tan, Wenjie

    2016-06-01

    To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed. PMID:27084706

  1. Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

    Science.gov (United States)

    Nagalingam, Mohandoss; Thirumalesh, Sushma Rahim Assadi; Kalleshamurthy, Triveni; Niharika, Nakkala; Balamurugan, Vinayagamurthy; Shome, Rajeswari; Sengupta, Pinaki Prasad; Shome, Bibek Ranjan; Prabhudas, Krishnamsetty; Rahman, Habibur

    2015-10-01

    This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population. PMID:26065562

  2. Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

    Science.gov (United States)

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-05-01

    Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta. PMID:8705668

  3. Use of recombinant gonadotropin-releasing hormone antigens for immunosterilization of beef heifers.

    Science.gov (United States)

    Geary, T W; Grings, E E; MacNeil, M D; de Avila, D M; Reeves, J J

    2006-02-01

    The objectives of this study were to evaluate the effects of immunization against recombinant GnRH fusion proteins and growth promotants on onset of puberty, feedlot performance, and carcass characteristics of beef heifers. Heifers were immunized against an ovalbumin fusion protein containing 7 GnRH peptides (oGnRH, n = 12), a thioredoxin fusion protein containing 7 GnRH peptides (tGnRH, n = 12), a combination of oGnRH plus tGnRH (otGnRH, n = 12), or a combination of ovalbumin and thioredoxin (control, n = 11). Each heifer received a primary immunization containing 1 mg of protein in 1 mL of adjuvant injected into the mammary gland at wk 0 (mean age = 38 wk) and booster immunizations at wk 6 and 12. Six heifers within each treatment received Synovex H implants at wk -2. Weekly blood samples were collected from wk -2 to 26 for determination of serum progesterone concentrations and GnRH antibody titers. In GnRH-immunized heifers, GnRH antibody titers increased after the first booster injection, peaked after the second booster injection, and remained elevated through the end of the study (P spaying of heifers. PMID:16424262

  4. The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

    OpenAIRE

    Raquel de Andrade Lima Coêlho; Hiroshi Yamasaki; Emília Perez; Luiz Bezerra de Carvalho Jr

    2003-01-01

    Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparabl...

  5. Establishment of an in vivo potency assay for the recombinant hepatit is B surface antigen in monovalent and combined vaccines

    Directory of Open Access Journals (Sweden)

    Mabel Izquierdo-López

    2014-12-01

    Full Text Available In this paper the development of potency assay in animals (mice was made, with the objective of demonstrating the immunogenic power of the recombinant Hepatitis B surface antigen in monovalent and combined vaccines, produced at the Center of Genetic Engineering and Biotechnology. The potency test is a parameter in quality control and it is also a tool to demonstrate the consistency of the production process. Parameters such as duration of the test, number of animals in the test, as well as different areas for the maintenance of the animals were evaluated. The results on the applicability of the potency test, to two presentations of the vaccines; monovalent Heberbiovac HB and pentavalent liquid in one vial Heberpenta-L are shown, for which specificity studies, evaluating different vaccine lots, the behavior of linearity, and parallelism, as well as establishing quality specification of the test were performed. This assay led to the obtainment of reliable results for the vaccines evaluated, the consistent evaluation of the immunogenic power and the monitoring of different production processes.

  6. Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

    Directory of Open Access Journals (Sweden)

    Rodríguez Islay

    2002-01-01

    Full Text Available A recombinant-antigen enzyme immunoassay (EIA, BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38, secondary (n = 10, early latent (n = 28 and congenital syphilis (n = 2, patients with leptospirosis ( n= 8, infectious mononucleosis (n = 7, hepatitis (n = 9, diabetes mellitus (n = 11, rheumatoid arthritis (n = 13, leprosy (n = 11, tuberculosis (n = 9, HIV/Aids ( n= 12, systemic lupus erythematosus (n = 4, rheumatic fever (n = 3, old-persons (n = 9, pregnant women (n = 29 and blood donors (n = 164. The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  7. Phase I Study of Safety and Immunogenicity of an Escherichia coli-Derived Recombinant Protective Antigen (rPA) Vaccine to Prevent Anthrax in Adults

    OpenAIRE

    Brown, Bruce K.; Josephine Cox; Anita Gillis; VanCott, Thomas C.; Mary Marovich; Mark Milazzo; Tanya Santelli Antonille; Lindsay Wieczorek; Mckee, Kelly T.; Karen Metcalfe; Mallory, Raburn M.; Deborah Birx; Polonis, Victoria R.; Merlin L Robb

    2010-01-01

    BACKGROUND: The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). METHODOLOGY/P...

  8. Oral Immunization with Recombinant Mycobacterium smegmatis Expressing the Outer Membrane Protein 26-Kilodalton Antigen Confers Prophylactic Protection against Helicobacter pylori Infection ▿ †

    OpenAIRE

    Lü, Lin; Zeng, Han-qing; Wang, Pi-Long; Shen, Wei; Xiang, Ting-xiu; Mei, Zhe-chuan

    2011-01-01

    Helicobacter pylori infection is prevalent worldwide and results in chronic gastritis, which may lead to gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. We have previously reported that oral immunization with recombinant Mycobacterium smegmatis expressing the H. pylori outer membrane protein 26-kilodalton (Omp26) antigen affords therapeutic protection against H. pylori infection in mice. In the present study, we investigated the prophylactic effects of this vaccine cand...

  9. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

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    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  10. Detection of IgG antibody against Crimean-Congo haemorrhagic fever virus using ELISA with recombinant nucleoprotein antigens from genetically diverse strains.

    Science.gov (United States)

    Rangunwala, A; Samudzi, R R; Burt, F J

    2014-10-01

    Crimean-Congo haemorrhagic fever virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Handling the virus requires biosafety level-4 facilities, limiting accessibility for many laboratories. Advances in molecular techniques have allowed preparation of safe recombinant antigens that have application in diagnosis and serosurveillance of CCHFV. The aim of this study was to determine genetic diversity in CCHFV based on all available complete sequence data for the S gene encoding CCHFV nucleoprotein (NP) and antibody cross-reactivity between the NP of a South African isolate and the NP of a Greek isolate (AP92), the most genetically diverse CCHFV strain. The nucleotide sequence diversity and amino-acid diversity between genotypes, within genotypes and the pairwise distances were calculated for a dataset of 45 CCHFV isolates retrieved from GenBank. The most diverse virus, AP92, isolated from a tick in Greece, displayed the highest amino-acid difference (8·7%) with SPU415/85, isolated from a human infection in South Africa. Recombinant NP encoded for by codon-optimized S genes of SPU415/85 and AP92 were expressed in a bacterial host system and used to develop an in-house ELISA to detect IgG antibody against CCHFV in South African patients who survived infection. A total of 14/14 sera reacted with the South African recombinant NP and 13/14 reacted with the Greek recombinant NP. The serological cross-reactivity of the two NP antigens suggests that recombinant antigens prepared from geographically distinct CCHFV will have diagnostic and epidemiological applications worldwide. PMID:24330947

  11. Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

    Science.gov (United States)

    Virginio, Veridiana Gomes; Gonchoroski, Taylor; Paes, Jéssica Andrade; Schuck, Desirée Cigaran; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2014-10-01

    Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP. PMID:25148775

  12. The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

    Directory of Open Access Journals (Sweden)

    Raquel de Andrade Lima Coêlho

    2003-04-01

    Full Text Available Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

  13. 重组抗原在弓形虫病诊断中的应用%Use of recombinant antigens to diagnose toxoplasmosis

    Institute of Scientific and Technical Information of China (English)

    黄泽智; 赵晋英; 李艳伟; 侯玉英

    2011-01-01

    Toxoplasma gondii infection can cause severe harm. Thus, highly sensitive and highly specific detection of T. Gondii infection is crucial to the treatment of toxoplasmosis. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to Toxoplasma antigens in a patient's serum sample. Most of the commercially available tests use T. Gondii native antigens and vary widely in accuracy. Recombinant antigens have been developed as diagnostic reagents for assays to detect toxoplasmosis. Thus, this review discusses recent use of recombinant antigens to diagnose toxoplasmosis.%刚地弓形虫感染危害严重,研究敏感性高和特异性好的弓形虫感染检测方法对于弓形虫病的诊断至关重要.弓形虫病的诊断一般依赖于检测患者血清中弓形虫特异性IgM和IgG.目前,商业试剂盒大多采用天然抗原,其检测精确性较差.重组抗原已经被发展用来诊断弓形虫病,且具有潜在的应用价值.本综述主要介绍重组抗原在弓形虫病诊断中的应用.

  14. Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved Influenza A antigens.

    Science.gov (United States)

    Antrobus, Richard D; Coughlan, Lynda; Berthoud, Tamara K; Dicks, Matthew D; Hill, Adrian Vs; Lambe, Teresa; Gilbert, Sarah C

    2014-03-01

    Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection. PMID:24374965

  15. The use of /sup 125/I recombinant DNA/sub 125/ derived human erythropoietin (R-HuEPO) as a replacement for /sup 125/I human urinary epo as tracer antigen in a radioimmunoassay for human epo

    International Nuclear Information System (INIS)

    This paper represents evidence that in a radioimmunoassay for human erythropoietin, recombinant DNA derived human erythropoietin can replace highly purified human urinary erythropoietin in the preparation of radioiodinated tracer antigen

  16. Immunoreactivity Analysis of Toxoplasma gondii Recombinant Antigen rSAG3 in Sera from Immunized BALB/c Mice and Toxoplasmosis Patients

    Science.gov (United States)

    MOTAZEDIAN, Mohammad Hossein; KAZEMI, Bahram; SHAHRIARI, Bahador; BANDEHPOUR, Mojgan; KHANALIHA, Khadijeh

    2016-01-01

    Background: The coccidian protozoa Toxoplasma gondii is an obligate intracellular parasite of humans and other warm-blooded animals. Diagnosis of toxoplasmosis is of considerable medical importance for human, especially pregnant women and immunocompromised individuals. The apply of an Escherichia coli recombinant antigen(s) would be significantly useful in developing standardization of the diagnostic tests and reducing their costs. In this study, immunoreactivity of recombinant SAG3 against sera from immunized mice and human anti-T. gondii IgG positive patients was evaluated by western-blotting and enzyme immunoassay (EIA) in Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences in 2013. Methods: Three inbreed BALB/c female mice were obtained. Two mice were injected with rSAG3 and one was remained untreated, as control. Sera from immunized mice and also pooled sera from IgG positive toxoplasmosis cases were evaluated with western-blotting. IgG antibody responses to recombinant SAG3 was measured by indirect ELISA against the negative control group. Results: The rSAG3 protein reacted with sera of immunized mice and sera from patients with anti-Toxoplasma IgG antibodies in western-blot analysis. The result of ELISA showed that, there was marked differences in the absorbance values between the recombinant SAG3 immunized mice and control group. Conclusion: The rSAG3 showed IgG reactivity with sera from immunized mice and anti-Toxoplasma IgG patients.

  17. Detection of GAD-Ab index in diabetic patients using 35S labeled recombinant human GAD65 antigen

    International Nuclear Information System (INIS)

    Objective: To establish a novel method for measuring glutamic acid decarboxylase autoanti-bodies(GAD-Ab). Methods: Recombinant human GAD65 was used as the antigen, in vitro transcribed and translated 35S-GAD65 as the tracer, a self-designed rotating incubation apparatus as the incubator, protein-A sepharose as the precipitator, and the liquid scintillation counter was used to measure radioactive count value to detect GAD-Ab. The positive cut-off point of GAD-Ab index was determined as > 0.05 by the 99.5% percentile in 109 healthy individuals. GAD-Ab levels were determined in 43 type 1 and 226 type 2 diabetic patients. Results: The optimized working conditions included SJ1515 35S-methionine for in vitro transcription and translation, 20-30 r/min setup of rotating incubation apparatus, test temperature 4-25 degree C, freshly prepared buffer of pH 7.2-7.4, and horizontal rotor centrifuge. The new method was better than original one, with intra-assay CV of 4.9%-8.3% and inter-assay CV of 7.1%-10.8 %, specificity of 98.2%. The results were comparable with the figures issued by an international standardized laboratory (concordance was 98.3%, Kappa value 0.971). The positive rate of GAD-Ab was 58.1% (25 of 43) in type 1 and 10.2%(23 of 226) in type 2 diabetes patients, but only 1.8% (2 of 109) in healthy individuals. Conclusion: The new assay for GAD-Ab is a highly sensitive, accurate, specific and reproducible method for clinical use

  18. Silencing of T lymphocytes by antigen-driven programmed death in recombinant adeno-associated virus vector–mediated gene therapy

    Science.gov (United States)

    Velazquez, Victoria M.; Bowen, David G.

    2009-01-01

    Recombinant adeno-associated virus (rAAV) vectors are considered promising for human gene replacement because they facilitate stable expression of therapeutic proteins in transduced tissues. Whether the success of gene therapy will be influenced by cellular immune responses targeting transgene-encoded proteins that are potentially immunogenic is unknown. Here we characterized CD8+ T-cell activity against β-galactosidase and enhanced green fluorescent protein, model antigens containing major histocompatibility complex (MHC) class I epitopes that are constitutively produced in murine skeletal muscle after rAAV vector transduction. Antigen-specific CD8+ T cells were detected in the spleen and liver of mice within 7 days of muscle transduction. CD8+ T-cell frequencies in these organs were stable, and effector functions were intact for months despite ongoing antigen production in muscle. CD8+ T cells also infiltrated transduced muscle, where frequencies were at least 5-fold higher than in untransduced spleen and liver. Significantly, the majority of antigen-specific CD8+ T cells in vector-transduced muscle were not functional. Loss of function in the muscle was associated with programmed death of the effector cells. Stable gene expression therefore depended on selective death of CD8+ T cells at the site of antigen production, an effective mechanism for subverting immunity that is also potentially reversible. PMID:18566327

  19. Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

    Science.gov (United States)

    Liang, Jinping; Teng, Xindong; Yuan, Xuefeng; Zhang, Ying; Shi, Chunwei; Yue, Tingting; Zhou, Lei; Li, Jianrong; Fan, Xionglin

    2015-08-01

    Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation. PMID:25974877

  20. Control of ticks resistant to immunization with Bm86 in cattle vaccinated with the recombinant antigen Bm95 isolated from the cattle tick, Boophilus microplus.

    Science.gov (United States)

    García-García, J C; Montero, C; Redondo, M; Vargas, M; Canales, M; Boue, O; Rodríguez, M; Joglar, M; Machado, H; González, I L; Valdés, M; Méndez, L; de la Fuente, J

    2000-04-28

    The recombinant Bm86-containing vaccine Gavac(TM) against the cattle tick Boophilus microplus has proved its efficacy in a number of experiments, especially when combined with acaricides in an integrated manner. However, tick isolates such as the Argentinean strain A, show low susceptibility to this vaccine. In this paper we report on the isolation of the Bm95 gene from the B. microplus strain A, which was cloned and expressed in the yeast Pichia pastoris producing a glycosylated and particulated recombinant protein. This new antigen was effective against different tick strains in a pen trial, including the B. microplus strain A, resistant to vaccination with Bm86. A Bm95-based vaccine was used to protect cattle against tick infestations under production conditions, lowering the number of ticks on vaccinated animals and, therefore, reducing the frequency of acaricide treatments. The Bm95 antigen from strain A was able to protect against infestations with Bm86-sensitive and Bm86-resistant tick strains, thus suggesting that Bm95 could be a more universal antigen to protect cattle against infestations by B. microplus strains from different geographical areas. PMID:10717348

  1. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    OpenAIRE

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length ...

  2. Isolation and Evaluation of Specific Human Recombinant Antibodies from a Phage Display Library against HER3 Cancer Signaling Antigen

    Directory of Open Access Journals (Sweden)

    Foroogh Nejatollahi

    2014-07-01

    Full Text Available Background: The human epidermal growth factor receptor family comprises four homologous members: EGFR (ErbB1, ErbB2 (HER2, ErbB3 (HER3 and ErbB4 (HER4.This family plays an important role in the signaling pathway and cell proliferation. The heterodimerization of HER2 with HER3 leads to tumor cell proliferation. Monoclonal antibody to the human HER3 receptor blocks HER3 heterodimerization and inhibits the growth of breast cancer cells. Due to their human origin, small size, rapid penetration and high affinity properties, recombinant single chain antibodies (scFv have been introduced as the most desired agents for cancer immunotherapy. In this study, we use a phage display system to select specific scFvs against HER3 for their use in cancer targeted therapy. Methods: A phage antibody display library of scFv was panned against an immunodominant epitope of HER3. Phage rescue was performed on the library. The supernatant that contained the appropriate scFv (109 PFU/ml was added to an immunotube which was coated with the peptide. Elution was done using log phase E. coli TG1. The clones were amplified by PCR and DNA fingerprinted to select the specific clones against the epitope. The specificity of the selected antibodies was tested in ELISA. Results: The results represented two predominant patterns with the frequency of 25%. The other patterns showed the frequencies of 5%-10%. scFv1 and scFv2 demonstrated positive ELISA with absorbances of 0.63 and 0.46, respectively while the absorbances of wells without peptide were 0.19 and 0.11, respectively. Conclusion: In this study two specific scFvs were selected against HER3 antigen in a successful panning process. Phage ELISA represented the specific binding of scFvs against HER3.The selected scFvs reacted only with the corresponding peptides. However, no reaction with the other peptides was detected. The selected anti-HER3 scFvs have suggested that these human high affinity and small antibodies that bind

  3. Vaccination with replication-deficient recombinant adenoviruses encoding the main surface antigens of toxoplasma gondii induces immune response and protection against infection in mice.

    Science.gov (United States)

    Caetano, Bráulia C; Bruña-Romero, Oscar; Fux, Blima; Mendes, Erica A; Penido, Marcus L O; Gazzinelli, Ricardo T

    2006-04-01

    We have generated recombinant adenoviruses encoding three genetically modified surface antigens (SAGs) of the parasite Toxoplasma gondii, that is, AdSAG1, AdSAG2, and AdSAG3. Modifications included the removal of their glycosylphosphatidylinositol (GPI) anchoring motifs and, in some cases, the exchange of the native signal peptide for influenza virus hemagglutinin signal sequence. Adenovirus immunization of BALB/c mice elicited potent antibody responses against each protein, displaying a significant bias toward a helper T cell type 1 (Th1) profile in animals vaccinated with AdSAG1. Furthermore, the presence of parasite-specific IFN-gamma-producing T cells was analyzed by proliferation assays and enzyme-linked immunospot assays in the same animals. Splenocytes from immunized mice secreted IFN-gamma after in vitro stimulation with tachyzoite lysate antigen or with a fraction enriched for membrane-purified GPI-anchored proteins (F3) from the T. gondii tachyzoite surface. Epitopes recognized by CD8+ T cells were identified in SAG1 and SAG3, but not SAG2, sequences, although this protein also induced a specific response. We also tested the capacity of the immune responses detected to protect mice against a challenge with live T. gondii parasites. Although no protection was observed against tachyzoites of the highly virulent RH strain, a significant reduction in cyst loads in the brain was observed in animals challenged with the P-Br strain. Thus, up to 80% of the cysts were eliminated from animals vaccinated with a mixture of the three recombinant viruses. Because adenoviruses seemed capable of inducing Th1-biased protective immune responses against T. gondii antigens, other parasite antigens should be tested alone or in combination with those described here to further develop a protective vaccine against toxoplasmosis. PMID:16610929

  4. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    Directory of Open Access Journals (Sweden)

    Jake E Lowry

    Full Text Available Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA. All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence

  5. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    Science.gov (United States)

    Lowry, Jake E; Isaak, Dale D; Leonhardt, Jack A; Vernati, Giulia; Pate, Jessie C; Andrews, Gerard P

    2011-01-01

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of

  6. Diagnostic Value of Proteins of Three Borrelia Species (Borrelia burgdorferi Sensu Lato) and Implications for Development and Use of Recombinant Antigens for Serodiagnosis of Lyme Borreliosis in Europe

    OpenAIRE

    Hauser, Ulrike; Lehnert, Gisela; Wilske, Bettina

    1998-01-01

    More and more assays for the serodiagnosis of Lyme borreliosis (LB) are based on recombinant antigens. However, so far, there is no consensus as to which are the most specific and sensitive proteins and how they should be used in combination to obtain tests with the best discrimination abilities. The present study was preceded by a detailed analysis of Western blots (WB) using whole-cell lysates of Borrelia burgdorferi sensu stricto strain PKa2, B. afzelii PKo, and B. garinii PBi (U. Hauser, ...

  7. Development and Clinical Evaluation of a Recombinant-Antigen-Based Cytomegalovirus Immunoglobulin M Automated Immunoassay Using the Abbott AxSYM Analyzer

    OpenAIRE

    Maine, G T; Stricker, R; Schuler, M.; Spesard, J.; Brojanac, S.; Iriarte, B.; Herwig, K.; Gramins, T.; Combs, B.; Wise, J.; Simmons, H.; Gram, T.; Lonze, J.; Ruzicki, D.; Byrne, B

    2000-01-01

    A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1,608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical condit...

  8. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites

    OpenAIRE

    Michal Sima; Blanka Ferencova; Alon Warburg; Iva Rohousova; Petr Volf

    2016-01-01

    Background Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replaceme...

  9. Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23)

    OpenAIRE

    Montserrat Cuquerella; José María Alunda; Concepción de la Fuente; Enrique Martínez; Francisco Angulo-Cubillán; Basilio Valladares; Leticia García-Coiradas

    2010-01-01

    Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein...

  10. Influence of culture medium on the production of eif antigen from Leishmania chagasi in recombinant Escherichia coli

    OpenAIRE

    Vaz, Michelle Rossana Ferreira; de França, Ricardo Luiz Soares; de Andrade, Sirtys Santos Lessa; Sousa Junior, Francisco Canindé de; dos Santos, Everaldo Silvino; Martins, Daniella Regina Arantes; Macedo, Gorete Ribeiro de

    2011-01-01

    With the advent of recombinant DNA technology, recombinant protein expression has become an important tool in the study of the structure, function and identification of new proteins, especially those with therapeutic functions. Escherichia coli has been the predominant prokaryote used in genetic engineering studies due to the abundance of information about its metabolism. Despite significant advances in molecular biology and immunology of infections, there are as yet no prophylactic drugs cap...

  11. Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies.

    Science.gov (United States)

    Ridgwell, K; Dixey, J; Scott, M L

    2007-10-01

    The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms. PMID:17725551

  12. Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice.

    Science.gov (United States)

    Sabio y García, Julia V; Bigi, Fabiana; Rossetti, Osvaldo; Campos, Eleonora

    2010-12-01

    Immunodominant MPB83 antigen from Mycobacterium bovis was expressed as a chimeric protein fused to either β-galactosidase, outer membrane lipoprotein OMP19 or periplasmic protein BP26 in gram-negative Brucella abortus S19, in all cases driven by each gene's own promoter. All fusion proteins were successfully expressed and localized in the expected subcellular fraction. Moreover, OMP19-MPB83 was processed as a lipoprotein when expressed in B. abortus. Splenocytes from BALB/c mice immunized with the recombinant S19 strains carrying the genes coding for the heterologous antigens in replicative plasmids, showed equally specific INF-γ production in response to MPB83 stimulation. Association to the lipid moiety of OMP19 presented no advantage in terms of immunogenicity for MPB83. In contrast, fusion to BP26, which was encoded by an integrative plasmid, resulted in a weaker immune response. None of the constructions affected the survival rate or the infection pattern of Brucella. We concluded that B. abortus S19 is an appropriate candidate for the expression of M. bovis antigens both associated to the membrane or cytosolic fraction and may provide the basis for a future combined vaccine for bovine brucellosis and tuberculosis. PMID:20888425

  13. Cytokine switch and bystander suppression of autoimmune responses to multiple antigens in experimental autoimmune encephalomyelitis by a single recombinant T-cell receptor ligand.

    Science.gov (United States)

    Sinha, Sushmita; Subramanian, Sandhya; Miller, Lisa; Proctor, Thomas M; Roberts, Chris; Burrows, Gregory G; Vandenbark, Arthur A; Offner, Halina

    2009-03-25

    Recombinant T-cell receptor ligands (RTLs) can reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner, and are currently in clinical trials for treatment of subjects with multiple sclerosis (MS). Antigen specificity of RTL raises the question as to whether this treatment would be successful in MS patients where target antigens are unknown. Using spinal cord homogenate or combinations of two different peptides to induce disease, we found that treatment with single RTL could reverse EAE as long as targeted T-cells were present. Therapy with three different RTLs each caused a significant reduction in IL-17 and increases in IL-10 and IL-13 in peptide-activated splenocytes, reduced proliferation of both cognate and bystander specificities of lymph node cells, and reduced inflammatory lesions and secreted IL-17 and IL-2 from peptide-activated spinal cord cells. These results show that treatment with single RTLs can induce a cytokine switch in cognate T-cells that inhibits both the target and bystander T-cells, providing new evidence for the potential applicability of RTL therapy in MS. PMID:19321778

  14. Development of a Recombinant Cell-Based Indirect Immunofluorescence Assay for the Determination of Autoantibodies against Soluble Liver Antigen in Autoimmune Hepatitis

    Directory of Open Access Journals (Sweden)

    Christiane Radzimski

    2013-01-01

    Full Text Available Autoantibodies against soluble liver antigen (SLA are specific markers for autoimmune hepatitis (AIH type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA, detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors. Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

  15. The Immunomodulatory Role of Adjuvants in Vaccines Formulated with the Recombinant Antigens Ov-103 and Ov-RAL-2 against Onchocerca volvulus in Mice.

    Directory of Open Access Journals (Sweden)

    Jessica A Hess

    2016-07-01

    Full Text Available In some regions in Africa, elimination of onchocerciasis may be possible with mass drug administration, although there is concern based on several factors that onchocerciasis cannot be eliminated solely through this approach. A vaccine against Onchocerca volvulus would provide a critical tool for the ultimate elimination of this infection. Previous studies have demonstrated that immunization of mice with Ov-103 and Ov-RAL-2, when formulated with alum, induced protective immunity. It was hypothesized that the levels of protective immunity induced with the two recombinant antigens formulated with alum would be improved by formulation with other adjuvants known to enhance different types of antigen-specific immune responses.Immunizing mice with Ov-103 and Ov-RAL-2 in conjunction with alum, Advax 2 and MF59 induced significant levels of larval killing and host protection. The immune response was biased towards Th2 with all three of the adjuvants, with IgG1 the dominant antibody. Improved larval killing and host protection was observed in mice immunized with co-administered Ov-103 and Ov-RAL-2 in conjunction with each of the three adjuvants as compared to single immunizations. Antigen-specific antibody titers were significantly increased in mice immunized concurrently with the two antigens. Based on chemokine levels, it appears that neutrophils and eosinophils participate in the protective immune response induced by Ov-103, and macrophages and neutrophils participate in immunity induced by Ov-RAL-2.The mechanism of protective immunity induced by Ov-103 and Ov-RAL-2, with the adjuvants alum, Advax 2 and MF59, appears to be multifactorial with roles for cytokines, chemokines, antibody and specific effector cells. The vaccines developed in this study have the potential of reducing the morbidity associated with onchocerciasis in humans.

  16. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S; Blancher, A; Dickmeiss, E; Engberg, J

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  17. Use of Novel Recombinant Antigens in the Interferon Gamma Assay for Detection of Mycobacterium Avium Subsp. Paratuberculosis Infection in Cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose;

    . The study included blood samples from 26 heifers of a MAP infected herd, collected three times with 4 and 5 week interval and blood samples from 60 heifers of a MAP non-infected herd collected once. The IFN-γ responses of the non-infected heifers were used to establish cut-off values for each antigen...

  18. Extensively variable surface antigens of Sarcocystis spp. infecting Brazilian marsupials in the genus Didelphis occur in myriad allelic combinations, suggesting sexual recombination has aided their diversification.

    Science.gov (United States)

    Monteiro, R M; Keid, L B; Richtzenhain, L J; Valadas, S Y; Muller, G; Soares, R M

    2013-09-01

    Sarcocystis neurona and Sarcocystis falcatula are very similar species of Apicomplexan protozoa that use marsupials of the genus Didelphis as definitive hosts. These mammals can serve as definitive hosts not only for these two parasites, but for other Sarcocystis such as Sarcocystis speeri and Sarcocystis lindsayi. Sarcocystis shed by opossums (with the exception of S. neurona) can cause disease in a great variety of birds, being commonly associated with acute pulmonary sarcocystosis in zoos. S. neurona is the most commonly associated parasite with the equine protozoal myeloencephalitis in horses. Herein we assessed the variability of Sarcocystis spp. isolated from opossums of the state of Rio Grande do Sul, Brazil, by sequencing fragments of genes coding for glycosylphosphatidylinositol-anchored surface antigens (termed surface antigen or SAG), SAG2, SAG3 and SAG4. Two genetic groups were identified, one of them related to S. falcatula and the other related to S. neurona. Various allelic combinations of SAG2, SAG3 and SAG4 occur among S. falcatula related isolates and strong evidences suggest that such isolates may exchange high divergent alleles in possible sexual recombination processes. Regarding the group S. neurona-like (isolates G37 and G38), none of the individuals in this group share alleles with individuals of the other group. Comparing G37 and G38 strains and North American strains of S. neurona, four polymorphisms were identified at SAG-3, five at SAG-2 and three at SAG-4. Gene sequences of locus SAG-3 from isolates G37 and G38 differed from the other sequences by an insertion 81bp long. This insertion contains several dinucleotide repeats of AT, resembling a microsatellite locus and has already been detected in SAG3 sequences of S. neurona from North America. When aligned against North American strains of S. neurona, G37 and G38 isolates have a deletion of 8 nucleotides within this intron which indicate that S. neurona strains of South America are

  19. Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

    Directory of Open Access Journals (Sweden)

    Xiaodong Weng

    2011-03-01

    Full Text Available Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA. Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4% and CD86 (80.13 ± 2.81%] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL and IL-12 (249.57 ± 12.51 pg/mL production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05 than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018, indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

  20. Production and Evaluation of Toxoplasma gondii Recombinant Sur­face Antigen 1 (SAG1 for Serodiagnosis of Acute and ChronicToxop­lasma Infection in Human Sera

    Directory of Open Access Journals (Sweden)

    M(Monavar Selseleh

    2012-09-01

    Full Text Available Background: The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis. Methods: This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii (RH Strain was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D - thiogalactopyrano­side (IPTG. A total of 204 sera were tested using a commercial IgG and IgM ELISA kit (Trinity, USA as gold standard prior to testing them with the recombinant antigen. Results: Tested sera were divided into the following groups:(a The 74 T. gondii IgG positive (b 70 T.gondii IgM positive (c 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis (N=5, malaria (N=14, leishmania­sis (N=7,fasciolasis (N=4 , sterengyloidiasis (N=1 . Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (Com ELISA were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively. Conclusion: The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis.

