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Sample records for candidate vaccine sequences

  1. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing

    NARCIS (Netherlands)

    Pizza, M; Scarlato, [No Value; Masignani, [No Value; Giuliani, MM; Arico, B; Comanducci, M; Jennings, GT; Baldi, L; Bartolini, E; Capecchi, B; Galeotti, CL; Luzzi, E; Manetti, R; Marchetti, E; Mora, M; Nuti, S; Ratti, G; Santini, L; Savino, S; Scarselli, M; Storni, E; Zuo, PJ; Broeker, M; Hundt, E; Knapp, B; Blair, E; Mason, T; Tettelin, H; Hood, DW; Jeffries, AC; Saunders, NJ; Granoff, DM; Venter, JC; Moxon, ER; Grandi, G; Rappuoli, R

    2000-01-01

    Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the en

  2. Leishmaniasis: vaccine candidates and perspectives.

    Science.gov (United States)

    Singh, Bhawana; Sundar, Shyam

    2012-06-06

    Leishmania is a protozoan parasite and a causative agent of the various clinical forms of leishmaniasis. High cost, resistance and toxic side effects of traditional drugs entail identification and development of therapeutic alternatives. The sound understanding of parasite biology is key for identifying novel drug targets, that can induce the cell mediated immunity (mainly CD4+ and CD8+ IFN-gamma mediated responses) polarized towards a Th1 response. These aspects are important in designing a new vaccine along with the consideration of the candidates with respect to their ability to raise memory response in order to improve the vaccine performance. This review is an effort to identify molecules according to their homology with the host and their ability to be used as potent vaccine candidates.

  3. New Zika Vaccine Candidate Provides Powerful Protection

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_163384.html New Zika Vaccine Candidate Provides Powerful Protection Made without live ... HealthDay News) -- A single dose of an experimental Zika vaccine protected mice and monkeys from the virus, ...

  4. High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E.

    Science.gov (United States)

    Homolka, Susanne; Ubben, Tanja; Niemann, Stefan

    2016-01-01

    The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies.

  5. Disruption of the Putative Vascular Leak Peptide Sequence in the Stabilized Ricin Vaccine Candidate RTA1-33/44-198

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    Charles B. Millard

    2013-01-01

    Full Text Available Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP consensus sequence in recombinant ricin toxin A-chain (RTA that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N and V76 (to I, or M had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP(− variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the Tm was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond.

  6. Towards the rational design of a candidate vaccine against pregnancy associated malaria: conserved sequences of the DBL6epsilon domain of VAR2CSA.

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    Cyril Badaut

    Full Text Available BACKGROUND: Placental malaria is a disease linked to the sequestration of Plasmodium falciparum infected red blood cells (IRBC in the placenta, leading to reduced materno-fetal exchanges and to local inflammation. One of the virulence factors of P. falciparum involved in cytoadherence to chondroitin sulfate A, its placental receptor, is the adhesive protein VAR2CSA. Its localisation on the surface of IRBC makes it accessible to the immune system. VAR2CSA contains six DBL domains. The DBL6epsilon domain is the most variable. High variability constitutes a means for the parasite to evade the host immune response. The DBL6epsilon domain could constitute a very attractive basis for a vaccine candidate but its reported variability necessitates, for antigenic characterisations, identifying and classifying commonalities across isolates. METHODOLOGY/PRINCIPAL FINDINGS: Local alignment analysis of the DBL6epsilon domain had revealed that it is not as variable as previously described. Variability is concentrated in seven regions present on the surface of the DBL6epsilon domain. The main goal of our work is to classify and group variable sequences that will simplify further research to determine dominant epitopes. Firstly, variable sequences were grouped following their average percent pairwise identity (APPI. Groups comprising many variable sequences sharing low variability were found. Secondly, ELISA experiments following the IgG recognition of a recombinant DBL6epsilon domain, and of peptides mimicking its seven variable blocks, allowed to determine an APPI cut-off and to isolate groups represented by a single consensus sequence. CONCLUSIONS/SIGNIFICANCE: A new sequence approach is used to compare variable regions in sequences that have extensive segmental gene relationship. Using this approach, the VAR2CSA DBL6 domain is composed of 7 variable blocks with limited polymorphism. Each variable block is composed of a limited number of consensus types

  7. Nonclinical Development of BCG Replacement Vaccine Candidates

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    Bernd Eisele

    2013-04-01

    Full Text Available The failure of current Mycobacterium bovis bacille Calmette–Guérin (BCG vaccines, given to neonates to protect against adult tuberculosis and the risk of using these live vaccines in HIV-infected infants, has emphasized the need for generating new, more efficacious and safer replacement vaccines. With the availability of genetic techniques for constructing recombinant BCG (rBCG strains containing well-defined gene deletions or insertions, new vaccine candidates are under evaluation at both the preclinical and clinical stages of development. Since most BCG vaccines in use today were evaluated in clinical trials decades ago and are produced by outdated processes, the development of new BCG vaccines offers a number of advantages that include a modern well-defined manufacturing process along with state-of-the-art evaluation of safety and efficacy in target populations. We provide a description of the preclinical development of two novel rBCGs, VPM1002 that was constructed by adding a modified hly gene coding for the protein listeriolysin O (LLO from Listeria monocytogenes and AERAS-422, which carries a modified pfoA gene coding for the protein perfringolysin O (PFO from Clostridium perfringens, and three genes from Mycobacterium tuberculosis. Novel approaches like these should be helpful in generating stable and effective rBCG vaccine candidates that can be better characterized than traditional BCG vaccines.

  8. Vaccines for leishmaniasis: from proteome to vaccine candidates.

    Science.gov (United States)

    Schroeder, Juliane; Aebischer, Toni

    2011-01-01

    Leishmania spp. cause a wide spectrum of tropical diseases which are threatening an estimated 350 million people around the globe. While in most cases non-fatal, the disease is associated with high morbidity, social stigmata and poverty. However, the most severe form visceral leishmaniasis can be fatal if left untreated. Chemotherapeutics are available but show high toxicity, costs and are prone to resistance development due to prolonged treatment periods. Healing is associated with a life-long resistance to re-infection and this argues for the feasibility of vaccination. However, despite much effort, no such vaccine has become available yet. Here, the status of vaccine development in this field is briefly summarized before the focus is set on the promise of reverse vaccinology for anti-Leishmania vaccine development in the post-genomic era. We report on our own experience with this approach using an instructive example of successful candidate vaccine antigen identification.

  9. Top Zika Vaccine Candidate Moves Closer to Field Testing

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_161274.html Top Zika Vaccine Candidate Moves Closer to Field Testing DNA- ... MONDAY, Oct. 3, 2016 (HealthDay News) -- The leading Zika vaccine candidate should be ready for field testing ...

  10. Vaccine candidates for leishmaniasis: a review.

    Science.gov (United States)

    Nagill, Rajeev; Kaur, Sukhbir

    2011-10-01

    Leishmaniasis is a diverse group of clinical syndromes caused by protozoan parasites of the genus Leishmania. The clinical manifestation of the disease varies from self-limiting cutaneous lesions to progressive visceral disease. It is estimated that 350 million people are at risk in 88 countries, with a global incidence of 1-1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. The key control measures mainly rely on early case detection and chemotherapy which has been hampered by the toxicity of drugs, side-effects and by the emergence of drug resistance in parasites. Control of reservoir host and vector is difficult due to operational difficulties and frequent relapses in the host. Therefore, the development of effective and affordable vaccine against leishmaniasis is highly desirable. Although considerable progress has been made over the last decade in understanding immune mechanisms underlying potential candidate antigens, including killed, live attenuated parasites, crude parasites, pure or recombinant Leishmania proteins or DNA encoding leishmanial proteins, as well as immunomodulators from sand fly saliva, very few candidate vaccines have progressed beyond the experimental stage. As such there is no vaccine against any form of human leishmaniasis. In recent years, however, much interest has been stimulated towards vaccination against leishmaniasis focused mainly on cutaneous leishmaniasis with fewer attempts against visceral leishmaniasis.

  11. Advanced Vaccine Candidates for Lassa Fever

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    Igor S. Lukashevich

    2012-10-01

    Full Text Available Lassa virus (LASV is the most prominent human pathogen of the Arenaviridae. The virus is transmitted to humans by a rodent reservoir, Mastomys natalensis, and is capable of causing lethal Lassa Fever (LF. LASV has the highest human impact of any of the viral hemorrhagic fevers (with the exception of Dengue Fever with an estimated several hundred thousand infections annually, resulting in thousands of deaths in Western Africa. The sizeable disease burden, numerous imported cases of LF in non-endemic countries, and the possibility that LASV can be used as an agent of biological warfare make a strong case for vaccine development. Presently there is no licensed vaccine against LF or approved treatment. Recently, several promising vaccine candidates have been developed which can potentially target different groups at risk. The purpose of this manuscript is to review the LASV pathogenesis and immune mechanisms involved in protection. The current status of pre-clinical development of the advanced vaccine candidates that have been tested in non-human primates will be discussed. Major scientific, manufacturing, and regulatory challenges will also be considered.

  12. Advanced vaccine candidates for Lassa fever.

    Science.gov (United States)

    Lukashevich, Igor S

    2012-10-29

    Lassa virus (LASV) is the most prominent human pathogen of the Arenaviridae. The virus is transmitted to humans by a rodent reservoir, Mastomys natalensis, and is capable of causing lethal Lassa Fever (LF). LASV has the highest human impact of any of the viral hemorrhagic fevers (with the exception of Dengue Fever) with an estimated several hundred thousand infections annually, resulting in thousands of deaths in Western Africa. The sizeable disease burden, numerous imported cases of LF in non-endemic countries, and the possibility that LASV can be used as an agent of biological warfare make a strong case for vaccine development. Presently there is no licensed vaccine against LF or approved treatment. Recently, several promising vaccine candidates have been developed which can potentially target different groups at risk. The purpose of this manuscript is to review the LASV pathogenesis and immune mechanisms involved in protection. The current status of pre-clinical development of the advanced vaccine candidates that have been tested in non-human primates will be discussed. Major scientific, manufacturing, and regulatory challenges will also be considered.

  13. Production of EV71 vaccine candidates.

    Science.gov (United States)

    Chong, Pele; Hsieh, Shih-Yang; Liu, Chia-Chyi; Chou, Ai-Hsiang; Chang, Jui-Yuan; Wu, Suh-Chin; Liu, Shih-Jen; Chow, Yen-Hung; Su, Ih-Jen; Klein, Michel

    2012-12-01

    Enterovirus 71 (EV71) is now recognized as an emerging neurotropic virus in Asia and with Coxsackie virus (CV) it is the other major causative agent of hand-foot-mouth diseases (HFMD). Effective medications and/or prophylactic vaccines against HFMD are urgently needed. From a scientific (the feasibility of bioprocess, immunological responses and potency in animal challenge model) and business development (cost of goods) points of view, we in this review address and discuss the pros and cons of different EV71 vaccine candidates that have been produced and evaluated in animal models. Epitope-based synthetic peptide vaccine candidates containing residues 211-225 of VP1 formulated with Freund's adjuvant (CFA/IFA) elicited low EV71 virus neutralizing antibody responses, but were protective in the suckling mouse challenge model. Among recombinant EV71 subunits (rVP1, rVP2 and rVP3) expressed in E. coli, purified and formulated with CFA/IFA, only VP1 elicited mouse antibody responses with measurable EV71-specific virus neutralization titers. Immunization of mice with either a DNA plasmid containing VP1 gene or VP1 expressed in Salmonella typhimurium also generated neutralizing antibody responses and protected animals against a live EV71 challenge. Recombinant EV71 virus-like particles (rVLP) produced from baculovirus formulated either with CFA/IFA or alum elicited good virus neutralization titers in both mice and non-human primates, and were found to be protective in the suckling mouse EV71 challenge model. Synthetic peptides or recombinant EV71 subunit vaccines (rVP1 and rVLP) formulated in alum were found to be poorly immunogenic in rabbits. Only formalin-inactivated (FI) EV71 virions formulated in alum elicited cross-neutralizing antibodies against different EV71 genotypes in mice, rabbits and non-human primates but induced weak neutralizing responses against CAV16. From a regulatory, economic and market acceptability standpoint, FI-EV71 virion vaccines are the most

  14. Ascaris suum enolase is a potential vaccine candidate against ascariasis.

    Science.gov (United States)

    Chen, Ning; Yuan, Zi-Guo; Xu, Min-Jun; Zhou, Dong-Hui; Zhang, Xiu-Xiang; Zhang, Yan-Zhong; Wang, Xiao-Wei; Yan, Chao; Lin, Rui-Qing; Zhu, Xing-Quan

    2012-05-14

    Ascariasis caused by Ascaris is the most common parasite problem in humans and pigs worldwide. No vaccines are available for the prevention of Ascaris infections. In the present study, the gene encoding Ascaris suum enolase (As-enol-1) was amplified, cloned and sequenced. Amino acid sequence alignment indicated that As-enol-1 was highly conserved between different nematodes and shared the highest identity (87%) with enolase from Anisakis simplex s.l. The recombinant pVAX-Enol was successfully expressed in Marc-145 cells. The ability of the pVAX-Enol for inducing immune protective responses against challenge infection with A. suum L3 was evaluated in Kunming mice. The immune response was evaluated by lymphoproliferative assay, cytokine and antibody measurements, and the reduction rate of recovery larvae. The results showed that the mice immunized with pVAX-Enol developed a high level of specific antibody responses against A. suum, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or blank controls, respectively. There was a 61.13% reduction (P<0.05) in larvae recovery compared with that in the blank control group. Our data indicated that A. suum enolase is a potential vaccine candidate against A. suum infection.