  1. Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum

    OpenAIRE

    Mohamad, Khalil Yousef; Rekiki, Abdessalem; Berri, Mustapha; Rodolakis, Annie

    2010-01-01

    Abstract Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected w...

  2. Onchocerciasis hyperendemic in the Unturán Mountains: the value of recombinant antigens in describing a new transmission area in southern Venezuela.

    Science.gov (United States)

    Botto, C; Gillespie, A J; Vivas-Martínez, S; Martínez, N; Planchart, S; Basáñez, M G; Bradley, J E

    1999-01-01

    A recently described hyperendemic onchocerciasis area, located in the Unturán Mountains (between the Siapa and Orinoco basins) of southern Venezuela was studied using a cocktail of 3 low molecular weight onchocercal recombinant antigens (OvMBP/10, OvMBP/11, and OvMBP/29). The resulting seroepidemiological data were compared with those from a hypoendemic community (Altamira) situated in the northern coastal mountain range. Parasitological (skin biopsy) and serological (enzyme-linked immunosorbent assay, ELISA) methods for the specific diagnosis of Onchocerca volvulus in these 2 very different endemic areas were, respectively, 88% and 96% sensitive in Unturán, and 57% and 91% sensitive in Altamira. The mean microfilarial load, the mean optical density (OD), and the seropositivity rates all increased significantly with age in both communities. The serological variables (mean OD and prevalence of anti-O. volvulus antibodies) were both significantly higher in Unturán than in Altamira for children and young adults (aged Altamira for the same age-class. The prevalence of specific antibodies (mainly a marker of exposure to risk of infection) exceeded 85% in the remaining age-categories at the hyperendemic area. This is in agreement with the high community microfilarial load recorded in Unturán (> 20 mf/mg) and the presence of sclerosing keratitis and hanging groin, suggesting that onchocerciasis is a public health problem in this community. The ELISA test used here, based on a cocktail of 3 low molecular weight onchocercal recombinant antigens, appears, therefore, to constitute a practical tool for the description of endemicity levels in remote areas, particularly given the fact that finger-prick blood samples are routinely taken from children in the Upper Orinoco region for surveys of malaria incidence. Such studies could aid in defining the true extent of the Amazon focus (still unknown) and providing priority indicators for the selection of communities where

  3. Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice

    Institute of Scientific and Technical Information of China (English)

    WANG Li-mei; SHI Chang-hong; FAN Xiong-lin; XUE Ying; BAI Yin-lai; XU Zhi-kai

    2007-01-01

    Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.Methods Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 μl normal saline containing 106 CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses.Results There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P<0.05). The elicited IFN-γ level of rBCG group was (1993±106) pg/ml, which was also significantly higher than that in BCG group ((1463±105) pg/ml, P<0.05). The splenocyte proliferation index of rBCG group reached 4.34±0.31, which was higher than that of BCG group (3.79±0.24, P<0.05).Conclusion rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.

  4. Development of Recombinant Flagellar Antigens for Serological Detection of Salmonella enterica Serotypes Enteritidis, Hadar, Heidelberg, and Typhimurium in Poultry

    OpenAIRE

    Hofacre, Charles L.; Peter S. Holt; Lee, Margie D.; Susan Sanchez; Joseph Minicozzi; Maurer, John J.

    2013-01-01

    Accurate and fast detection of harmful Salmonella is a major concern of food safety. Common Salmonella serotypes responsible for human associated foodborne outbreaks are S. Enteritidis, S. Hadar, S. Heidelberg, and S. Typhimurium are also commonly isolated from poultry. Serology is commonly used to monitor disease in poultry, therefore application of Salmonella serotype-specific test will have added value in Salmonella surveillance or monitoring vaccine efficacy. Recombinant flagellins were p...

  5. Immuno-epidemiology of human Schistosoma haematobium infection: preferential IgG3 antibody responsiveness to a recombinant antigen dependent on age and parasite burden

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    Fernandez Cecilia

    2006-06-01

    Full Text Available Abstract Background Schistosomiasis is a major parasitic disease affecting over 200 million people in the developing world with a further 400 million people at risk of infection. The aim of this study was to identify a single antigen from adult Schistosoma haematobium worms and subsequently use this antigen to study the development of schistosome-acquired immunity in a human population. Methods The full-length cDNA sequence of a S. haematobium protein, a putative orthologue of the S. mansoni tegumental antigen Sm13, was obtained from a cDNA library of adult S. haematobium worms and named Sh13 following a small-scale expressed sequence tags (EST project. The recombinant Sh13 protein expressed in E. coli, was used to investigate immuno-epidemiological patterns in 147 Zimbabweans (7–18 years old exposed to S. haematobium. Results Sequence analysis of the full-length cDNA sequence of the S. haematobium protein Sh13, indicated that the protein has an N-terminal signal peptide and encodes an 85-amino acid mature protein with a highly conserved predicted transmembrane domain (86 % identity with the S. mansoni tegumental antigen Sm13. The recombinant Sh13 protein was used in ELISA assays to determine the reactivity of sera from the study participants. Antibody responses against Sh13 were predominantly IgG3 isotype compared to responses against crude worm antigens which were predominantly IgG1 and IgG4. The relationship between anti-Sh13 IgG3 levels and infection intensity varied significantly with host age. The youngest children (7–10 years old had relatively low levels of both infection and anti-Sh13 IgG3. In older children (11–12 years old rising infection levels were accompanied by a significant increase in anti-Sh13 IgG3 levels. Subsequently, infection intensity declined significantly in 13–18 year olds but levels of the antibody continued to rise. The changing relationship between infection intensity and anti-Sh13 IgG3 levels with host age

  6. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    Directory of Open Access Journals (Sweden)

    Matthew D Reed

    Full Text Available Protective antigen (PA, one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax. Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel, elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  7. Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.

    Science.gov (United States)

    Gurramkonda, Chandrasekhar; Zahid, Maria; Nemani, Satish Kumar; Adnan, Ahmad; Gudi, Satheesh Kumar; Khanna, Navin; Ebensen, Thomas; Lünsdorf, Heinrich; Guzmán, Carlos A; Rinas, Ursula

    2013-12-01

    Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B. PMID:24141044

  8. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection...

  9. Evaluation of immune effect of recombinant fusion protein targeting the prostate stem cell antigen based on PSCA and HSP70

    Directory of Open Access Journals (Sweden)

    Lei DONG

    2014-10-01

    Full Text Available Objective To explore the immune effect and antitumor activity of recombinant prostate stem cell protein (PSCA and heat shock protein 70 (HSP70 in a murine model of prostate cancer. Methods Twenty-five healthy male C57BL/6 mice were randomly divided into 5 groups (5 each: PSCA, HSP, PSCA+HSP, PSCA-HSP and control group. Mice in the first 4 groups were vaccinated with the corresponding proteins, and those in control group were faked with injection of phosphate buffer saline (PBS. After immunization with recombinant proteins, the PSCA-specific cellular immune responses were monitored with ELISPOT, intracellular cytokine staining assay, and flow cytometry, and ELISA assay was used to detect humoral immune responses. The tumor growth and survival of vaccined mice were observed. Results ELISPOT revealed that the mice in PSCA-HSP group generated much more IFN-γ spot-forming cells than those in other groups (P<0.05, and they could generate strong anti-PSCA antibody response. Results of flow cytometry showed that the number of CD8+/IFN-γ+ T cells was significantly higher in PSCAHSP group than that in other groups (P<0.05. ELISA results revealed that all the mice in PSCA, PSCA+HSP and PSCA-HSP group were induced to generate the PSCA-specific humoral immune response, and no statistical difference was found on the antibody levels among the three groups. Animal experiment showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. Conclusion HSP70 is a chaperone with significant effect for protein vaccines, and the recombinant fusion protein PSCA-HSP70 could be of potential value for prostate cancer treatment. DOI: 10.11855/j.issn.0577-7402.2014.09.08

  10. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

    OpenAIRE

    Both, L.; van Dolleweerd, C.; Wright, E.; Banyard, A. C.; Bulmer-Thomas, B.; Selden, D.; Altmann, F.; Fooks, A.R.; Ma, J. K.- C.

    2013-01-01

    Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed ...

  11. Evaluation of immune effect of recombinant fusion protein targeting the prostate stem cell antigen based on PSCA and HSP70

    OpenAIRE

    Dong, Lei; Liu, Juan; Ma, Hong-Yu; Shou-zhen QUAN; Jian-xiao LV; Ying LI; Zhu, Mei-Cai

    2014-01-01

    Objective To explore the immune effect and antitumor activity of recombinant prostate stem cell protein (PSCA) and heat shock protein 70 (HSP70) in a murine model of prostate cancer. Methods Twenty-five healthy male C57BL/6 mice were randomly divided into 5 groups (5 each): PSCA, HSP, PSCA+HSP, PSCA-HSP and control group. Mice in the first 4 groups were vaccinated with the corresponding proteins, and those in control group were faked with injection of phosphate buffer saline (PBS). After immu...

  12. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    OpenAIRE

    2014-01-01

    Virus-like particles (VLPs) of chimeric porcine circovirus type 2 (PCV2) were generated by replacing the nuclear localization signal (NLS; at 1–39 aa) of PCV2 capsid protein (Cap) with classical swine fever virus (CSFV) T-cell epitope (1446–1460 aa), CSFV B-cell epitope (693–716 aa) and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The ab...

  13. Evaluation of an ELISA using recombinant Ssλ20ΔB3 antigen for the serological diagnosis of Sarcoptes scabiei infestation in domestic and wild rabbits.

    Science.gov (United States)

    Casais, Rosa; Millán, Javier; Rosell, Joan Maria; Dalton, Kevin P; Prieto, José Miguel

    2015-12-15

    An ELISA, based on the Sarcoptes scabiei Ssλ20ΔB3 inmunodominant antigen, was evaluated for the detection of antibodies to S. scabiei in experimentally infested (n=10), farm (n=109), and wild (n=78) rabbit sera. The S. scabiei antigen Ssλ20ΔB3, a major structural protein present over the entire mite's body, was produced as a recombinant protein in Escherichia coli and purified for its use in the ELISA. The resulting ELISA showed, in experimentally infested domestic rabbits, detectable specific antibody responses (IgG) above the cut off level from week three post-infestation indicating that the assay is able to detect positive rabbits very early during the course of the infestation. The ELISA was validated on a panel of 109 domestic breeding rabbit sera collected from 26 Spanish farms, of which 41 were obtained from rabbits with skin lesions compatible with sarcoptic mange, 26 with skin lesions compatible with psoroptic mange, and 42 from unexposed individuals from mange-free farms. The ELISA in this group was characterized by 95% sensitivity, 97% specificity, and a high degree of repeatability. In the psoroptic mange compatible lesions group, included in the study as control group for cross-reactivity with the closely related mite Psoroptes cuniculi, cross-reacting antibodies to Ssλ20ΔB3 S. scabiei antigen were detected in 42.30% of the rabbit sera. However, mean% OD values of the sarcoptic-mange group (55.61 ± 39.20%) were significantly higher (pmange (3.64% ± 5.4%) and also of the free-mange (0.21% ± 0.67%) groups. In addition, the ELISA was also evaluated in serum samples obtained from both naturally infested and non-infested wild rabbits from Mallorca Island. The sensitivity of the assay for this group was 100% (4 out of the 4 rabbits with sarcoptic mange compatible lesions and presence of S. scabiei mites were seropositive) and the specificity was 90% (67 out of 74 wild rabbits without detectable mange lesions were seronegative). Although, the total

  14. Assessing drivers of the IgG4 antibody reactivity to recombinant antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa.

    Science.gov (United States)

    Damgaard, Johanne; Meyrowitsch, Dan W; Rwegoshora, Rwehumbiza T; Magesa, Stephen M; Mukoko, Dunstan A; Simonsen, Paul E

    2016-09-01

    A high proportion of the human population in lymphatic filariasis (LF) endemic areas is positive for filarial specific IgG4 antibodies, including many individuals without microfilariae (mf; circulating larvae in the human blood) or circulating filarial antigens (CFA; marker of adult worm infection). The antibodies are commonly regarded as markers of infection and/or exposure to filarial larvae, but a direct association between the antibodies and these indices has not been well documented. The present study assessed the role and relative effect of potential drivers of the human IgG4 antibody reactivity to the recombinant filarial antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa. Sera collected during previous studies from 395 well characterized individuals with regard to age, sex, mf, CFA, household vector biting and household exposure to infective filarial larvae were tested for IgG4 antibodies to Bm14, and associations between antibody reactivity and the different variables were statistically analyzed. IgG4 reactivity to Bm14 was highly positively associated with CFA, and to a lesser extent with age. However, an expected association with household exposure to infective filarial larvae was not found. Bm14 antibody reactivity thus appeared mainly to reflect actual infection of individuals with adult filarial worms rather than ongoing exposure to transmission. The analyses moreover suggested that many of the CFA negative but Bm14 positive individuals had early or low level infections where antibodies had been induced but where CFA was not (yet?) measurable. Although the study indicated that IgG4 reactivity to Bm14 is a marker of filarial infection, assessment of this reactivity, especially in children, will still be useful for indirect monitoring of changes in transmission intensity, including break of transmission and post-elimination surveillance, in LF control. PMID:27172877

  15. Immunogenicity and efficacy of recombinant 78 kDa antigen of Leishmania donovani formulated in various adjuvants against murine visceral leishmaniasis

    Institute of Scientific and Technical Information of China (English)

    Rajeev; Nagill; Tejinder; Kaur; Jyoti; Joshi; Sukhbir; Kaur

    2015-01-01

    Objective: To analyze the protective efficacy of recombinant 78 kDa antigen of Leishmania donovani in combination with two adjuvants, that is, cationic liposomes or MPL-A against visceral leishmaniasis in BALB/c mice. Methods: The genomic DNA of promastigotes was isolated and 583 bp of T cell epitopes of gene encoding 78 k Da was amplified using specific primers. The amplified gene was cloned into p ET28 c, transformed into Escherichia coli BL21(DE3) and got expressed after IPTG induction. The recombinant protein was then purified using Ni-NTA and named r78. Three groups of mice were immunized with 10 μg of r78 plus MPL-A, r78 encapsulated in positively charged liposomes and control animals immunized with PBS. Two booster doses were given with the respective vaccine at an interval of 2 weeks each. Mice were challenged with 1×107 Leishmania promastigotes and sacrificed on different post infection/challenge days. Results: Immunization with r78 along with MPL-A and liposomeencapsulated r78 brought a significant reduction in parasite load. In comparison to the infected controls, the parasite load declined by 96.2% in mice immunized with r78 plus MPL-A and 97.23% in animals immunized with liposome-encapsulated r78. The immunized animals also exhibited profound DTH response. The serum antibody responses increased from 15 to 90 days post infection/challenge. Immunized animals showed greater IgG2 a levels and lesser Ig G1 levels in comparison to the infected controls. The splenocytes from immunized mice were cultured, stimulated with r78 and analyzed for cytokine profile. The levels of IL-2 and IFN-γ were greater in immunized animals as compared to control mice. Conclusions: The study proves that r78 in combination with suitable adjuvants is a potential vaccine candidate and may be instrumental in control of visceral leishmaniasis.

  16. Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

    Directory of Open Access Journals (Sweden)

    Ross Anton

    2009-02-01

    Full Text Available Abstract Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs, an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.

  17. Simulation of control strategies for the cattle tick Boophilus microplus employing vaccination with a recombinant Bm86 antigen preparation.

    Science.gov (United States)

    Labarta, V; Rodríguez, M; Penichet, M; Lleonart, R; Luaces, L L; de la Fuente, J

    1996-05-01

    Current strategies for the control of the cattle tick Boophilus microplus include the use of chemicals as the principal control method. These methods, however, have met with partially successful results. The recent development of immunological methods for the control of the cattle tick has opened new possibilities for the design of control strategies. Employing the results obtained by us in experiments testing the effect of vaccination with the recombinant vaccine, Gavac (Heber Biotec S.A.), on tick populations, we have developed a model to evaluate, through a computer program, the efficacy of the vaccine as a control method. The action of the vaccine on the control of tick populations was simulated and the specific serum antibody titers required to decrease the tick population in the field were calculated. The specific serum antibody titer required to decrease the tick population in the field after the first vaccination scheme was found to be > or = 57,200 and the antibody titer required to maintain this effect when the vaccine is already acting and after successive revaccinations was found to be > or = 27,500. Considerations about revaccination schemes and combination between vaccination and acaricide treatments as possible control strategies are discussed. PMID:8792587

  18. Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.

    Science.gov (United States)

    Hartleben, Cláudia P; Leal, Fernanda M A; Monte, Leonardo G; Hartwig, Daiane D; Seixas, Fabiana K; Vasconcellos, Sílvio A; Brihuega, Bibiana; Dellagostin, Odir A

    2013-02-01

    Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys. PMID:23064970

  19. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

    Directory of Open Access Journals (Sweden)

    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  20. Bovine immunoprotection against Rhipicephalus (Boophilus) microplus with recombinant Bm86-Campo Grande antigen Imunoproteção de bovinos contra Rhipicephalus (Boophilus) microplus com antígeno recombinante Bm86-Campo Grande

    OpenAIRE

    Rodrigo Casquero Cunha; Adalberto Angel Pérez de León; Fábio Pereira Leivas Leite; Luciano da Silva Pinto; Alceu Gonçalves dos Santos Júnior; Renato Andreotti

    2012-01-01

    The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strai...

  1. Live recombinant Salmonella Typhi vaccines constructed to investigate the role of rpoS in eliciting immunity to a heterologous antigen.

    Science.gov (United States)

    Shi, Huoying; Santander, Javier; Brenneman, Karen E; Wanda, Soo-Young; Wang, Shifeng; Senechal, Patti; Sun, Wei; Roland, Kenneth L; Curtiss, Roy

    2010-01-01

    We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (chi9639 and chi9640) were derived from the rpoS mutant strain Ty2 and one (chi9633) from the RpoS(+) strain ISP1820. In chi9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS(+) vaccines induced a balanced Th1/Th2 immune response while the RpoS(-) strain chi9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS(+) strain chi9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, chi9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile

  2. Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.

    Directory of Open Access Journals (Sweden)

    Alexandra Schwarz

    Full Text Available BACKGROUND: Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. METHODOLOGY/PRINCIPAL FINDINGS: T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6 in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. CONCLUSIONS/SIGNIFICANCE: The recombinant rTiSP14.6 is a suitable and promising

  3. Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

    Institute of Scientific and Technical Information of China (English)

    XIN Jiu-qing; GAO Yun-long; LI Yuan; WANG Yan-fan; QIAN Ai-dong

    2007-01-01

    Mycoplasma mycoides subsp mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI Ⅹ strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL-1, and coated to microtiter enzyme-linked immunosorbent assay (ELISA) plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 (0.934/0.193), the sensitivity to be 95.8% (46/48), and the specificity to be 98.9% (161/163). 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

  4. Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA vaccine to prevent anthrax in adults.

    Directory of Open Access Journals (Sweden)

    Bruce K Brown

    Full Text Available BACKGROUND: The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA. METHODOLOGY/PRINCIPAL FINDINGS: A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29 was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. CONCLUSIONS/SIGNIFICANCE: The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. TRIAL REGISTRATION: ClinicalTrials.gov NCT00057525.

  5. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata).

    Science.gov (United States)

    SongLin, Guo; PanPan, Lu; JianJun, Feng; JinPing, Zhao; Peng, Lin; LiHua, Duan

    2015-04-01

    The immogenicity of a novel vaccine antigen was evaluated after immunized American eels (Anguilla rostrata) with a recombinant bivalent expressed outer membrane protein (OMP) of Vibrio vulnificus and Aeromonas hydrophila. Three groups of eels were intraperitoneal (i.p) injected with phosphate-buffered saline (PBS group), formaline-killed-whole-cell (FKC) of A. hydrophila and V. vulnificus (FKC group) or the bivalent OMP (OMP group). On 14, 21, 28 and 42 days post-vaccination respectively, proliferation of the whole blood cells, titers of specific antibody and lysozyme activities of experimental eels were detected. On 28 day post-vaccination, eels from three groups were challenged by i.p injection of live A. hydrophila or V. vulnificus. The results showed that, compared with the PBS group, proliferation of whole blood cells in OMP group was significant enhanced on 28 days, and the serum titers of anti-A.hydrophila and anti-V. vulnificus antibody in eels of FKC and OMP group were significant increased on 14, 21 and 28d. Lysozyme Activities in serum, skin mucus, liver and kidney were significant changed between the three groups. Relative Percent Survival (RPS) after challenged A. hydrophila in KFC vs. PBS group and OMP vs. PBS group were 62.5% and 50% respectively, and the RPS challenged V. vulnificus in FKC and OMP vs. PBS group were 37.5% and 50% respectively. These results suggest that American eels immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect against infection by the two pathogens in fresh water farming. PMID:25655329

  6. Preparation of a K39sub Recombinant Antigen for the Detection of Leishmania infantum Antibodies in Human: a Comparative Study with an Immunochromatographic Test and Direct Agglu-tina¬tion

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    M Mahmoudi

    2007-05-01

    Full Text Available Background: The Mediterranean type of kala-azar is occurred in different parts of Iran and caused by Leishmania infan¬tum. A rapid and valid test for early detection of visceral leishmaniasis in human would be highly desirable because it could de¬crease mortality rate of the disease. In this study, we aimed to compare the results of K39sub antigen with an commercial immu¬nochromatographic dipstick rk39 test (Cypress Diagnostic Company, Belgium for early detection of L. infantum infec¬tion in human. Methods: K39sub recombinant antigen of L. infantum LON49 was expressed in prokaryotic system and evaluated for the diagno¬sis of human visceral leishmaniasis. This study evaluated the performance of recombinant K39sub antigen by ELISA and an commercial immunochromatographic dipstick rk39 test for the detection of L. infantum antibodies in 43 clinically in¬fected patients with direct agglutination test (DAT at a 1: 3200 cut off titer and higher. Controls included 69 healthy volun¬teers and 28 patients with other diseases including malaria (n=5, tuberculosis (n= 3, toxoplasmosis (n= 4, cystic hydatido¬sis (n= 5 and cutaneous leishmaniasis (n= 11. Results: The sensitivity of the K39sub antigen and an immunochromatographic dipstick rk39 test was 90.7%, and 97.7%, respec¬tively, while the specificity was 95.6% and 97.9%, correspondingly. A good concordance was found between k39sub anti¬gen and commercial dipstick rk39 strips (k= 96.4%. Conclusion: The accuracy of the K39sub antigen in the detection of L. infantum antibodies in human infecetion is confirmed.

  7. Recombinant Antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays in Dogs

    Science.gov (United States)

    Ye, Cuilian; Yan, Weiwei; Xiang, Hua; He, Hongxuan; Yang, Maosheng; Ijaz, Muhammad; Useh, Nicodemus; Hsieh, Ching-Lin; McDonough, Patrick L.; McDonough, Sean P.; Mohamed, Hussni; Yang, Zhibang; Chang, Yung-Fu

    2014-01-01

    Animal leptospirosis is one of the most common zoonotic diseases in the United States and around the world. In a previous study, we applied four recombinant antigens, rLipL21, rLoa22, rLipL32 and rLigACon4-8 of Leptospira interrogans (L. interrogans) for the serological diagnosis of equine leptospirosis (Ye et al, Serodiagnosis of equine leptospirosis by ELISA using four recombinant protein markers, Clin. Vaccine. Immunol. 21:478–483). In this study, the same four recombinant antigens were evaluated for their potential to diagnose canine leptospirosis by ELISA. A total of 305 canine sera that were Leptospira microscopic agglutination test (MAT)-negative (n = 102) and MAT-positive (n = 203) to 5 serovars (Pomona, Grippotyphosa, Icterohaemorrhagiae, Canicola and Hardjo) were tested. When individual recombinant antigens were used, the sensitivity and specificity of ELISA were 97.5% and 84.3% for rLigACon4-8; 89.7% and 81.4% for rLoa22; 92.6% and 84.3% for rLipL32 and 99.5% and 84.3% for rLipL21, respectively compared to the MAT. The sensitivity and specificity of ELISA were, 92.6% and 91.2% for rLigACon4-8 and rLipL32, 97.5% and 84.3% for rLigACon4-8 and rLipL21, 89.7% and 87.3% for rLigACon4-8 and rLoa22, 89.7% and 87.3% to rLipL21 and rLoa22, 92.6% and 91.2% for rLipL21 and rLipL32 and 89.2% and 94.1% for rLoa22 and rLipL32 when one of the two antigens was test positive. The use of all four antigens in the ELISA assay was found to be sensitive and specific, easy to perform, and agreed with the results of the standard Leptospira Microscopic Agglutination test (MAT) for the diagnosis of canine leptospirosis. PMID:25526513

  8. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

    Science.gov (United States)

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant. PMID:25750999

  9. Induction of anti-human immunodeficiency virus (HIV) neutralizing antibodies in rabbits immunized with recombinant HIV--hepatitis B surface antigen particles.

    OpenAIRE

    Michel, M L; M. Mancini; Sobczak, E.; Favier, V.; Guetard, D; Bahraoui, E M; Tiollais, P

    1988-01-01

    Fragments of the human immunodeficiency virus (HIV) envelope coding region have been fused with the hepatitis B virus envelope middle protein. In this system, HIV antigenic determinants are exposed at the surface of a highly antigenic structure, the hepatitis B surface antigen particle. Immunization of rabbits with these particles elicited antibodies directed against both parts of the hybrid protein. One of the rabbit antisera not only exhibited a neutralizing effect on the original HIV1 isol...

  10. Immunogenicity of three recombinant hepatitis B vaccines administered to students in three doses containing half the antigen amount routinely used for adult vaccination

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    Baldy José Luís da Silveira

    2004-01-01

    Full Text Available We evaluated the immunogenicity of three recombinant hepatitis B vaccines, one Brazilian (Butang, Instituto Butantan and two Korean vaccines (Euvax-B, LG Chemical Ltd. and Hepavax-Gene, Greencross Vaccine Corp., administered intramuscularly to students aged 17 to 19 years in three 10-µg doses (corresponding to half the amount of antigen routinely used for adult vaccination at intervals of one month between the first and second dose, and of four months between the second and third dose. A total of 316 students non-reactive for any serological marker of hepatitis B virus infection were vaccinated: 77 (24.4% with the Butang vaccine, 71 (22.5% with Euvax-B, 85 (26.9% with Hepavax-Gene and, for comparison, 83 (26.2% with Engerix-B (GlaxoSmithKline, whose efficacy in young adults at the dose used here has been confirmed in previous studies. Similar seroconversion rates (anti-HBs > 10 mIU/mL about one month after application of the third dose were obtained for the Butang, Euvax-B, Hepavax-Gene and Engerix-B vaccines (96.2%, 98.6%, 96.5% and 97.6%, respectively. The frequency of good responders (anti-HBs > 100 mIU/mL was also similar among students receiving the four vaccines (85.8%, 91.6%, 89.4% and 89.2%, respectively. The geometric mean titers (GMT of anti-HBs about one month after the third dose obtained with these vaccines were 727.78 ± 6.46 mIU/mL, 2009.09 ± 7.16 mIU/mL, 1729.82 ± 8.85 mIU/mL and 2070.14 ± 11.69 mIU/mL, respectively. The GMT of anti-HBs induced by the Euvax-B and Engerix-B vaccines were higher than those obtained with the Butang vaccine (p < 0.05; this difference was not significant when comparing the other vaccines two-by-two. No spontaneous adverse effects attributable to the application of any dose of the four vaccines were reported.

  11. Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Li, Min; Deng, Guangcun; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2015-08-01

    Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection. PMID:25962477

  12. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    Science.gov (United States)

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (Phyopneumoniae infection. PMID:25091529

  13. Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

    OpenAIRE

    Ross Anton; Lünsdorf Heinrich; Gäbel Thomas; Adnan Ahmad; Gurramkonda Chandrasekhar; Nemani Satish; Swaminathan Sathyamangalam; Khanna Navin; Rinas Ursula

    2009-01-01

    Abstract Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product ...

  14. Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis

    OpenAIRE

    Sinha, Sushmita; Miller, Lisa; Subramanian, Sandhya; McCarty, Owen; Proctor, Thomas; Meza-Romero, Roberto; Burrows, Gregory G.; Vandenbark, Arthur A.; Offner, Halina

    2010-01-01

    Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in antigen specific manner. We evaluated effects of RTL401 (I-As α1β1 + PLP-139-151) on splenocytes from mice with EAE to study RTL- T cell-tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-α1β1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RT...

  15. 泰勒焦虫重组蛋白 TaSP-Tams1-SPAG1间接ELISA 检测方法的建立%Development of ELISA with Recombinant Protein Antigens for Theileria annulata

    Institute of Scientific and Technical Information of China (English)

    魏玉荣; 吴国梁; 祁巧芬; 埃尼瓦尔·太外库力; 黄炯; 易忠; 马文戈; 郭会玲; 向银珍; 米晓云; 陈智; 魏婕; 苗书魁

    2013-01-01

    以真核表达的焦虫表面抗原重组蛋白 TaSP-Tams1-SPAG1作为 ELISA 板包被抗原,通过方阵实验确定抗原包被浓度,探索抗原包被时间、温度、一抗及二抗血清稀释度及作用时间,建立牛环形泰勒焦虫表面抗原重组蛋白 ELISA 方法.结果得出 TaSP-Tams1-SPAG1的最佳包被浓度为10μg/mL,血清最佳稀释度为1∶100, ELISA 阳性反应的临界值为 OD 492nm ≥0.348,重复试验变异系数小于12%,所建立的 ELISA 方法不与牛巴贝斯虫及牛瑟氏泰勒虫血清发生反应.TaSP-Tams1-SPAG1为抗原建立的 ELISA 方法特异性强,敏感度高,重复性好,为牛环形泰勒焦虫病的准确检测及防治提供技术支持.%The objective of this paper was to discuss the antigen coating time,temperature,serum dilution of first antibody and second antibody and action time,and develop antigen recombinant protein ELISA method against Theileria annulata in cattle,to determine antigen coating concentration on the square ex-periment by using antigen recombinant protein TaSP-Tams1-SPAG1 against Theileria annulata expressed by eukarya as ELISA coating antigen.The results indicated that the optimal concentration of coating anti-gen was 10 μg/mL and the optimal blocking dilution of serum sample was 1 ∶100 in the cross assay.The critical value reacted by ELISA positive was an OD 492nm ≥ 0.348.The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than 12%.No cross reactions were found among the piro-plasms.The improved indirect TaSP-Tams1-SPAG1 ELISA Was highly sensitive specific and reproducible, which provided a technical basis for accurate detection and control of Theileria annulata diseases in cattle.

  16. Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens.

    Science.gov (United States)

    Ziethlow, V; Favre, D; Viret, J-F; Frey, J; Stoffel, M H

    2008-03-01

    Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a. PMID:18093230

  17. Human T Cell and Antibody-Mediated Responses to the Mycobacterium tuberculosis Recombinant 85A, 85B, and ESAT-6 Antigens

    OpenAIRE

    Oliveira, Sergio C.; Macedo, Gilson C.; Teixeira, Henrique C.; Andre Bafica; Adriana Bozzi; Helena Rachel Weinreich

    2011-01-01

    Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis. In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P, n = 13) or extrapulmonary (EP, n = 12) tuberculosis, patients u...

  18. HIV-1 Gp41 recombinant antigen: expression and immunoreactivity analysis%HIV-1gp41重组抗原的表达及其免疫反应性检测

    Institute of Scientific and Technical Information of China (English)

    张昕; 梁权; 王缦

    2012-01-01

    为了在大肠杆菌中表达具有良好免疫反应性的HIV-1 gp41重组抗原,本实验运用基因工程技术,经PCR扩增gp41的主要抗原表位序列,BamH I、XhoI双酶切后与E3质粒连接,转化克隆宿主菌DH5α,再提取重组质粒进一步转化表达宿主菌BL21(DE3),经IPTG诱导表达重组蛋白,纯化后标记HRP,通过双抗原夹心酶联免疫方法检测其免疫反应性和特异性.结果表明,获得的HIV-1 gp41重组抗原能够与相应抗体特异性结合,与多种无关抗体间无交叉反应,对825份HIV阴性标本检测无错检.检测结果说明该重组抗原具有良好的免疫反应性,在HIV-1抗体诊断试剂中具有潜在的应用价值,为进一步研究gp41抗原奠定了基础.%To express HIV-1 transmembrane glycoprotein gp41 that has fine immunoreactivity in prokaryotic system of E. coli, the target DNA sequence encoding for major epitopes of HIV-1 gp41 was amplified through PCR. The product was digested by BamH I and Xho I , and inserted into E3 vector. Then the joint product was transported into cloning host DH5a from which we extracted recombinant plasmid that was further transported into BL21(DE3) host. The recombinant protein was expressed when induced with IPTG. And after being purified and labelled with HRP, the immunoreactivity and specificity of the protein was evaluated through double-antigen sandwich ELISA. Results proved that, the HIV-1 gp41 recombinant antigen bound corresponding antibody specifically, and had no cross reactivity with various irrelevant antibodies and showed no false positive results when testing with 825 HIV negative specimens. So, this recombinant antigen has fine immunoreactivity and potentcial application value in HIV antibody detection kit.

  19. Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris Imunogenicidade e antigenicidade da proteína recombinante EMA-1 de Theileria equi expressa em Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Leandro Q. Nizoli

    2009-06-01

    Full Text Available The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg.L-1 of recombinant protein. The protein was concentrated and detected in Dot blot. The recombinant product was antigenically similar to the native protein as determined using monoclonal antibodies, and polyclonal antibodies obtained from equines naturally infected with T. equi. The immunogenicity of rEMA-1 protein was demonstrated by IFAT using sera from recombinant-protein-immunized mice using aluminum hydroxide as adjuvant. All animals vaccinated with rEMA-1 developed a high specific antibody response. This results suggest that rEMA-1expressed in P. pastoris might be a strong candidate to be used as an antigen for immune diagnostics as well as a vaccine antigen.A piroplasmose equina causada por Theileria equi é uma das mais importantes doenças parasitárias de equídeos, causando danos a saúde animal e perdas econômicas. Em T. equi, 2 proteínas de superfície de merozoítos, equi merozoite antigen EMA-1 e EMA-2, têm sido identificadas como antígenos imunodominantes. Sugerindo que estes antígenos possam ser usados como produtos imunobiológicos. O gene EMA-1 de T. equi foi clonado e expressado na levedura Pichia pastoris. As leveduras transformadas foram cultivadas a altas densidades celulares expressando 389 mg.L-1 de proteína recombinante. A proteína foi concentrada e detectada em Dot blot. O produto recombinante foi antigenicamente similar à proteína nativa quando determinado usando anticorpo

  20. Recombinant Adenovirus Delivery of Calreticulin-ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    NARCIS (Netherlands)

    Esparza-Gonzalez, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro Hernandez, Julio; Torres-Lopez, E.; Ancer-Rodriguez, J.; Gutierrez-Puente, Y.; Munoz-Maldonado, G.; Saucedo-Cardenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2012-01-01

    Bacillus CalmetteGuerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuber

  1. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. PMID:27156997

  2. Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Sinha, Sushmita; Miller, Lisa; Subramanian, Sandhya; McCarty, Owen J T; Proctor, Thomas; Meza-Romero, Roberto; Huan, Jianya; Burrows, Gregory G; Vandenbark, Arthur A; Offner, Halina

    2010-08-25

    Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. We evaluated effects of RTL401 (I-A(s) alpha1beta1+PLP-139-151) on splenocytes from SJL/J mice with EAE to study RTL-T cell tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-alpha1beta1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RTL- incubated splenocytes to transfer EAE was likely mediated through macrophages/DC, since B cells were unnecessary for RTL treatment of EAE. These results demonstrate a novel pathway of T cell regulation by RTL-bound APCs. PMID:20546940

  3. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection

    OpenAIRE

    Reed, Matthew D.; Wilder, Julie A.; Mega, William M.; Hutt, Julie A.; Kuehl, Philip J.; Valderas, Michelle W.; Chew, Lawrence L.; Liang, Bertrand C.; Squires, Charles H.

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse inject...

  4. Whole recombinant Pichia pastoris expressing HPV16 L1 antigen is superior in inducing protection against tumor growth as compared to killed transgenic Leishmania

    OpenAIRE

    Bolhassani, Azam; Muller, Martin; Roohvand, Farzin; Motevalli, Fatemeh; Agi, Elnaz; Shokri, Mehdi; Rad, Mahdieh Motamedi; Hosseinzadeh, Sahar

    2015-01-01

    The development of an efficient vaccine against high-risk HPV types can reduce the incidence rates of cervical cancer by generating anti-tumor protective responses. Traditionally, the majority of prophylactic viral vaccines are composed of live, attenuated or inactivated viruses. Among them, the design of an effective and low-cost vaccine is critical. Inactivated vaccines especially heat-killed yeast cells have emerged as a promising approach for generating antigen-specific immunotherapy. Rec...

  5. Mucosal Delivery of Murine Interleukin-2 (IL-2) and IL-6 by Recombinant Strains of Lactococcus lactis Coexpressing Antigen and Cytokine

    OpenAIRE

    Steidler, Lothar; Robinson, K.; Chamberlain, L.; SCHOFIELD, KM; Remaut, Erik; LE PAGE, RWF; Wells, JM

    1998-01-01

    Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized i...

  6. Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.

    Science.gov (United States)

    Schroeder, Matías Nicolás; Tribulatti, María Virginia; Carabelli, Julieta; André-Leroux, Gwenaëlle; Caramelo, Julio Javier; Cattaneo, Valentina; Campetella, Oscar

    2016-04-01

    Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects. PMID:26795039

  7. Antigen-free bovine cancellous bone loaded with recombinant human bone morphogenetic protein-2 for the repair of tibial bone defects in goat model.

    Science.gov (United States)

    Li, Donghai; Deng, Liqing; Yang, Zhouyuan; Xie, Xiaowei; Kang, Pengde; Tan, Zhen

    2016-04-01

    Antigen-free bovine cancellous bone has good performances of porous network structures and mechanics with antigen extracted. To develop a bioactive scaffold for enhancing bone repair and evaluate its biological property, rhBMP-2 loaded with antigen-free bovine cancellous bone was used to treat tibial bone defect. Twenty-four healthy adult goats were chosen to establish goat defects model and randomly divided into four groups. The goats were treated with rhBMP-2/antigen-free bovine cancellous bone scaffolds (group A), autogenous cancellous bone graft (group B), porous tricalciumphosphate scaffolds (group C) and nothing (group D). Animals were evaluated with radiological and histological methods at 4, 8 and 12 weeks after surgery. The gray value of radiographs was used to evaluate the healing of the defects, which revealed that the group A had a better outcome of defect healing compared with group C at 4, 8 and 12 weeks, respectively (p difference between groups A and B was without significance at each time (p > 0.05). The newly formed bone area was calculated from histological sections, and the results indicated that the amount of new bone in group A increased significantly compared with that in group C (p  0.05) at 4, 8 and 12 weeks, respectively. In addition, the expression of collagen I and vascular endothelial growth factor by real-time polymerase chain reaction at 12 weeks in group A was significantly higher than that in group C (p = 0.034, p = 0.032, respectively), but no significant differences were found when compared with that in group B (p = 0.36, p = 0.54, respectively). At the same time, group C presented better results than group D on bone defects healing. Therefore, the composites of antigen-free bovine cancellous bone loaded with rhBMP-2 have a good osteoinductive activity and capacity to promote the repair of bone defects. PMID:26801475

  8. Murine antibody response to oral infection with live aroA recombinant Salmonella dublin vaccine strains expressing filamentous hemagglutinin antigen from Bordetella pertussis.

    OpenAIRE

    Molina, N C; Parker, C D

    1990-01-01

    Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (Tcr) gene were transformed into Salmonella dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition,...

  9. Enhanced specific immune responses by CpG DNA in mice immunized with recombinant hepatitis B surface antigen and HB vaccine

    Directory of Open Access Journals (Sweden)

    Wang Xingtai

    2011-02-01

    Full Text Available Abstract Background Hepatitis B vaccine adjuvant, alum, is generally used for vaccination although it does not stimulate Th1 immunity and 10% of the population has low or no antibody response. Efforts have been continued to find more efficient vaccine adjuvants for better antibody response as well as stimulation of Th1 immunity. Methods CpG DNA was used as an adjuvant for recombinant HBsAg to immunize 6- to 8-week-old female BALB/c mice with or without alum for different dosages. The production of HBsAb, CD80 and CD86 from dendritic cells, and cytokines IL-10, IL12, etc., were analyzed and compared for the performance of immunization. Results 5-20 μg CpG DNA had the best co-stimulation effect of HBsAb serum conversion for mice vaccinated with recombinant expressed HBsAg. The mice vaccinated with recombinant 20 μg CpG DNA and regular vaccine (containing alum adjuvant had the highest concentration of antibody production. IL-12b, IL-12a and IL10 mRNA reached to the peak level between 3 and 6 hours after the CpG DNA induction in splenocytes. The expression levels of CD80 and CD86 leucocyte surface molecules were increased with 20 μg CpG DNA alone or with 20 μg CpG DNA and 4 μg HBsAg. Conclusions Our results confirmed the adjuvant effect of CpG DNA for HBsAg in the mouse model. The increase of IL10 and IL12 production suggested the involvement of Th1 cell activation. The activation of CD80 and CD86 molecules by CpG-ODN might be part of the mechanism of T/B cells coordination and the enhancement of recombinant HBsAg induced immune response.

  10. 猪囊尾蚴重组抗原和基因工程疫苗的研究进展%Research Progress for Recombinant Antigen and Genetic Engineering Vaccine of Cysticercosis

    Institute of Scientific and Technical Information of China (English)

    冯金瑞; 刘立军

    2012-01-01

    猪囊尾蚴病(Cysticercosis)是由猪带绦虫的幼虫囊尾蚴寄生于人或猪等而引起的人畜共患寄生虫病,是公认的世界经济病之一。严重威胁着人体健康,并给畜牧业造成重大经济损失,猪囊尾蚴病的免疫防治势在必行。然而在猪囊尾蚴病疫苗研究中,疫苗抗原的选择和来源一直困扰着兽医工作者。该文就近年来猪囊尾蚴病诊断重组抗原和基因工程疫苗的分子生物学研究进展进行了综述。%Cysticercosis,which is known as a social-economic disease in the world,is an important zoonosis infecting human and pig caused by Taenia solium larval Cysticercus cellulosae.This disease must be prevented for its influence on international competition of the meat product and the great economic loss.However,the choice and source of vaccinal antigen always puzzles veterinarians.This paper reviewed recent research progress in molecular biology of vaccine for Recombinant antigen and genetic engineering vaccine of cysticercosis.

  11. Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen.

    Science.gov (United States)

    Goud, Gaddam Narsa; Deumic, Vehid; Gupta, Richi; Brelsford, Jill; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan L; Tsao, Eric I; Hotez, Peter J; Bottazzi, Maria Elena

    2012-06-01

    The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing. PMID:22503665

  12. Advances in the study of multivalent recombinant DNA vaccines utilizing the hepatitis B virus surface antigen gene%乙肝表面抗原载体多价重组核酸疫苗研究进展

    Institute of Scientific and Technical Information of China (English)

    肖婷; 郭根灵; 辛宪云; 魏庆宽

    2012-01-01

    研究表明,乙肝病毒的包膜蛋白HBsAg不仅可以作为疫苗的理想候选分子,还可作为基因工程疫苗的理想载体,用来成功构建多种重组核酸疫苗.本文概述了以乙肝表面抗原为载体,重组或联合其他病毒、寄生虫、细胞因子等其他基因制作多价核酸疫苗的研究进展.%Studies have shown that hepatitis B virus envelope protein HBsAg can be used as an ideal candidate molecule for vaccines and also as an ideal vehicle for genetically engineered vaccines to successfully build a variety of recombinant DNA vaccines. This article provides an overview of advances in recombinant DNA vaccines prepared by using hepatitis B virus surface antigen as a carrier to restructure or join it to other viruses, parasites, cell factors, or other genes.

  13. A novel recombinant Mycobacterium bovis bacillus Calmette-Guerin strain expressing human granulocyte macrophage colony-stimulating factor and Mycobacterium tuberculosis early secretory antigenic target 6 complex augments Th1 immunity

    Institute of Scientific and Technical Information of China (English)

    Xiaoling Yang; Lang Bao; Yihao Deng

    2011-01-01

    Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacteriutn tuberculosis, named rBCG:GE (expressing GMCSFESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF)and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Thl cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4+ and CD8+ T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.

  14. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    Science.gov (United States)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  15. Recombinant Rhipicephalus appendiculatus gut (Ra86 and salivary gland cement (Trp64 proteins as candidate antigens for inclusion in tick vaccines: protective effects of Ra86 on infestation with adult R. appendiculatus

    Directory of Open Access Journals (Sweden)

    Saimo M

    2011-11-01

    Full Text Available Margaret Saimo1,2,*, David O Odongo3,4,*, Stephen Mwaura3, Just M Vlak1, Anthony J Musoke5, George W Lubega2, Richard P Bishop3, Monique M van Oers11Laboratory of Virology, Wageningen University, Wageningen, The Netherlands; 2School of Veterinary Medicine, Makerere University, Kampala, Uganda; 3International Livestock Research Institute, Nairobi, Kenya; 4School of Biological Sciences, University of Nairobi, Nairobi, Kenya; 5Onderstepoort Veterinary Institute, Onderstepoort, Pretoria, South Africa *These two authors made an equal contribution to this workAbstract: Rhipicephalus appendiculatus gut protein Ra86 (variants Ra85A and Ra92A and the salivary gland cement protein (Trp64 were expressed in the baculovirus-insect cell system. The recombinant gut proteins expressed as soluble proteins and the recombinant cement protein, as insoluble inclusion bodies, were used to immunize rabbits, which were then challenged with larval, nymphal, and adult stages of R. appendiculatus ticks. High tick mortality (23.3% occurred on adult ticks that fed on rabbits vaccinated with the gut proteins, compared with 1.9% mortality in ticks that fed on unvaccinated naïve control rabbits. The mean weight of engorged female ticks was significantly reduced by 31.5% in rabbits vaccinated with the Ra86 recombinant protein compared with controls, as was egg production. Marked effects on these parameters were also observed in adult ticks as a result from vaccination using Trp64, but these were not statistically significant. For both antigens, there was no demonstrable effect on larval or nymphal ticks. This study demonstrates for the first time the protective efficacy of a homolog of Boophilus microplus Bm86 in reducing tick infestation by the adult stage of the three-host tick R. appendiculatus. The results demonstrate the potential of Ra86 for vaccine development against this tick and for the control of East Coast fever.Keywords: baculovirus, Ra85A, Ra92A, Boophilus

  16. Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

    OpenAIRE

    Jake E Lowry; Isaak, Dale D.; Leonhardt, Jack A.; Giulia Vernati; Jessie C Pate; Andrews, Gerard P.

    2011-01-01

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for ...

  17. Human T Cell and Antibody-Mediated Responses to the Mycobacterium tuberculosis Recombinant 85A, 85B, and ESAT-6 Antigens

    Directory of Open Access Journals (Sweden)

    Gilson C. Macedo

    2011-01-01

    Full Text Available Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis. In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P, n=13 or extrapulmonary (EP, n=12 tuberculosis, patients undergoing chemotherapy (PT, n=23, and noninfected healthy individuals (NI, n=7. Compared to NI, we observed increased levels of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB patients from controls by IgG1, IFN-γ and TNF-α production.

  18. Virulence, Immunogenicity, and Protective Efficacy of Two Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Strains Expressing the Antigen ESAT-6 from Mycobacterium tuberculosis

    OpenAIRE

    Bao, Lang; Chen, Wei; Zhang, Huidong; Wang, Xiaoying

    2003-01-01

    We constructed two recombinant Mycobacterium bovis BCG (rBCG) strains expressing ESAT-6 of Mycobacterium tuberculosis, named rBCG-1 and rBCG-2. rBCG-1 contained the ESAT-6 gene linked to BCG hsp60 and expressed a fusion protein, while rBCG-2, with a secretory sequence, could secret ESAT-6 into the culture medium. There was no evidence for increased virulence of the two rBCG strains when we made a comparison between them and BCG with regard to organ bacterial loads, lung histology, and surviva...

  19. Enhanced specific immune responses by CpG DNA in mice immunized with recombinant hepatitis B surface antigen and HB vaccine

    OpenAIRE

    Wang Xingtai; Hu Zhongyu; He Peng; Zhang Xiancheng; Liang Zhenglun

    2011-01-01

    Abstract Background Hepatitis B vaccine adjuvant, alum, is generally used for vaccination although it does not stimulate Th1 immunity and 10% of the population has low or no antibody response. Efforts have been continued to find more efficient vaccine adjuvants for better antibody response as well as stimulation of Th1 immunity. Methods CpG DNA was used as an adjuvant for recombinant HBsAg to immunize 6- to 8-week-old female BALB/c mice with or without alum for different dosages. The producti...

  20. Clonging and Expression of Leptospiral Outer Membrane Protein LipL32 Gene and Application of Recombinant Antigen in Enzyme-linked Immunosorbent Assays%钩端螺旋体外膜脂蛋白LipL32基因的克隆和表达及其在ELISA检测中的应用

    Institute of Scientific and Technical Information of China (English)

    范薇; 于长明; 杨敬; 隋丽华; 战大伟; 贺争鸣; 孙岩松

    2003-01-01

    Objective To construct L32-pQE32 recombinant expression vectors, and to induce the expression of recombinant Leptospiral outer membrane protein LipL32. Establish method of recombinant Leptospiral outer membrane proteinbased ELISA. Method Gene coding of Leptospiral LipL32 protein was amplified by PCR, then recombinant cloning vectors pGEM-T/L32 and expression vectors L32-pQE32 were constructed. Recombinant expression vector was transformed into the competent host E. coli. DH-5α and E. coli. M15. Recombinant Leptospiral LipL32 protein was expressed by IPTG induced method. Immulon microtiter plates were coated at 37℃ overnight with 100 ng of purified recombinant protein per well, 3 positive and 4 negative sera were used in indirect ELISA. Results Mature Leptospiral LipL32 gene fragment about 750 bp was amplified by PCR. LipL32 gene was inserted into expression vectors pQE32, the molecular weight of fusion protein was corresponding to the estimated molecular size of mature Leptospiral LipL32 protein. Results of Western-blot and ELISA demonstrated intense LipL32 reactivity with anti-Leptospira sera. Conclusion findings indicate that recombinant Leptospiral LipL32 may be an important, useful antigen for the serodiagnosis of Leptospira.

  1. Two amino acid substitutions in apolipoprotein B are in complete allelic association with the antigen group (x/y) polymorphism: Evidence for little recombination in the 3 prime end of the human gene

    Energy Technology Data Exchange (ETDEWEB)

    Dunning, A.M.; Renges, H.H.; Xu, Chunfang; Peacock, R.; Humphries, S.E.; Talmud, P.; Laxer, G. (Bickbeck Coll., London (England)); Brasseur, R. (Free Univ. of Brussels (Belgium)); Tikkanen, M.J. (Univ. of Helsinki (Switzerland)); Buetler, R. (Swiss Red Cross, Berne (Switzerland)); Saha, N. (National Univ. of Singapore (Singapore)); Hamsten, A. (Karolinska Hospital, Stockholm (Sweden)); Rosseneu, M. (A.Z. St-Jan, Brugge (Belgium))

    1992-01-01

    The authors report the identification of an A-to-G base change, in exon 29 of the apolipoprotein B (apo B) gene, that results in the substitution of serine for asparagine at residue 4,311 of mature apo B100. In a recent publication, Huang et al. have reported a C-to-T base change in exon 26 that causes the substitution of leucine for proline at residue 2712 of apo B. The authors have found complete linkage disequilibrium between the alleles at both these sites and an immunochemical polymorphism of LDL designated antigen group (x/y) (Ag(x/y)) in a sample of 118 Finnish individuals. This implies that either one of these substitutions - or both of them combined - could be the molecular basis of the Ag(x/y) antigenic determinants, with the allele encoding serine{sub 4311} plus leucine{sub 2,712} representing the Ag(x) epitope, and that encoding asparagine{sub 4,311} plus proline{sub 2,712} the Ag(y) epitope. In a sample of 90 healthy Swedish individuals the Leu{sub 2,712}/Ser{sub 4,311} allele is associated both with reduced serum levels of LDL cholesterol and apo B and with raised levels of HDL. They have also genotyped 523 individuals from European, Asian, Chinese, and Afro-Caribbean populations and have found complete association between the sites encoding residues 2,712 and 4,311 in all of these samples, although there are large allele frequency differences between these populations. Taken together, these data suggest that, since the divergence of the major ethnic groups, there has been little or no recombination in the 3' end of the human apo B gene.

  2. 弓形虫病免疫诊断用重组抗原及检测方法研究进展%Research progress on recombinant antigen and diagnostic technique for immunological diagnosis of toxoplasmosis

    Institute of Scientific and Technical Information of China (English)

    杨永刚; 曹利民; 朱荫昌

    2010-01-01

    Toxoplasma is a kind of parasitic protozoa that can infect all warm blooded animals including humans and cause toxoplasmosis manifesting various symptoms. Diagnosis for toxoplasmosis include etiology,immunology, and molecular biology methods. Immunological diagnostic methods are common for Toxoplasma infection. This article reviewed research progress on recombinant antigen and diagnostic technique development for immunological diagnosis of toxoplasmosis.%弓形虫是一种寄生原虫,可感染人在内的所有温血动物,并会引发表现为多种症状的弓形虫病.弓形虫病的诊断方法有病原学、免疫学、分子生物学等方法.免疫学诊断是进行弓形虫感染检测的常用方法.该文就弓形虫感染的免疫诊断用重组抗原及检测方法的应用和研究进展作一综述.

  3. Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein.

    OpenAIRE

    Hosein, B; Fang, C T; Popovsky, M A; J. Ye; Zhang, M; WANG, C. Y.

    1991-01-01

    Cloning and expression of hepatitis C virus have allowed the development of immunoassays to detect hepatitis C virus infection. However, currently available recombinant fusion protein C100-3 assays, based on a nonstructural protein of the virus, are limited in sensitivity, particularly for detecting acute infection. In this report seroconversion panels showed that an assay based on synthetic peptides, derived from immunodominant regions of both capsid and nonstructural proteins, accelerated h...

  4. Comparing Enterovirus 71 with Coxsackievirus A16 by analyzing nucleotide sequences and antigenicity of recombinant proteins of VP1s and VP4s

    Directory of Open Access Journals (Sweden)

    Sun Yu

    2011-11-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 and Coxsackievirus A16 (CA16 are two major etiological agents of Hand, Foot and Mouth Disease (HFMD. EV71 is associated with severe cases but not CA16. The mechanisms contributed to the different pathogenesis of these two viruses are unknown. VP1 and VP4 are two major structural proteins of these viruses, and should be paid close attention to. Results The sequences of vp1s from 14 EV71 and 14 CA16, and vp4s from 10 EV71 and 1 CA16 isolated in this study during 2007 to 2009 HFMD seasons were analyzed together with the corresponding sequences available in GenBank using DNAStar and MEGA 4.0. Phylogenetic analysis of complete vp1s or vp4s showed that EV71 isolated in Beijing belonged to C4 and CA16 belonged to lineage B2 (lineage C. VP1s and VP4s from 4 strains of viruses expressed in E. coli BL21 cells were used to detect IgM and IgG in human sera by Western Blot. The detection of IgM against VP1s of EV71 and CA16 showed consistent results with current infection, while none of the sera were positive against VP4s of EV71 and CA16. There was significant difference in the positive rates between EV71 VP1 and CA16 VP1 (χ2 = 5.02, P 2 = 15.30, P 2 = 26.47, P 2 = 16.78, P Conclusions EV71 and CA16 were highly diverse in the nucleotide sequences of vp1s and vp4s. The sera positive rates of VP1 and VP4 of EV71 were lower than those of CA16 respectively, which suggested a less exposure rate to EV71 than CA16 in Beijing population. Human serum antibodies detected by Western blot using VP1s and VP4s as antigen indicated that the immunological reaction to VP1 and VP4 of both EV71 and CA16 was different.

  5. Development of a Procedure for Preparation of Recombinant Yersinia pestis LcrV Antigen%重组鼠疫耶尔森菌LcrV抗原制备工艺的建立

    Institute of Scientific and Technical Information of China (English)

    胡丽娜; 王秉翔; 常娅莉; 魏东; 万艳; 马维民; 傅林锋; 王革; 吴智远; 韩少波

    2011-01-01

    目的 建立稳定、高效的重组鼠疫耶尔森菌LcrV抗原工程菌的发酵与纯化工艺.方法 研究LcrV工程菌pET-V/BL21 (DE3)在试管和三角摇瓶中的生长和表达规律,对种子培养基、IPTG诱导时机、诱导时间及诱导浓度进行优化,并放大至30 L发酵罐培养,建立稳定的发酵工艺.收获菌体,经冻融、离心收集蛋白溶液,高压匀浆处理后,分别采用Q.Sepharose HP阴离子交换层析、Phenyl Sepharose 6 FF(hs)疏水层析及Superdex 75 pg凝胶过滤层析三步柱层析纯化,建立稳定的纯化工艺,连续纯化3批,并按《中国药典》三部(2010版)的相关要求进行全面检定.结果 经优化的条件培养及诱导表达,工程菌的收菌密度(A600值)可达34,LcrV抗原的表达量达36%,含量为1.6 g/L.发酵产物经柱层析纯化,LcrV产量高于140 mg,纯度大于95%,蛋白总回收率达8.5%以上.各项检定指标均符合《中国药典》三部(2010版)的相关要求.结论 已建立了稳定的、适合规模化生产的LcrV制备工艺,为新型鼠疫组分疫苗的研制奠定了基础.%Objective To develop a stable and effective procedure for fermentation of recombinant Yersinia pestis and purification of LcrV antigen. Methods The regularity of growth of recombinant E. Coli strain pET-V/BL21 (DE3) in tube and in conical flask as well as expression of LcrV antigen were investigated, based on which the medium, time point and duration for induction with IPTG and IPTG concentration were optimized and scaled up to a 30 L fermenter to develop a stable fermentation procedure. The harvested bacteria were subjected to freeze-thawing, from which protein solution was collected by centrifugation and treated by high pressure homogenization, the purified by Q. Sepharose HP anion exchange chromatography, Phenyl Sepharose 6 FF (hs) hydrophobic chromatography and Superdex 75 pg gel filtration chromatography, based on which a stable purification procedure was developed

  6. Bovine immunoprotection against Rhipicephalus (Boophilus microplus with recombinant Bm86-Campo Grande antigen Imunoproteção de bovinos contra Rhipicephalus (Boophilus microplus com antígeno recombinante Bm86-Campo Grande

    Directory of Open Access Journals (Sweden)

    Rodrigo Casquero Cunha

    2012-09-01

    Full Text Available The southern cattle fever tick, Rhipicephalus (Boophilus microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B. microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B. microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG strain of R. (B. microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B. microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B. microplus in the region that includes Mato Grosso do Sul.O carrapato Rhipicephalus (Boophilus microplus é, sem dúvidas, o ectoparasito economicamente mais importante para o gado a nível mundial. A utilização inadequada de acaricidas tem impulsionado a evolução da resistência em populações de R. (B. microplus. Vacinas contra o carrapato representam uma tecnologia que pode ser combinada com acaricidas em programas de controle integrado para diminuir o impacto de R. (B. microplus. A forma recombinante da Bm86 da cepa Campo Grande (rBm86-CG de R. (B. microplus foi produzido utilizando o sistema de expressão em Pichia pastoris para testar sua capacidade de imunoproteção ao gado contra a infestação de

  7. Recombinant influenza vaccines.

    Science.gov (United States)

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  8. RECOMBINANT INFLUENZA VACCINES

    OpenAIRE

    Sedova, E.; Shcherbinin, D.; Migunov, A.; Smirnov, Iu; Logunov, D.; Shmarov, M.; Tsybalova, L.; Naroditskiĭ, B.; O. Kiselev; Gintsburg, A.