  15. A novel live-attenuated vaccine candidate for mayaro Fever.

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    William J Weise

    2014-08-01

    Full Text Available Mayaro virus (MAYV is an emerging, mosquito-borne alphavirus that causes a dengue-like illness in many regions of South America, and which has the potential to urbanize. Because no specific treatment or vaccine is available for MAYV infection, we capitalized on an IRES-based approach to develop a live-attenuated MAYV vaccine candidate. Testing in infant, immunocompetent as well as interferon receptor-deficient mice demonstrated a high degree of attenuation, strong induction of neutralizing antibodies, and efficacy against lethal challenge. This vaccine strain was also unable to infect mosquito cells, a major safety feature for a live vaccine derived from a mosquito-borne virus. Further preclinical development of this vaccine candidate is warranted to protect against this important emerging disease.

  16. A novel live-attenuated vaccine candidate for mayaro Fever.

    Science.gov (United States)

    Weise, William J; Hermance, Meghan E; Forrester, Naomi; Adams, A Paige; Langsjoen, Rose; Gorchakov, Rodion; Wang, Eryu; Alcorn, Maria D H; Tsetsarkin, Konstantin; Weaver, Scott C

    2014-08-01

    Mayaro virus (MAYV) is an emerging, mosquito-borne alphavirus that causes a dengue-like illness in many regions of South America, and which has the potential to urbanize. Because no specific treatment or vaccine is available for MAYV infection, we capitalized on an IRES-based approach to develop a live-attenuated MAYV vaccine candidate. Testing in infant, immunocompetent as well as interferon receptor-deficient mice demonstrated a high degree of attenuation, strong induction of neutralizing antibodies, and efficacy against lethal challenge. This vaccine strain was also unable to infect mosquito cells, a major safety feature for a live vaccine derived from a mosquito-borne virus. Further preclinical development of this vaccine candidate is warranted to protect against this important emerging disease.

  17. Shigella sonnei Vaccine Candidates WRSs2 and WRSs3 Are as Immunogenic as WRSS1, a Clinically Tested Vaccine Candidate, in a Primate Model of Infection

    Science.gov (United States)

    2011-01-01

    Please cite this article in press as: Barnoy S, et al. Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically... Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate...o Article history: Received 15 October 2010 Accepted 28 April 2011 Available online Keywords: Shigella sonnei Live vaccine candidates WRSs2

  18. Novel approaches to identify protective malaria vaccine candidates

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    Wan Ni eChia

    2014-11-01

    Full Text Available Efforts to develop vaccines against malaria have been the focus of substantial research activities for decades. Several categories of candidate vaccines are currently being developed for protection against malaria, based on antigens corresponding to the pre-erythrocytic, blood-stage or sexual stages of the parasite. Long lasting sterile protection from Plasmodium falciparum sporozoite challenge has been observed in human following vaccination with whole parasite formulations, clearly demonstrating that a protective immune response targeting predominantly the pre-erythrocytic stages can develop against malaria. However, most of vaccine candidates currently being investigated, which are mostly subunits vaccines, have not been able to induce substantial (>50% protection thus far. This is due to the fact that the antigens responsible for protection against the different parasite stages are still yet to be known and relevant correlates of protection have remained elusive. For a vaccine to be developed in a timely manner, novel approaches are required. In this article, we review the novel approaches that have been developed to identify the antigens for the development of an effective malaria vaccine.

  19. Molecular Diversity of Vaccine Candidates in Leptospira spp.

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    Patricia Hernández-Rodríguez

    2015-09-01

    Full Text Available The aim of this study was to determine the molecular diversity of OmpL1, LipL32, LipL41, LigA and LigB proteins and that of the genes that encode them using bioinformatic analysis in different pathogenic strains of Leptospira spp. based on the information available in databases. The amino acid sequences of OmpL1, LipL32, LipL41, LigA and LigB proteins were used, as well as the genes encoding them in strains of Leptospira spp. reported at The National Center for Biotechnology Information (NCBI. The analysis of proteins and genes were performed using the Protein, Nucleotide and Gene resources from the NCBI. The alignment of the consensus sequences was performed using the PSI-BLAST and BLASTn tools. The coverage percentage of the selected sequences of the ompL1, lipL32, lipL41, ligA and ligB genes in pathogenic strains of Leptospira spp. is 100% for ompL1, lipL32 and lipL41, 75% for ligA and 99% for ligB with identity percentages of 85, 98, 88, 90 and 80% respectively; the coverage percentage of the selected protein sequences is 100, 77, 99, 100 and 100% with identity percentages of 90, 99, 92, 63 and 60% respectively, indicating that genes and proteins, except LigA and LigB proteins, are highly conserved in various pathogenic serovars of Leptospira spp. According to these results, it is recommended that further analysis of these proteins be made in order to determine the feasibility of its use as vaccine candidates.

  20. Moving candidate vaccines into development from research: lessons from HIV.

    Science.gov (United States)

    Sullivan, Mark

    2009-07-01

    There is a logarithmic increase in the cost and complexity of the research and development process when transitioning a promising candidate vaccine from the laboratory into the clinic. Managing complex development programs involving people from diverse technical, cultural and geographical backgrounds is a specialised skill. It is essential that the group is clear on their objectives and how their activities affect others, that communication is open, inclusive and effective, and that the most rigorous, scientific approach based on statistical principles in compliance with regulatory requirements is used. Applying these standards to all vaccine development programs will filter out inappropriate candidates more readily and enhance the efficiency of vaccine development. The challenges of developing a new vaccine are illustrated in human immunodeficiency virus (HIV) vaccinology. Selecting vaccine candidates for HIV requires the ability to evaluate the large number of potential antigens in imperfect and non-standardised animal models. Further, using these models to evaluate questions such as dose scaling to humans, optimal route of administration, the use of adjuvants and potential formulation improvements adds variable to variable, making the interpretation of results particularly challenging. This may lead to the promotion of a poor candidate or the elimination of a good one. The absence of precise immunological correlates of protection and the prohibitive cost of confirmatory clinical trials are further significant barriers. However, there are practical steps that can be taken to standardise early vaccine evaluation, which would result in more efficient development of new vaccines for HIV and other disease areas with similarly challenging development issues (such as hepatitis C virus, influenza, Mycobacterium tuberculosis and malaria).

  1. Identification of Novel Potential Vaccine Candidates against Tuberculosis Based on Reverse Vaccinology.

    Science.gov (United States)

    Monterrubio-López, Gloria P; González-Y-Merchand, Jorge A; Ribas-Aparicio, Rosa María

    2015-01-01

    Tuberculosis (TB) is a chronic infectious disease, considered as the second leading cause of death worldwide, caused by Mycobacterium tuberculosis. The limited efficacy of the bacillus Calmette-Guérin (BCG) vaccine against pulmonary TB and the emergence of multidrug-resistant TB warrants the need for more efficacious vaccines. Reverse vaccinology uses the entire proteome of a pathogen to select the best vaccine antigens by in silico approaches. M. tuberculosis H37Rv proteome was analyzed with NERVE (New Enhanced Reverse Vaccinology Environment) prediction software to identify potential vaccine targets; these 331 proteins were further analyzed with VaxiJen for the determination of their antigenicity value. Only candidates with values ≥0.5 of antigenicity and 50% of adhesin probability and without homology with human proteins or transmembrane regions were selected, resulting in 73 antigens. These proteins were grouped by families in seven groups and analyzed by amino acid sequence alignments, selecting 16 representative proteins. For each candidate, a search of the literature and protein analysis with different bioinformatics tools, as well as a simulation of the immune response, was conducted. Finally, we selected six novel vaccine candidates, EsxL, PE26, PPE65, PE_PGRS49, PBP1, and Erp, from M. tuberculosis that can be used to improve or design new TB vaccines.

  2. Identification of Novel Potential Vaccine Candidates against Tuberculosis Based on Reverse Vaccinology

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    Gloria P. Monterrubio-López

    2015-01-01

    Full Text Available Tuberculosis (TB is a chronic infectious disease, considered as the second leading cause of death worldwide, caused by Mycobacterium tuberculosis. The limited efficacy of the bacillus Calmette-Guérin (BCG vaccine against pulmonary TB and the emergence of multidrug-resistant TB warrants the need for more efficacious vaccines. Reverse vaccinology uses the entire proteome of a pathogen to select the best vaccine antigens by in silico approaches. M. tuberculosis H37Rv proteome was analyzed with NERVE (New Enhanced Reverse Vaccinology Environment prediction software to identify potential vaccine targets; these 331 proteins were further analyzed with VaxiJen for the determination of their antigenicity value. Only candidates with values ≥0.5 of antigenicity and 50% of adhesin probability and without homology with human proteins or transmembrane regions were selected, resulting in 73 antigens. These proteins were grouped by families in seven groups and analyzed by amino acid sequence alignments, selecting 16 representative proteins. For each candidate, a search of the literature and protein analysis with different bioinformatics tools, as well as a simulation of the immune response, was conducted. Finally, we selected six novel vaccine candidates, EsxL, PE26, PPE65, PE_PGRS49, PBP1, and Erp, from M. tuberculosis that can be used to improve or design new TB vaccines.

  3. Vaccine Candidates against Nontypeable Haemophilus influenzae: a Review

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    Behrouzi, Ava; Vaziri, Farzam; Rahimi-Jamnani, Fatemeh; Afrough, Parviz; Rahbar, Mohammad; Satarian, Fereshteh; Siadat, Seyed Davar

    2017-01-01

    Nonencapsulated, nontypeable Hemophilus influenzae (NTHi) remains an important cause of acute otitis and respiratory diseases in children and adults. NTHi bacteria are one of the major causes of respiratory tract infections, including acute otitis media, cystic fibrosis, and community-acquired pneumonia among children, especially in developing countries. The bacteria can also cause chronic diseases such as chronic bronchitis and chronic obstructive pulmonary disease in the lower respiratory tract of adults. Such bacteria express several outer membrane proteins, some of which have been studied as candidates for vaccine development. Due to the lack of effective vaccines as well as the spread and prevalence of NTHi worldwide, there is an urgent need to design and develop effective vaccine candidates against these strains. PMID:28088130

  4. SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C; Nielsen, M; Lamberth, K;

    2004-01-01

    of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.......An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T-lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)-restricted presentation of SARS-derived peptide epitopes......-CoV) was isolated and full-length sequenced (Marra et al., Science 2003: 300: 1399-404). Here, we have combined advanced bioinformatics and high-throughput immunology to perform an HLA supertype-, genome-wide scan for SARS-specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99...

  5. SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C.; Nielsen, Morten; Lamberth, K.;

    2004-01-01

    of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.......An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T-lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)-restricted presentation of SARS-derived peptide epitopes......-CoV) was isolated and full-length sequenced (Marra et al., Science 2003: 300: 1399404). Here, we have combined advanced bioinformatics and high-throughput immunology to perform an HLA supertype-, genome-wide scan for SARS-specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99...

  6. Anti-Lyme Subunit Vaccines: Design and Development of Peptide-Based Vaccine Candidates.

    Science.gov (United States)

    Small, Christina M; Mwangi, Waithaka; Esteve-Gassent, Maria D

    2016-01-01

    Vaccinology today has been presented with several avenues to improve protection against infectious disease. The recent employment of the reverse vaccinology technique has changed the face of vaccine development against many pathogens, including Borrelia burgdorferi, the causative agent of Lyme disease. Using this technique, genomics and in silico analyses come together to identify potentially antigenic epitopes in a high-throughput fashion. The forward methodology of vaccine development was used previously to generate the only licensed human vaccine for Lyme disease, which is no longer on the market. Using reverse vaccinology to identify new antigens and isolate specific epitopes to protect against B. burgdorferi, subunit vaccines will be generated that lack reactogenic and nonspecific epitopes, yielding more effective vaccine candidates. Additionally, novel epitopes are being utilized and are presently in the commercialization pipeline both for B. burgdorferi and other spirochaetal pathogens. The versatility and methodology of the subunit protein vaccine are described as it pertains to Lyme disease from conception to performance evaluation.

  7. A Natural Vaccine Candidate Strain Against Cholera

    Institute of Scientific and Technical Information of China (English)

    LIUYAN-QING; QIGUO-MING; 等

    1995-01-01

    El Tor Vibrio cholerae(EVC)strains may be classified into two kinds-epidemigenic(EEVC)strains and non-epidemigenic(NEEVC)strains-based on a phage-biotyping system.A large number of EEVC strains have been screened for toxigenic and putative colonization attributes.One such naturally occurring strain(designated IEM101)has been found which is devoid of genes encoding cholera toxin(CT),accessory cholera enterotoxin(ACE),zonula occludens toxin(ZOT),but possesses RS1 sequences and toixn-coregulated pilus A gene(tcpA)although tcpA is poorly expressed.It expresses type B pili but does not posses type C pili.It is an El Tor Ogawa strain and does not cause fluid accumulation in rabbit ileal loop tests.Active immunization of rabbits with strain IEM101 elicited good protection against challenge with virulent strains of V.cholerae Ol.Oral administration cased no side effects in 15 human volunteers.colonized the gut for four to ten days and elicited good immune responses.

  8. Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens.

    Directory of Open Access Journals (Sweden)

    Christian M Parobek

    2014-04-01

    Full Text Available Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens--Plasmodium vivax merozoite surface protein 1 (pvmsp-1 and Plasmodium vivax circumsporozoite protein (pvcsp. Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n = 44 and the complete gene of pvcsp (n = 47 from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines.

  9. Characterization nanoparticles-based vaccines and vaccine candidates: a Transmission Electron Microscopy study

    Directory of Open Access Journals (Sweden)

    I. Menéndez I

    2016-05-01

    Full Text Available Transmission Electron Microscopy (TEM is a valuable tool for the biotech industry. This paper summarizes some of the contributions of MET in the characterization of the recombinant antigens are part of vaccines or vaccine candidates obtained in the CIGB. It mentions the use of complementary techniques MET (Negative staining, and immunoelectron that enhance visualization and ultrastructural characterization of the recombinant proteins obtained by Genetic Engineering.

  10. Identification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology

    Directory of Open Access Journals (Sweden)

    Marine Meunier

    2016-01-01

    Full Text Available Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due to Campylobacter jejuni or Campylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacteria’s main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for this purpose. However, despite many studies, there is currently no vaccine available on the market to reduce the intestinal Campylobacter load in chickens. It is essential to identify and characterize new vaccine antigens. This study applied the reverse vaccinology approach to detect new vaccine candidates. The main criteria used to select immune proteins were localization, antigenicity, and number of B-epitopes. Fourteen proteins were identified as potential vaccine antigens. In vitro and in vivo experiments now need to be performed to validate the immune and protective power of these newly identified antigens.

  11. Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates

    Science.gov (United States)

    Dellagostin, Odir A.; Grassmann, André A.; Rizzi, Caroline; Schuch, Rodrigo A.; Jorge, Sérgio; Oliveira, Thais L.; McBride, Alan J. A.; Hartwig, Daiane D.

    2017-01-01

    Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV) has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis. PMID:28098813

  12. Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates

    Directory of Open Access Journals (Sweden)

    Odir A. Dellagostin

    2017-01-01

    Full Text Available Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis.

  13. Evaluation of Lethal Giant Larvae as a Schistosomiasis Vaccine Candidate

    Directory of Open Access Journals (Sweden)

    Yufan Cao

    2016-01-01

    Full Text Available Schistosomiasis is a neglected tropical disease of humans, and it is considered to be the second most devastating parasitic disease after malaria. Eggs produced by normally developed female worms are important in the transmission of the parasite, and they responsible for the pathogenesis of schistosomiasis. The tumor suppressor gene lethal giant larvae (lgl has an essential function in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. In our earlier study, downregulation of the lgl gene induced a significant reduction in the egg hatching rate of Schistosoma japonicum (Sj eggs. In this study, the Sjlgl gene was used as a vaccine candidate against schistosomiasis, and vaccination achieved and maintained a stable reduction of the egg hatching rate, which is consistent with previous studies, in addition to reducing the worm burden and liver egg burden in some trials.

  14. Evaluation of Lethal Giant Larvae as a Schistosomiasis Vaccine Candidate

    Science.gov (United States)

    Cao, Yufan; Qiao, Hongbin; Shi, Yanli; Han, Yu; Liu, Jinming; Li, Hao; Lu, Ke; Lin, Jiaojiao

    2016-01-01

    Schistosomiasis is a neglected tropical disease of humans, and it is considered to be the second most devastating parasitic disease after malaria. Eggs produced by normally developed female worms are important in the transmission of the parasite, and they responsible for the pathogenesis of schistosomiasis. The tumor suppressor gene lethal giant larvae (lgl) has an essential function in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. In our earlier study, downregulation of the lgl gene induced a significant reduction in the egg hatching rate of Schistosoma japonicum (Sj) eggs. In this study, the Sjlgl gene was used as a vaccine candidate against schistosomiasis, and vaccination achieved and maintained a stable reduction of the egg hatching rate, which is consistent with previous studies, in addition to reducing the worm burden and liver egg burden in some trials. PMID:27957496

  15. Genetic diversity of vaccine candidate antigens in Plasmodium falciparum isolates from the Amazon basin of Peru

    Directory of Open Access Journals (Sweden)

    Lucas Carmen M

    2008-05-01

    Full Text Available Abstract Background Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. In this study, characterization of genetic variability was performed in major B and T-cell epitopes within vaccine candidate antigens in isolates of P. falciparum from Peru. Methods DNA sequencing analysis was completed on 139 isolates of P. falciparum collected from endemic areas of the Amazon basin in Loreto, Peru from years 1998 to 2006. Genetic diversity was determined in immunological important regions in circumsporozoite protein (CSP, merozoite surface protein-1 (MSP-1, apical membrane antigen-1 (AMA-1, liver stage antigen-1 (LSA-1 and thrombospondin-related anonymous protein (TRAP. Alleles identified by DNA sequencing were aligned with the vaccine strain 3D7 and DNA polymorphism analysis and FST study-year pairwise comparisons were done using the DnaSP software. Multilocus analysis (MLA was performed and average of expected heterozygosity was calculated for each loci and haplotype over time. Results Three different alleles for CSP, seven for MSP-1 Block 2, one for MSP-1 Block 17, three for AMA-1 and for LSA-1 each and one for TRAP were identified. There were 24 different haplotypes in 125 infections with complete locus typing for each gene. Conclusion Characterization of the genetic diversity in Plasmodium isolates from the Amazon Region of Peru showed that P. falciparum T and B cell epitopes in these antigens have polymorphisms more similar to India than to Africa. These findings are helpful in the formulation of a vaccine considering restricted repertoire populations.

  16. Vaccination coverage and out-of-sequence vaccinations in rural Guinea-Bissau

    DEFF Research Database (Denmark)

    Hornshøj, Linda; Benn, Christine Stabell; Fernandes, Manuel

    2012-01-01

    this study was conducted. The WHO assesses coverage by 12 months of age. The sequence of vaccines may have an effect on child mortality, but is not considered in official statistics or assessments of programme performance. We assessed vaccination coverage and frequency of out-of-sequence vaccinations by 12...... to ascertain vaccination status. Between 2003 and 2009 vaccination status by 12 months of age was assessed for 5806 children aged 12-23 months; vaccination status by 24 months of age was assessed for 3792 children aged 24-35 months. OUTCOME MEASURES: Coverage of EPI vaccinations and frequency of out......-of-sequence vaccinations. RESULTS: Half of 12-month-old children and 65% of 24-month-old children had completed all EPI vaccinations. Many children received vaccines out of sequence: by 12 months of age 54% of BCG-vaccinated children had received DTP with or before BCG and 28% of measles-vaccinated children had received...

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

    Science.gov (United States)

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

    2009-08-13

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

  18. Development of a recombinant, chimeric tetravalent dengue vaccine candidate.

    Science.gov (United States)

    Osorio, Jorge E; Partidos, Charalambos D; Wallace, Derek; Stinchcomb, Dan T

    2015-12-10

    Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24-67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8(+) T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.

  19. Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants.

    Science.gov (United States)

    Poot, J; Janssen, L H M; van Kasteren-Westerneng, T J; van der Heijden-Liefkens, K H A; Schijns, V E J C; Heckeroth, A

    2009-07-16

    Chimerical protein "Q", composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant-antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs.

  20. Three Candidate Epitope-Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV-1

    Institute of Scientific and Technical Information of China (English)

    刘祖强; 田海军; 王颖; 陈应华

    2003-01-01

    HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines.So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies.Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HIV-1.According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated E1, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2: C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits.After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies.An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide.Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine.Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response.This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 μg.Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.

  1. The Vaccine Candidate Vibrio cholerae 638 Is Protective against Cholera in Healthy Volunteers

    Science.gov (United States)

    García, Luis; Jidy, Manuel Díaz; García, Hilda; Rodríguez, Boris L.; Fernández, Roberto; Año, Gemma; Cedré, Bárbara; Valmaseda, Tania; Suzarte, Edith; Ramírez, Margarita; Pino, Yadira; Campos, Javier; Menéndez, Jorge; Valera, Rodrigo; González, Daniel; González, Irma; Pérez, Oliver; Serrano, Teresita; Lastre, Miriam; Miralles, Fernando; del Campo, Judith; Maestre, Jorge Luis; Pérez, José Luis; Talavera, Arturo; Pérez, Antonio; Marrero, Karen; Ledón, Talena; Fando, Rafael

    2005-01-01

    Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXΦ prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 109 CFU of freshly harvested 638 buffered with 1.3% NaHCO3, while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 109 CFU of ΔCTXΦ attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 × 105 CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO3. Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (109 CFU) of strain

  2. HMGA1a recognition candidate DNA sequences in humans.

    Directory of Open Access Journals (Sweden)

    Takayuki Manabe

    Full Text Available High mobility group protein A1a (HMGA1a acts as an architectural transcription factor and influences a diverse array of normal biological processes. It binds AT-rich sequences, and previous reports have demonstrated HMGA1a binding to the authentic promoters of various genes. However, the precise sequences that HMGA1a binds to remain to be clarified. Therefore, in this study, we searched for the sequences with the highest affinity for human HMGA1a using an existing SELEX method, and then compared the identified sequences with known human promoter sequences. Based on our results, we propose the sequences "-(G/A-G-(A/T-(A/T-A-T-T-T-" as HMGA1a-binding candidate sequences. Furthermore, these candidate sequences bound native human HMGA1a from SK-N-SH cells. When candidate sequences were analyzed by performing FASTAs against all known human promoter sequences, 500-900 sequences were hit by each one. Some of the extracted genes have already been proven or suggested as HMGA1a-binding promoters. The candidate sequences presented here represent important information for research into the various roles of HMGA1a, including cell differentiation, death, growth, proliferation, and the pathogenesis of cancer.

  3. Proteins of Bartonella bacilliformis: Candidates for Vaccine Development

    Directory of Open Access Journals (Sweden)

    Cesar Henriquez-Camacho

    2015-01-01

    Full Text Available Bartonella bacilliformis is the etiologic agent of Carrión’s disease or Oroya fever. B. bacilliformis infection represents an interesting model of human host specificity. The notable differences in clinical presentations of Carrión’s disease suggest complex adaptations by the bacterium to the human host, with the overall objectives of persistence, maintenance of a reservoir state for vectorial transmission, and immune evasion. These events include a multitude of biochemical and genetic mechanisms involving both bacterial and host proteins. This review focuses on proteins involved in interactions between B. bacilliformis and the human host. Some of them (e.g., flagellin, Brps, IalB, FtsZ, Hbp/Pap31, and other outer membrane proteins are potential protein antigen candidates for a synthetic vaccine.