    2012-01-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery pla...

  9. ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE Análise de antígenos recombinantes de Treponema pallidum no diagnóstico da sífilis utilizando a técnica de Western Blotting

    OpenAIRE

    SATO Neuza Satomi; HIRATA Mário H.; HIRATA Rosário D.C.; Lia Carmen M.S. ZERBINI; Edilene P.R. SILVEIRA; MELO Carmem Silvia de; Ueda, Mirthes

    1999-01-01

    Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rT...

  10. Recombinant Rhipicephalus appendiculatus gut (Ra86) and salivary gland cement (Trp64) proteins as candidate antigens for inclusion in tick vaccines: protective effetcs of Ra86 on infestation with adult R. appendiculatus.

    NARCIS (Netherlands)

    Saimo, M.; Odongo, D.O.; Mwaura, S.; Vlak, J.M.; Musoke, A.J.; Lubega, G.W.; Bishop, R.P.; Oers, van M.M.

    2011-01-01

    Rhipicephalus appendiculatus gut protein Ra86 (variants Ra85A and Ra92A) and the salivary gland cement protein (Trp64) were expressed in the baculovirus-insect cell system. The recombinant gut proteins expressed as soluble proteins and the recombinant cement protein, as insoluble inclusion bodies, w

  11. Modulation of Recombinant Antigenic Constructs Containing Multi-Epitopes towards Effective Reduction of Atherosclerotic Lesion in B6;129S-Ldlrtm1HerApobtm2Sgy/J Mice

    OpenAIRE

    Xia, Min; Chen, Daxin; Endresz, Valeria; Lantos, Ildiko; Szabo, Andrea; Kakkar, Vijay; Lu, Xinjie

    2015-01-01

    Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease. Many more studies have extended vaccination against atherosclerosis by using epitopes from self-antigens or beyond and demonstrated that vaccination with antigens or derivatives could reduce the extent of the lesions in atherosclerosis-prone mice. Our previous study has demonstrated that construct AHHC [ApoB100688-707 + hHSP60303-312 + hHSP60153-163 + Cpn derived peptide (C)] significantly reduced atheroscle...

  12. RAPID SCREENING WITH A RECOMBINANT ANTIGEN (rK39) FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS USING DIPSTICK%用重组rK39抗原试纸条快速诊断内脏利什曼病

    Institute of Scientific and Technical Information of China (English)

    瞿靖琦; S.G.Reed; K.-P.Chang; 管立人; 依马木苏里单; 左新平; 柴君杰; 陈生邦; Shinichiro Kawazu; Ken Katakura; Yoshigeru Matsumoto

    2000-01-01

    [Objective] To evaluate the diagnostic value of the recombinant antigen of 39 amino acid repeats encoded by a kinesin-like gene of Leishmania changasi (rK39) in serodiagnosis of visceral leishmaniasis (VL). [Methods] In Kashi, Xinjiang, 13 VL patients with splenomegaly and bone marrow aspirate culture positive were subjected to dipstick assay. A drop of whole blood or serum from patient was placed at the absorbing pad at the bottom of the dipstick.Flooding of the bottom protein with buffer allows serum proteins to migrate upwards, producing the positive band and Western blot analysis of rK39 subsequently performed with the sera collected. [Results] The end-point titers of antirK39 antibodies of these sera were determined by ELISA and found to fall within the range of 10-2 to 10-4, which were consistent with the intensity of their reaction with rK39 in dipstick assay. The positive sera could also recognize the specific rK39 band as analyzed by Western blot analysis. [Conclusion] The rK39 dipstick assay is more rapid, specific, sensitive and less invasive than the conventional methods of diagnosis for VL in the areas of low endemicity.%[目的]评价以恰氏利氏曼原虫类kinesin基因中编码39个氨基酸的基因片段(rK39)为重组抗原,用于血清学诊断内脏利什曼病的价值.[方法]在新疆喀什地区对13例经脾检和骨髓穿刺阳性的内脏利什曼病患者,取一滴病人全血或血清滴在rK39抗原试纸条底部的吸收垫上,血清中蛋白随缓冲液向试纸条上部移动,其中相应特异抗体可与rK39抗原带结合,而产生阳性条带.同时,本文亦用相同阳性血清作了关于rK39抗原的Western印迹分析对照.[结果]ELISA分析显示病人血清抗体滴度在10-2~10-4,与所见到的rK39试纸条上的反应强度符合.Western印迹分析亦显示阳性血清可识别rK39蛋白条带.[结论]与传统诊断内脏利什曼病方法相比较,rK39试纸条更快速,特异,灵敏和低损伤性,可用

  13. Immunization of Aotus monkeys with Plasmodium falciparum blood-stage recombinant proteins.

    OpenAIRE

    S Herrera; Herrera, M. A.; Perlaza, B L; Burki, Y; Caspers, P; Döbeli, H; Rotmann, D; Certa, U

    1990-01-01

    The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falciparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen pr...

  14. A High Through-put Platform for Recombinant Antibodies to Folded Proteins*

    OpenAIRE

    Hornsby, Michael; Paduch, Marcin; Miersch, Shane; Sääf, Annika; Matsuguchi, Tet; Lee, Brian; Wypisniak, Karolina; Doak, Allison; King, Daniel; Usatyuk, Svitlana; Perry, Kimberly; Lu, Vince; Thomas, William; Luke, Judy; Goodman, Jay

    2015-01-01

    Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had ...

  15. A competitive-inhibiton radioimmunoassay for influenza virus envelope antigens

    International Nuclear Information System (INIS)

    A double-antibody competitive-inhibition radioimmunoassay for influenza virus envelope antigens is described. A viral antigen preparation from influenza A virus recombinant MRC11 [antigenically identical to A/Port Chalmers/1/73 (H3N2)] consisting of haemagglutinin and neuraminidase was labelled with radioiodine. Rabbit antisera were allowed to react with the labelled antigen and the resultant antigen-antibody complexes were precipitated with the appropriate antiglobulin. The competitive-inhibition radioimmunoassay very sensitively elucidated differences even among closely related influenza virus strains. Attempts have been made to eliminate neuraminidase from radioimmunoprecipitation to obtain a competitive-inhibition radioimmunoassay system for haemagglutinin alone. (author)

  16. Evaluating the Use of Commercial West Nile Virus Antigens as Positive Controls in the Rapid Analyte Measurement Platform West Nile Virus Assay.

    Science.gov (United States)

    Burkhalter, Kristen L; Savage, Harry M

    2015-12-01

    We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them. PMID:26675461

  17. Expression of Treponema pallidum antigens in Escherichia coli K-12.

    OpenAIRE

    Stamm, L V; Folds, J D; Bassford, P J

    1982-01-01

    A colony bank of recombinant plasmids harboring Treponema pallidum DNA inserts has been established in Escherichia coli K-12. By using an in situ immunoassay, we identified four E. coli clones that expressed T. pallidum antigens. Thus, recombinant DNA technology may provide powerful new tools for studying the pathogenesis of T. pallidum infection.

  18. The next wave of recombinant and synthetic anticancer vaccines

    OpenAIRE

    Irvine, Kari R.; Restifo, Nicholas P

    1995-01-01

    The identification of tumor-associated antigens (TAA) recognized by T lymphocytes makes the development of antigen-specific synthetic and recombinant vaccines possible. The expression of TAA within a recombinant vector increases control over the kinetics and quantity, the molecular form, and the subcellular location of the immunogen delivered. The next generation of antitumor vaccines employs cytokines and costimulatory molecules expressed in concert with TAA that are capable of augmenting th...

  19. ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE Análise de antígenos recombinantes de Treponema pallidum no diagnóstico da sífilis utilizando a técnica de Western Blotting

    Directory of Open Access Journals (Sweden)

    Neuza Satomi SATO

    1999-03-01

    Full Text Available Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.Os antígenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusão com glutationa S-transferase (GST em E. coli, foram analisados quanto ao potencial diagnóstico da sífilis pela técnica de Western blotting. Foram testadas 53 amostras, sendo 25 de pacientes em diferentes estágios clínicos da sífilis, com resultados positivos no teste treponêmico clássico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doença sexualmente transmissível não relacionado à sífilis. Todas as amostras de pacientes com sífilis apresentaram alta reatividade com o antígeno GST-rTp17. Quanto aos antígenos GST-rTp47 e GST-Tp15 verificou-se uma variação na presença ou na intensidade da reação em diferentes amostras de pacientes com sífilis, sem mostrar correlação com o estágio da doença. Nenhuma reatividade contra quaisquer desses antígenos foi observada com as amostras do grupo controle. Nenhuma das amostras testadas apresentaram reatividade com a GST purificada. A

  20. A Functional Analysis of the Spacer of V(D)J Recombination Signal Sequences

    OpenAIRE

    Lee Alfred Ian; Fugmann Sebastian D; Cowell Lindsay G; Ptaszek Leon M; Kelsoe Garnett; Schatz David G

    2003-01-01

    During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jbeta2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jbeta2.6 RSS demonstrates that the sequences of the ...

  1. Carcinoma-associated antigens

    International Nuclear Information System (INIS)

    This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125I-labeled forms of said antigens and methods of detecting said antigens in serum or plasma. The invention also relates to a diagnostic kit containing standardised antigens or antisera or marked forms thereof for the detection of said antigens in human blood, serum or plasma. (author)

  2. Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen induced strong recall vaccine effects against Streptococcus iniae infection in olive flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Kim, Daniel; Beck, Bo Ram; Lee, Sun Min; Jeon, Jongsu; Lee, Dong Wook; Lee, Jae Il; Song, Seong Kyu

    2016-08-01

    The aim of this study was to develop a fish feed vaccine that provides effective disease prevention and convenient application. A lactic acid bacterium (LAB), Lactococcus lactis BFE920, was modified to express the SiMA antigen, a membrane protein of Streptococcus iniae. The antigen was engineered to be expressed under the nisin promoter, which is induced by nisin produced naturally by the host LAB. Various sizes (40 ± 3.5 g, 80 ± 2.1 g, and 221 ± 2.4 g) of olive flounder (Paralichthys olivaceus) were vaccinated by feeding the extruded pellet feed, onto which the SiMA-expressing L. lactis BFE920 (1.0 × 10(7) CFU/g) was adsorbed. Vaccine-treated feed was administered twice a day for 1 week, and priming and boosting were performed with a 1-week interval in between. The vaccinated fish had significantly elevated levels of antigen-specific serum antibodies and T cell marker mRNAs: CD4-1, CD4-2, and CD8a. In addition, the feed vaccine significantly induced T cell effector functions, such as the production of IFN-γ and activation of the transcription factor that induces its expression, T-bet. When the flounder were challenged by intraperitoneal infection and bath immersion with S. iniae, the vaccinated fish showed 84% and 82% relative percent survival (RPS), respectively. Furthermore, similar protective effects were confirmed even 3 months after vaccination in a field study (n = 4800), indicating that this feed vaccine elicited prolonged duration of immunopotency. In addition, the vaccinated flounder gained 21% more weight and required 16% less feed to gain a unit of body weight compared to the control group. The data clearly demonstrate that the L. lactis BFE920-SiMA feed vaccine has strong protective effects, induces prolonged vaccine efficacy, and has probiotic effects. In addition, this LAB-based fish feed vaccine can be easily used to target many different pathogens of diverse fish species. PMID:27302864

  3. Use of Recombinant Envelope Proteins for Serological Diagnosis of Dengue Virus Infection in an Immunochromatographic Assay

    OpenAIRE

    Cuzzubbo, Andrea J.; Endy, Timothy P.; Nisalak, Ananda; Kalayanarooj, Siripen; Vaughn, David W.; Ogata, Steven A.; Clements, David E.; Devine, Peter L.

    2001-01-01

    An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay wer...

  4. Influence of protein formulation and carrier solution on asymmetrical flow field-flow fractionation: a case study of the plant-produced recombinant anthrax protective antigen pp-PA83.

    Science.gov (United States)

    Palais, Caroline; Chichester, Jessica A; Manceva, Slobodanka; Yusibov, Vidadi; Arvinte, Tudor

    2015-02-01

    Asymmetrical flow field-flow fractionation (afFFF) was used to investigate the properties of a plant-produced anthrax toxin protective antigen, pp-PA83. The afFFF fractogram consisted of two main peaks with molar masses similar to the molecular mass of pp-PA83 monomer. afFFF carrier solutions strongly influenced the ratio and the intensity of the two main peaks. These differences indicate that conformation changes in the pp-PA83 molecule occurred during the afFFF analysis. Similar fractograms were obtained for different pp-PA83 formulations when the afFFF carrier solution and the protein formulation were the same (or very similar). The data show that in specific cases, afFFF could be used to study protein conformation and document the importance of studying the influence of the carrier solution on afFFF. PMID:25417936

  5. [Study on recombinant BCG to prevent infections of intracellular pathogens].

    Science.gov (United States)

    Takeshi, Y

    2000-07-01

    Studies on recombinant BCG (rBCG) which my group carried out so far were reviewed. Recombinant BCG which secreted alpha antigen-fused foreign antigen was constructed and tested for its ability to induce protective immunity. Thus, rBCG secreting merozoite surface protein 1 (MSP1) of Plasmodium yoelii efficiently protected the infection more than recombinant MSP 1 mixed with artificial adjuvant RAS or IFA did. rBCG which secreted excess amounts of antigen 85 complex A inhibited the multiplication of M. leprae in the footpads of mice. rBCG which secreted alpha antigen-fused IL-2 stimulated peritoneal exudate cells of mice resulting in enhancing killing a bladder cancer cell line in vitro. PMID:10979272

  6. Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged 3 antigens: Preparation, structural study and immune response towards rHsp90

    Czech Academy of Sciences Publication Activity Database

    Mašek, J.; Bartheldyová, E.; Turánek-Knotigová, P.; Škrabalová, M.; Korvasová, Z.; Plocková, J.; Koudelka, Š.; Škodová, P.; Kulich, P.; Křupka, M.; Zachová, K.; Czerneková, L.; Horynová, M.; Kratochvílová, Irena; Miller, A. D.; Zyka, Daniel; Michálek, J.; Vrbová, J.; Šebela, M.; Ledvina, Miroslav; Raška, M.; Turánek, J.

    2011-01-01

    Roč. 151, č. 2 (2011), s. 193-201. ISSN 0168-3659 R&D Projects: GA ČR(CZ) GAP304/10/1951; GA AV ČR KAN200520703; GA AV ČR KAN200100801 Institutional research plan: CEZ:AV0Z10100520; CEZ:AV0Z40550506 Keywords : recombinant vaccine * liposome Subject RIV: EC - Immunology Impact factor: 5.732, year: 2011 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3D-520M1P7-1&_user=625012&_coverDate=01%2F21%2F2011&_rdoc=1&_fmt=high&_orig=gateway&_origi

  7. Effect of vaccination with a recombinant Bm86 antigen preparation on natural infestations of Boophilus microplus in grazing dairy and beef pure and cross-bred cattle in Brazil.

    Science.gov (United States)

    Rodríguez, M; Massard, C L; da Fonseca, A H; Ramos, N F; Machado, H; Labarta, V; de la Fuente, J

    1995-12-01

    Current methods for the control of the cattle tick Boophilus microplus infestations are not effective and the parasite remains a serious problem for the cattle industry in tropical and sub-tropical areas. Recent advances have introduced the possibility for the immunological control of the parasite through the use of recombinant vaccines. Recently, it was shown that the recombinant vaccine Gavac (Heber Biotec S.A.) is able to control B. microplus populations in artificially infected grazing dairy cattle in Cuba. To assay the effect of the vaccine on a different B. microplus strain and under different ecological conditions, we conducted a trial in Brazil on grazing dairy and beef pure and cross-bred cattle under natural infestation conditions. A farm in the northeast of the state of Sao Paulo was selected and two groups of animals per breed were included in the experiment and were maintained grazing on separate but similar pastures. For each breed, one group was vaccinated with the vaccine Gavac and the second group was not vaccinated and was employed as a control. In vaccinated cattle, during 36 weeks of experiment, the average infestation rate was maintained below 78 ticks per animal while average infestation peaks (mean +/- S.E.) of 144 +/- 44 ticks per animal (for dairy cross-bred cattle) and 195 +/- 42 ticks per animal (for beef cross-bred cattle) were recorded in the control groups. Tick infestation rates showed statistical significant differences (p = 0.04) between both experimental groups throughout the experiment. These results clearly showed, as in the Cuban study, that the vaccine controlled tick numbers in successive generations in the field. PMID:8701597

  8. Molecular identification and antigenic characterization of a merozoite surface antigen and a secreted antigen of Babesia canis (BcMSA1 and BcSA1)

    OpenAIRE

    Zhou, Mo; Cao, Shinuo; Luo, Yuzi; Liu, Mingming; Wang, Guanbo; Moumouni, Paul Franck Adjou; Jirapattharasate, Charoonluk; IGUCHI, Aiko; Vudriko, Patrick; Terkawi, Mohamad Alaa; Löwenstein, Mario; Kern, Angela; Nishikawa, Yoshifumi; Suzuki, Hiroshi; Igarashi, Ikuo

    2016-01-01

    Background Babesia canis is an apicomplexan tick-transmitted hemoprotozoan responsible for causing canine babesiosis in Europe and west Asia. Despite its importance, there is no known rapid diagnostic kit detection of B. canis infection in dogs. The present study identified two novel antigens of B. canis and used the recombinant antigens to establish a rapid, specific and sensitive serodiagnostic technique for detection of B. canis infection. Methods A complementary DNA (cDNA) expression libr...

  9. Evaluation of the immune response to CRA and FRA recombinant antigens of Trypanosoma cruzi in C57BL/6 mice Avaliação da resposta imune em camundongos C57BL/6 imunizados com os antígenos recombinantes CRA e FRA de Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Valéria Rêgo Alves Pereira

    2003-07-01

    Full Text Available Humoral and cellular immune responses were evaluated in 44 C57BL/6 mice immunized with the Trypanosoma cruzi recombinant antigens CRA and FRA. Both antigens induced cutaneous immediate-type hypersensitivity response. The levels of IgG1, IgG2a, IgG2b and IgG3 were high in CRA immunized mice. IgG3 was the predominant isotype. Although no difference in antibody levels was observed in FRA-immunized mice when compared to control mice, both antigens were able to induce lymphoproliferation in immunized mice. Significant differences were observed between incorporation of [³H]- thymidine by spleen cell stimulated in vitro with CRA or FRA and the control group. These results suggest that CRA and FRA could be involved in mechanisms of resistance to Trypanosoma cruzi infection.As respostas imune humoral e celular foram avaliadas em 44 camundongos C57Bl/6 imunizados com os antígenos recombinantes CRA e FRA de Trypanosoma cruzi. Ambos antígenos induziram reação de hipersensibilidade do tipo imediato. Os níveis de IgG1, IgG2a, IgG2b e IgG3 foram elevados nos camundongos imunizados com CRA. IgG3 foi o isotipo predominante. Nenhuma diferença nos níveis de anticorpos foi observada em camundongos imunizados com FRA em relação aos animais controle. No entanto, ambos antígenos foram capazes de induzir proliferação de linfócitos em camundongos imunizados. Diferenças significativas foram observadas entre a incorporação da timidina - [³H] pelas células esplênicas estimuladas com CRA ou FRA e o grupo controle. Esses resultados sugerem que CRA e FRA poderão estar envolvidos nos mecanismos de resistência à infecção pelo Trypanosoma cruzi.

  10. Metallization of the {\\beta}-SiC(100) 3\\times2 Surface: a DFT Investigation

    OpenAIRE

    Westover, James; Oughaddou, Hamid; Enriquez, Hanna; Kara, Abdelkader

    2011-01-01

    Using density functional theory (DFT) we report results for the electronic structure and vibrational dynamics of hydrogenated {\\beta} reconstructed Silicon Carbide (001) (3x2) surfaces with various levels of hydrogenation. These results were obtained using density functional theory with a generalized gradient exchange correlation function. The calculations reveal that metallization can be achieved via hydrogen atoms occupying the second silicon layer. Further increases of hydrogen occupation ...

  11. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in...... induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed....

  12. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens

    International Nuclear Information System (INIS)

    We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/Kb transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8+ T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolΔFsΔPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8+ T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolΔFsΔPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses

  13. Sequence analysis of secretory antigen P53 and immunological identification of the recombinant product of Trichinella spiralis%旋毛形线虫分泌抗原P53的序列分析及重组表达产物的免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    徐鸿绪; 武伟华; 毛玉玲; 梁健; 谌嘉嘉; 胡旭初

    2010-01-01

    Objective To analyze the immunological characteristics of Trichinella spiralis secretory antigen P53 and to evaluate its value in diagnosis of trichinellosis. Methods An open read frame of secretory antigen P53 was cloned from Trichinella spiralis by reverse transcriptasepolymerase chain reaction (RT-PCR) and then sequenced. Bioinformatics analysis was performed to search for its homologues in other helminths and predict its potential linear B cell epitopes and T cell epitopes. The sequence coding mature peptide was inserted into prokaryotic expression vector pET28a(+) and the purified recombinant product was identified by Western blot using serum samples of patients infected with Trichinella spiralis or other helminth. Results Bioinformaties analysis results showed that there was no P53 homologue in other helminths, which indicated that there were many linear B cell epitopes and T cell epitopes in TsP53. The recombinant P53 antigen only reacted with the serum samples of patients infected with Trichinella spiralis without any cross-reaction with the serum of patients infected with other helminths. Conclusion P53 has strong immunogenicity and immunoreactivity, which may be a promising candidate for developing Trichinella spiralis specific diagnostic method.%目的 分析旋毛形线虫分泌抗原P53的免疫学特性,评价其免疫诊断价值.方法 克隆并测序分析旋毛形线虫P53的编码区序列,应用生物信息学分析其与其他蠕虫同源蛋白的相似性以及潜在的线性B细胞和T细胞表位;并将成熟肽编码区克隆至pET28a(+)原核表达质粒中,纯化的重组蛋白用旋毛形线虫感染血清和其他寄生蠕虫感染血清经Western印迹法鉴定其免疫反应性.结果 生物信息学分析未发现其他寄生蠕虫的P53同源基因,表明TsP53具有多个T细胞和B细胞识别表位,Western印迹法显示TsP53抗原只与旋毛形线虫感染血清反应,与其他几种蠕虫感染血清无反应.结论 P53具有

  14. Histocompatibility antigen test

    Science.gov (United States)

    ... more common in certain autoimmune diseases . For example, HLA-B27 antigen is found in many people (but not ... More Ankylosing spondylitis Autoimmune disorders Bone marrow transplant HLA-B27 antigen Kidney transplant Reactive arthritis Update Date 2/ ...

  15. Effects of Supplementation of Various Medium Components on Chinese Hamster Ovary Cell Cultures Producing Recombinant Antibody

    OpenAIRE

    Kim, Do Yun; Lee, Joon Chul; Chang, Ho Nam; Oh, Duk Jae

    2005-01-01

    Thirteen vitamins, twenty amino acids, hormones, inorganic salts, and other chemical agents, which constitute typical serum-free media, were evaluated for the development of fortified medium to enhance cell growth and productivity of recombinant antibody in the cultures of the recombinant Chinese hamster ovary (rCHO) cells. Two different rCHO cell lines, rCHO-A producing recombinant antibodies against the human platelet and rCHO-B secreting recombinant antibodies against the S surface antigen...

  16. Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3

    OpenAIRE

    Amanda R Bitencourt; Elaine C Vicentin; Jimenez, Maria C.; Ricardo Ricci; Leite, Juliana A.; Fabio T Costa; Luis C Ferreira; Bruce Russell; François Nosten; Laurent Rénia; Galinski, Mary R.; Barnwell, John W.; Rodrigues, Mauricio M; Soares, Irene S

    2013-01-01

    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...

  17. Immunogenicity of transgenic plant-derived hepatitis B surface antigen.

    OpenAIRE

    Thanavala, Y; Yang, Y. F.; Lyons, P; Mason, H S; Arntzen, C

    1995-01-01

    The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves...

  18. SEREX技术筛选及鉴定食管癌肿瘤抗原%Human Esophageal Carcinoma Antigens Screened by Serologic Analysis of Recombinant cDNA Expression Libraries (SEREX)

    Institute of Scientific and Technical Information of China (English)

    遇珑; 胡海; 冉宇靓; 彭良平; 李江伟; 杨治华

    2007-01-01

    背景与目的:正常细胞向癌细胞转化过程中,突变的基因或各种异常表达的蛋白可以成为肿瘤抗原诱导机体的免疫反应,因此肿瘤患者的血清中存在着与肿瘤相关的自身抗体.重组cDNA表达文库血清学分析法(serological analysis of recombinant cDNA expression libraries,SEREX)是利用肿瘤患者血清中的自身抗体筛选、鉴定肿瘤抗原的技术.本研究拟采用SEREX的方法寻找食管癌自身抗体的相关肿瘤抗原,鉴定与食管癌发生、发展相关的基因和免疫治疗分子靶点,并为食管癌的诊断提供候选血清标志物.方法:用食管癌组织建立库容量达1.6×106 pfu的cDNA表达文库,SEREX筛选获得21个不同cDNA序列的阳性克隆,进一步使用SADA法分析其中4个抗原在10例食管癌及10例正常人血清中的反应.结果:在Homosapiens desmin(DES)等21个阳性克隆中,4个克隆与已知EST序列明显无同源性,另外17个克隆与已知基因高度同源.Ribosomal protein S4等4个抗原与食管癌患者和正常人血清反应阳性率分别为40%和0%、60%和10%、70%和20%、30%和20%.结论:Ribosomal protein S4等4个抗原普遍参与了食管癌患者的体液免疫反应,与食管癌患者血清的反应阳性率明显高于正常人的血清.本研究发现的21个食管癌抗原可作为食管癌治疗的潜在分子靶点和食管癌诊断新的候选血清学标志物.

  19. Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Hai-Jie Yang; Ning-Shao Xia; Min Chen; Tong Cheng; Shui-Zhen He; Shao-Wei Li; Bao-Quan Guan; Zi-Heng Zhu; Ying Gu; Jun Zhang

    2005-01-01

    AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier.METHODS: One to 6 tandem copies of HBV preS1 (21-47)fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E. coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric partides was detected with immuno-capture PCR.RESULTS: Recombinant antigens CⅠ, CⅡ, CⅢ carrying 1-3 copies of HBV preS1 (21-47) individually could form viruslike particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CⅠ, CⅡ, CⅢ could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CⅠ, CⅡ and CⅢ) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CⅠ, CⅡ and CⅢ were able to capture HBV virions in immuno-capture PCR assay in vitro.CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

  20. Polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.

    OpenAIRE

    Moore, J. L.; Chowdhury, K.; Martin, M A; Israel, M A

    1980-01-01

    The arrangement of viral DNA sequences in a hamster cell line derived from a tumor induced by a recombinant plasmid DNA preparation containing the entire polyoma virus genome was examined. In the recombinant plasmid employed, viral DNA sequences specifying the large species of polyoma tumor antigen but not the small and middle tumor antigens were interrupted by the insertion of plasmid DNA at the EcoRI restriction endonuclease site. Blot-hybridization analyses of tumor cell DNA indicated that...

  1. Immunohistochemical localization of granzyme B antigen in cytotoxic cells in human tissues.

    OpenAIRE

    Hameed, A.; Truong, L D; Price, V; Kruhenbuhl, O.; Tschopp, J

    1991-01-01

    Human granzyme B antigen is expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme B was generated using a prokaryotic expression vector under the control of T7 transcription and translation signals. The 25-kd recombinant protein (granzyme B) was used to develop a rabbit polyclonal antiserum. Purified anti-granzyme B antibodies were used to detect the antigen expression in cytotoxic cells in human tissues. Using the avidin-biotin-...

  2. Antigenic variation with a twist--the Borrelia story.

    Science.gov (United States)

    Norris, Steven J

    2006-06-01

    A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface-exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid-encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes. PMID:16796669

  3. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

    Science.gov (United States)

    Bitencourt, Amanda R; Vicentin, Elaine C; Jimenez, Maria C; Ricci, Ricardo; Leite, Juliana A; Costa, Fabio T; Ferreira, Luis C; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R; Barnwell, John W; Rodrigues, Mauricio M; Soares, Irene S

    2013-01-01

    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. PMID:23457498

  4. 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.

  5. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  6. Recombinant shark natural antibodies to thyroglobulin.

    Science.gov (United States)

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL. PMID:15954089

  7. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    Elizabeth A Specht

    2014-02-01

    Full Text Available Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for molecular pharming in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae are poised to become the next candidate in recombinant subunit vaccine production, and they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally-delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and system immune reactivity.

  8. RECOMBINANT PRODUCTION OF HORSERADISH PEROXIDASE CONJUGATES WITH FAB ANTIBODIES IN PICHIA PASTORIS FOR ANALYTICAL APPLICATIONS

    OpenAIRE

    Koliasnikov, O.; Grigorenko, V.; Egorov, A.; S. Lange(Justus Liebig-Universität Gie\\ssen II Physikalisches Institut, Germany); Schmid, R

    2011-01-01

    Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) with Fab fragments of anti...

  9. Antibody responses to recombinant and plasma derived hepatitis B vaccines.

    OpenAIRE

    Brown, S E; Stanley, C.; Howard, C. R.; Zuckerman, A J; Steward, M W

    1986-01-01

    The antibody response to hepatitis B surface antigen (anti-HBs) induced in 25 recipients of a recombinant hepatitis B vaccine derived from yeast was compared with that induced in 25 recipients of a vaccine prepared from hepatitis B surface antigen (HBsAg) derived from plasma. Anti-HBs affinity and specificity were compared using assays of antibody affinity with two different antigens, a complex of the major polypeptide of HBsAg (p25; molecular weight 25 000 daltons) covalently linked to its g...