  4. Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Fenimore, Paul W [Los Alamos National Laboratory; Fischer, William M [Los Alamos National Laboratory; Kuiken, Carla [Los Alamos National Laboratory; Foley, Brian T [Los Alamos National Laboratory; Thurmond, J R [Los Alamos National Laboratory; Yusim, K [Los Alamos National Laboratory; Korber, B T [Los Alamos National Laboratory

    2008-01-01

    than is possible with a wild-type protein, (2) reducing the number of low-prevalence k-mers minimizes the likelihood of undesirable immunodominance, and (3) excluding exogenous k-mers will result in mosaic proteins whose processing for presentation is close to what occurs with wild-type proteins. The first and second applications of the mosaic method were to HIV and Hepatitis C Virus (HCV). HIV is the virus with the largest number of known sequences, and consequently a plethora of information for the CTL vaccine designer to incorporate into their mosaics. Experience with HIV and HCV mosaics supports the validity of the three conjectures above. The available FILV sequences are probably closer to the minimum amount of information needed to make a meaningful mosaic vaccine candidate. There were 532 protein sequences in the National Institutes of Health GenPept database in November 2007 when our reference set was downloaded. These sequences come from both Ebola and Marburg viruses (EBOV and MARV), representing transcripts of all 7 genes. The coverage of viral diversity by the 7 genes is variable, with genes 1 (nucleoprotein, NP), 4 (glycoprotein, GP; soluble glycoprotein, sGP) and 7 (polymerase, L) giving the best coverage. Broadly-protective vaccine candidates for diverse viruses, such as HIV or Hepatitis C virus (HCV) have required pools of antigens. FILV is similar in this regard. While we have designed CTL mosaic proteins using all 7 types of filoviral proteins, only NP, GP and L proteins are reported here. If it were important to include other proteins in a mosaic CTL vaccine, additional sequences would be required to cover the space of known viral diversity.

  5. Immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Objective: To describe the expression and immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections. Methods: The hybrid protein OprF (aa 190-342)-Opr I (aa 21-83) was modified N-terminally, either with a minimal histidine tag or with a homologous sequence of OprF. Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions. Results :This produced three suitable candidates for a vaccination trial: protein His-F- I , which was purified in its native as well as in its refolded form,and the native purified N-terminally extended protein ex-F- I . In mice, significantly higher antibody titers and survival rates after challenge with P. aeruginosa were observed, following immunization with protein His-F- I purified under native conditions. Conclusion: A hybrid OprF-Opr I molecule was cloned and a purification method which yields a protective vaccine against P. aeruginosa infections was established.

  6. Candidate Multi-Peptide-Vaccine Against Classical Swine Fever Virus Induces Strong Antibody Response with Predefined Specificity

    Institute of Scientific and Technical Information of China (English)

    张耿; 董晓楠; 陈应华

    2002-01-01

    Previous investigations demonstrated that the envelope glycoprotein E2 (gp55) of classical swine fever virus (CSFV) is the most immunogenic protein. Interestingly, recombinant protein E2 that contains only one structural antigenic unit (unit B/C or A) could protect pigs from a lethal challenge of CSFV. Based on these findings, we designed and prepared five overlapping synthetic peptides that covered the sequence unit B/C (aa 693-777) of Shimen E2 and conjugated individual peptides with bovine serum albumin (BSA). After the vaccination, the specificity of the rabbit sera was analyzed in the enzyme-linked immunosorbent assay (ELISA) and the fast protein liquid chromatography (FPLC). The results show that each of the five candidate peptide-vaccines can successfully induce a high titer of specific antibodies in New Zealand White Rabbits (n=3). Subsequently, the five candidate peptide-vaccines were applied in combination for immunization of pigs (n=10) and induced specific and strong humoral responses against all of the five designed peptides in pigs. Our studies indicate that the candidate multi-peptide-vaccine would prove an excellent marker vaccine against CSFV and provide a model for developing effective synthetic peptide vaccines to stop viral epidemics in humans and animals.

  7. Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate.

    Science.gov (United States)

    Andreoni, Francesca; Boiani, Romina; Serafini, Giordano; Amagliani, Giulia; Dominici, Sabrina; Riccioni, Giulia; Zaccone, Renata; Mancuso, Monique; Scapigliati, Giuseppe; Magnani, Mauro

    2013-01-21

    Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

  8. Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

    Science.gov (United States)

    Knight, J M; Zingales, B; Bottazzi, M E; Hotez, P; Zhan, B

    2014-12-01

    Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1.

  9. Reduced antibody responses against Plasmodium falciparum vaccine candidate antigens in the presence of Trichuris trichiura

    DEFF Research Database (Denmark)

    Esen, Meral; Mordmüller, Benjamin; de Salazar, Pablo Martinez;

    2012-01-01

    subjects compared to positive participants, whereas immunoglobulin subclass distribution was similar. Memory B-cell response was moderately increased in T. trichiura negative individuals, although the difference was not significant. CONCLUSIONS: Future malaria vaccine development programs need to account......BACKGROUND: Helminth infections are highly prevalent in the tropics and may have an effect on immune responses to vaccines due to their immunomodulatory effect. The prevalence of helminth infections in young children, the target group for malaria and most other vaccines, is high. Therefore we...... assessed the influence of helminth infection on vaccine-induced immune responses in a phase I clinical trial of the malaria vaccine candidate GMZ2. METHODS: Twenty Gabonese preschool-age children were vaccinated with GMZ2, a blood stage malaria vaccine candidate. Humoral immune response against the vaccine...

  10. Evaluation of the potential of Mycobacterium smegmatis as vaccine Candidate against tuberculosis by in silico and in vivo studies

    Directory of Open Access Journals (Sweden)

    Le Thuy Nguyen Thi

    2010-04-01

    Full Text Available In this study, we scanned multiple published databases of gene expression in vivo of M. tuberculosis at different phases of infection in animals and humans, to select 38 proteins that are highly expressed in the active, latent and reactivation phases. The selected proteins were predicted for T and B epitopes. For each proteins, the regions containing T and B epitopes were selected at the same time to look for identical epitopes on M. smegmatis based on sequence alignments. Preliminary studies of humoral immunogenicity and cross-reactivity with M. tuberculosis in mice using two M. smegmatis-derived experimental vaccines were carried out, demonstrating the immunogenicity of M. smegmatis proteoliposomes and the recognition of M. tuberculosis proteins by the sera of animals immunized with this vaccine candidate. The conjunction of in silico and in vivo studies suggested the potential for future evaluation of M. smegmatis as vaccine candidate against tuberculosis using different strategies

  11. DNA vaccine prime and recombinant FPV vaccine boost: an important candidate immunization strategy to control bluetongue virus type 1.

    Science.gov (United States)

    Li, Junping; Yang, Tao; Xu, Qingyuan; Sun, Encheng; Feng, Yufei; Lv, Shuang; Zhang, Qin; Wang, Haixiu; Wu, Donglai

    2015-10-01

    Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

  12. Evaluation of Novel Oral Vaccine Candidates and Validation of a Caprine Model of Johne's Disease

    Directory of Open Access Journals (Sweden)

    Murray E. Hines

    2014-03-01

    Full Text Available Johne’s disease (JD caused by Mycobacterium avium subspecies paratuberculosis (MAP is a major threat to the dairy industry and possibly some cases of Crohn’s disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were 1 to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne’s Disease Integrated Program (JDIP Animal Model Standardization Committee (AMSC, and 2 to validate the AMSC Johne’s disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis, or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 X 10^9 CFU divided in 2 consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate. All kids were necropsied at 13 months post challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318 do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329 reduced fecal shedding and tissue

  13. Evaluation of novel oral vaccine candidates and validation of a caprine model of Johne's disease.

    Science.gov (United States)

    Hines, Murray E; Turnquist, Sue E; Ilha, Marcia R S; Rajeev, Sreekumari; Jones, Arthur L; Whittington, Lisa; Bannantine, John P; Barletta, Raúl G; Gröhn, Yrjö T; Katani, Robab; Talaat, Adel M; Li, Lingling; Kapur, Vivek

    2014-01-01

    Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn's disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne's Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne's disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 10(9) CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.

  14. A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

    Science.gov (United States)

    Govindarajan, Dhanasekaran; Guan, Liming; Meschino, Steven; Fridman, Arthur; Bagchi, Ansu; Pak, Irene; ter Meulen, Jan; Casimiro, Danilo R; Bett, Andrew J

    2016-01-01

    Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

  15. The next generation recombinant human cytomegalovirus vaccine candidates-beyond gB.

    Science.gov (United States)

    Lilja, Anders E; Mason, Peter W

    2012-11-19

    Human cytomegalovirus (HCMV) infects the majority of the global population and persists within the infected host for life; infection of healthy adults rarely leads to severe acute clinical symptoms. In contrast, HCMV is a leading infectious cause of congenital disease and a common cause of complications in transplant recipients. A vaccine to prevent HCMV disease in these populations is a widely recognized medical need. We review recent advances in our understanding of the candidate vaccine antigens and published clinical trial data for the four most recent HCMV vaccine candidates: a gB subunit adjuvanted with MF59, a DNA vaccine expressing gB and pp65, alphavirus replicon particles (VRPs) expressing gB and a pp65-IE1 fusion protein, and a pp65 peptide vaccine. The candidates are safe, although some adverse events were reported for an adjuvanted variant of the pp65 peptide vaccine. The gB/MF59 vaccine elicited strong humoral responses with limited durability. The gB/pp65 DNA vaccine elicited cellular immunity, and the pp65 peptide vaccine elicited modest cellular immunity, but only when formulated with an adjuvant. Only the VRP vaccine expressing gB and pp65-IE1 elicited both humoral and cellular immunity. The gB/MF59 vaccine showed a short-term 50% efficacy at preventing infection of seronegative women and significantly reduced viremia and need for antivirals in solid organ transplant recipients, and the gB/pp65 DNA vaccine showed signs of clinical benefit in hematopoietic stem cell transplant recipients. Importantly, the partial efficacy of the subunit and DNA vaccines is new evidence that both humoral and cellular immunity contribute to controlling HCMV-related disease. These data show the clinical feasibility of a recombinant HCMV vaccine. We discuss areas for potential improvements in the next generation of vaccine candidates.

  16. Immunological Evaluation and Comparison of Different EV71 Vaccine Candidates

    Directory of Open Access Journals (Sweden)

    Ai-Hsiang Chou

    2012-01-01

    Full Text Available Enterovirus 71 (EV71 and coxsackievirus A16 (CVA16 are major causative agents of hand, foot, and mouth diseases (HFMDs, and EV71 is now recognized as an emerging neurotropic virus in Asia. Effective medications and/or prophylactic vaccines against HFMD are not available. The current results from mouse immunogenicity studies using in-house standardized RD cell virus neutralization assays indicate that (1 VP1 peptide (residues 211–225 formulated with Freund’s adjuvant (CFA/IFA elicited low virus neutralizing antibody response (1/32 titer; (2 recombinant virus-like particles produced from baculovirus formulated with CFA/IFA could elicit good virus neutralization titer (1/160; (3 individual recombinant EV71 antigens (VP1, VP2, and VP3 formulated with CFA/IFA, only VP1 elicited antibody response with 1/128 virus neutralization titer; and (4 the formalin-inactivated EV71 formulated in alum elicited antibodies that cross-neutralized different EV71 genotypes (1/640, but failed to neutralize CVA16. In contrast, rabbits antisera could cross-neutralize strongly against different genotypes of EV71 but weakly against CVA16, with average titers 1/6400 and 1/32, respectively. The VP1 amino acid sequence dissimilarity between CVA16 and EV71 could partially explain why mouse antibodies failed to cross-neutralize CVA16. Therefore, the best formulation for producing cost-effective HFMD vaccine is a combination of formalin-inactivated EV71 and CAV16 virions.

  17. Limited variation in vaccine candidate Plasmodium falciparum Merozoite Surface Protein-6 over multiple transmission seasons

    Directory of Open Access Journals (Sweden)

    Branch OraLee H

    2010-05-01

    same community. By contrast, PfMSP6 was highly stable at the sequence level, with no SNPs detected in the 506 samples analysed. This limited diversity supports further investigation of PfMSP6 as a blood stage vaccine candidate, with the clear caveat that any such vaccine must either contain both alleles or generate cross-protective responses that react against both allele classes. Detailed immunoepidemiology studies are needed to establish the viability of these approaches before PfMSP6 advances further down the vaccine development pipeline.

  18. Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

    Directory of Open Access Journals (Sweden)

    Caroline Kulangara

    Full Text Available In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1. In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

  19. Attenuated strains of Mycobacterium avium subspecies paratuberculosis as vaccine candidates against Johne's disease.

    Science.gov (United States)

    Settles, Erik W; Kink, John A; Talaat, Adel

    2014-04-11

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. Johne's disease has a severe economic impact on the dairy industry in the USA and worldwide. In an effort to combat this disease, we screened several transposon mutants that were attenuated in the murine model of paratuberculosis for the potential use as live attenuated vaccines. Using the murine model, two vaccine candidates (pgs1360, pgs3965 with mutations of fabG2_2 and umaA1, respectively) were at or below the limit of detection for tissue colonization suggesting their low level persistence and hence safety. Prior to challenge, both candidates induced a M. paratuberculosis-specific IFN-γ, an indication of eliciting cell-mediated immunity. Following challenge with a virulent strain of M. paratuberculosis, the two vaccine candidates significantly reduced bacterial colonization in organs with reduced histological scores compared to control animals. In addition, one of the vaccine candidates (pgs3965) also induced IL-17a, a cytokine associated with protective immunity in mycobacterial infection. Our analysis suggested that the pgs3965 vaccine candidate is a potential live-attenuated vaccine that could be tested further in ruminant models of paratuberculosis. The analysis also validated our screening strategy to identify effective vaccine candidates against intracellular pathogens.

  20. [Candid#1 vaccine against Argentine hemorrhagic fever produced in Argentina. Immunogenicity and safety].

    Science.gov (United States)

    Enria, Delia A; Ambrosio, Ana M; Briggiler, Ana M; Feuillade, María Rosa; Crivelli, Eleonora

    2010-01-01

    A clinical study in 946 human volunteers was done to compare Candid #1 vaccine manufactured in Argentina with the vaccine produced in USA that had been previously used. The efficacy was evaluated using immunogenicity measured by the detection of neutralizing antibodies as a subrogate marker. Safety was evaluated comparing the rate of adverse events. Both vaccines showed a comparable rate of seroconversion, slightly higher than the efficacy estimated from previous studies (95.5%). There were no severe adverse events related to the vaccines. The general events considered related to the vaccines were not clinically relevant and disappeared either spontaneously or with symptomatic treatment. Similar rates of adverse events (29.9% for the Argentine vaccine and 35.0% for the USA vaccine) were found for both vaccines. These included: headache, weakness, myalgias, mild low blood cell (ANMAT).

  1. Leishmania genome analysis and high-throughput immunological screening identifies tuzin as a novel vaccine candidate against visceral leishmaniasis.

    Science.gov (United States)

    Lakshmi, Bhavana Sethu; Wang, Ruobing; Madhubala, Rentala

    2014-06-24

    Leishmaniasis is a neglected tropical disease caused by Leishmania species. It is a major health concern affecting 88 countries and threatening 350 million people globally. Unfortunately, there are no vaccines and there are limitations associated with the current therapeutic regimens for leishmaniasis. The emerging cases of drug-resistance further aggravate the situation, demanding rapid drug and vaccine development. The genome sequence of Leishmania, provides access to novel genes that hold potential as chemotherapeutic targets or vaccine candidates. In this study, we selected 19 antigenic genes from about 8000 common Leishmania genes based on the Leishmania major and Leishmania infantum genome information available in the pathogen databases. Potential vaccine candidates thus identified were screened using an in vitro high throughput immunological platform developed in the laboratory. Four candidate genes coding for tuzin, flagellar glycoprotein-like protein (FGP), phospholipase A1-like protein (PLA1) and potassium voltage-gated channel protein (K VOLT) showed a predominant protective Th1 response over disease exacerbating Th2. We report the immunogenic properties and protective efficacy of one of the four antigens, tuzin, as a DNA vaccine against Leishmania donovani challenge. Our results show that administration of tuzin DNA protected BALB/c mice against L. donovani challenge and that protective immunity was associated with higher levels of IFN-γ and IL-12 production in comparison to IL-4 and IL-10. Our study presents a simple approach to rapidly identify potential vaccine candidates using the exhaustive information stored in the genome and an in vitro high-throughput immunological platform.

  2. A 52 Kilodalton Protein Vaccine Candidate for Francisella tularensis

    Science.gov (United States)

    2004-12-01

    du vaccin vivant F. tularensis (LVS). Soixante pourcent (60%) des souris vaccindes ont survdcu la dose ltale multiple alors que toutes les souris non...le lysat des cellules de cultures vivantes du vaccin vivant F. tularensis. Plusieurs composants de Francisella tularensis ont dt6 identifids par cet...antiserum. Le s6rum de souris provenant de souris vaccin6es avec F. tularensis non- vivant n’a pas identifid ces composants. A partir de ces prot6ines

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

    Science.gov (United States)

    Pore, Debasis; Chakrabarti, Manoj K

    2016-01-01

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

  4. Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Wilson Mayrink

    2010-02-01

    Full Text Available For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac as well as to phenol (PhVac. The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05. The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05. The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

  5. An assessment of enterotoxigenic Escherichia coli and Shigella vaccine candidates for infants and children.

    Science.gov (United States)

    Walker, Richard I

    2015-02-18

    Despite improvements to water quality, sanitation, and the implementation of current prevention and treatment interventions, diarrhea remains a major cause of illness and death, especially among children less than five years of age in the developing world. Rotavirus vaccines have already begun making a real impact on diarrhea, but several more enteric vaccines will be necessary to achieve broader reductions of illness and death. Among the many causes of diarrheal disease, enterotoxigenic Escherichia coli (ETEC) and Shigella are the two most important bacterial pathogens for which there are no currently licensed vaccines. Vaccines against these two pathogens could greatly reduce the impact of disease caused by these infections. This review describes the approaches to ETEC and Shigella vaccines that are currently under development, including a range of both cellular and subunit approaches for each pathogen. In addition, the review discusses strategies for maximizing the potential benefit of these vaccines, which includes the feasibility of co-administration, consolidation, and combination of vaccine candidates, as well as issues related to effective administration of enteric vaccines to infants. Recent impact studies indicate that ETEC and Shigella vaccines could significantly benefit global public health. Either vaccine, particularly if they could be combined together or with another enteric vaccine, would be an extremely valuable tool for saving lives and promoting the health of infants and children in the developing world, as well as potentially providing protection to travelers and military personnel visiting endemic areas.

  6. Preclinical testing of a vaccine candidate against tularemia.

    Science.gov (United States)

    Suresh, Ragavan Varadharajan; Ma, Zhuo; Sunagar, Raju; Bhatty, Vivek; Banik, Sukalyani; Catlett, Sally V; Gosselin, Edmund J; Malik, Meenakshi; Bakshi, Chandra Shekhar

    2015-01-01

    Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia.

  7. Speeding disease gene discovery by sequence based candidate prioritization

    Directory of Open Access Journals (Sweden)

    Porteous David J

    2005-03-01

    Full Text Available Abstract Background Regions of interest identified through genetic linkage studies regularly exceed 30 centimorgans in size and can contain hundreds of genes. Traditionally this number is reduced by matching functional annotation to knowledge of the disease or phenotype in question. However, here we show that disease genes share patterns of sequence-based features that can provide a good basis for automatic prioritization of candidates by machine learning. Results We examined a variety of sequence-based features and found that for many of them there are significant differences between the sets of genes known to be involved in human hereditary disease and those not known to be involved in disease. We have created an automatic classifier called PROSPECTR based on those features using the alternating decision tree algorithm which ranks genes in the order of likelihood of involvement in disease. On average, PROSPECTR enriches lists for disease genes two-fold 77% of the time, five-fold 37% of the time and twenty-fold 11% of the time. Conclusion PROSPECTR is a simple and effective way to identify genes involved in Mendelian and oligogenic disorders. It performs markedly better than the single existing sequence-based classifier on novel data. PROSPECTR could save investigators looking at large regions of interest time and effort by prioritizing positional candidate genes for mutation detection and case-control association studies.

  8. Defined neoglycoproteins as candidate vaccines against Streptococcus pneumoniae type 3

    NARCIS (Netherlands)

    Lefeber, Dirk Jaap

    2002-01-01

    Several bacteria that are surrounded by a polysaccharide coat can cause severe diseases like meningitis, pneumonia and otitis media, especially in young children. Against the bacterium Streptococcus pneumoniae, a polysaccharide vaccine exists. However, it does not effectively protect high-risk group

  9. Hsp70 as a candidate subunit vaccine for paratuberculosis

    NARCIS (Netherlands)

    Santema, W.J.

    2011-01-01

    This thesis focuses on vaccination-based control of bovine paratuberculosis, a chronic mycobacterial infection of the small intestine. Bovine paratuberculosis is a highly prevalent disease affecting ruminants worldwide, leading to substantial economic losses. There are concerns that the causative ag

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  11. Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis.

    Science.gov (United States)

    Mayrink, Wilson; Tavares, Carlos Alberto Pereira; Deus, Rosangela Barbosa de; Pinheiro, Melina Barros; Guimarães, Tânia Mara Pinto Dabés; Andrade, Hélida Monteiro de; Costa, Carlos Alberto da; Toledo, Vicente de Paulo Coelho Peixoto da

    2010-02-01

    For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p leishmaniasis.

  12. Molecular signature of high yield (growth influenza a virus reassortants prepared as candidate vaccine seeds.

    Directory of Open Access Journals (Sweden)

    Manojkumar Ramanunninair

    Full Text Available BACKGROUND: Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8, to provide the high yield reassortant (HYR viral 'seeds' for vaccine production. HYR must contain the hemagglutinin (HA and neuraminidase (NA genes of wt virus and one to six 'internal' genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth in ovo. METHODOLOGY: The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2 and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry. RESULTS: Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA. SIGNIFICANCE: In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth in ovo may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine 'seed' viruses.

  13. Optimization and Characterization of Candidate Strain for Coxsackievirus A16 Inactivated Vaccine

    Directory of Open Access Journals (Sweden)

    Jingliang Li

    2015-07-01

    Full Text Available Coxsackievirus A16 (CA16 and enterovirus 71 (EV71, both of which can cause hand, foot and mouth disease (HFMD, are responsible for large epidemics in Asian and Pacific areas. Although inactivated EV71 vaccines have completed testing in phase III clinical trials in Mainland China, CA16 vaccines are still under development. A Vero cell-based inactivated CA16 vaccine was developed by our group. Screening identified a CA16 vaccine strain (CC024 isolated from HFMD patients, which had broad cross-protective abilities and satisfied all requirements for vaccine production. Identification of the biological characteristics showed that the CA16CC024 strain had the highest titer (107.5 CCID50/mL in Vero cells, which would benefit the development of an EV71/CA16 divalent vaccine. A potential vaccine manufacturing process was established, including the selection of optimal time for virus harvesting, membrane for diafiltration and concentration, gel-filtration chromatography for the down-stream virus purification and virus inactivation method. Altogether, the analyses suggested that the CC-16, a limiting dilution clone of the CC024 strain, with good genetic stability, high titer and broad-spectrum immunogenicity, would be the best candidate strain for a CA16 inactivated vaccine. Therefore, our study provides valuable information for the development of a Vero cell-based CA16 or EV71-CA16 divalent inactivated vaccine.

  14. Identification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology

    OpenAIRE

    Meunier, Marine; Guyard-Nicodème, Muriel; Hirchaud, Edouard; Parra, Alberto; Chemaly, Marianne; Dory, Daniel

    2016-01-01

    Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due to Campylobacter jejuni or Campylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacteria’s main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for thi...

  15. A novel dengue virus serotype 1 vaccine candidate based on Japanese encephalitis virus vaccine strain SA14-14-2 as the backbone.

    Science.gov (United States)

    Yang, Huiqiang; Li, Zhushi; Lin, Hua; Wang, Wei; Yang, Jian; Liu, Lina; Zeng, Xianwu; Wu, Yonglin; Yu, Yongxin; Li, Yuhua

    2016-06-01

    To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.

  16. Cellular immune responses to nine Mycobacterium tuberculosis vaccine candidates following intranasal vaccination.

    Directory of Open Access Journals (Sweden)

    Suraj B Sable

    Full Text Available BACKGROUND: The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis. METHODS AND PRINCIPAL FINDINGS: In this study, a comparison of intranasal (i.n. and subcutaneous (s.c. vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860 was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study. CONCLUSION AND SIGNIFICANCE: Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.