  10. Recombinant DNA in Medicine

    OpenAIRE

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  11. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  12. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  13. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  14. Yeast retrotransposon particles as antigen delivery systems.

    Science.gov (United States)

    Kingsman, A J; Burns, N R; Layton, G T; Adams, S E

    1995-05-31

    The development of technologies to produce recombinant proteins for use in the pharmaceutical industry has made substantial advances, in particular in the area of generating antigens containing multiple copies of important immunological regions. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles. Ty-fusion proteins retain this ability to form particles, and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to induce potent immune responses. In particular, hybrid VLPs carrying the core protein p24 of HIV (p24-VLPs) have been shown to induce antibody and T-cell proliferative responses in both experimental animals and human volunteers, and immunization of rabbits with VLPs carrying the principal neutralizing determinant of HIV (V3-VLPs) resulted in the induction of neutralizing antibody responses and T-cell proliferation. Further studies with V3-VLPs have shown that this particulate antigen stimulates enhanced V3-specific lymphoproliferative responses as compared to whole recombinant gp120 or to V3 peptide conjugated to albumin. The V3-VLPs also induce potent CTL responses following immunization of mice in the absence of adjuvant. These responses are MHC class I restricted and are mediated by CD8-positive cells. These observations therefore demonstrate that hybrid Ty-VLPs induce both humoral and cellular immune responses against HIV and suggest that these immunogens may be important in combatting AIDS and other infections. PMID:7625653

  15. Enhancing the recognition of tumor associated antigens

    OpenAIRE

    Restifo, Nicholas P; Irvine, Kari R.; Minev, Boris R.; Taggarse, Akash S.; McFariand, Barbra J.; Wang, Michael

    1994-01-01

    Activated CD8+ T cells (TCD8+) can directly recognize malignant cells because processed fragments of tumor associated antigens (TAA), 8-10 amino acids in length and complexed with MHC class I molecules, are displayed on tumor cell surfaces. Tumor cells have been genetically modified in a variety of ways in efforts to enhance the immune recognition of TAA. An alternative strategy is the expression of TAA in recombinant or synthetic form. This has been made possible by the recent cloning of TAA...

  16. Pesquisa de IgA contra o antígeno recombinante HspX de Mycobacterium tuberculosis no diagnóstico de tuberculose pleural Determination of levels of specific IgA to the HspX recombinant antigen of Mycobacterium tuberculosis for the diagnosis of pleural tuberculosis

    Directory of Open Access Journals (Sweden)

    Loanda Carvalho Sant' Ana Limongi

    2011-06-01

    Full Text Available OBJETIVO: Avaliar a acurácia da dosagem de IgA contra o antígeno recombinante HspX no líquido pleural e no soro de pacientes com derrame pleural para o diagnóstico de tuberculose pleural. MÉTODOS: Estudo transversal de teste diagnóstico. Amostras de líquido pleural e de soro de pacientes com derrame pleural e suspeita de tuberculose pleural foram avaliadas para a determinação da densidade óptica de IgA contra HspX utilizando ELISA indireto. RESULTADOS: Foram avaliadas amostras de líquido pleural e de soro de 132 pacientes: 97 com tuberculose pleural (grupo de estudo e 35 com derrame pleural por outras causas (grupo controle. A dosagem de IgA em líquido pleural foi capaz de discriminar os pacientes com tuberculose pleural dos controles. A sensibilidade do teste em líquido pleural e em soro foi, respectivamente, de 69% e 30%, enquanto a especificidade foi de 83% e 84%, respectivamente. CONCLUSÕES: Os dados sugerem o potencial da utilização deste teste no diagnóstico de tuberculose pleural. Estudos com amostras maiores e em diferentes cenários epidemiológicos são necessáriosOBJECTIVE: To evaluate the accuracy of determining specific IgA to HspX recombinant antigen in pleural fluid and serum samples for the diagnosis of pleural tuberculosis in patients with pleural effusion. METHODS: This was a cross-sectional study. Serum and pleural fluid samples of patients with pleural effusion and suspected of having pleural tuberculosis were tested with indirect ELISA in order to determine the optical density of specific IgA to HspX. RESULTS: We evaluated serum and pleural fluid samples from 132 patients: 97 diagnosed with pleural tuberculosis (study group and 35 diagnosed with pleural effusion due to other causes (control group. The determination of IgA in pleural fluid satisfactorily discriminated between pleural tuberculosis patients and control patients. The sensitivity of the test in pleural fluid and in serum was 69% and 30

  17. Potential Serological Use of a Recombinant Protein That Is a Replica of a Mycobacterium tuberculosis Protein Found in the Urine of Infected Mice

    OpenAIRE

    Mukherjee, Sandeep; Daifalla, Nada; Zhang, Yanni; Douglass, John; Brooks, Lisa; Vedvick, Thomas; Houghton, Raymond; Reed, Steven G.; Campos-Neto, Antonio

    2004-01-01

    The recent availability of numerous well-characterized Mycobacterium tuberculosis recombinant proteins has revived interest in the serological diagnosis of tuberculosis. Several promising results have been reported, particularly when more than one antigen is used in the test. However, thus far these antigens have not been used in routine diagnostic tests because they lack sufficient sensitivity. In addition, with the exception of one antigen, most recombinant M. tuberculosis proteins do not i...

  18. The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants

    OpenAIRE

    Jul-Larsen, Åsne; Madhun, Abdullah S.; Brokstad, Karl A.; Montomoli, Emanuele; Yusibov, Vidadi; Cox, Rebecca J.

    2012-01-01

    Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine p...

  19. Serological diagnosis of infectious mononucleosis using three anti-Epstein-Barr virus recombinant ELISAs

    OpenAIRE

    Färber, I.; Wutzler, P.; Wohlrabe, P.; Wolf, Hans J.; Hinderer, W.; Sonneborn, H H

    1993-01-01

    A new Epstein-Barr virus (EBV) ELISA system (Biotest Anti-EBV recombinant) was evaluated for usefulness for routine diagnosis of EBV primary infection. The assay system is composed of three different microtest plates coated with three highly purified recombinant EBV antigens. The early antigens p138 (BALF2, truncated) and p54 (BMRF1, whole sequence) are used as a mixture for testing IgM (assay 1) and IgG (assay 2) antibodies. In addition, the EBNA-1 antigen p72 (BKRF1, carboxy-half) is used f...

  20. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  1. Recombinant Yellow Fever Vaccine Virus 17D Expressing Simian Immunodeficiency Virus SIVmac239 Gag Induces SIV-Specific CD8+ T-Cell Responses in Rhesus Macaques ▿

    OpenAIRE

    Bonaldo, Myrna C.; Martins, Mauricio A.; Rudersdorf, Richard; Mudd, Philip A.; Sacha, Jonah B.; Piaskowski, Shari M.; Costa Neves, Patrícia C.; Veloso de Santana, Marlon G.; Vojnov, Lara; Capuano, Saverio; Rakasz, Eva G.; Wilson, Nancy A.; Fulkerson, John; Sadoff, Jerald C.; Watkins, David I.

    2010-01-01

    Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8+ T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8+ T-cell responses after recombinant YF17D boosting. These recombinan...

  2. Isolation of an Intertypic Poliovirus Capsid Recombinant from a Child with Vaccine-Associated Paralytic Poliomyelitis

    OpenAIRE

    Martín, Javier; Samoilovich, Elena; Dunn, Glynis; Lackenby, Angie; Feldman, Esphir; Heath, Alan; Svirchevskaya, Ekaterina; Cooper, Gill; Yermalovich, Marina; Minor, Philip D.

    2002-01-01

    The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5′-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigeni...

  3. Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

    Science.gov (United States)

    Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

    2008-06-22

    Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats. PMID:18182256

  4. Vaxvec: The first web-based recombinant vaccine vector database and its data analysis.

    Science.gov (United States)

    Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

    2015-11-27

    A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development. PMID:26403370

  5. Recombineering Homologous Recombination Constructs in Drosophila

    OpenAIRE

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A.; Williams, Nathan David; Hiesinger, P. Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineeri...

  6. A Viral Vectored Prime-Boost Immunization Regime Targeting the Malaria Pfs25 Antigen Induces Transmission-Blocking Activity

    OpenAIRE

    Goodman, Anna L.; Blagborough, Andrew M.; Sumi Biswas; Yimin Wu; Hill, Adrian V.; Sinden, Robert E.; Draper, Simon J

    2011-01-01

    The ookinete surface protein Pfs25 is a macrogamete-to-ookinete/ookinete stage antigen of Plasmodium falciparum, capable of exerting high-level anti-malarial transmission-blocking activity following immunization with recombinant protein-in-adjuvant formulations. Here, this antigen was expressed in recombinant chimpanzee adenovirus 63 (ChAd63), human adenovirus serotype 5 (AdHu5) and modified vaccinia virus Ankara (MVA) viral vectored vaccines. Two immunizations were administered to mice in a ...

  7. Fundamental study of recombination and recombineering in Escherichia coli

    OpenAIRE

    Sun, Xiaohang; Huang, Yang

    2008-01-01

    Recombination and recombineering systems have been used in Escherichia coli to recombinant DNA sequences. With endonuclease and DNA lipase the bacterial plasmid and target DNA fragment can bind together and recombinant for a new DNA sequences. Red Proteins have been used in recombineering system to perform the function as the enzymes in recombination system, and faster and easier than the other way of recombinant new DNA sequences in E.coli. In this report we get to know the pr...

  8. An efficient fusion protein system for expression ofBacillus anthracis protective antigen as immunogenic and diagnostic antigen

    Institute of Scientific and Technical Information of China (English)

    Vahid Bagheri; Hossein Motamedi; Masoud Reza Seifiabad Shapouri

    2010-01-01

    Objective:To produce high quantities of recombinant protective antigen (rPA) for human vaccine and diagnosis.Methods: ThePAgene was amplified byPCR with pXO1 plasmid as template. ThePCR product was cloned into pMAL-c2X vector using theBamHI andSalI restriction enzymes. The recombinant plasmid was transformed intoEscherichia coliDH5α strain and then screened for transformation. The expression of protective antigen was analyzed bySDS-PAGE and Western blotting after isopropyl β-D-thiogalactopyranoside(IPTG) induction.Results:The full-length PA gene (2.2kb) was cloned into pMAL vector system. The recombinant vector was confirmed by restriction enzyme andPCRanalysis. The expression of cytoplasmic maltose-binding protein-protective (MBP-P) antigen fusion protein was detected bySDS-PAGE and Western blotting, and obtained a125 kDa protein band, which was similar to expected size of fusion protein.Conclusions: This expression system can be used in the high production of rPA. After purification and immunization studies, the purified rPA may be used in the development of the human recombinant anthrax vaccine and also in diagnosis of anthrax disease.

  9. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    Science.gov (United States)

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  10. Targeted Delivery of VP1 Antigen of Foot-and-mouth Disease Virus to M Cells Enhances the Antigen-specific Systemic and Mucosal Immune Response

    OpenAIRE

    Kim, Sae-Hae; Lee, Ha-Yan; Jang, Yong-Suk

    2013-01-01

    Application of vaccine materials through oral mucosal route confers great economical advantage in animal farming industry due to much less vaccination cost compared with that of injection-based vaccination. In particular, oral administration of recombinant protein antigen against foot-and-mouth disease virus (FMDV) is an ideal strategy because it is safe from FMDV transmission during vaccine production and can induce antigen-specific immune response in mucosal compartments, where FMDV infecti...

  11. Combination of Two Candidate Subunit Vaccine Antigens Elicits Protective Immunity to Ricin and Anthrax Toxin in Mice

    OpenAIRE

    David J Vance; Rong, Yinghui; Brey, Robert N.; Mantis, Nicholas J.

    2014-01-01

    In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.

  12. Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice.

    Science.gov (United States)

    Vance, David J; Rong, Yinghui; Brey, Robert N; Mantis, Nicholas J

    2015-01-01

    In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population. PMID:25475957

  13. Comparative Study of rK39 Leishmania Antigen for Serodiagnosis of Visceral Leishmaniasis: Systematic Review with Meta-Analysis

    OpenAIRE

    Maia, Zuinara; Lírio, Monique; Mistro, Sóstenes; Mendes, Carlos Maurício Cardeal; Mehta, Sanjay R; Badaro, Roberto

    2012-01-01

    Background The rK39 recombinant protein is derived from a specific antigen produced by the Leishmania donovani complex, and has been used in the last two decades for the serodiagnosis of visceral leishmaniasis. We present here a systematic review and meta-analysis of studies evaluating serologic assays to diagnose visceral leishmaniasis to determine the accuracy of rK39 antigen in comparison to the use of other antigen preparations. Methodology/Principal Findings A systematic review with meta...

  14. European collaborative evaluation of Enzygnost HBsAg 6.0: performance on hepatitis B virus surface antigen variants

    OpenAIRE

    Avellón, Ana; Echevarría, Jose-Manuel; Weber, Bernard; Weik, Michael; Schobel, Uwe-Peter; Willems, Wulf R.; Gerlich, Wolfram

    2010-01-01

    Abstract Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may eventually modify the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its capability to detect these mutants. One-hundred and forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant H...

  15. HA03 as an Iranian Candidate Concealed Antigen for Vaccination against Hyalomma anatolicum anatolicum: Comparative Structural and In silico Studies

    OpenAIRE

    Mohammadi, A.; Aghaiypour, K; Keywanfar, H.; Paykari, H.,; Tajbakhsh, H.; Jalali, A.H.,; Safavieh, S.; Foroghi, A.

    2013-01-01

    In the last decades researchers had focused on developing a vaccine against tick based on protective antigen. Recombinant vaccines based on concealed antigen from Boophilus microplus have been developed in Australia and Cuba by the name of TICKGARD and GAVAC (De La Fuente and Kocan, 2006). Further studies on this antigen have shown some extent of protection against other species (De Vos et al., 2001). In Iran most important species is Hyalomma anatolicum and limited information about its cont...

  16. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    Directory of Open Access Journals (Sweden)

    Mark W. Jackwood

    2011-09-01

    Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

  17. Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

    Science.gov (United States)

    Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-07-01

    Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. PMID:27101782

  18. Cancer vaccine--Antigenics.

    Science.gov (United States)

    2002-01-01

    Antigenics is developing a therapeutic cancer vaccine based on heat-shock proteins (HSPs). The vaccine [HSPPC-96, Oncophage] is in a pivotal phase III clinical trial for renal cancer at 80 clinical sites worldwide. The trial is enrolling at least 500 patients who are randomised to receive surgical removal of the primary tumour followed by out-patient treatment with Oncophage((R)) or surgery only. This study was initiated on the basis of results from a pilot phase I/II study and preliminary results from a phase II study in patients with renal cell cancer. In October 2001, Oncophage was designated as a fast-track product by the Food and Drug Administration in the US for the treatment of renal cell carcinoma. Oncophage is in phase I/II trials in Italy for colorectal cancer (30 patients) and melanoma. The trials in Italy are being conducted at the Istituto dei Tumouri, Milan (in association with Sigma-Tau). Preliminary data from the phase II trial for melanoma was presented at the AACR-NCI-EORTC International Conference in Florida, USA, in October 2001. Oncophage is also in a phase I/II (42 patients) and a phase II trial (84 patients) in the US for renal cell cancer, a phase II trial in the US for non-Hodgkin's lymphoma (35 patients), a phase II trial in the US for sarcoma (20-35 patients), a phase I/II trial in the US for melanoma (36 patients), and phase I/II trials in Germany for gastric (30 patients) and pancreatic cancers. A pilot phase I trial in patients with pancreatic cancer began in the US in 1997 with 5 patients enrolled. In November 2000, Antigenics announced that this trial had been expanded to a phase I/II study which would now include survival as an endpoint and would enroll 5 additional patients. The US trials are being performed at Memorial Sloan-Kettering Cancer Center and the M.D. Anderson Cancer Center. The trials in Germany are being carried out at Johannes Gutenberg-University Hospital, Mainz. Oncophage is an autologous vaccine consisting of

  19. Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

    Science.gov (United States)

    Farahmand, Mahin; Nahrevanian, Hossein

    2016-07-01

    Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs. PMID:26883952

  20. Expression of hepatitis B surface antigen in transgenic plants.

    OpenAIRE

    Mason, H S; Lam, D M; Arntzen, C J

    1992-01-01

    Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBs...

  1. THE SEARCH OF OPTIMAL COMBINATION OF ANTIGENS FOR SEROLOGICAL DIAGNOSTICS OF TUBERCULOSIS

    Directory of Open Access Journals (Sweden)

    E. V. Vasilyeva

    2013-01-01

    Full Text Available Abstract. The four chimeric recombinant antigens CBD-CFP10, CBD-ESAT6, ESAT6-CFP10 and CBD-P38 contained aminoacid sequences of full-size proteins ESAT6, CFP10 and matured protein P38 of M. tuberculosis, joined with aminoacid sequences of cellulose bind domain of endogluconase A (CBD from Cellumonas fimi have been obtained by gene engineering methods. Recombinant proteins were purified by affine chromatography in column with Ni-NTA-sepharose 6В-CL and as PPDN-3 were used for detection of their antigenic activity in indirect ELISA for TB serological diagnostics. The sera from patients with lung tuberculosis (n = 321, from persons who had professional contacts with TB patients (n = 42, from healthy blood donors (n = 366 and from patients with lung diseases of non-TB etiology were tested. It was detected that there was positive correlation between antibodies level for all studied antigens compared by pair. It has been demonstrated that although antigens were different by antigenic and immunobiological characteristics they add each other in the content of antigenic diagnostics compositions. Thus, all these antigens can be used in the test kits for serological diagnostics of TB. Using of these antigens will allow to detect persons infected by TB and patients with active tuberculosis. 

  2. Expression in yeast (Pichia pastoris) of recombinant Cb-peritrophin-42 and Cbperitrophin- 48 isolated from Chrysomya bezziana (the Old World Screwworm fly)

    OpenAIRE

    2000-01-01

    Pichia pastoris has been investigated as a means to express recombinant forms of two putative peritrophic membrane antigens from Chrysomya bezziana, Cb-peritrophin-42 (Cb42) and Cb-peritrophin-48 (Cb48). Recombinant Cb48 was expressed as a secreted and glycosylated protein. The yield of recombinant protein was 8 mg per litre of culture. In contrast, recombinant Cb42 was not expressed at detectable levels in Pichia pastoris, probably due to A + T rich sequence which may cause premature transcr...

  3. Antigen smuggling in tuberculosis.

    Science.gov (United States)

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. PMID:24922567

  4. Antigen detection systems

    Science.gov (United States)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  5. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G; Abal, A T; Ravn, P; Oftung, F; Andersen, P

    1998-01-01

    We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r......GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen...

  6. Aspergillus antigen skin test (image)

    Science.gov (United States)

    The aspergillus antigen skin test determines whether or not a person has been exposed to the mold aspergillus. It is performed by injecting an aspergillus antigen under the skin with a needle. After 48 ...

  7. Use of recombinant DNA in the diagnosis and control of babesiosis

    International Nuclear Information System (INIS)

    The potential of a vaccine against babesiosis using antigens produced through recombinant DNA technology has been experimentally demonstrated in the control of Babesia bovis infections in beef cattle. To develop the vaccine, the antigens had to be identified by fractionation. In the penultimate step of purification, a series of monoclonal antibodies were generated and used as affinity ligands to obtain homogeneous antigen. In this manner, several protective antigens have been identified and characterized. These monoclonal antibodies, and/or synthetic oligonucleotides based on amino acid sequence information, were used to isolate the appropriate genes from cDNA libraries prepared from B. bovis mRNA. Fusion proteins were effective in reducing the development of babesiosis in cattle. Notably, vaccination with each of the antigens led to at least a 10-fold decrease in the maximal parasitaemia developed. When these antigens were used together in a small field trial, only 10% of the animals required treatment to prevent severe illness, compared with 40% of control animals. Using DNA hybridization techniques, one of these antigens (the 12D3 antigen) has been found in other species of Babesia, including B. bigemina. Thus a candidate protective antigen for use in controlling B. bigemina infections has been identified without the large scale and costly purification procedures used to identify the B. bovis antigen. The ability to produce the protective antigens in quantity in bacteria provides the prospect for overcoming the limitations of the existing live vaccine. 19 refs, 3 figs

  8. A Novel Treponema pallidum Antigen, TP0136, Is an Outer Membrane Protein That Binds Human Fibronectin▿

    OpenAIRE

    Brinkman, Mary Beth; McGill, Melanie A.; Pettersson, Jonas; Rogers, Arthur; Matějková, Petra; Šmajs, David; Weinstock, George M.; Norris, Steven J; Palzkill, Timothy

    2008-01-01

    The antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidum subsp. pallidum were investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial ...

  9. Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12.

    OpenAIRE

    Clayton, C L; Wren, B. W.; Mullany, P.; Topping, A; Tabaqchali, S

    1989-01-01

    A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsor...

  10. Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

    OpenAIRE

    Murakami Akihiro; Hachiya Takahisa; Kurei Shunsuke; Nishimori Takanori; Yasuraoka Mari; Nakashima-Fujita Kazue; Kuboshima Mari; Shiratori Tooru; Shimada Hideaki; Kagaya Akiko; Tamura Yutaka; Nomura Fumio; Ochiai Takenori; Matsubara Hisahiro; Takiguchi Masaki

    2011-01-01

    Abstract Background Diagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available. Results Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of the...

  11. Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

    OpenAIRE

    Jackson Marcondes; Ekkehard Hansen

    2008-01-01

    The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons wit...

  12. Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

    Directory of Open Access Journals (Sweden)

    Jackson Marcondes

    2008-12-01

    Full Text Available The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L. using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety "Vitória de Verão" genetically modified.

  13. Stool Test: H. Pylori Antigen

    Science.gov (United States)

    ... All About Food Allergies Stool Test: H. Pylori Antigen KidsHealth > For Parents > Stool Test: H. Pylori Antigen Print A A A Text Size What's in ... sample is used to determine if H. pylori antigens are present in your child's gastrointestinal (GI) system. ...

  14. Cloning and Expression of Hepatitis B Surface Antigen

    Directory of Open Access Journals (Sweden)

    Bahram Kazemi

    2008-02-01

    Full Text Available Background and Aims: Hepatitis B virus (HBV is a major cause of both acute and chronic liver disease. It is estimated that there are 350 million carriers of the virus in the world, and a high proportion will develop serious liver disease, including hepatocellular carcinoma. The aim of this study was cloning and expression hepatitis B surface antigen (HBsAg gene to design a DNA vaccine.Methods: In this study, we amplified the HBsAg gene from Iranian patients. The gene was cloned in pGEMEX-1 expression vector and recombinant plasmid was transformed in to JM109 E. coli strain and induced by IPTG.Results: We amplified, cloned and expressed hepatitis B virus surface antigen successfully and expressed protein was serologically assayed using gel diffusion and western blot analysis. Gene was sequenced and submitted to GenBank. Conclusions: The cloned HBsAg gene is ready for using in experimental DNA vaccine animal study. There are some mutations on this recombinant protein (T45D, Y206C and S207R which will affect on folding and function of recombinant protein.Keywords: Hepatitis B Virus, HBsAg, Recombinant Protein, Vaccine

  15. Murine T-lymphocyte activation by mycobacterial antigens

    International Nuclear Information System (INIS)

    There has been renewed interest in the diagnosis of tuberculosis and other mycobacterial infections in the United States. Effective immunity to mycobacterial infections, as well as diagnosis by the skin test, involves T-cells rather than antibodies. Studies currently underway use the new technologies of monoclonal antibodies and recombinant DNA to define better mycobacterial antigens for T-cell activation, in the hope of identifying species specific antigens. Lymph node cells from mice sensitized to Mycobacterium intracellulare and Mycobacterium avium were assayed for activation by mycobacterial fractions, and cell lines and clones were generated. Comparing BALB/c and B10 mice indicated better responses to M. avium sonicate by B10 mice. A recombinant gene product containing a M. intracellulare peptide was assayed with lymph node cells and indicated excellent T-cell stimulation in BALB/c lymph node cells and cell lines. However, assays using B10 T-cell clones have yet to detect responders to the recombinant protein. Future studies using synthetic epitopes produced by recombinant DNA techniques and defined by monoclonal antibodies are necessary for the identification of reactive T-cell epitopes that are potentially species specific. 4 refs, 7 figs, 1 tab

  16. Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.

    OpenAIRE

    Goldberg, J B; Hatano, K; Meluleni, G S; Pier, G B

    1992-01-01

    As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Tra...

  17. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

    OpenAIRE

    Nagendra Suryanarayana; Vanlalhmuaka,; Bharti Mankere; Monika Verma; Kulanthaivel Thavachelvam; Urmil Tuteja

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression cons...

  18. Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells.

    Science.gov (United States)

    Betting, David J; Mu, Xi Y; Kafi, Kamran; McDonnel, Desmond; Rosas, Francisco; Gold, Daniel P; Timmerman, John M

    2009-01-01

    Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources. PMID:19000731

  19. Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Yan Chen; Hong Tian; Jian-Hui He; Jin-Yin Wu; You-jun Shang; Xiang-tao Liu

    2011-01-01

    Porcine reproductive and respiratory syndrome is caused by the PRRS virus(PRRSV), which has six structural proteins(GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay(ELISA)and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7%(266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.

  20. Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol in mucosal epithelial cells (specifically Caco-2 cell layers and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies.

  1. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    Science.gov (United States)

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  2. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  3. Immunoassay of antigens

    International Nuclear Information System (INIS)

    A method is described of immunoassay of an antigen in a liquid sample wherein a complex is formed between antigen contained in the said sample and two or more antibody reagents, and the said complex is bound to a solid support by non-covalent bonding as defined herein: and the amount of complex becoming bound to the support is determined; the process employing at least one monoclonal antibody reagent. Labelling methods including radioactive, fluorimetric and enzyme labelling may be used to effect determination of the binding ofthe complex to the solid support. The solid support may take the form of particles, beads, wall-coatings on the reaction vessel or an insert of large surface area. The method is particularly applicable to the assay of TSH, CEA, HCG, alphafeto protein, immunoglobulins, viruses, allergens, bacteria, toxins, drugs and vitamins. Use of monoclonal reagents improves the specificity of the process, and also decreases non-specific binding

  4. Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Collins, M T; Høiby, N;

    1989-01-01

    To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a...

  5. CTLA-4 recombinant protein genetically fused to canine Fcepsilon receptor Ialpha enhances allergen specific lymphocyte responses in experimentally sensitized dogs.

    Science.gov (United States)

    Yasunaga, Sho; Tsukui, Toshihiro; Masuda, Kenichi; Ohno, Koichi; Tsujimoto, Hajime

    2004-06-01

    Vaccination with a recombinant antigen fused to a targeting molecule is a potential strategy for inducing efficient immune responses. For the therapeutic purpose of allergic diseases in dogs, a DNA construct which expresses recombinant fusion protein with two functional domains, cytotoxic T lymphocyte antigen (CTLA-4) and Fcepsilon receptor Ialpha, was developed to bridge antigen-presenting cells and IgE-allergen complex. The recombinant fusion protein expressed by the DNA construct was demonstrated to retain the ability to bind monocytes in PBMC and dog IgE, respectively. Additionally, the recombinant protein induced enhancement of allergen-induced lymphoproliferation in experimentally sensitized dogs under conditions of suboptimal allergen stimulation. These results indicated that the DNA construct could enhance allergen-induced immune responses in vivo, implying its usefulness for perspective application in immunotherapy in dogs. PMID:15240934

  6. Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

    Institute of Scientific and Technical Information of China (English)

    WANG Fang-kun; YUAN Xiu-fang; WANG Yi-cheng; ZHANG Cun; XU Li-huan; LIU Si-dang

    2008-01-01

    To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and cloned into a prokaryotic expression vector, pET-28a(+), and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay (ELISA) was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

  7. Role for Gr-1+ Cells in the Control of High-Dose Mycobacterium bovis Recombinant BCG

    OpenAIRE

    Panas, Michael W.; Letvin, Norman L.

    2014-01-01

    Mycobacterium bovis bacillus Calmette-Guérin (BCG) is an attractive target for development as a live vaccine vector delivering transgenic antigens from HIV and other pathogens. Most studies aimed at defining the clearance of BCG have been performed at doses between 102 and 104 CFU. Interestingly, however, recombinant BCG (rBCG) administered at doses of >106 CFU effectively generates antigen-specific T-cell responses and primes for heterologous boost responses. Thus, defining clearance at high...

  8. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  9. A novel recombinant multi-epitope protein against Brucella melitensis infection.

    Science.gov (United States)

    Yin, Dehui; Li, Li; Song, Dandan; Liu, Yushen; Ju, Wen; Song, Xiuling; Wang, Juan; Pang, Bo; Xu, Kun; Li, Juan

    2016-07-01

    Live, attenuated Brucella vaccines are considered effective but can induce abortions in pregnant animals and are potentially infectious to humans. There is a strong need to improve the immunoprotective effects and safety of vaccines against Brucella. Currently, subunit vaccines have been demonstrated to be safe and efficacious alternatives in both humans and animals. In this study, we employed bioinformatics tools to predict B and T cell epitopes to aid development of a novel recombinant multi-epitope antigen for brucellosis vaccination. To evaluate the protective capacity of the recombinant antigen, the antigen's efficacy was studied in a mouse model of brucellosis. Our results indicated that BALB/c mice immunized with this recombinant multi-epitope antigen showed mixed Th1-Th2 immune responses with high levels of specific IgG and exhibited high degrees of IFN-γ and IL-6 and significantly higher CD3, CD4, and CD8 frequencies compared to the control group. The recombinant antigen and vaccine strain M5-90 also provided protection against Brucella melitensis 16 M infection. Using bioinformatics tools to develop candidate vaccines is a promising strategy for the development of Brucella vaccines. PMID:27133932

  10. Development of Recombinant Nucleoprotein-Based Diagnostic Systems for Lassa Fever▿

    OpenAIRE

    Saijo, Masayuki; Georges-Courbot, Marie-Claude; Marianneau, Philippe; Romanowski, Victor; Fukushi, Shuetsu; Mizutani, Tetsuya; Georges, Alain-Jean; Kurata, Takeshi; Kurane, Ichiro; Morikawa, Shigeru

    2007-01-01

    Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa ce...