  17. Evaluation of Three Live Attenuated H2 Pandemic Influenza Vaccine Candidates in Mice and Ferrets

    Science.gov (United States)

    Chen, Grace L.; Lamirande, Elaine W.; Cheng, Xing; Torres-Velez, Fernando; Orandle, Marlene; Jin, Hong; Kemble, George

    2014-01-01

    ABSTRACT H2 influenza viruses have not circulated in humans since 1968, and therefore a significant portion of the population would be susceptible to infection should H2 influenza viruses reemerge. H2 influenza viruses continue to circulate in avian reservoirs worldwide, and these reservoirs are a potential source from which these viruses could emerge. Three reassortant cold-adapted (ca) H2 pandemic influenza vaccine candidates with hemagglutinin (HA) and neuraminidase (NA) genes derived from the wild-type A/Japan/305/1957 (H2N2) (Jap/57), A/mallard/6750/1978 (H2N2) (mal/78), or A/swine/MO/4296424/2006 (H2N3) (sw/06) viruses and the internal protein gene segments from the A/Ann Arbor/6/60 ca virus were generated by plasmid-based reverse genetics (Jap/57 ca, mal/78 ca, and sw/06 ca, respectively). The vaccine candidates exhibited the in vitro phenotypes of temperature sensitivity and cold adaptation and were restricted in replication in the respiratory tract of ferrets. In mice and ferrets, the vaccines elicited neutralizing antibodies and conferred protection against homologous wild-type virus challenge. Of the three candidates, the sw/06 ca vaccine elicited cross-reactive antibodies and provided significant protection against the greatest number of heterologous viruses. These observations suggest that the sw/06 ca vaccine should be further evaluated in a clinical trial as an H2 pandemic influenza vaccine candidate. IMPORTANCE Influenza pandemics arise when novel influenza viruses are introduced into a population with little prior immunity to the new virus and often result in higher rates of illness and death than annual seasonal influenza epidemics. An influenza H2 subtype virus caused a pandemic in 1957, and H2 viruses circulated in humans till 1968. H2 influenza viruses continue to circulate in birds, and the development of an H2 influenza vaccine candidate is therefore considered a priority in preparing for future pandemics. However, we cannot predict whether a

  18. A candidate H1N1 pandemic influenza vaccine elicits protective immunity in mice.

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    Julia Steitz

    Full Text Available BACKGROUND: In 2009 a new pandemic disease appeared and spread globally. The recent emergence of the pandemic influenza virus H1N1 first isolated in Mexico and USA raised concerns about vaccine availability. We here report our development of an adenovirus-based influenza H1N1 vaccine tested for immunogenicity and efficacy to confer protection in animal model. METHODS: We generated two adenovirus(Ad5-based influenza vaccine candidates encoding the wildtype or a codon-optimized hemagglutinin antigen (HA from the recently emerged swine influenza isolate A/California/04/2009 (H1N1pdm. After verification of antigen expression, immunogenicity of the vaccine candidates were tested in a mouse model using dose escalations for subcutaneous immunization. Sera of immunized animals were tested in microneutalization and hemagglutination inhibition assays for the presence of HA-specific antibodies. HA-specific T-cells were measured in IFNgamma Elispot assays. The efficiency of the influenza vaccine candidates were evaluated in a challenge model by measuring viral titer in lung and nasal turbinate 3 days after inoculation of a homologous H1N1 virus. CONCLUSIONS/SIGNIFICANCE: A single immunization resulted in robust cellular and humoral immune response. Remarkably, the intensity of the immune response was substantially enhanced with codon-optimized antigen, indicating the benefit of manipulating the genetic code of HA antigens in the context of recombinant influenza vaccine design. These results highlight the value of advanced technologies in vaccine development and deployment in response to infections with pandemic potential. Our study emphasizes the potential of an adenoviral-based influenza vaccine platform with the benefits of speed of manufacture and efficacy of a single dose immunization.

  19. Development and characterization of candidate rotavirus vaccine strains derived from children with diarrhoea in Vietnam.

    Science.gov (United States)

    Luan, Le T; Trang, Nguyen V; Phuong, Nguyen M; Nguyen, Huong T; Ngo, Huong T; Nguyen, Huong T M; Tran, Hanh B; Dang, Ha N; Dang, Anh D; Gentsch, Jon R; Wang, Yuhuan; Esona, Mathew D; Glass, Roger I; Steele, A Duncan; Kilgore, Paul E; Nguyen, Man V; Jiang, Baoming; Nguyen, Hien D

    2009-11-20

    In Vietnam, rotavirus infection accounts for more than one-half of all hospitalizations for diarrhoea among children less than 5 years of age. While new vaccines to prevent rotavirus diarrhoea have been developed and introduced into some countries by multinational manufacturers, the ability for developing countries such as Vietnam to introduce several new and important vaccines into the routine infant immunization schedule may be challenging. In order to be partially self-sufficient in vaccine production, Vietnam has pursued the development of several rotavirus strains as candidate vaccines using isolates obtained from Vietnamese children with diarrhoea. This paper describes the origin, isolation and characterization of 3 human rotavirus strains being considered for further vaccine development in Vietnam. The goal is to prepare a monovalent G1P [8] rotavirus vaccine using one of these strains obtained in Vietnam and naturally attenuated by multiple passages in cell culture. While this is an ambitious project that will require several years' work, we are using the lessons learned to improve the overall quality of vaccine production including the use of Vero cell techniques for the manufacture of other vaccines in Vietnam.

  20. Gonadotrophin releasing hormone-based vaccine, an effective candidate for prostate cancer and other hormone-sensitive neoplasms.

    Science.gov (United States)

    Junco, Jesús A; Basalto, Roberto; Fuentes, Franklin; Bover, Eddy; Reyes, Osvaldo; Pimentel, Eulogio; Calzada, Lesvia; Castro, Maria D; Arteaga, Niurka; López, Yovisleidis; Hernández, Héctor; Bringas, Ricardo; Garay, Hilda; Peschke, Peter; Bertot, José; Guillén, Gerardo

    2008-01-01

    Prostate growth, development, functions, and neoplastic transformation is androgen dependent. Estrogens have similar effects in the ovary and breast. Previous studies using gonadotrophin releasing hormone (GnRH/LHRH) vaccines have shown the usefulness of immunization against this hormone in prostate (PC) and breast cancer (BC). We have synthesized a peptide mutated at position 6 and attached to the 830-844 tetanic toxoid (TT) helper T cell sequence in the same synthesis process. After repeated pig immunizations, we have demonstrated a vaccine that significantly decreased testes size (p < 0.001), prostate (p < 0.01), seminal vesicles (p < 0.01), and testosterone (T) castration [0.05 nM ml(-1) (p < 0. 01)]. Similar results were obtained in adult male and female healthy dogs and Macaca fascicularis models. These data indicate that this GnRHm1-TT vaccine is safe and able to induce significant tumor growth inhibition in the Dunning R3327-H rat androgen responsive prostate tumor model. In these rats, the immunization induced high anti-GnRH titers concomitant with T castration reduction (p < 0.01) in 90% of the animals tested. In addition, 70% of the responders exhibited tumor growth inhibition (p = 0.02) and a survival rate approximately three times longer that those of untreated rats. These data indicate that GnRHm1-TT vaccine may be a potential candidate in the treatment of PC, BC, and other hormone-dependent cancers.

  1. Three Candidate Peptide-Vaccines in Combination To Induce High Levels of Multiantibodies Against HIV-1

    Institute of Scientific and Technical Information of China (English)

    王颖; 田海军; 秦莉; 朱梅; 陈应华

    2001-01-01

    N-and C-domains of human immunodeficiency virus type 1 (HIV-1) gp41 are demonstrated to play an important role in HIV-entry and prevention. In addition, the V3 loop on gp120 was identified as the principal neutralizing determinant (PND). Based on the fact that a combination of several monoclonal antibodies to different neutralizing epitopes showed great protection against intravenous challenge and vaginal transmission of pathogenic HIV-1/Simian immunodeficiency virus (SIV) chimeric virus on macaques, three candidate peptide-vaccines were prepared and used in combination to induce high levels of multiantibodies against HIV-1. The three peptides contained important functional regions on HIV-1 gp160. The N-domain peptide (P1: aa550-579) and C-domain peptide (P2: aa633-662) of gp41 and V3 peptide (P3: aa301-328) of gp120 were conjugated with bovine serum albumin (BSA) using the glutaraldehyde method. After the vaccination course, each of the three candidate peptide-vaccines induced strong antibody response in rabbits. The three vaccines used in combination induced high levels of multiantibodies against the peptides of the N-and C-domains and the V3 Ioop, with the titer of antibodies up to 1: 6400-1: 25 600 in rabbit sera in comparison with the titer of 1: 800-1:3200 induced by rgp41 or rgp160. Our results indicate that immunogenicities of the N-and C-domains and the V3 loop in these three candidate peptide-vaccines were clearly stronger than those induced by rgp41 or rgp160, and these peptide-vaccines used in combination synchronously induced high levels of multiantibodies against HIV-1, suggesting that used in combination they may provide a new vaccine-strategy to induce strong multi-antiviral activity.

  2. Complete Genome Sequences of Bordetella pertussis Vaccine Reference Strains 134 and 10536

    Science.gov (United States)

    Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Burroughs, Mark; Johnson, Taccara; Juieng, Phalasy; Rowe, Lori; Tondella, M. Lucia; Williams, Margaret M.

    2016-01-01

    Vaccine formulations and vaccination programs against whooping cough (pertussis) vary worldwide. Here, we report the complete genome sequences of two divergent Bordetella pertussis reference strains used in the production of pertussis vaccines. PMID:27635001

  3. Complete Genome Sequences of Bordetella pertussis Vaccine Reference Strains 134 and 10536.

    Science.gov (United States)

    Weigand, Michael R; Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Burroughs, Mark; Johnson, Taccara; Juieng, Phalasy; Rowe, Lori; Tondella, M Lucia; Williams, Margaret M

    2016-09-15

    Vaccine formulations and vaccination programs against whooping cough (pertussis) vary worldwide. Here, we report the complete genome sequences of two divergent Bordetella pertussis reference strains used in the production of pertussis vaccines.

  4. Evaluation of Mdh1 protein as an antigenic candidate for a vaccine against candidiasis.

    Science.gov (United States)

    Shibasaki, Seiji; Aoki, Wataru; Nomura, Takashi; Karasaki, Miki; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

    2014-01-01

    Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.

  5. Lipoprotein NMB0928 from Neisseria meningitidis serogroup B as a novel vaccine candidate.

    Science.gov (United States)

    Delgado, Maité; Yero, Daniel; Niebla, Olivia; González, Sonia; Climent, Yanet; Pérez, Yusleydis; Cobas, Karem; Caballero, Evelín; García, Darien; Pajón, Rolando

    2007-12-01

    Polysaccharide-based vaccines for serogroup B Neisseria meningitidis have failed to induce protective immunity. As a result, efforts to develop vaccines for serogroup B meningococcal disease have mostly focused on outer membrane proteins (OMP). Vaccine candidates based on meningococcal OMP have emerged in the form of outer membrane vesicles (OMVs) or, more recently, purified recombinant proteins, as alternative strategies for serogroup B vaccine development. In our group, the protein composition of the Cuban OMVs-based vaccine VA-MENGOC-BC was elucidated using two-dimensional gel electrophoresis and mass spectrometry. The proteomic map of this product allowed the identification of new putative protective proteins not previously reported as components of an antimeningococcal vaccine. In the present study, we have determined the immunogenicity and protective capacity of NMB0928, one of those proteins present in the OMVs. The antigen was obtained as a recombinant protein in Escherichia coli, purified and used to immunize mice. The antiserum produced against the protein was capable to recognize the natural protein in different meningococcal strains by whole-cell ELISA and Western blotting. After immunization, recombinant NMB0928 induced bactericidal antibodies, and when the protein was administered inserted into liposomes, the elicited antibodies were protective in the infant rat model. These results suggest that NMB0928 is a novel antigen worth to be included in a broadly protective meningococcal vaccine.

  6. In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers.

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    Sandhya Vasan

    Full Text Available BACKGROUND: DNA-based vaccines have been safe but weakly immunogenic in humans to date. METHODS AND FINDINGS: We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. CONCLUSIONS: This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. TRIAL REGISTRATION: ClinicalTrials.gov NCT00545987.

  7. Cloning and sequencing of the gene encoding LipL21 in the vaccinal leptospira serovars

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    Rasoul Hoseinpur

    2016-01-01

    Full Text Available Background: Leptospirosis is a zoonotic disease in humans and animals, caused by the bacterium Leptospira interrogans. Gene expressing LipL21 is one of the genes identified in the bacterium, existing only in the pathogenic strains. The aim of this study was to cloning and analyzing the sequence of the gene encoding surface lipoprotein, LipL21, in five vaccinal leptospira serovars in Iran. Material and Methods: Pathogenic Leptospira interrogans serovars were cultured in EMJH medium with 10% rabbit serum. After genomic DNA extraction, PCR with specific primers was employed and the resulting product inserted in a vector then transferred into E. Coli DH5&alpha. The recombinant plasmids were finally sent for sequencing. Results: The analysis of gene lipL21 in domestic vaccinal serovars and comparison of them with other serovars in the GenBank database revealed that three vaccinal serovars serjo hardjo, canicola and pomona had 100% similarity with each other and grippotyphosa serovar had the highest difference with the vaccinal serovars. In general, the results showed that this gene is a highly conserved gene in the domestic vaccinal serovars and serovars in the GenBank database with more than 95.7 percent similarity. Conclusion: These results showed that the gene, lipL21, is highly conserved in the vaccinal serovars (similarities > 96.4 %. Therefore, the gene encoding surface protein LipL21 can serve as a useful serologic test with high specificity and sensitivity for diagnosis of leptospirosis in clinical samples and in future as an effective subunit vaccine candidate to be used.

  8. Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope candidates for epitope-based vaccines.