  11. A method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy.

    Science.gov (United States)

    Yu, Bin; Yang, Mei; Wong, Ho Yin Bosco; Watt, Rory M; Song, Erwei; Zheng, Bo-Jian; Yuen, Kwok-Yung; Huang, Jian-Dong

    2011-07-01

    Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains. PMID:21611798

  12. [Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

    Science.gov (United States)

    Esmagambetov, I B; Sedova, E S; Shcherbinin, D N; Lysenko, A A; Garas, M N; Shmarov, M M; Logunov, D Iu

    2014-01-01

    Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice. PMID:25080815

  13. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  14. Vaccine testing of a recombinant activation-associated secreted protein (ASP1) from Ostertagia ostertagi

    OpenAIRE

    Geldhof, Peter; Meyvis, Yves; Vercruysse, Jozef; Claerebout, Edwin

    2008-01-01

    Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation-associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo-asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challeng...

  15. Vaccination against hepatitis B: comparison of intradermal and intramuscular administration of plasma derived and recombinant vaccines.

    OpenAIRE

    Payton, C. D.; Scarisbrick, D A; Sikotra, S.; Flower, A J

    1993-01-01

    A retrospective analysis of levels of antibody to hepatitis B surface antigen in 1419 health care workers was carried out to compare the efficacy of intramuscular and intradermal administration of plasma derived and recombinant hepatitis B vaccines. No significant difference was detected between the response to intradermal and intramuscular plasma derived vaccine. However of those who received intramuscular recombinant vaccine 81.6%, 13.8% and 4.7% were good (> or = 100 miu/ml), low (10-99 mi...

  16. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli

    DEFF Research Database (Denmark)

    Hiszczynska-Sawicka, E.; Brillowska-Dabrowska, A.; Dabrowski, Slawomir; Pietkiewicz, H.; Myjak, P.; Kur, J.

    2003-01-01

    This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed in...

  17. Comparative testing of six antigen-based malaria vaccine candidates directed toward merozoite-stage Plasmodium falciparum

    DEFF Research Database (Denmark)

    Arnot, David E; Cavanagh, David R; Remarque, Edmond J;

    2008-01-01

    Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-1(19) and five Pichia pastoris candidates, including two versions of MSP-1(19), AMA-1 (domains I and II), AMA-1...

  18. Enhanced and Enduring Protection against Tuberculosis by Recombinant BCG-Ag85C and Its Association with Modulation of Cytokine Profile in Lung

    OpenAIRE

    Jain, Ruchi; Dey, Bappaditya; Dhar, Neeraj; Rao, Vivek; Singh, Ramandeep; Gupta, Umesh D.; Katoch, V. M.; Ramanathan, V. D.; Tyagi, Anil K.

    2008-01-01

    Background The variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB. Methods/Principal Findings A recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretor...

  19. Carcino-Embryonic Antigen

    International Nuclear Information System (INIS)

    Tumour marker analysis has increased our understanding of the presence of tumours in the body. Carcino-embryonic antigen, CEA, is one of the best studied tumour markers and has proved an ideal diagnostic adjuvant. It has helped in quantifying the amount of disease present in a patient and thence to make accurate prognosis on the various diagnosed ailments. At UCH, it is observed that there is an increase in cancer related ailments and therefore the need for early diagnosis is more compelling in our environment to mitigate future cost of managing advanced manifestation

  20. Recombination in ionized gases

    International Nuclear Information System (INIS)

    In this paper it is shown how capture-stabilized methodology (both macroscopic and microscopic) can provide a generic basis for a unified treatment of all of the above recombination mechanisms. A new semiclassical theory of dissociative recombination is also presented in an effort to gain further insight into the physics not included in the first-order treatment and difficult to extract from numerical quantal treatments based on configuration mixing and on multichannel quantum defect theory. A simple analytical expression more accurate than the standard first-order result is obtained for the cross section σ and rate coefficient α. (author)

  1. Human leucocyte antigens in tympanosclerosis.

    Science.gov (United States)

    Dursun, G; Acar, A; Turgay, M; Calgüner, M

    1997-02-01

    This study was designed to evaluate the association between certain HLA antigens and tympanosclerosis. The serum concentrations of HLA antigens were measured by a microlymphocytotoxicity technique in patients with tympanosclerosis and compared with a healthy control group. The serum levels of HLA-B35 and -DR3 were significantly higher in the patients with tympanosclerosis. This result suggests that certain types of HLA antigens may play an important role as an indicator or mediator in the pathogenesis of tympanosclerosis. PMID:9088683

  2. Antigenic variants of rabies virus

    OpenAIRE

    Wiktor, TJ; Koprowski, H

    1980-01-01

    Antigenic variants of CVS-11 strain of rabies virus were selected after treatment of virus populations with monoclonal antibodies directed against the glycoprotein antigen of the virus. These variants resisted neutralization by the hybridoma antibody used for their selection. Two independently mutating antigenic sites could be distinguished when five variants were tested with nine hybridoma antibodies. The frequency of single epitope variants in a cloned rabies virus seed was approximately 1:...

  3. Recombinant DNA for Teachers.

    Science.gov (United States)

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  4. Recombineering Pseudomonas syringae

    Science.gov (United States)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  5. 9 CFR 113.407 - Pullorum antigen.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pullorum antigen. 113.407 Section 113... and Reagents § 113.407 Pullorum antigen. Pullorum Antigen shall be produced from a culture of... standard for stained antigen K's and 50 ±10 times McFarland No. 1 standard for tube antigen....

  6. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

    OpenAIRE

    Peter J Bredenbeek; Molenkamp, Richard; Spaan, Willy J. M.; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S.; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S.

    2006-01-01

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa F...

  7. Antigen antibody interactions

    CERN Document Server

    DeLisi, Charles

    1976-01-01

    1. 1 Organization of the Immune System One of the most important survival mechanisms of vertebrates is their ability to recognize and respond to the onslaught of pathogenic microbes to which they are conti- ously exposed. The collection of host cells and molecules involved in this recognition­ 12 response function constitutes its immune system. In man, it comprises about 10 cells 20 (lymphocytes) and 10 molecules (immunoglobulins). Its ontogenic development is c- strained by the requirement that it be capable of responding to an almost limitless variety of molecular configurations on foreign substances, while simultaneously remaining inert to those on self components. It has thus evolved to discriminate, with exquisite precision, between molecular patterns. The foreign substances which induce a response, called antigens, are typically large molecules such as proteins and polysaccharides. The portions of these with which immunoglobulins interact are called epitopes or determinants. A typical protein epitope m...

  8. Trypanosoma cruzi: circulating antigens

    Directory of Open Access Journals (Sweden)

    V. Bongertz

    1981-03-01

    Full Text Available Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.Antígenos circulantes foram detectados em soros de camundongos infectados experimentalmente com elevadas doses de Trypanosoma cruzi pela reação com soros obtidos de camundongos em fase crônica de infecção. A reação de imunodifusão entre soros homólogos agudo e crônico produziu quatro linhas de precipitação. Por reação com soro crônico de camundongo antígenos circulantes foram detectados em soros de crícetos, cães e coelhos infectados com doses elevadas de Trypanosoma cruzi e em soros de pacientes chagásicos. Uma reação foi também observada com urina de camundongos e cães infectados de forma aguda. Exoantígeno de Trypanosoma cruzi foi detectado em meio de cultura de tripanosomas e em sobrenadantes de culturas de células infectadas. Tentativas de isolamento dos antigenos são descritas.

  9. Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Aline Michelle Barbosa Bafica

    2011-11-01

    Full Text Available Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA from cutaneous leishmaniasis (CL patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ levels in 37-50% of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α. We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.

  10. HA03 as an Iranian Candidate Concealed Antigen for Vaccination against Hyalomma anatolicum anatolicum: Comparative Structural and In silico Studies

    Directory of Open Access Journals (Sweden)

    Mohammadi, A.

    2013-12-01

    Full Text Available In the last decades researchers had focused on developing a vaccine against tick based on protective antigen. Recombinant vaccines based on concealed antigen from Boophilus microplus have been developed in Australia and Cuba by the name of TICKGARD and GAVAC (De La Fuente and Kocan, 2006. Further studies on this antigen have shown some extent of protection against other species (De Vos et al., 2001. In Iran most important species is Hyalomma anatolicum and limited information about its control are available. This paper reports structural and polymorphic analysis of HA03 as an Iranian candidate concealed antigen of H. a. anatolicum deposited in Gen-Bank .(Aghaeipour et al. GQ228820. The comparison between this antigen and other mid gut concealed antigen that their characteristics are available in GenBank showed there are high rate of similarity between them. The HA03 amino acid sequence had a homology of around 89%, 64%, 56% with HA98, BM86, BM95 respectively. Potential of MHC class I and II binding region indicated a considerable variation between BM86 antigen and its efficiency against Iranian H. a. anatolicum. In addition, predicted major of hydrophobisity and similarity in N-glycosylation besides large amount of cystein and seven EGF like regions presented in protein structure revealed that value of HA03 as a new protective antigen and the necessity of the development, BM86 homolog of H. a. anatolicum HA03 based recombinant vaccine.

  11. Cancer antigen 125 and prognosis

    DEFF Research Database (Denmark)

    Høgdall, Estrid Vilma Solyom

    2008-01-01

    PURPOSE OF REVIEW: This review addresses recently reported progress in cancer antigen 125 as a prognostic marker in patients with ovarian cancer. RECENT FINDINGS: Serum cancer antigen 125 levels measured preoperatively in both early and late stage ovarian cancer may be of prognostic value. Before...... cancer antigen 125 determination may be implemented into clinical practice, cut-off levels must be evaluated and internationally defined. Studies examining serum cancer antigen 125 levels after surgery but before, during, or after treatment confirmed that changes in serum levels are of prognostic value....... Furthermore, recent studies have shown that the level of expression of cancer antigen 125 in tissue may be an independent prognostic indicator in late stage ovarian cancer. SUMMARY: Prognostic markers may potentially help to individualize treatment within subgroups of patients. In a recent study the level of...

  12. Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations.

    Science.gov (United States)

    Almazán, Consuelo; Lagunes, Rodolfo; Villar, Margarita; Canales, Mario; Rosario-Cruz, Rodrigo; Jongejan, Frans; de la Fuente, José

    2010-01-01

    The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant Rhipicephalus microplus Bm86 antigen has been shown to protect cattle against tick infestations. However, variable efficacy of Bm86-based vaccines against geographic tick strains has encouraged the research for additional tick-protective antigens. Herein, we describe the analysis of R. microplus glutathione-S transferase, ubiquitin (UBQ), selenoprotein W, elongation factor-1 alpha, and subolesin (SUB) complementary DNAs (cDNAs) by RNA interference (RNAi) in R. microplus and Rhipicephalus annulatus. Candidate protective antigens were selected for vaccination experiments based on the effect of gene knockdown on tick mortality, feeding, and fertility. Two cDNA clones encoding for UBQ and SUB were used for cattle vaccination and infestation with R. microplus and R. annulatus. Control groups were immunized with recombinant Bm86 or adjuvant/saline. The highest vaccine efficacy for the control of tick infestations was obtained for Bm86. Although with low immunogenic response, the results with the SUB vaccine encourage further investigations on the use of recombinant subolesin alone or in combination with other antigens for the control of cattle tick infestations. The UBQ peptide showed low immunogenicity, and the results of the vaccination trial were inconclusive to assess the protective efficacy of this antigen. These experiments showed that RNAi could be used for the selection of candidate tick-protective antigens. However, vaccination trials are necessary to evaluate the effect of recombinant antigens in the control of tick infestations, a process that requires efficient recombinant protein production and formulation systems. PMID:19943063

  13. Recombinant T-cell receptors : An immunologic link to cancer therapy

    NARCIS (Netherlands)

    Calogero, A; de Leij, YFMH; Mulder, NH; Hospers, GAP

    2000-01-01

    Cytotoxic T cells can specifically kill target cells that express antigens recognized by the T-cell receptor. These are membrane-bound proteins that are not ubiquitous and thus are difficult to purify and study at the protein level. The advent of recombinant DNA technology has facilitated these obje

  14. Adjuvant and immunostimulating properties of the recombinant Bm86 protein expressed in Pichia pastoris.

    Science.gov (United States)

    García-García, J C; Soto, A; Nigro, F; Mazza, M; Joglar, M; Hechevarría, M; Lamberti, J; de la Fuente, J

    1998-01-01

    The cattle tick Boophilus microplus has remained a latent problem to the cattle industry. The recombinant vaccine GAVAC against the cattle tick has proved its efficacy and, conveniently, combined with the use of chemicals could be the solution to this problem. As this vaccine is based in the recombinant concealed antigen Bm86, it has to be given periodically to the animal to maintain an adequate level of antibodies. Some other commercially available vaccines for cattle also have to be given periodically, which creates the possibility of combining vaccines for cattle. In an attempt to evaluate the possible interactions of the Bm86 with other vaccine antigens, a potent stimulatory effect was demonstrated of the recombinant Bm86 on the humoral immune response to the recombinant Hepatitis B surface antigen in mice, and to the inactivated Infectious Bovine Rhinothraqueitis virus in cattle. These results make the Bm86 antigen expressed in Pichia pastoris a good candidate for combining vaccines for cattle because of its dual role, immunogen and adjuvant. PMID:9682358

  15. Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LIU Hui Xin; ZHANG Lin; HOU Xue Xia; WAN Kang Lin; HAO Qin

    2016-01-01

    ObjectiveIn this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB. MethodsSix recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. ResultsTwo IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%,P ConclusionThree recombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

  16. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

    Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

  17. Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations

    OpenAIRE

    Almazan, C.; Lagunes, R.; Villar, M.; Canales, M.; R Rosario-Cruz; Jongejan, F; de la Fuente, J.

    2009-01-01

    The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant Rhipicephalus microplus Bm86 antigen has been shown to protect cattle against tick infestations. However, variable efficacy of Bm86-based vaccines against geographic tick strains has encouraged the research for additional tick-protective antigens. Herein, ...

  18. Cultivation of Inducible Escherichi a Coli RB791 Hepatitis B Core Antigen Producer: Screening and Modelling of Fermentation Process

    OpenAIRE

    Grīgs, O; Ruģele, K

    2010-01-01

    Recombinant Hepatitis B core antigen (HBcAg) expressed in Escherichia coli, exhibits similar morphology and antigenicity to the native core particles of Hepatitis B virus (HBV). Also core particle has become one of the most frequently studied systems, as a carrier for various foreign epitopes. This aspect leads to great interest of HBcAg as a component for discovery of new type HBV vaccine, as therapeutic and diagnostic tool. Also core particles are examined as vectors for gene engineering. ...

  19. Detecting variable (V, diversity (D and joining (J gene segment recombination using a two-colour fluorescence system

    Directory of Open Access Journals (Sweden)

    Scott Gina B

    2010-03-01

    Full Text Available Abstract Background Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG-mediated rearrangement of variable (V, diversity (D and joining (J gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(DJ recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. Results This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(DJ recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. Conclusions This system will be useful in the analysis and exploitation of the V(DJ recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.

  20. Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations.

    Science.gov (United States)

    Canales, Mario; Labruna, Marcelo B; Soares, João F; Prudencio, Carlos R; de la Fuente, José

    2009-12-01

    The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. PMID:19835826

  1. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    Institute of Scientific and Technical Information of China (English)

    葛猛; 徐俊杰; 李冰; 董大勇; 宋小红; 郭强; 赵剑; 陈薇

    2004-01-01

    The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E. coli. Signal sequence of the outer membrane protein A (OmpA) of E. coli was attached to the 5' end of the gene encoding protective antigen receptor-binding domain (the 4th domain of PA, PALM). The plasmid carrying the fusion gene was then transformed into E. coli and induced to express recombinant PAlM by IFFG. The recombinant protein was purified by chromatography and then identified by N-terrainal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ionexchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E. coli. The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

  2. Soluble recombinant influenza vaccines.

    OpenAIRE

    Fiers, W; Neirynck, S; Deroo, T; Saelens, X; Jou, W M

    2001-01-01

    Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the...

  3. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  4. Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies

    OpenAIRE

    Cottingham, Matthew G; van Maurik, Andre; Zago, Manola; Newton, Angela T.; Anderson, Richard J.; Howard, M. Keith; Schneider, Jörg; Skinner, Michael A.

    2006-01-01

    The FP9 strain of Fowlpox virus has been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recom...

  5. [Antigenic response against PPD and antigen 60 in tubercular patients: single antigen versus the combined test].

    Science.gov (United States)

    Máttar, S; Broquetas, J M; Gea, J; Aran, X; el-Banna, N; Sauleda, J; Torres, J M

    1992-05-01

    We analyze serum samples from 70 patients with pulmonary tuberculosis and 50 healthy individuals. The antigenic activity (IgG) against protein purified antigen (PPD) and antigen 60 (A60) from M. tuberculosis. Thirteen patients were also HIV infected, and three patients had AIDS defined by the presence of disseminated tuberculosis. The test using antigen alone showed a 77% sensitivity and 74% specificity when PPD is used. When A60 was used, both values improved (81% sensitivity, 94% specificity). The use of a combined test (PPD and A60) improves the sensitivity (89%) but reduces the specificity (82%). The HIV infected patients showed similar responses to those of other patients. The combined use of different antigens might be useful for diagnosing tuberculosis. PMID:1390996

  6. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P;

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  7. Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination.

    Science.gov (United States)

    Bolland, Daniel J; Koohy, Hashem; Wood, Andrew L; Matheson, Louise S; Krueger, Felix; Stubbington, Michael J T; Baizan-Edge, Amanda; Chovanec, Peter; Stubbs, Bryony A; Tabbada, Kristina; Andrews, Simon R; Spivakov, Mikhail; Corcoran, Anne E

    2016-06-14

    Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency. PMID:27264181

  8. COLONOSCOPY AND CARCINOEMBRYONIC ANTIGEN VARIATIONS

    Directory of Open Access Journals (Sweden)

    Rita G SOUSA

    2014-03-01

    Full Text Available Context Colonoscopy is essential for synchronous and metachronous cancer detection. Carcinoembryonic antigen is a colorectal cancer tumor marker, important as a follow-up tool in patients with previous colorectal cancer. False-positive carcinoembryonic antigen elevation results in multiples exams and in patient anxiety. In literature, there is reference to transient carcinoembryonic antigen increase with colonoscopy. Objective To evaluate the influence of bowel preparation and colonoscopy in carcinoembryonic antigen blood levels. Methods We prospectively studied subjects that underwent routine colonoscopy in our institution. Blood samples were collected (1 before bowel cleaning, (2 before colonoscopy and (3 immediately after colonoscopy. Blood carcinoembryonic antigen levels were determined by “Sandwich” immunoassay. The statistical methods used were the paired t-test and ANOVA. Results Thirty-seven patients (22M/15F were included; age range 28-84 (mean 56 years. Mean carcinoembryonic antigen values were 1.9, 2 and 1.8 for (1, (2 and (3, respectively. An increase in value (2 compared with (1 was observed in 20/37 patients (P = 0.018, mainly in younger patients and in patients requiring more endoluminal interventions. In 29/37 patients, the CEA value decreased from (2 to (3 (P = 1.3x10-7. Conclusions A trend for carcinoembryonic antigen increase after bowel cleaning was observed, especially in younger patients and in patients with more endoluminal interventions, but without clinical meaning.

  9. Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents.

    Science.gov (United States)

    Barr, P J; Inselburg, J; Green, K M; Kansopon, J; Hahm, B K; Gibson, H L; Lee-Ng, C T; Bzik, D J; Li, W B; Bathurst, I C

    1991-03-01

    We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae. Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies. Two recombinant SERA antigens were selected for purification and immunological analysis. The first (SERA 1), corresponding to amino acids 24-285 of the natural SERA precursor, was expressed by the ubiquitin fusion method. This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide. The second, larger protein (SERA N), encompassing amino acids 24-506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human gamma-interferon (gamma-IFN). Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P. falciparum. The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine. PMID:2052035

  10. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants

    OpenAIRE

    Chen, Qiang; He, Junyun; Phoolcharoen, Waranyoo; Mason, Hugh S.

    2011-01-01

    Expression of recombinant vaccine antigens and monoclonal antibodies using plant viral vectors has developed extensively during the past several years. The approach benefits from high yields of recombinant protein obtained within days after transient delivery of viral vectors to leaves of Nicotiana benthamiana, a tobacco relative. Modified viral genomes of both RNA and DNA viruses have been created. Geminiviruses such as bean yellow dwarf virus (BeYDV) have a small, single stranded DNA genome...

  11. 'Nothing is permanent but change'- antigenic variation in persistent bacterial pathogens.

    Science.gov (United States)

    Palmer, Guy H; Bankhead, Troy; Lukehart, Sheila A

    2009-12-01

    Pathogens persist in immunocompetent mammalian hosts using various strategies, including evasion of immune effectors by antigenic variation. Among highly antigenically variant bacteria, gene conversion is used to generate novel expressed variants from otherwise silent donor sequences. Recombination using oligonucleotide segments from multiple donors is a combinatorial mechanism that tremendously expands the variant repertoire, allowing thousands of variants to be generated from a relatively small donor pool. Three bacterial pathogens, each encoded by a small genome (Treponema pallidum TprK and Anaplasma marginale Msp2 expression sites and donors are chromosomally encoded. Both T. pallidum and A. marginale generate antigenic variants in vivo in individual hosts and studies at the population level reveal marked strain diversity in the variant repertoire that may underlie pathogen strain structure and the capacity for re-infection and heterologous strain superinfection. Here, we review gene conversion in bacterial antigenic variation and discuss the short- and long-term selective pressures that shape the variant repertoire. PMID:19709057

  12. Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis.

    Science.gov (United States)

    Matsuba, Takashi; Siddiqi, Umme Ruman; Hattori, Toshio; Nakajima, Chie; Fujii, Jun; Suzuki, Yasuhiko

    2016-05-01

    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains. PMID:27190237

  13. Recombinant vaccines and the development of new vaccine strategies

    International Nuclear Information System (INIS)

    Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks

  14. Recombinant vaccines and the development of new vaccine strategies

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, I.P.; Leite, L.C.C. [Centro de Biotecnologia, Instituto Butantan, São Paulo, SP (Brazil)

    2012-09-07

    Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

  15. Production of Brugia malayi BmSXP Recombinant Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Khoo, T. K.

    2010-01-01

    Full Text Available A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd. had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5, with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

  16. Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

    Science.gov (United States)

    Duthie, Malcolm S; Favila, Michelle; Hofmeyer, Kimberley A; Tutterrow, Yeung L; Reed, Steven J; Laurance, John D; Picone, Alessandro; Guderian, Jeffrey; Bailor, H Remy; Vallur, Aarthy C; Liang, Hong; Mohamath, Raodoh; Vergara, Julie; Howard, Randall F; Coler, Rhea N; Reed, Steven G

    2016-05-27

    Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs. PMID:27142329

  17. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

    International Nuclear Information System (INIS)

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP

  18. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta.

    Science.gov (United States)

    Brodzik, R; Bandurska, K; Deka, D; Golovkin, M; Koprowski, H

    2005-12-16

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP. PMID:16236249

  19. Oncogenic cancer/testis antigens

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Andersen, Mads H; Ditzel, Henrik J

    2015-01-01

    Recent developments have set the stage for immunotherapy as a supplement to conventional cancer treatment. Consequently, a significant effort is required to further improve efficacy and specificity, particularly the identification of optimal therapeutic targets for clinical testing. Cancer....../testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor...... immunology and immune escape suggests that targeting oncogenic antigens may be beneficial, meaning that identification of cancer/testis antigens with oncogenic properties is of high priority. Recent work from our lab and others provide evidence that many cancer/testis antigens, in fact, have oncogenic...

  20. Production and purification of avian antibodies (igys) from inclusion bodies of a recombinant protein central in nad+ metabolism

    OpenAIRE

    Moreno-González, Paula A.; Diaz, Gonzalo J.; María H. Ramírez-Hernández

    2013-01-01

    The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recom...

  1. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  2. Natural Selection Promotes Antigenic Evolvability

    OpenAIRE

    Graves, C.J.; Ros, V.I.D.; Stevenson, B.; Sniegowski, P. D.; Brisson, D.

    2013-01-01

    The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed ‘cassettes...

  3. Primordial magnetogenesis before recombination

    CERN Document Server

    Fabre, Ophélia

    2015-01-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order $10^{-49}$ G. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications.

  4. Design and Construction of Chimeric VP8-S2 Antigen for Bovine Rotavirus and Bovine Coronavirus

    Science.gov (United States)

    Nasiri, Khadijeh; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza; Zibaee, Saeed

    2016-01-01

    Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure. PMID:27123423

  5. Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development.

    Directory of Open Access Journals (Sweden)

    Joao C Aguiar

    Full Text Available Nearly 100% protection against malaria infection can be achieved in humans by immunization with P. falciparum radiation-attenuated sporozoites (RAS. Although it is thought that protection is mediated by T cell and antibody responses, only a few of the many pre-erythrocytic (sporozoite and liver stage antigens that are targeted by these responses have been identified.Twenty seven P. falciparum pre-erythrocytic antigens were selected using bioinformatics analysis and expression databases and were expressed in a wheat germ cell-free protein expression system. Recombinant proteins were recognized by plasma from RAS-immunized subjects, and 21 induced detectable antibody responses in mice and rabbit and sera from these immunized animals were used to characterize these antigens. All 21 proteins localized to the sporozoite: five localized to the surface, seven localized to the micronemes, cytoplasm, endoplasmic reticulum or nucleus, two localized to the surface and cytoplasm, and seven remain undetermined. PBMC from RAS-immunized volunteers elicited positive ex vivo or cultured ELISpot responses against peptides from 20 of the 21 antigens.These T cell and antibody responses support our approach of using reagents from RAS-immunized subjects to screen potential vaccine antigens, and have led to the identification of a panel of novel P. falciparum antigens. These results provide evidence to further evaluate these antigens as vaccine candidates.ClinicalTrials.gov NCT00870987 ClinicalTrials.gov NCT00392015.

  6. Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development

    Science.gov (United States)

    Aguiar, Joao C.; Bolton, Jessica; Wanga, Joyce; Sacci, John B.; Iriko, Hideyuki; Mazeika, Julie K.; Han, Eun-Taek; Limbach, Keith; Patterson, Noelle B.; Sedegah, Martha; Cruz, Ann-Marie; Tsuboi, Takafumi; Hoffman, Stephen L.; Carucci, Daniel; Hollingdale, Michael R.; Villasante, Eileen D.; Richie, Thomas L.

    2015-01-01

    Background Nearly 100% protection against malaria infection can be achieved in humans by immunization with P. falciparum radiation-attenuated sporozoites (RAS). Although it is thought that protection is mediated by T cell and antibody responses, only a few of the many pre-erythrocytic (sporozoite and liver stage) antigens that are targeted by these responses have been identified. Methodology Twenty seven P. falciparum pre-erythrocytic antigens were selected using bioinformatics analysis and expression databases and were expressed in a wheat germ cell-free protein expression system. Recombinant proteins were recognized by plasma from RAS-immunized subjects, and 21 induced detectable antibody responses in mice and rabbit and sera from these immunized animals were used to characterize these antigens. All 21 proteins localized to the sporozoite: five localized to the surface, seven localized to the micronemes, cytoplasm, endoplasmic reticulum or nucleus, two localized to the surface and cytoplasm, and seven remain undetermined. PBMC from RAS-immunized volunteers elicited positive ex vivo or cultured ELISpot responses against peptides from 20 of the 21 antigens. Conclusions These T cell and antibody responses support our approach of using reagents from RAS-immunized subjects to screen potential vaccine antigens, and have led to the identification of a panel of novel P. falciparum antigens. These results provide evidence to further evaluate these antigens as vaccine candidates. Trial Registration ClinicalTrials.gov NCT00870987 ClinicalTrials.gov NCT00392015 PMID:26292257

  7. Serological identification of Tektin5 as a cancer/testis antigen and its immunogenicity

    International Nuclear Information System (INIS)

    Identification of new cancer antigens is necessary for the efficient diagnosis and immunotherapy. A variety of tumor antigens have been identified by several methodologies. Among those antigens, cancer/testis (CT) antigens have became promising targets. The serological identification of antigens by the recombinant expression cloning (SEREX) methodology has been successfully used for the identification of cancer/testis (CT) antigens. We performed the SEREX analysis of colon cancer. We isolated a total of 60 positive cDNA clones comprising 38 different genes. They included 2 genes with testis-specific expression profiles in the UniGene database, such as TEKT5 and a CT-like gene, A kinase anchoring protein 3 (AKAP3). Quantitative real-time RT-PCR analysis showed that the expression of TEKT5 was restricted to the testis in normal adult tissues. In malignant tissues, TEKT5 was aberrantly expressed in a variety of cancers, including colon cancer. A serological survey of 101 cancer patients with different cancers by ELISA revealed antibodies to TEKT5 in 13 patients, including colon cancer. None of the 16 healthy donor serum samples were reactive in the same test. We identified candidate new CT antigen of colon cancer, TEKT5. The findings indicate that TEKT5 is immunogenic in humans, and suggest its potential use as diagnostic as well as an immunotherapeutic reagent for cancer patients

  8. Anvendelse af prostataspecifikt antigen. En oversigt

    DEFF Research Database (Denmark)

    Brasso, K; Skaarup, P; Roosen, Jens Ulrik; Iversen, Peter

    1998-01-01

    Since it was first introduced, measurement of prostate specific antigen has gained increasing interest, and prostate specific antigen is regarded as being the best tumour marker available. The antigen lacks cancer specificity, limiting the usefulness in early diagnosis, The use of prostate specific...... antigen in early diagnosis, staging, and in monitoring patients with prostate cancer is reviewed....

  9. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

    OpenAIRE

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2013-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel ...

  10. Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations

    OpenAIRE

    Jongejan Frans; Naranjo Victoria; Almazán Consuelo; Canales Mario; de la Fuente José

    2009-01-01

    Abstract Background The cattle ticks, Boophilus spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant B. microplus Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for B. annulatus Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast Pichia past...

  11. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip

    OpenAIRE

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Background Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Methods Multiplex fluorescent immunoassay of 139 patients’ blood ser...

  12. Expression of Recombinant Streptokinase from Streptococcus Pyogenes and Its Reaction with Infected Human and Murine Sera

    Directory of Open Access Journals (Sweden)

    Neda Molaee

    2013-09-01

    Full Text Available   Objective(s: Streptokinase (SKa is an antigenic protein which is secreted by Streptococcus pyogenes. Streptokinase induces inflammation by complement activation, which may play a role in post infectious diseases. In the present study, recombinant streptokinase from S. pyogenes was produced and showed that recombinant SKa protein was recognized by infected human sera using Western blot analysis.   Materials and Methods: In this study, the ska gene from S. pyogenes was amplified and cloned into pET32a which is a prokaryotic expression vector. pET32a-ska was transformed to Escherichia coli BL21 (DE3 pLysS and gene expression was induced by IPTG. Protein production was improved by modification of composition of the bacterial culture media and altering the induction time by IPTG. The expressed protein was purified by affinity chromatography using the Ni-NTA resin. The integrity of the product was confirmed by Westernblot analysis using infected mice. Serum reactivity of five infected individuals was further analyzed against the recombinant SKa protein. Results: Data indicated that recombinant SKa protein from S. pyogenes can be recognized by patient and mice sera. The concentration of the purified recombinant protein was 3.2 mg/L of initial culture. The highest amount of the expressed protein after addition of IPTG was obtained in a bacterial culture without glucose with the culture optical density of 0.8 (OD600 = 0.8. Conclusion : Present data shows, recombinant SKa protein has same epitopes with natural form of this antigen. Recombinant SKa also seemed to be a promising antigen for the serologic diagnosis of S. pyogenes infections.

  13. CRMAGE: CRISPR Optimized MAGE Recombineering

    OpenAIRE

    Carlotta Ronda; Lasse Ebdrup Pedersen; Sommer, Morten O. A.; Alex Toftgaard Nielsen

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulat...

  14. Recombinant constructs of Borrelia burgdorferi

    Energy Technology Data Exchange (ETDEWEB)

    Dattwyler, Raymond J. (Setauket, NY); Gomes-Solecki, Maria J. C. (New York, NY); Luft, Benjamin J. (East Setauket, NY); Dunn, John J.(Bellport, NY)

    2007-02-20

    Novel chimeric nucleic acids, encoding chimeric Borrelia proteins comprising OspC or an antigenic fragment thereof and OspA or an antigenic fragment thereof, are disclosed. Chimeric proteins encoded by the nucleic acid sequences are also disclosed. The chimeric proteins are useful as vaccine immunogens against Lyme borreliosis, as well as for immunodiagnostic reagents.

  15. Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

    Directory of Open Access Journals (Sweden)

    Hafez Heydari Zarnagh

    2015-04-01

    Full Text Available Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3 cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

  16. Commercialisation of a recombinant vaccine against Boophilus microplus.

    Science.gov (United States)

    Willadsen, P; Bird, P; Cobon, G S; Hungerford, J

    1995-01-01

    Increasingly, there is need for methods to control cattle tick (Boophilus microplus) infestations by the use of non-chemical technology. This need is brought about by a mixture of market forces and the failure or inadequacy of existing technology. A recombinant vaccine has now been developed against the tick. This vaccine relies on the uptake with the blood meal of antibody directed against a critical protein in the tick gut. The isolation of the vaccine antigen, Bm86, and its production as a recombinant protein is briefly described. The vaccine has been tested in the field, has been taken through the full registration process and is now in commercial use in Australia. A related development has occurred in Cuba. The potential for improvement of the current vaccine and for the development of similar vaccines against other haematophagous parasites is discussed. PMID:7784128

  17. Characterization of recombinant Raccoonpox Vaccine Vectors in Chickens

    Science.gov (United States)

    Hwa, S.-H.; Iams, K.P.; Hall, J.S.; Kingstad, B.A.; Osorio, J.E.

    2010-01-01

    Raccoonpox virus (RCN) has been used as a recombinant vector against several mammalian pathogens but has not been tested in birds. The replication of RCN in chick embryo fibroblasts (CEFs) and chickens was studied with the use of highly pathogenic avian influenza virus H5N1 hemagglutinin (HA) as a model antigen and luciferase (luc) as a reporter gene. Although RCN replicated to low levels in CEFs, it efficiently expressed recombinant proteins and, in vivo, elicited anti-HA immunoglobulin yolk (IgY) antibody responses comparable to inactivated influenza virus. Biophotonic in vivo imaging of 1-wk-old chicks with RCN-luc showed strong expression of the luc reporter gene lasting up to 3 days postinfection. These studies demonstrate the potential of RCN as a vaccine vector for avian influenza and other poultry pathogens. ?? American Association of Avian Pathologists 2010.

  18. Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

    OpenAIRE

    Zarucki-Schulz, T; Kulomaa, M S; Headon, D R; N.L. Weigel; Baez, M; Edwards, D.P.; McGuire, W L; Schrader, W T; O'Malley, B W

    1984-01-01

    A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 10...

  19. Antigenicity and Diagnostic Potential of Vaccine Candidates in Human Chagas Disease

    OpenAIRE

    Shivali Gupta; Xianxiu Wan; Zago, Maria P.; Martinez Sellers, Valena C.; Silva, Trevor S.; Dadjah Assiah; Monisha Dhiman; Sonia Nuñez; Petersen, John R; Vázquez-Chagoyán, Juan C.; Jose G Estrada-Franco; Nisha Jain Garg

    2013-01-01

    BACKGROUND: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. METHODS AND RESULTS: Sera samples f...

  20. Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein.

    Science.gov (United States)

    Chalayon, Piyanart; Chanket, Phanita; Boonchawalit, Toungporn; Chattanadee, Siriporn; Srimanote, Potjanee; Kalambaheti, Thareerat

    2011-05-01

    The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test. PMID:21353274

  1. Development of an Aeromonas hydrophila recombinant extracellular protease vaccine.

    Science.gov (United States)

    Wu, Lei; Jiang, Ya-nan; Tang, Qian; Lin, Hui-xing; Lu, Cheng-ping; Yao, Huo-chun

    2012-01-01

    Aeromonas hydrophila (Ah) exists widely in the aquatic environment and infects a variety of animals. Extracellular protease (EPR) is an important protective antigen that induces a specific antibody response to resist Ah infection. In this study, two genes encoding extracellular protease epr2 and epr3 were linked within the expression vector pET32a to construct a recombinant pET-epr2-3 plasmid. The immunogenicity of the fusion protein epr2-3 was investigated as a subunit vaccine in ICR mice. The recombinant epr2-3 protein induced the production of high antibody titers. The survival rate against homogenous Ah J-1 challenge was significantly higher in the epr2-3 vaccinated group (≥80%) compared with the inactivated Ah vaccinated group and the challenge control group (P < 0.01), thus indicating that the recombinant epr2-3 protein provided significant protection against Ah infection. Therefore, the recombinant epr2-3 is a promising candidate for development as a vaccine against Ah infections. PMID:22874879

  2. A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

    OpenAIRE

    Bonafé, Nathalie; Rininger, Joseph A.; Chubet, Richard G.; Foellmer, Harald G.; Fader, Stacey; Anderson, John F.; Bushmich, Sandra L.; Anthony, Karen; Ledizet, Michel; Fikrig, Erol; Koski, Raymond A.; Kaplan, Paul

    2008-01-01

    In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hyd...

  3. Effects of dexamethasone on human natural killer cell cytotoxicity, interferon production, and interleukin-2 receptor expression induced by microbial antigens.

    OpenAIRE

    E. Piccolella; Lombardi, G.; Vismara, D; Del Gallo, F; Colizzi, V; Dolei, A; Dianzani, F

    1986-01-01

    Dexamethasone inhibits the expression of the interleukin-2 receptor, the synthesis of immune interferon, and the development of natural killer cells when added to peripheral blood mononuclear cells cultured with soluble microbial antigens (purified protein derivative and a polysaccharide extract from Candida albicans [MPPS]) or human recombinant interleukin-2.

  4. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  5. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    International Nuclear Information System (INIS)

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems

  6. The effects of a partitioned var gene repertoire of Plasmodium falciparum on antigenic diversity and the acquisition of clinical immunity

    Directory of Open Access Journals (Sweden)

    Arinaminpathy Nimalan

    2008-01-01

    Full Text Available Abstract Background The human malaria parasite Plasmodium falciparum exploits antigenic diversity and within-host antigenic variation to evade the host's immune system. Of particular importance are the highly polymorphic var genes that encode the family of cell surface antigens PfEMP1 (Plasmodium falciparum Erythrocyte Membrane Protein 1. It has recently been shown that in spite of their extreme diversity, however, these genes fall into distinct groups according to chromosomal location or sequence similarity, and that recombination may be confined within these groups. Methods This study presents a mathematical analysis of how recombination hierarchies affect diversity, and, by using simple stochastic simulations, investigates how intra- and inter-genic diversity influence the rate at which individuals acquire clinical immunity. Results The analysis demonstrates that the partitioning of the var gene repertoire has a limiting effect on the total diversity attainable through recombination and that the limiting effect is strongly influenced by the respective sizes of each of the partitions. Furthermore, by associating expression of one of the groups with severe malaria it is demonstrated how a small number of infections can be sufficient to protect against disease despite a seemingly limitless number of possible non-identical repertoires. Conclusion Recombination hierarchies within the var gene repertoire of P. falciparum have a severe effect on strain diversity and the process of acquiring immunity against clinical malaria. Future studies will show how the existence of these recombining groups can offer an evolutionary advantage in spite of their restriction on diversity.

  7. Successful vaccination against Boophilus microplus and Babesia bovis using recombinat antigens

    Directory of Open Access Journals (Sweden)

    P. Willadsen

    1992-01-01

    Full Text Available Current methods for the control of the cattle tick Boophils microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials.

  8. [Farmer's lung antigens in Germany].

    Science.gov (United States)

    Sennekamp, J; Joest, M; Sander, I; Engelhart, S; Raulf-Heimsoth, M

    2012-05-01

    Recent studies suggest that besides the long-known farmer's lung antigen sources Saccharopolyspora rectivirgula (Micropolyspora faeni), Thermoactinomyces vulgaris, and Aspergillus fumigatus, additionally the mold Absidia (Lichtheimia) corymbifera as well as the bacteria Erwinia herbicola (Pantoea agglomerans) and Streptomyces albus may cause farmer's lung in Germany. In this study the sera of 64 farmers with a suspicion of farmer's lung were examined for the following further antigens: Wallemia sebi, Cladosporium herbarum, Aspergillus versicolor, and Eurotium amstelodami. Our results indicate that these molds are not frequent causes of farmer's lung in Germany. PMID:22477566

  9. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  10. Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis.

    Science.gov (United States)

    Carrillo, Eugenia; Crusat, Martín; Nieto, Javier; Chicharro, Carmen; Thomas, Maria del Carmen; Martínez, Enrique; Valladares, Basilio; Cañavate, Carmen; Requena, Jose María; López, Manuel Carlos; Alvar, Jorge; Moreno, Javier

    2008-03-28

    Zoonotic visceral leishmaniasis (ZVL) is a parasitic disease caused by Leishmania infantum/L. chagasi that is emerging as an important medical and veterinary problem. Dogs are the domestic reservoir for this parasite and, therefore, the main target for controlling the transmission to humans. In the present work, we have evaluated the immunogenicity of the Leishmania infantum heat shock protein (HSP)-70, paraflagellar rod protein (PFR)-2 and kinetoplastida membrane protein (KMP)-11 recombinant proteins in dogs experimentally infected with the parasite. We have shown that peripheral blood mononuclear cells (PBMC) from experimentally infected dogs proliferated in response to these recombinant antigens and against the soluble leishmanial antigen (SLA). We have also quantified the mRNA expression level of the cytokines induced in PBMC upon stimulation with the HSP-70, PFR-2 and KMP-11 proteins. These recombinant proteins induced an up-regulation of IFN-gamma. HSP-70 and PFR-2 also produced an increase of the TNF-alpha transcripts abundance. No measurable induction of IL-10 was observed and low levels of IL-4 mRNA were produced in response to the three mentioned recombinant antigens. Serum levels of specific antibodies against HSP-70, PFR-2 and KMP-11 recombinant proteins were also determined in these animals. Our study showed that HSP-70, KMP-11 and PFR-2 proteins are recognized by infected canines. Furthermore, these antigens produce a Th1-type immune response, suggesting that they may be involved in protection. The identification as vaccine candidates of Leishmania antigens that elicit appropriate immune responses in the canine model is a key step in the rational approach to generate a vaccine for canine visceral leishmaniasis. PMID:18321614

  11. Protective immunity induced in Aotus monkeys by recombinant SERA proteins of Plasmodium falciparum.

    OpenAIRE

    Inselburg, J; Bzik, D J; Li, W B; Green, K M; Kansopon, J; Hahm, B K; Bathurst, I C; Barr, P J; Rossan, R N

    1991-01-01

    We describe the vaccination of Panamanian monkeys (Aotus sp.) with two recombinant blood stage antigens that each contain a portion of the N-terminal region of the SERA (serine repeat antigen) protein of the malaria parasite Plasmodium falciparum. We immunized with either a 262-amino-acid SERA fragment (SERA I) that contains amino acids 24 to 285 of the 989-amino-acid protein or a 483-amino-acid SERA fragment (SERA N) that contains amino acids 24 to 506 as part of a fusion protein with human ...

  12. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

    Directory of Open Access Journals (Sweden)

    Chung Nam-Jun

    2011-04-01

    Full Text Available Abstract Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1, a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP, a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR, beta-glucuronidase reporter gene (GUS assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38 and a clinical specificity of 100% (n = 24 as assessed by enzyme-linked immunosorbent assay (ELISA. Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40, TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

  13. Transcriptional Regulation of the Borrelia burgdorferi Antigenically Variable VlsE Surface Protein

    OpenAIRE

    Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P.; Norris, Steven J; Stevenson, Brian

    2006-01-01

    The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that...

  14. Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis

    OpenAIRE

    Cao, Liang; Chen, Da-Liang; Lee, Cindy; Chan, Che-Man; Chan, King-Man; Vanittanakom, Nongnuch; Tsang, Dominic N.C.; Yuen, Kwok-Yung

    1998-01-01

    The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marne...

  15. Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

    OpenAIRE

    Medina, Marcela; Vintiñi, Elisa; Villena, Julio; Raya, Raul; Alvarez, Susana

    2010-01-01

    Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent...

  16. Oral immunization with hepatitis B surface antigen expressed in transgenic plants

    OpenAIRE

    Kong, Qingxian; Richter, Liz; Yang, Yu Fang; Arntzen, Charles J.; Mason, Hugh S.; Thanavala, Yasmin

    2001-01-01

    Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic ...

  17. Antigen capture ELISA for the heat shock protein (hsp60) of Chlamydia trachomatis.

    OpenAIRE

    Horner, P J; Ali, M.; Parker, D.; Weber, J. N.; Taylor-Robinson, D.; McClure, M O

    1996-01-01

    AIMS: To develop an indirect ELISA using the heat shock protein (hsp60) of Chlamydia trachomatis as antigen. METHODS: The hsp60 gene was amplified by PCR, expressed in the vector pDEV-107 and transformed into Escherichia coli. The recombinant protein, expressed as a beta-galactosidase fusion product, was captured onto a solid phase using a monoclonal antibody directed against beta-galactosidase. Following incubation with goat anti-human antibody conjugated to peroxidase and colour development...

  18. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

    Directory of Open Access Journals (Sweden)

    Pacífico Lucila G

    2007-01-01

    Full Text Available Abstract Background Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL in the presence of Polymyxin B (10 μg/mL. Results The levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves. Conclusion This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

  19. Delayed recombination and standard rulers

    International Nuclear Information System (INIS)

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  20. Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

    Directory of Open Access Journals (Sweden)

    Paula A. Moreno-González

    2013-08-01

    Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

  1. Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA

    Science.gov (United States)

    Orubu, Toritse; Alharbi, Naif Khalaf; Lambe, Teresa; Gilbert, Sarah C.; Cottingham, Matthew G.

    2012-01-01

    CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens. PMID:22761956

  2. Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA.

    Directory of Open Access Journals (Sweden)

    Toritse Orubu

    Full Text Available CD8(+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8(+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens.

  3. Commercial bacterins did not induce detectable levels of antibodies in mice against Mycoplasma hyopneumoniae antigens strongly recognized by swine immune system

    Directory of Open Access Journals (Sweden)

    Andressa Fisch

    2016-01-01

    Full Text Available Enzootic Pneumonia (EP caused by Mycoplasma hyopneumoniae results in major economic losses to the swine industry. Hence, the identification of factors that provide protection against EP could help to develop effective vaccines. One such factor that provides partial protection are bacterins. Therefore, the aim of this study was to verify the induction of antibodies against fifteen M. hyopneumoniae antigens, strongly recognized by the swine immune system during natural infection, in mice vaccinated with six commercial bacterins. Each group of mice was inoculated with one bacterin, and seroconversion was assessed by indirect ELISA using recombinant antigens and M. hyopneumoniae 7448 whole cell extract. Sera from one inoculated group recognized antigen MHP_0067, and sera from four inoculated groups recognized antigens MHP_0513 and MHP_0580. None of the bacterins was able to induce seroconversion against the twelve remaining antigens. This absence of a serological response could be attributed to the lack of antigen expression in M. hyopneumoniae strains used in bacterin production. Additionally the partial protection provided by these vaccines could be due to low expression or misfolding of antigens during vaccine preparation. Therefore, the supplementation of bacterins with these recombinant antigens could be a potential alternative in the development of more effective vaccines.

  4. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    International Nuclear Information System (INIS)

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

  5. Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUS.

    Science.gov (United States)

    Preuss, Klaus-Dieter; Held, Gerhard; Kubuschok, Boris; Hung, Chun-Zhu; Malatsidze, Natalia; Wagner, Mathias; Pfreundschuh, Michael

    2007-07-15

    Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 x 10(8) paraprotein-antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely. PMID:17373665

  6. Major histocompatibility complex (MHC) antigens

    Czech Academy of Sciences Publication Activity Database

    Hála, K.; Plachý, Jiří; Kaufman, J.

    New York : Academic Press, 1998 - (Pastoret, P.; Griebel, P.; Bazin, H.; Govaerts, A.), s. 92-95 ISBN 0-12-546401-0 R&D Projects: GA ČR GA523/96/0670 Keywords : chicken MHC * histocompatibility antigens * disease resistance Subject RIV: EB - Genetics ; Molecular Biology

  7. Production and Characterization of ZFP36L1 Antiserum against Recombinant Protein from Escherichia coli

    OpenAIRE

    Cao, Heping; LIN, RUI; Ghosh, Sanjukta; Anderson, Richard A.; Urban, Joseph F.

    2008-01-01

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over-expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were us...

  8. Serological identification of tumor antigens of esophageal squamous cell carcinoma.

    Science.gov (United States)

    Shimada, Hideaki; Nakashima, Kazue; Ochiai, Takenori; Nabeya, Yoshihiro; Takiguchi, Masaki; Nomura, Fumio; Hiwasa, Takaki

    2005-01-01

    Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma. PMID:15586227

  9. Biological role of surface Toxoplasma gondii antigen in development of vaccine

    Institute of Scientific and Technical Information of China (English)

    Ke-Yi Liu; Dian-Bo Zhang; Qing-Kuan Wei; Jin Li; Gui-Ping Li; Jin-Zhi Yu

    2006-01-01

    AIM: To analyze the biological role of the surface antigen of Toxoplasma gondii (T gondii) in development of vaccine.METHODS: The surface antigen of Tgondii (SAG1)was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1and challenged with lethal strain of T gondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on T gondii tachyzoites under electromicroscope.RESULTS:The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group.The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-γ, IL-2 and IL-4cytokines in mice. In contrast, IL-12, IL-6 and TNF-αwere undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed,swollen, and holes and gaps formed on the surface.CONCLUSION: SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against Tgondii may be regulated by both hormone- and cell-mediated immune response.

  10. Production and characterization of a Brazilian candidate antigen for Hepatitis E Virus genotype 3 diagnosis.

    Science.gov (United States)

    de Almeida Ramos, Denise; Miani, Michela; Pandolfi, Rafael; Tondo, Luis; Colli, Maikel L; Rosado Spilki, Fernando; Rovaris Gardinali, Noemi; Alves Pinto, Marcelo; Kreutz, Luiz C; Frandoloso, Rafael

    2016-03-01

    Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2((394-661)) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets' feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p. PMID:26832642

  11. Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen.

    Science.gov (United States)

    Flexner, C; Murphy, B R; Rooney, J F; Wohlenberg, C; Yuferov, V; Notkins, A L; Moss, B

    1988-09-15

    A global vaccination strategy must take into account production and delivery costs as well as efficacy and safety. A heat-stable, polyvalent vaccine that requires only one inoculation and induces a high level of humoral and cellular immunity against several diseases is therefore desirable. A new approach is to use live microorganisms such as mycobacteria, enteric bacteria, adenoviruses, herpesviruses and poxviruses as vaccine vectors. A potential limitation of live polyvalent vaccines, however, is existing immunity within the target population not only to the vector, but to any of the expressed antigens. This could restrict replication of the vector, curtail expression of antigens, and reduce the total immune response to the vaccine. Recently acquired immunity to vaccinia virus can severely limit the efficacy of a live recombinant vaccinia-based vaccine, so a strategy involving closely spaced inoculations with the same vector expressing different antigens may present difficulties. We have constructed a recombinant vaccinia virus that expresses surface proteins from two diverse pathogens, influenza A virus haemagglutinin and herpes simplex virus type 1 (HSV-1) glycoprotein D. Mice that had recently recovered from infection with either HSV-1 or influenza A virus could still be effectively immunized with the double recombinant. PMID:2842693

  12. Contrasting roles of interallelic recombination at the HLA-A and HLA-B loci

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, A.L.; Hughes, M.K. (Pennsylvania State Univ., University Park (United States)); Watkins, D.I. (Univ. of Wisconsin, Madison (United States))

    1993-03-01

    A statistical study of DNA sequences of alleles at the highly polymorphic class I MHC loci of humans, HLA-A and HLA-B, showed evidence of both large-scale recombination events(involving recombination of exons 1-2 of one allele with exons 3-8 of another) and small scale recombination events (involving apparent exchange of short DNA segments). The latter events occurred disproportionately in the region of the gene encoding the antigen recognition site (ARS) of the class I molecule. Furthermore, they involved the ARS codons which are under the strongest selection favoring allelic diversity at the amino acid level. Thus, the frequency of recombinant alleles appears to have been increased by some form of balancing selection (such as overdominant selection) favoring heterozygosity in the ARS. These analyses also revealed a striking difference between the A and B loci. Recombination events appear to have occurred about twice as frequently at the B locus, and recombinants at the B locus were significantly more likely to affect polymorphic sites in the ARS. At the A locus, there are well-defined allelic lineages that have persisted since prior to the human-chimpanzee divergence; but at the B locus, there is no evidence for such long-lasting allelic lineages. Thus, relatively frequent interallelic recombination has apparently been a feature of the long-term evolution of the B locus but not of the A locus. 45 refs., 4 figs., 5 tabs.

  13. Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity.

    Science.gov (United States)

    Machado, Alexandre V; Caetano, Bráulia C; Barbosa, Rafael P; Salgado, Ana Paula C; Rabelo, Renata H; Garcia, Cristiana C; Bruna-Romero, Oscar; Escriou, Nicolas; Gazzinelli, Ricardo T

    2010-04-19

    In this work, we explored an original vaccination protocol using recombinant influenza and adenovirus. We constructed recombinant influenza viruses harboring dicistronic NA segments containing the surface antigen 2 (SAG2) from Toxoplasma gondii under control of the duplicated 3' promoter. Recombinant influenza viruses were able to drive the expression of the foreign SAG2 sequence in cell culture and to replicate efficiently both in cell culture and in lungs of infected mice. In addition, mice primed with recombinant influenza virus and boosted with a recombinant adenovirus encoding SAG2 elicited both humoral and cellular immune responses specific for SAG2. Moreover, when immunized animals were challenged with the cystogenic P-Br strain of T. gondii, they displayed up to 85% of reduction in parasite burden. These results demonstrate the potential use of recombinant influenza vectors harboring the dicistronic segments in the development of vaccines against infectious diseases. PMID:20189485

  14. Suppression of the antigen-specific T cell immune response by co-immunization with the HBV DNA vaccine and recombinant HbsAg%重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

    Institute of Scientific and Technical Information of China (English)

    杜小刚; 王军朋; 康有敏; 王肖; 赵干; 王宾

    2009-01-01

    [Objective] To explore a new therapeutic strategy against acute hepatitis B and fulminant hepatitis B, we studied effect of co-immunization with HBV DNA and HBsAg on the T cell proliferation reaction. [Methods] We immunized the BALB/ c mice with HBV DNA vaccine (pcDS2) plus HBsAg by intramuscular injection. The immunization was performed on week 0, 2 and 4. The anti-HBs(IgC)antibody titer, T lymphocyte proliferation reaction , and the expression of IL-10 and Foxp3 in CD3 + T cell were detected on week 6. [Results] The anti-HBs IgC titer induced by pcDS2 plus HBsAg group was higher than that induced by pcDS2, or HBsAg alone. Compared to mice immunized with pcDS2, or HBsAg alone, the stimulated index (SI) of T cell proliferation induced by the pcDS2 plus HBsAg group tested by MTT methods decreased. Besides, the immune suppression of T cell proliferation response induced by co-immunization group was further confirmed by flow cytometry. Finally, the expression of IL-10 and Foxp3 in CD3+ T cell was up-regulated in the co-immunization group significantly. [Conclusion] The co-immunization of HBV DNA vaccine and HBsAg can induce the humoral immune response, but cannot induce antigen specific T cell proliferation reaction. Besides, the immune suppression induced by co-immunization may be correlated with the expression of IL-10 and Foxp3.%[目的]为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响.[方法]我们将HBV DNA疫苗(pcDS2)和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTr和流式细胞仪检测T细胞增殖反应及流式细胞仪检测细胞因子表达水平.[结果]pcDs2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后,联

  15. Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody

    Institute of Scientific and Technical Information of China (English)

    LIU Ming-ming; LI Qing; ZHAO Lan-lan; GAO Ying; HUANG You-yuan; LU Gui-zhi; GAO Yan-ming

    2013-01-01

    Background Thyroid peroxidase (TPO) is an important autoantigen in Hashimoto's thyroiditis (HT),and almost all epitopes are located in TPO ectodomain.The glycosylation of TPO might contribute to breaking self-tolerance,therefore,purified glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT.The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO.Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain.The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays (ELISAs).The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs.Results TPO ectodomain was recovered from the culture media as a soluble protein,and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography.The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies.Fucose,sialic acid and galactose were all detected on the recombinant TPO ectodomain.Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid.Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites,which might have little effect on recognition by serum TPOAb.

  16. A plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with Bacillus anthracis Ames spores

    OpenAIRE

    Chichester, Jessica A.; Manceva, Slobodanka D; Rhee, Amy; Coffin, Megan V.; Musiychuk, Konstantin; Mett, Vadim; Shamloul, Moneim; Norikane, Joey; Streatfield, Stephen J.; Yusibov, Vidadi

    2013-01-01

    The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our la...

  17. Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

    Directory of Open Access Journals (Sweden)

    AH ahmadi

    2015-02-01

    Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

  18. Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

    OpenAIRE

    Griffiths, J C; Harris, S. J.; Layton, G T; Berrie, E L; French, T J; Burns, N R; Adams, S E; Kingsman, A J

    1993-01-01

    In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that ...

  19. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

    Science.gov (United States)

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Ling, Shigan; Zhang, Heqiu

    2011-01-01

    A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies. PMID:21029749

  20. Genome Scale Identification of Treponema pallidum Antigens

    OpenAIRE

    McKevitt, Matthew; Brinkman, Mary Beth; McLoughlin, Melanie; Perez, Carla; Howell, Jerrilyn K.; Weinstock, George M.; Norris, Steven J; Palzkill, Timothy

    2005-01-01

    Antibody responses for 882 of the 1,039 proteins in the proteome of Treponema pallidum were examined. Sera collected from infected rabbits were used to systematically identify 106 antigenic proteins, including 22 previously identified antigens and 84 novel antigens. Additionally, sera collected from rabbits throughout the course of infection demonstrated a progression in the breadth and intensity of humoral immunoreactivity against a representative panel of T. pallidum antigens.

  1. Therapeutic Use of Native and Recombinant Enteroviruses

    Directory of Open Access Journals (Sweden)

    Jani Ylä-Pelto

    2016-02-01

    Full Text Available Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these “viral” receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy.

  2. Therapeutic Use of Native and Recombinant Enteroviruses.

    Science.gov (United States)

    Ylä-Pelto, Jani; Tripathi, Lav; Susi, Petri

    2016-03-01

    Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these "viral" receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy. PMID:26907330

  3. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  4. Three Decades of Recombinant DNA.

    Science.gov (United States)

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  5. Stable recombination hotspots in birds.

    Science.gov (United States)

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  6. Detection of O antigens in Escherichia coli

    Science.gov (United States)

    Lipopolysaccharide on the surface of Escherichia coli constitute the O antigens, which are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in host-pathogen interactions. O antigens that are responsible for antigenic specificity of the ...

  7. Plague virulence antigens from Yersinia enterocolitica.

    OpenAIRE

    Carter, P B; Zahorchak, R J; Brubaker, R R

    1980-01-01

    The virulence of Yersinia enterocolitica, biotype 2, serotype O:8, in mice is related to its ability to produce plague V and W antigens. V and W antigens in Y. enterocolitica are shown to be immunologically identical to the previously described V and W antigens of Yersinia pestis and Yersinia pseudotuberculosis.

  8. Molecular characterization of common treponemal antigens.

    OpenAIRE

    Hanff, P A; Miller, J N; Lovett, M A

    1983-01-01

    A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.

  9. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus

    International Nuclear Information System (INIS)

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis virus nucleocapsid

  10. Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens

    Science.gov (United States)

    Stone, Brad C.; Kas, Arnold; Billman, Zachary P.; Fuller, Deborah H.; Fuller, James T.; Shendure, Jay; Murphy, Sean C.