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    Paul Thiamjoo Tan

    Full Text Available BACKGROUND: The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. METHODOLOGY/PRINCIPAL FINDINGS: HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54 peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-gamma ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. CONCLUSIONS/SIGNIFICANCE: Seventeen (17 T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.

  9. Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

    Directory of Open Access Journals (Sweden)

    William E. Gillanders

    2011-11-01

    Full Text Available New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.

  10. Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lijin [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); Goedegebuure, Peter [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Mardis, Elaine R. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); The Genome Institute at Washington University School of Medicine, St. Louis, MO 63108 (United States); Ellis, Matthew J.C. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 (United States); Zhang, Xiuli; Herndon, John M. [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); Fleming, Timothy P. [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Carreno, Beatriz M. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 (United States); Hansen, Ted H. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Gillanders, William E., E-mail: gillandersw@wudosis.wustl.edu [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States)

    2011-11-25

    New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.

  11. Evaluation of avian influenza virus isolated from ducks as a potential live vaccine candidate against novel H7N9 viruses.

    Science.gov (United States)

    Jiang, Wen-Ming; Wang, Su-Chun; Liu, Hua-Lei; Yu, Jian-Min; Du, Xiang; Hou, Guang-Yu; Li, Jin-Ping; Liu, Shuo; Wang, Kai-Cheng; Zhuang, Qing-Ye; Liu, Xiang-Ming; Chen, Ji-Ming

    2014-11-12

    Recent outbreaks of a novel H7N9 avian influenza virus in humans in China raise pandemic concerns and underscore an urgent need to develop effective vaccines. Theoretically, live influenza vaccines are of multiple advantages over traditional inactivated influenza vaccines to be used in a pandemic, because they can be produced rapidly, safely, and inexpensively. However, studies on live vaccines against the novel H7N9 virus are limited. In this study, we evaluated a potential live influenza vaccine candidate using an H7N3 avian influenza virus isolated from ducks with controls of two recombinant viruses generated through reverse genetics. The potential candidate could be produced efficiently using chicken embryonated eggs, and is homogenous to the novel H7N9 virus in their viral hemagglutinin genes. The potential candidate is likely low pathogenic to birds and mammals, and likely sensitive to oseltamivir and amantadine, as suggested by its genomic sequences. Its low pathogenicity was further supported through inoculation in mice, chicken embryonated eggs and chickens. Specific antibodies elicited in mice were detectable at least during the period between day 14 and day 56 after intranasal administration of the candidate for one time. Titers of the specific antibodies increased significantly with a boost intranasal administration or a higher inoculation dose. The induced specific antibodies were of substantial cross-reactivity with the novel H7N9 virus. These primary but promising evaluation data suggest that the duck influenza virus could be used as a potential live vaccine candidate, favorably through a prime-boost route, to mitigate the severity of the possible pandemic caused by the newly emerging H7N9 virus, and is valuable to be further evaluated.

  12. A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; Ye Ye; You-Hua Xie; Yu-Ying Kong; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine.METHODS: Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes.These cells were subjected to HCVantigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals.RESULTS: Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-γ secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-γ but did not change the profile of IL-4 secretion.CONCLUSION: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

  13. Recombinant feline coronaviruses as vaccine candidates confer protection in SPF but not in conventional cats.

    Science.gov (United States)

    Bálint, Ádám; Farsang, Attila; Szeredi, Levente; Zádori, Zoltán; Belák, Sándor

    2014-03-14

    Feline infectious peritonitis virus (FIPV) is a major pathogen of Felidae. Despite the extensive efforts taken in the past decades, development of the "ideal" live attenuated FIPV vaccine was not successful yet. In the present study, we provide data of immunisation experiments with a recombinant FCoV pair differing only in the truncation (PBFIPV-DF-2) or completion (PBFIPV-DF-2-R3i) of their ORF3abc regions. In our previous in vivo studies, these viruses proved to show the characters of low virulent or avirulent FCoV phenotypes, respectively. Therefore, we hypothesised the ability of these viruses, as possible vaccine candidates, in conferring protection in specific pathogen free (SPF) Domestic Shorthair as well as in conventional purebred British Shorthair cats. In SPF cats, after two oronasal and two intramuscular vaccinations with two weeks intervals, both vaccine candidates provided 100% protection against lethal homologous challenge with the highly virulent FIPV DF-2 strain. In contrast, the conventional purebred British Shorthair cats did not develop protection when they were immunised with the same vaccination regimes. In these groups 100% of the PBFIPV-DF-2-R3i immunised animals developed antibody-dependent enhancement (ADE). Prolonged survival was observed in 40% of the animals, while 60% showed fulminant disease course. Genetic and more probably immunological differences between the SPF and non-SPF purebred kittens can explain the different outcome of the vaccination experiment. Our data highlight the diverse immune responses between SPF and conventional cats and suggest a decisive role of previous infection by heterologous causative agents in the outcome of the vaccination against FIP.

  14. Phase 1/2a Trial of Plasmodium vivax Malaria Vaccine Candidate VMP001/AS01B in Malaria-Naive Adults: Safety, Immunogenicity, and Efficacy.

    Directory of Open Access Journals (Sweden)

    Jason W Bennett

    2016-02-01

    Full Text Available A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001, a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP and a truncated repeat region that contains repeat sequences from both the VK210 (type 1 and the VK247 (type 2 parasites, was developed as a vaccine candidate for global use.We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI of VMP001 formulated in the GSK Adjuvant System AS01B. A total of 30 volunteers divided into 3 groups (10 per group were given 3 intramuscular injections of 15 μg, 30 μg, or 60 μg respectively of VMP001, all formulated in 500 μL of AS01B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls.The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period.This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials.

  15. Generation and Characterization of Live Attenuated Influenza A(H7N9 Candidate Vaccine Virus Based on Russian Donor of Attenuation.

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    Svetlana Shcherbik

    Full Text Available Avian influenza A (H7N9 virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV to provide temperature sensitive, cold-adapted and attenuated phenotypes.LAIV candidate A/Anhui/1/2013(H7N9-CDC-LV7A (abbreviated as CDC-LV7A, based on the Russian MDV, A/Leningrad/134/17/57 (H2N2, was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9RG-LV1 and A(H7N9RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9 virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7.Our data indicate that the A/Anhui/1/2013(H7N9-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for

  16. Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation

    Science.gov (United States)

    Shcherbik, Svetlana; Pearce, Nicholas; Balish, Amanda; Jones, Joyce; Thor, Sharmi; Davis, Charles Todd; Pearce, Melissa; Tumpey, Terrence; Cureton, David; Chen, Li-Mei; Villanueva, Julie; Bousse, Tatiana L.

    2015-01-01

    Background Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes. Methodology/Principal Findings LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7. Conclusions/Significance Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and

  17. Preliminary Evaluation of a Candidate Multi-Epitope-Vaccine Against the Classical Swine Fever Virus

    Institute of Scientific and Technical Information of China (English)

    YING Jian; DONG Xiaonan; CHEN Yinghua

    2008-01-01

    A multi-epitope-vaccine MEVABC consisting of two linear neutralizing determinants (BC1: aa693-716; A6: aa844-865) located on antigenic unit B/C and unit A of glycoprotein E2 was prepared to evaluate whether a combination strategy is effective in the design of peptide vaccines.After immunization,pig sera collected every one to two weeks were evaluated by enzyme linked immunosorbent assay.C-strain- induced anti-sera and hyper-immune sera cannot recognize overlapping peptides that cover the E2 N-terminus,while MEVABC is able to elicit high levels of peptide-specific antibody response.When compared with previously studied peptide vaccines PV-BC1 and PV-A6,the same dose of either component in the MEVABC increases the BC1- or A6-specific antibodies (to 1/3-1/2 of the levels of the separate vaccines).However,the synergy between the antibodies may make MEVABC much more potent.Moreover,anti-C-strain immunity pre-existing in pigs does not disturb the sequent MEVABC vaccination.Thus,MEVABC can be ad- ministrated to pigs which already possess anti-classical swine fever virus immunity.MEVABC is a promising candidate marker vaccine.

  18. Nucleoprotein nanostructures combined with adjuvants adapted to the neonatal immune context: a candidate mucosal RSV vaccine.

    Directory of Open Access Journals (Sweden)

    Aude Remot

    Full Text Available BACKGROUND: The human respiratory syncytial virus (hRSV is the leading cause of severe bronchiolitis in infants worldwide. The most severe RSV diseases occur between 2 and 6 months-of-age, so pediatric vaccination will have to be started within the first weeks after birth, when the immune system is prone to Th2 responses that may turn deleterious upon exposure to the virus. So far, the high risk to prime for immunopathological responses in infants has hampered the development of vaccine. In the present study we investigated the safety and efficacy of ring-nanostructures formed by the recombinant nucleoprotein N of hRSV (N(SRS as a mucosal vaccine candidate against RSV in BALB/c neonates, which are highly sensitive to immunopathological Th2 imprinting. METHODOLOGY AND PRINCIPAL FINDINGS: A single intranasal administration of N(SRS with detoxified E. coli enterotoxin LT(R192G to 5-7 day old neonates provided a significant reduction of the viral load after an RSV challenge at five weeks of age. However, neonatal vaccination also generated an enhanced lung infiltration by neutrophils and eosinophils following the RSV challenge. Analysis of antibody subclasses and cytokines produced after an RSV challenge or a boost administration of the vaccine suggested that neonatal vaccination induced a Th2 biased local immune memory. This Th2 bias and the eosinophilic reaction could be prevented by adding CpG to the vaccine formulation, which, however did not prevent pulmonary inflammation and neutrophil infiltration upon viral challenge. CONCLUSIONS/SIGNIFICANCE: In conclusion, protective vaccination against RSV can be achieved in neonates but requires an appropriate combination of adjuvants to prevent harmful Th2 imprinting.

  19. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1.

    Science.gov (United States)

    Kenner, J R; Coster, T S; Taylor, D N; Trofa, A F; Barrera-Oro, M; Hyman, T; Adams, J M; Beattie, D T; Killeen, K P; Spriggs, D R

    1995-10-01

    Cholera vaccine candidate Peru-15 was derived from a Vibrio cholerae O1 El Tor Inaba strain by deleting the cholera toxin genetic element, introducing the gene encoding cholera toxin B subunit into recA, and screening for nonmotility. In a controlled study, Peru-15 (2 x 10(8) cfu) was administered to 11 volunteers. No vaccinee developed diarrhea, and 10 of 11 had > 4-fold rises in vibriocidal antibody titers. One month later, 5 vaccinees and 5 control volunteers were challenged with wild type V. cholerae O1. Four of 5 controls developed diarrhea (mean, 1.9 L). Two Peru-15 vaccinees developed diarrhea, 1 with volunteer had not developed a significant vibriocidal immune response to vaccination. Peru-15 shows promise as a single-dose, oral cholera vaccine that is safe, immunogenic, and protective.

  20. A defined syphilis vaccine candidate inhibits dissemination of Treponema pallidum subspecies pallidum

    Science.gov (United States)

    Lithgow, Karen V.; Hof, Rebecca; Wetherell, Charmaine; Phillips, Drew; Houston, Simon; Cameron, Caroline E.

    2017-01-01

    Syphilis is a prominent disease in low- and middle-income countries, and a re-emerging public health threat in high-income countries. Syphilis elimination will require development of an effective vaccine that has thus far remained elusive. Here we assess the vaccine potential of Tp0751, a vascular adhesin from the causative agent of syphilis, Treponema pallidum subsp. pallidum. Tp0751-immunized animals exhibit a significantly reduced bacterial organ burden upon T. pallidum challenge compared with unimmunized animals. Introduction of lymph nodes from Tp0751-immunized, T. pallidum-challenged animals to naive animals fails to induce infection, confirming sterile protection. These findings provide evidence that Tp0751 is a promising syphilis vaccine candidate. PMID:28145405

  1. Rotavirus VP6 preparations as a non-replicating vaccine candidates.

    Science.gov (United States)

    Jalilvand, Somayeh; Marashi, Sayed Mahdi; Shoja, Zabihollah

    2015-06-26

    Rotavirus (RV) structural proteins VP4 and VP7, located on the surface of viral particles, elicit neutralizing antibodies (Abs) and are therefore considered to be important components of RV vaccines. However, despite inducing neutralizing Abs, limits of cross-neutralizing activity and lack of full correlation with protection limit the usefulness of these proteins as protective agents against RV disease. VP6 protein, which forms the middle layer of RV particles, is discussed as an alternative vaccine candidate since it can induce cross-protective immune responses against different RV strains although the Ab raised is not neutralizing. This report reviews different functions of VP6 that can lead to considering it as an alternative vaccine against RV disease.