    2016-01-01

    Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens. PMID:27070430

  11. 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

  12. Combinatorics in Recombinational Population Genomics

    Science.gov (United States)

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  13. Progenitors of Recombining Supernova Remnants

    OpenAIRE

    Moriya, Takashi J.

    2012-01-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the superno...

  14. Do mitochondria recombine in humans?

    OpenAIRE

    Eyre-Walker, A

    2000-01-01

    Until very recently, mitochondria were thought to be clonally inherited through the maternal line in most higher animals. However, three papers published in 2000 claimed population-genetic evidence of recombination in human mitochondrial DNA. Here I review the current state of the debate. I review the evidence for the two main pathways by which recombination might occur: through paternal leakage and via a mitochondrial DNA sequence in the nuclear genome. There is no strong evidence for either...

  15. Recombinant snake venom prothrombin activators

    OpenAIRE

    Lövgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  16. Combining Viral Vectored and Protein-in-adjuvant Vaccines Against the Blood-stage Malaria Antigen AMA1: Report on a Phase 1a Clinical Trial

    OpenAIRE

    Susanne H. Hodgson; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Thomas W Rampling; Biswas, Sumi; Ian D Poulton; Miura, Kazutoyo; Douglas, Alexander D.; Alanine, Daniel GW; Illingworth, Joseph J.; de Cassan, Simone C.; ZHU, DAMING; Nicosia, Alfredo; Long, Carole A.

    2014-01-01

    The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines—chimpanzee adenovir...

  17. Speciifc T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese hIV Positive Individuals

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect speciifc T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were veriifed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity;the recombinant CFP-10 protein was taken as stimulus to detect speciifc T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94%in total bacterial protein of E. coli and the molecular weight is 31 kD;Western blot conifrmed the recombinant proteins had high antigenicity;our in-house ELISpot-IFN-γassay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

  18. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    OpenAIRE

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong; Yu, Xinglong

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 9...

  19. Delayed recombination and cosmic parameters

    International Nuclear Information System (INIS)

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: εαi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  20. Booster immunization with a partially purified citrus tristeza virus (CTV) preparation after priming with recombinant CTV coat protein enhances the binding capacity of capture antibodies by ELISA.

    Science.gov (United States)

    Bar-Joseph, M; Filatov, V; Gofman, R; Guang, Y; Hadjinicolis, A; Mawassi, M; Gootwine, E; Weisman, Y; Malkinson, M

    1997-08-01

    Groups of rabbits and young lambs were immunized subcutaneously and intramuscularly with a recombinant citrus tristeza virus (CTV) coat protein (rCTV-CP) antigen. Three weeks after primary immunization the animals were divided into two groups that were boosted either with rCTV-CP or with a partially purified preparation of CTV particles (ppCTV). Twelve and 15 days after the last injection, the animals were bled and the binding capacity of the antisera for CTV detection was examined for capture antibodies by the indirect ELISA. Considerably higher ELISA titers were obtained from animals that were boosted with ppCTV than with rCP. Boosting with partially purified native antigens after priming with recombinant antigens is expected to extend the applicability of the antisera for detecting other structural and non-structural viral antigens by trapping ELISA. PMID:9274814

  1. Recombinant human erythropoietin increases cerebral cortical width index and neurogenesis following ischemic stroke☆

    OpenAIRE

    Wen, Zhongmin; Wang, Peiji

    2012-01-01

    The cerebral cortical expansion index refers to the ratio between left and right cortex width and is recognized as an indicator for cortical hyperplasia. Cerebral ischemia was established in CB-17 mice in the present study, and the mice were subsequently treated with recombinant human erythropoietin via subcutaneous injection. Results demonstrated that cerebral cortical width index significantly increased. Immunofluorescence detection showed that the number of nuclear antigen antibody/5-bromo...

  2. The origins of the RAG genes – from transposition to V(D)J recombination

    OpenAIRE

    Fugmann, Sebastian D.

    2009-01-01

    The Recombination Activating Genes 1 and 2 (Rag1 and Rag2) encode the key enzyme that is required for the generation of the highly diversified antigen receptor repertoire central to adaptive immunity. The longstanding model proposed that this gene pair was acquired by horizontal gene transfer to explain its abrupt appearance in the vertebrate lineage. The analyses of the enormous amount of sequence data created by many genome sequencing projects now provides the basis for a more refined model...

  3. Induced HBs antigenemia in healthy adults after immunization with two different hepatitis B recombinant vaccines

    OpenAIRE

    Masoud ZIAEE; Saádatjoo, Alireza; Mohamadpour, Malihe; Namaei, Mohammad Hasan

    2010-01-01

    Background and Aims Currently, vaccination is the most effective protective tool against hepatitis B virus infection. Some studies have shown that positive results for a hepatitis B virus surface antigen (HBsAg) test may be seen after vaccination. Materials and Methods In this clinical trial study, 62 healthy adult volunteers were randomly assigned to receive either the Engerix-B or the Hepavax-Gene hepatitis B recombinant vaccine. Blood samples were drawn 1, 3, and 5 days after vaccination a...

  4. Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor

    OpenAIRE

    Liangliang Li; Qiang Guo; Ju Liu; Jun Zhang; Ying Yin; Dayong Dong; Ling Fu; Junjie Xu; Wei Chen

    2016-01-01

    Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, t...

  5. Experimental vaccination of sheep and cattle against tick infestation using recombinant 5′-nucleotidase

    OpenAIRE

    Hope, M.; Jiang, X.; Gough, J.; CADOGAN, L; JOSH, P; Jonsson, N.; P. Willadsen

    2010-01-01

    Limited prior evidence suggests that 5′-nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti-tick vaccine. To assess this, recombinant 5′-nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti-nucleotidase antibodies showed significant p...

  6. Recombinant human interleukin 2 acts as a B cell growth and differentiation promoting factor

    OpenAIRE

    Emmrich, F.; Moll, Heidrun; Markus M Simon

    2009-01-01

    Human B cells appropriately activated by a B cell mitogen are rendered susceptible to human Interleukin 2 (IL-2) as demonstrated with recombinant human IL-2 (rec. h IL-2). They show increased proliferation and drastically enhanced immunoglobulin secretion. Susceptibility to IL-2 is accompanied with the expression of the IL-2 receptor (Tac antigen) on B cells. The data suggest that IL-2 is one of the lymphokines directly involved in the activation of B lymphocytes.

  7. Recombinant T Cell Receptor Ligands Improve Outcome After Experimental Cerebral Ischemia

    OpenAIRE

    Akiyoshi, Kozaburo; Dziennis, Suzan; Palmateer, Julie; Ren, Xuefang; Vandenbark, Arthur A.; Offner, Halina; Herson, Paco S; Hurn, Patricia D.

    2011-01-01

    A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a ther...

  8. Designer genes. Recombinant antibody fragments for biological imaging

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs), with high specificy and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs or toxins in vivo. However, the implementation of radiolabeled antibodies as magic bullets for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-17 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-CH3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor: blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering and

  9. Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms

    Directory of Open Access Journals (Sweden)

    Michael R. Kierny

    2012-07-01

    Full Text Available The utility of biomarker detection in tomorrow's personalized health care field will mean early and accurate diagnosis of many types of human physiological conditions and diseases. In the search for biomarkers, recombinant affinity reagents can be generated to candidate proteins or post-translational modifications that differ qualitatively or quantitatively between normal and diseased tissues. The use of display technologies, such as phage-display, allows for manageable selection and optimization of affinity reagents for use in biomarker detection. Here we review the use of recombinant antibody fragments, such as scFvs and Fabs, which can be affinity-selected from phage-display libraries, to bind with both high specificity and affinity to biomarkers of cancer, such as Human Epidermal growth factor Receptor 2 (HER2 and Carcinoembryonic antigen (CEA. We discuss how these recombinant antibodies can be fabricated into nanostructures, such as carbon nanotubes, nanowires, and quantum dots, for the purpose of enhancing detection of biomarkers at low concentrations (pg/mL within complex mixtures such as serum or tissue extracts. Other sensing technologies, which take advantage of ‘Surface Enhanced Raman Scattering’ (gold nanoshells, frequency changes in piezoelectric crystals (quartz crystal microbalance, or electrical current generation and sensing during electrochemical reactions (electrochemical detection, can effectively provide multiplexed platforms for detection of cancer and injury biomarkers. Such devices may soon replace the traditional time consuming ELISAs and Western blots, and deliver rapid, point-of-care diagnostics to market.

  10. Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease.

    Directory of Open Access Journals (Sweden)

    Shivali Gupta

    Full Text Available BACKGROUND: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. METHODS AND RESULTS: Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd-phase for antibody response to the recombinant antigens (individually or mixed by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175. The IgG antibodies to TcG1, TcG2, and TcG4 (individually and TcG(mix were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%, TcG2- (96%, TcG4- (94.6% and TcG(mix- (98% based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8% in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. CONCLUSIONS: Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

  11. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund; Wiuf, Carsten

    the effect of a recombination event is the genealogical type of the event and whether SNP variation is present that can reveal the genealogical consequences of the recombination event. Recombination events that only change some branch lengths in the genealogy have a very small, but detectable, effect....... The more lineages left when the recombination event occurs, the larger effect it has, implying that it is mainly young recombination events that we detect when estimating the rate. If the population is growing, though, more lineages are present back in time and relatively more ancient recombination...... shared by these two populations are expected to contribute compared to the effect of private recombination events...

  12. Radioprotective activity of shigella antigens

    International Nuclear Information System (INIS)

    The possibility of using experimental microbe antigenous preparation out of Flexner and Zonne shigellas as a protector and a remedy in the case of gamma irradiation, is investigated. The experiments are carried out on mice of both sexes immunized before or after irradiation by two methods: subcutaneously and enerally. It is found that in most cases investigated, the introduction of the experimental preparation 3, 5, 7 and 10 days before irradiation increases the survivability of animals

  13. Investigations for designing catalytic recombiners

    International Nuclear Information System (INIS)

    In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m3 might be generated and released into the containments. The mixture consisting of hydrogen and oxygen may either burn or detonate, if ignited. In case of detonation the generated shock wave may endanger the components of the plant or the plant itself. Consequently, effective removal of hydrogen is required. The fact that hydrogen and oxygen react exo-thermally on catalytically acting surfaces already at low temperatures generating steam and heat is made use of in catalytic recombiners. They consist of substrates coated with catalyst (mainly platinum or palladium) which are arranged inside a casing. Being passively acting measures, recombiners do not need any additional energy supply. Experimental investigations on catalytic hydrogen recombination are conducted at FZJ (Forschungszentrum Juelich) using three test facilities. The results yield insight in the development potential of contemporary recombiner systems as well as of innovative systems. Detailed investigations on a recombiner section show strong temperature gradients over the surface of a catalytically coated sample. Dependent on the flow velocity, ignition temperature may be reached at the leading edge already at an inlet hydrogen concentration of about 5 vol.-%. The thermal strain of the substrate leads to considerable detachment of catalyst particles probably causing unintended ignition of the flammable mixture. Temperature peaks can be prevented effectively by leaving the first part of the plate uncoated. In order to avoid overheating of the catalyst elements of a recombiner even at high hydrogen concentrations a modular system of porous substrates is proposed. The metallic substrates are coated with platinum at low catalyst densities thus limiting the activity of the single specimen. A modular arrangement of these elements provides high recombination rates over a large hydrogen concentration

  14. Chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies against FMDV in guinea pigs.

    OpenAIRE

    Kitson, J D; Burke, K L; Pullen, L A; Belsham, G J; Almond, J W

    1991-01-01

    Five poliovirus recombinants containing sequences corresponding to foot-and-mouth disease virus (FMDV) antigenic sites were constructed. Viable virus was recovered from four of these plasmids, in which the VP1 beta B-beta C loop (antigenic site 1) of poliovirus type 1 Sabin had been replaced with sequences derived from the VP1 beta G-beta H loop (antigenic site 1) of FMDV O1 Kaufbeuren (O1K), chimera O1.1 (residues 141 to 154), chimera O1.2 (residues 147 to 156), and chimera O1.3 (residues 14...

  15. Homologous Recombination in Negative Sense RNA Viruses

    OpenAIRE

    Michael Worobey; Guan-Zhu Han

    2011-01-01

    Recombination is an important process that influences biological evolution at many different levels. More and more homologous recombination events have been reported among negative sense RNA viruses recently. While sporadic authentic examples indicate that homologous recombination does occur, recombination seems to be generally rare or even absent in most negative sense RNA viruses, and most of the homologous recombination events reported in the literature were likely generated artificially d...

  16. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    Science.gov (United States)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  17. Recombining WMAP: Constraints on ionizing and resonance radiation at recombination

    International Nuclear Information System (INIS)

    We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent cosmic microwave background temperature and polarization spectra coming from the Wilkinson Microwave Anisotropy Probe (WMAP). We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be weakly affected by a modified recombination scheme. However, it may affect the current WMAP constraints on inflationary parameters such as the spectral index ns and its running. Physically motivated models, such as those based on primordial black holes or super heavy dark matter decay, are able to provide a good fit to the current data. Future observations in both temperature and polarization will be needed to more stringently test these models

  18. Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

    Science.gov (United States)

    Rosenthal, Joseph A; Huang, Chung-Jr; Doody, Anne M; Leung, Tiffany; Mineta, Kaho; Feng, Danielle D; Wayne, Elizabeth C; Nishimura, Nozomi; Leifer, Cynthia; DeLisa, Matthew P; Mendez, Susana; Putnam, David

    2014-01-01

    Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion. PMID:25426709

  19. Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Joseph A Rosenthal

    Full Text Available Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs. Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.

  20. Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

    OpenAIRE

    Zhang, T.; Stanley, S L

    1994-01-01

    The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with vi...

  1. A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

    Science.gov (United States)

    Cortina, María E; Balzano, Rodrigo E; Rey Serantes, Diego A; Caillava, Ana J; Elena, Sebastián; Ferreira, A C; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

    2016-06-01

    Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans. PMID:26984975

  2. Recombinant toxin-coregulated pilus A (TcpA as a candidate subunit cholera vaccine.

    Directory of Open Access Journals (Sweden)

    Somayeh Kiaie

    2014-04-01

    Full Text Available The toxin co-regulated pilus A (TcpA has been described as a critical pathogenicity factor of Vibrio cholerae. TcpA is a candidate for making subunit vaccine against cholera. The aim of this study was to produce a candidate vaccine by expressing recombinant TcpA in E. coli.In this study, the toxin co-regulated pilus A gene from EL-Tor, V. cholerae subspecies, was amplified by PCR and sub-cloned into prokaryotic expression vector pGEX4T1. E. coli BL21 (DE3 was transformed with pGEX4T1- TcpA and gene expression was induced by IPTG and purified by GST resin. The integrity of the product was confirmed by Western blot analysis using a standard rabbit anti-V. cholerae antibody. Sera reactivity of infected individuals was further analyzed against the recombinant TcpA protein.The concentration of purified recombinant protein was calculated to be 8 mg/L of initial culture. The integrity of product was confirmed by Western blot analysis using a standard rabbit anti V. cholerae antibody. Sera reactivity of infected individual was further analyzed against the recombinant TcpA protein. The obtained data indicated that recombinant TcpA protein from V. cholerae was recognized by patient serum and animal sera.These results show that the recombinant TcpA is antigenic and could be used in a carrier host as an oral vaccine against cholera.

  3. EXPRESSION AND GLYCOSYLATION OF ROTAVIRUS STRAIN SA11 VP4 PROTEIN IN A RECOMBINANT ADENOVIRUS

    Institute of Scientific and Technical Information of China (English)

    孙茂盛; 昝云红; 马雁冰; 张光明; 杜秋江; 戴长柏

    2001-01-01

    Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain.``Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terninal.``The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co-transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation.``Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein.``Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.``

  4. Vaccination against influenza with recombinant hemagglutinin expressed by Schizochytrium sp. confers protective immunity.

    Directory of Open Access Journals (Sweden)

    Anne-Cécile V Bayne

    Full Text Available For the rapid production of influenza vaccine antigens in unlimited quantities, a transition from conventional egg-based production to cell-based and recombinant systems is required. The need for higher-yield, lower-cost, and faster production processes is critical to provide adequate supplies of influenza vaccine to counter global pandemic threats. In this study, recombinant hemagglutinin proteins of influenza virus were expressed in the microalga Schizochytrium sp., an established, fermentable organism grown in large scale for the manufacture of polyunsaturated fatty acids for animal and human health applications. Schizochytrium was capable of exporting the full-length membrane-bound proteins in a secreted form suitable for vaccine formulation. One recombinant hemagglutinin (rHA protein derived from A/Puerto Rico/8/34 (H1N1 influenza virus was evaluated as a vaccine in a murine challenge model. Protective immunity from lethal challenge with homologous virus was elicited by a single dose of 1.7, 5 or 15 µg rHA with or without adjuvant at survival rates between 80-100%. Full protection (100% was established at all dose levels with or without adjuvant when mice were given a second vaccination. These data demonstrate the potential of Schizochytrium sp. as a platform for the production of recombinant antigens useful for vaccination against influenza.

  5. Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins

    Directory of Open Access Journals (Sweden)

    Dong Jinhua

    2012-05-01

    Full Text Available Abstract Background Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry. Results Recombinant surface associated antigen 1 (NcSAG1, NcSAG1 related sequence 2 (NcSRS2 and the dense granule antigen 2 (NcGRA2 of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA. By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay. Conclusions A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.

  6. Intra HLA-D/DR region recombinant detected by primed lymphocyte typing (PLT)

    DEFF Research Database (Denmark)

    Jakobsen, B K; Kristensen, T; Lamm, L U;

    1983-01-01

    lymphocyte typing (PLT) for HLA-D/DR region associated DP antigens. None of these studies gave evidence that the recombinations had occurred within the HLA region. Mixed leucocyte culture (MLC) tests within the families showed no detectable stimulation between the HLA identical siblings in two of the...... to reactive reagents. One of these (GHx), reacted with a determinant which segregated within the GG family as if child G was a paternal recombinant between the HLA-D, DR, DP, and C4 loci, on the one hand, and on the other hand one or more loci governing other HLA-D/DR region controlled lymphocyte...... the panel it reacted with a determinant significantly associated with D/DR/DP2. Other PLT reagents could be generated within the family against lymphocyte activating determinants controlled by genes in the two paternal haplotypes telomeric to the assumed recombinational site. These reagents gave...

  7. Detection of TGEV Antibody by Enzyme-Linked Immunosorbent Assay Using Recombinant Nucleocapsid Proteins

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; HOU Xi-lin

    2005-01-01

    An enzyme linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of transmissible gastroenteritis virus (TGEV) infection.The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein migrated at 42 kDa and reacted with His6 tag specific monoclonal antibody by immunoblotting.Recombinant N protein ELISA (rnELISA) demonstrated 97.5% specificity among 80 TGEV-free individuals, and 97.3%sensitivity ranging among 110 clinical samples with TGEV. Taken together, these results indicated that nucleocapsid may be a useful antigen for the sera-diagnosis of TGEV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies against TGEV.

  8. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins.

    Science.gov (United States)

    Bredenbeek, Peter J; Molenkamp, Richard; Spaan, Willy J M; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S

    2006-02-20

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa. PMID:16412488

  9. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

    Science.gov (United States)

    Bredenbeek, Peter J.; Molenkamp, Richard; Spaan, Willy J.M.; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S.; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S.

    2006-01-01

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa. PMID:16412488

  10. Inhomogeneous recombinations during cosmic reionization

    CERN Document Server

    Sobacchi, Emanuele

    2014-01-01

    By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing photo-heating feedback on star-formation, we present large-scale, semi-numeric reionization simulations which self-consistently track the local (sub-grid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly-evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large HII regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reioniza...

  11. Recombinant snake venom prothrombin activators.

    Science.gov (United States)

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  12. Native and recombinant herpes simplex virus type 1 envelope proteins induce human immune T-lymphocyte responses.

    Science.gov (United States)

    Torseth, J W; Cohen, G H; Eisenberg, R J; Berman, P W; Lasky, L A; Cerini, C P; Heilman, C J; Kerwar, S; Merigan, T C

    1987-05-01

    The abilities of whole herpes simplex virus type 1 (HSV-1) antigen (HSV-ag) and purified HSV-1 native and recombinant envelope proteins to stimulate in vitro T-lymphocyte responses were compared in patients with recurrent herpes labialis. Immunochemically purified preparations of native glycoproteins B, C, and D (ngB, ngC, ngD) from cultured HSV-1 as well as expressed recombinant plasmid preparations of gD (rgD-1t, rgD-45K) elicited lymphocyte proliferation (LT) and production of gamma interferon (IFN-gamma) and interleukin-2 (IL-2) only in seropositive individuals. The IFN-gamma induced by rgD-1t correlated with the time to the next herpetic lesion in 19 volunteers followed to recurrence (r = 0.69, P less than 0.008), although the magnitude and frequency of LT and IFN-gamma responses were lower with either recombinant or native purified antigens than with the whole-virus antigen. Combinations of ngB plus ngD or ngB plus ngC plus ngD stimulated more IFN-gamma, equivalent to whole-virus-antigen responses. Recombinant-derived human IL-2 also specifically increased LT and IFN-gamma responses in antigen-driven cultures. ngD stimulated IL-2 and LT responses similar to those of whole-virus antigen and higher than those of ngC. HSV-ag and ngB induced significantly higher titers of total IFN than could be accounted for by IFN-gamma; this was not seen for the other antigens, which induced only IFN-gamma. HSV-ag-driven Leu 2a-, plastic-nonadherent blood cells, unlike whole peripheral blood mononuclear cells, showed evidence of an increase and then a decline in the frequency of HSV-responsive cells after a lesion recurrence. These studies suggest that HSV-1 envelope proteins are capable of stimulating an immune T-helper-cell response which is associated with the prevention of human herpes simplex lesion recurrence. Although the whole virus probably contains additional important antigens, increasing concentrations or combinations of certain purified glycoproteins or the

  13. Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

    Science.gov (United States)

    Anugraha, G; Madhumathi, J; Prince, P R; Prita, P J Jeya; Khatri, V K; Amdare, N P; Reddy, M V R; Kaliraj, P

    2015-10-01

    Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate. PMID:26179420

  14. Central role of the Holliday junction helicase RuvAB in vlsE recombination and infectivity of Borrelia burgdorferi.

    Directory of Open Access Journals (Sweden)

    Tao Lin

    2009-12-01

    Full Text Available Antigenic variation plays a vital role in the pathogenesis of many infectious bacteria and protozoa including Borrelia burgdorferi, the causative agent of Lyme disease. VlsE, a 35 kDa surface-exposed lipoprotein, undergoes antigenic variation during B. burgdorferi infection of mammalian hosts, and is believed to be a critical mechanism by which the spirochetes evade immune clearance. Random, segmental recombination between the expressed vlsE gene and adjacent vls silent cassettes generates a large number of different VlsE variants within the infected host. Although the occurrence and importance of vlsE sequence variation is well established, little is known about the biological mechanism of vlsE recombination. To identify factors important in antigenic variation and vlsE recombination, we screened transposon mutants of genes known to be involved in DNA recombination and repair for their effects on infectivity and vlsE recombination. Several mutants, including those in BB0023 (ruvA, BB0022 (ruvB, BB0797 (mutS, and BB0098 (mutS-II, showed reduced infectivity in immunocompetent C3H/HeN mice. Mutants in ruvA and ruvB exhibited greatly reduced rates of vlsE recombination in C3H/HeN mice, as determined by restriction fragment polymorphism (RFLP screening and DNA sequence analysis. In severe combined immunodeficiency (C3H/scid mice, the ruvA mutant retained full infectivity; however, all recovered clones retained the 'parental' vlsE sequence, consistent with low rates of vlsE recombination. These results suggest that the reduced infectivity of ruvA and ruvB mutants is the result of ineffective vlsE recombination and underscores the important role that vlsE recombination plays in immune evasion. Based on functional studies in other organisms, the RuvAB complex of B. burgdorferi may promote branch migration of Holliday junctions during vlsE recombination. Our findings are consistent with those in the accompanying article by Dresser et al., and together

  15. Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis.

    Science.gov (United States)

    Singh, Swati; Saraav, Iti; Sharma, Sadhna

    2014-02-01

    Tuberculosis remains a great health threat to the world among infectious diseases particularly with the advent of human immunodeficiency virus and emergence of drug resistant strains. In the light of the inconsistent efficacy imparted by the only currently available pre-exposure vaccine bacillus Calmette-Guerin BCG, the development of an improved TB vaccine is a very high international research priority. Vaccine candidates currently in clinical trials are also pre-exposure vaccines that aim to prevent active tuberculosis during an individual's lifetime. According to World Health Organization approximately a third of the world's population is latently infected with Mycobacterium tuberculosis. Dormancy or latency of Mycobacteria is associated with the formation of granuloma with poorly perfused interior leading to expression of genes which help them survive in this hostile environment. A group of about 50 genes belonging to the DosR regulon also known as latency antigens are expressed by Mycobacteria when they are persisting in the immuno-competent host. An understanding of the immunological effects produced by products of these latency induced genes may help in making a more potent vaccine. Incorporation of latency antigens into improved (live or subunit) vaccines may enhance the impact of these vaccines in which BCG priming can be followed by multisubunit protein boosting. These vaccines could act as post exposure vaccines for containment and prevention of latent TB activation. This heterologous boosting of BCG-primed immunity will be able to stimulate the known immune correlates of protective immunity against M. tuberculosis i.e. TH1 cells (CD4(+) and CD8(+) T cells) mediated immune responses with cytokines such as IFN-γ and TNF-α⋅ In our review we have analysed and compared the immunogenic potential of various latency-associated antigens of the DosR regulon in line with the current strategy of developing a recombinant post exposure booster vaccine. PMID

  16. Restoration of proliferative response to M. leprae antigens in lepromatous T cells against candidate antileprosy vaccines.

    Science.gov (United States)

    Mustafa, A S

    1996-09-01

    Several studies conducted in the last decade suggest that Mycobacterium lepraereactive T cells exist in lepromatous patients, but their number may be too few to yield a detectable response in cell-mediated immunity (CMI) assays. Immunizations with candidate antileprosy vaccines and stimulation of T cells with M. leprae + interleukin-2 restore the M. leprae-induced CMI response in lepromatous leprosy patients. These immunizations and stimulation may enrich the pre-existing M. leprae-responsive T cells in lepromatous patients and, thereby, induce a detectable CMI response to M. leprae antigens upon repeat testing. To verify this proposition, we carried out a study in a group of 10 lepromatous leprosy patients. Peripheral blood mononuclear cells (PBMC) obtained from these patients were anergic to M. leprae antigens in proliferative assays, but they responded to the antigens of candidate antileprosy vaccines, i.e., M. bovis BCG, M. bovis BCG + M. leprae, and Mycobacterium w. The enrichment of M. leprae-responsive T cells was performed by establishing T-cell lines from the PBMC after in vitro stimulation with M. leprae, M. bovis BCG, M. bovis BCG + M. leprae, and Mycobacterium w. When tested for their proliferative responses, 1/10, 3/10, 6/10 and 2/10 T-cell lines established against M. leprae, M. bovis BCG, M. bovis BCG + M. leprae, and Mycobacterium w, respectively, responded to M. leprae. These results suggest that enrichment of pre-existing M. leprae-responsive T cells may contribute to the restoration of the T-cell response to M. leprae in some lepromatous patients. Four of the 10 M. leprae-induced T-cell lines proliferated in response to the 65 kDa, 36 kDa, 28 kDa, and 12 kDa recombinant antigens of M. leprae, suggesting that the nonresponsiveness of T cells in some lepromatous patients may be overcome by using recombinant antigens of M. leprae. PMID:8862259

  17. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  18. Histocompatibility antigens on astrocytoma cells.

    OpenAIRE

    Hirschberg, H.; Endresen, L I; Wikeby, P

    1982-01-01

    Biopsies tumour cells from astrocytoma-bearing patients were grown in primary culture for 3-5 days. Both low and high grade tumours were represented in the study. The cultured cells could be shown to express the HLA-A and -B antigens using a multispecific allo-antiserum and a rabbit anti-beta-2 microglobulin antibody. The tumour cells were negative for the HLA-DR determinants when tested with either rabbit anti-Ia-like antisera or specific anti-HLA-DR allo-antisera. They also failed to stimul...

  19. The antigenic properties of human prolactin

    International Nuclear Information System (INIS)

    The antigenic properties of human prolactin (HPr) were studied using various methods of radio-immuno assay. The homologous system, the difficulty of which resides in the preparation of the tracer, easily permits measurement of physiological levels. In this system, blood prolactin in the monkey has an antigenicity comparable with that of human prolactin, whereas growth hormone and human chorionic somatotropin have feeble or nil antigenic relationship with HPr. Human, sheep and pig prolactins have variable antigenic cross-reactions depending on the immune serum used. These antigenic cross reactions may be applied to the isolation of amniotic prolactin. Human blood prolactin has several components of different molecular weight, but antigenicity comparable with that of pituitary HPr

  20. Yeast-recombinant hepatitis B vaccine: efficacy with hepatitis B immune globulin in prevention of perinatal hepatitis B virus transmission

    International Nuclear Information System (INIS)

    A yeast-recombinant hepatitis B vaccine was licensed recently by the Food and Drug administration and is now available. To assess the efficacy of the yeast-recombinant vaccine, the authors administered the vaccine in combination with hepatitis B immune globulin to high-risk newborns. If infants whose mothers were positive for both hepatitis B surface antigen and the e antigen receive no immunoprophylaxis, 70% to 90% become infected with the virus, and almost all become chronic carriers. Among infants in this study who received hepatitis B immune globulin at birth and three 5-+g doses of yeast-recombinant hepatitis B vaccine, only 4.8% became chronic carriers, a better than 90% level of protection and a rate that is comparable with that seen with immune globulin and plasma-derived hepatitis B vaccine. Hepatitis surface antigen and antibodies were detected by radioimmunoassay. These data suggest that, in this high-risk setting, the yeast-recombinant vaccine is as effective as the plasma-derived vaccine in preventing hepatitis B virus infection and the chronic carrier state