  2. Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

    Directory of Open Access Journals (Sweden)

    Ai-Hsiang Chou

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 has caused several epidemics of hand, foot and mouth diseases (HFMD in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration, a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice

  3. Chemically induced Salmonella enteritidis ghosts as a novel vaccine candidate against virulent challenge in a rat model.

    Science.gov (United States)

    Vinod, Nagarajan; Oh, Sung; Kim, Seongdae; Choi, Chang Won; Kim, Sei Chang; Jung, Cheong-Hwan

    2014-05-30

    Salmonella enteritidis ghosts (SEGs), non-living empty bacterial cell envelopes were generated by using the minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH) and investigated as a vaccine candidate in rats. To determine the immunogenicity and protective efficacy of SEG vaccine, rats were divided into four groups: group A (non-vaccinated control), group B (orally vaccinated), group C (intramuscularly vaccinated) and group D (intramuscularly vaccinated with complete Freund's adjuvant). Vaccination of rats with SEGs induced significant immune responses before and after virulent challenge. Rats vaccinated with SEGs showed significant increases in serum IgG antibodies after challenging with virulent S. enteritidis on week 8 and week 10 (P<0.01). During the vaccination period, groups B, C and D showed significantly higher serum bactericidal activity (SBA) compared to group A (P<0.01). Most importantly, bacterial loads in vaccinated groups were significantly lower than in the non-vaccinated group (P<0.01). In conclusion, these results show that the chemically induced SEGs as a vaccine candidate against virulent challenge.

  4. Scrub Typhus Vaccine Candidate Kp r56 Induces Humoral and Cellular Immune Responses in Cynomolgus Monkeys

    Science.gov (United States)

    2005-03-28

    binding of 100 l/well of horseradish peroxidase-conjugated goat antibodies directed to monkey immu- noglobulin G (IgG) or IgM (Kirkegaard & Perry...candidates. Vaccine 21:4550–4554. 8. Cheers, C., H. Pavlov, C. Riglar, and E. Madraso. 1980. Macrophage acti- vation during experimental murine brucellosis ...III. Do macrophages exert feedback control during brucellosis ? Cell. Immunol. 49:168–177. 9. Ching, W. M., H. Wang, C. Eamsila, D. J. Kelly, and G. A

  5. Evaluation of Three Live Attenuated H2 Pandemic Influenza Vaccine Candidates in Mice and Ferrets

    OpenAIRE

    2014-01-01

    H2 influenza viruses have not circulated in humans since 1968, and therefore a significant portion of the population would be susceptible to infection should H2 influenza viruses reemerge. H2 influenza viruses continue to circulate in avian reservoirs worldwide, and these reservoirs are a potential source from which these viruses could emerge. Three reassortant cold-adapted (ca) H2 pandemic influenza vaccine candidates with hemagglutinin (HA) and neuraminidase (NA) genes derived from the wild...

  6. Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

    Directory of Open Access Journals (Sweden)

    Svetlana V Shcherbik

    Full Text Available BACKGROUND: Live attenuated influenza vaccine viruses (LAIVs can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV and a seasonal wild-type (wt virus. The vaccine candidates contain hemagglutinin (HA and neuraminidase (NA genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. METHODOLOGY/PRINCIPAL FINDINGS: This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2 (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012 or influenza A (H7N9 (A/Anhui/1/2013 wt viruses with the MDV A/Leningrad/134/17/57(H2N2. Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

  7. Production of a Recombinant Vaccine Candidate against Burkholderia pseudomallei Exploiting the Bacterial N-Glycosylation Machinery

    Directory of Open Access Journals (Sweden)

    Fatima eGarcia-Quintanilla

    2014-07-01

    Full Text Available Vaccines developing immune responses towards surface carbohydrates conjugated to proteins are effective in preventing infection and death by bacterial pathogens. Traditional production of these vaccines utilizes complex synthetic chemistry to acquire and conjugate the glycan to a protein. However, glycoproteins produced by bacterial protein glycosylation systems are significantly easier to produce, and could possible be used as vaccine candidates. In this work we functionally expressed the B. pseudomallei O polysaccharide (OPS II, the C. jejuni oligosaccharyltransferase (OTase, and a suitable glycoprotein (AcrA in a designer E. coli strain with a higher efficiency for production of glycoconjugates. We were able to produce and purify the OPS II-AcrA glycoconjugate, and MS analysis confirmed correct glycan was produced and attached. We observed the attachment of the O-acetylated deoxyhexose directly to the acceptor protein, which expands the range of substrates utilized by the OTase PglB. Injection of the glycoprotein into mice generated an IgG immune response against B. pseudomallei, and this response was partially protective against an intranasal challenge. Our experiments show that bacterial engineered glycoconjugates can be utilized as vaccine candidates against B. pseudomallei. Additionally, our new E. coli strain SDB1 is more efficient in glycoprotein production, and could have additional applications in the future.

  8. 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.

  9. Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

    Institute of Scientific and Technical Information of China (English)

    芮贤良; 徐永强; 吴旭东; 苏国富; 黄翠芬

    1997-01-01

    A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd ’host/ Asd+ vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the

  10. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    Energy Technology Data Exchange (ETDEWEB)

    Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2016-03-15

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  11. Cloning and sequence analysis of the isoforms H11-4 of the vaccine candidate antigen H11 from Haemonchus contortus%捻转血矛线虫ZJ株疫苗候选抗原H11亚型基因H11-4的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    段丽君; 周前进; 张红丽; 杨怡; 闫宝龙; 杜爱芳

    2013-01-01

    微粒体氨肽酶H11天然提取物是目前捻转血矛线虫(Haemonchus contortus)防治研究中最好的疫苗候选抗原之一,但其重组形式均不能提供有效的免疫保护效果;同时报道H11蛋白存在多种亚型,推测其某种亚型或亚型组合可能在天然提取物参与免疫保护中起了关键作用.本试验参考NCBI公布的H.contortus H11-4基因序列,设计2对特异性引物,以H.contortus ZJ株总RNA为模板,利用RT-PCR技术分段扩增出该基因的部分片段,并进行T-A克隆.测序正确后,利用含有不同片段的阳性质粒经BamH Ⅰ/Nco Ⅰ消化,连接后获得H11-4基因的全长cDNA序列.测序结果显示成功获得H11 4基因,开放阅读框大小为2 916 bp,与NCBI中公布的核苷酸序列同源性为97.8%,氨基酸序列同源性为97.6%.生物信息学分析,已获得的H11-4与H11 (H11-3)亚型氨基酸序列高度同源,且具有保守糖基化位点、相对保守的跨膜区与微粒体氨肽酶活性中心锌指基序.为进一步分析H11天然提取物各亚型在参与免疫保护的机制和角色分工奠定了基础.%The recombinant microsomal aminopeptidase H11 antigen has not shown highly protective efficacy against Haemonchus contortus compared to its native extract which is considered to be the most efficient vaccine candidate antigen. It is reported that there exist several isoforms of native H11 and it was supposed that one or combined isoforms of native H11 play a key role in immune protection. Fragments of H11-4 gene were amplified by reverse transcription polymerase chain reaction (RT-PCR) with two pairs of primers designed according to the published gene sequence from NCBI. Then the fragments of H11-4 gene were ligated to the T-A cloning vector pMD18-T and sequenced. Positive clones representative the different two fragments were digested with BamH Ⅰ / Nco Ⅰ and then ligated to obtain the full-length cDNA. Sequence analysis shows that with 2 916 bp, H11-4 gene

  12. Development of Designed Site-Directed Pseudopeptide-Peptido-Mimetic Immunogens as Novel Minimal Subunit-Vaccine Candidates for Malaria

    Directory of Open Access Journals (Sweden)

    Luisa F. Carreño

    2010-12-01

    Full Text Available Synthetic vaccines constitute the most promising tools for controlling and preventing infectious diseases. When synthetic immunogens are designed from the pathogen native sequences, these are normally poorly immunogenic and do not induce protection, as demonstrated in our research. After attempting many synthetic strategies for improving the immunogenicity properties of these sequences, the approach consisting of identifying high binding motifs present in those, and then performing specific changes on amino-acids belonging to such motifs, has proven to be a workable strategy. In addition, other strategies consisting of chemically introducing non-natural constraints to the backbone topology of the molecule and modifying the α-carbon asymmetry are becoming valuable tools to be considered in this pursuit. Non-natural structural constraints to the peptide backbone can be achieved by introducing peptide bond isosters such as reduced amides, partially retro or retro-inverso modifications or even including urea motifs. The second can be obtained by strategically replacing L-amino-acids with their enantiomeric forms for obtaining both structurally site-directed designed immunogens as potential vaccine candidates and their Ig structural molecular images, both having immuno-therapeutic effects for preventing and controlling malaria.

  13. Limited potential for mosquito transmission of genetically engineered, live-attenuated western equine encephalitis virus vaccine candidates.

    Science.gov (United States)

    Turell, Michael J; O'Guinn, Monica L; Parker, Michael D

    2003-02-01

    Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.

  14. Genetic diversity and population structure of genes encoding vaccine candidate antigens of Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Chenet Stella M

    2012-03-01

    Full Text Available Abstract Background A major concern in malaria vaccine development is genetic polymorphisms typically observed among Plasmodium isolates in different geographical areas across the world. Highly polymorphic regions have been observed in Plasmodium falciparum and Plasmodium vivax antigenic surface proteins such as Circumsporozoite protein (CSP, Duffy-binding protein (DBP, Merozoite surface protein-1 (MSP-1, Apical membrane antigen-1 (AMA-1 and Thrombospondin related anonymous protein (TRAP. Methods Genetic variability was assessed in important polymorphic regions of various vaccine candidate antigens in P. vivax among 106 isolates from the Amazon Region of Loreto, Peru. In addition, genetic diversity determined in Peruvian isolates was compared to population studies from various geographical locations worldwide. Results The structured diversity found in P. vivax populations did not show a geographic pattern and haplotypes from all gene candidates were distributed worldwide. In addition, evidence of balancing selection was found in polymorphic regions of the trap, dbp and ama-1 genes. Conclusions It is important to have a good representation of the haplotypes circulating worldwide when implementing a vaccine, regardless of the geographic region of deployment since selective pressure plays an important role in structuring antigen diversity.

  15. Identification of vaccine candidate antigens of Staphylococcus pseudintermedius by whole proteome characterization and serological proteomic analyses.

    Science.gov (United States)

    Couto, Natacha; Martins, Joana; Lourenço, Ana Mafalda; Pomba, Constança; Varela Coelho, Ana

    2016-02-05

    The recent emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) has complicated considerably the treatment of infections caused by these bacteria. Therefore new treatment strategies are urgently needed, namely through the development of vaccines towards the control of bacterial infections. Our study describes an extensive characterization of the proteome of S. pseudintermedius through a 2-DE MALDI-TOF/TOF approach, followed by SERological Proteome Analysis (SERPA) to identify potential vaccine candidate antigens. We were able to identify 361 unique proteins, of which 39 are surface proteins. In order to assess the immunogenic potential of S. pseudintermedius proteins, a Western blot analysis of two-dimensional gels was carried out with serum from healthy dogs, dogs with atopic dermatitis infected and not infected with S. pseudintermedius. Only immunogenic areas detected by ≥ 50% of the dogs with atopic dermatitis infected with S. pseudintermedius sera and by proteins could induce hypersensitivity. We were able to identify 13 unique proteins after in-gel digestion of selected protein gel spots, with 4 antigenic proteins showing promising features for vaccine development. No specific antibodies were identified in the dogs with atopic dermatitis not infected with S. pseudintermedius sera that could contribute to prevention of infection. The SERPA approach employed in this study revealed novel candidate therapeutic targets for the control of S. pseudintermedius infections.

  16. Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism.

    Directory of Open Access Journals (Sweden)

    Kenneth Plante

    2011-07-01

    Full Text Available Chikungunya virus (CHIKV is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.

  17. Phage Library Screening for the Rapid Identification and In Vivo Testing of Candidate Genes for a DNA Vaccine against Mycoplasma mycoides subsp. mycoides Small Colony Biotype

    Science.gov (United States)

    March, John B.; Jepson, Catherine D.; Clark, Jason R.; Totsika, Makrina; Calcutt, Michael J.

    2006-01-01

    A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia

  18. In silico identification of genetically attenuated vaccine candidate genes for Plasmodium liver stage.

    Science.gov (United States)

    Kumar, Hirdesh; Frischknecht, Friedrich; Mair, Gunnar R; Gomes, James

    2015-12-01

    Genetically attenuated parasites (GAPs) that lack genes essential for the liver stage of the malaria parasite, and therefore cause developmental arrest, have been developed as live vaccines in rodent malaria models and recently been tested in humans. The genes targeted for deletion were often identified by trial and error. Here we present a systematic gene - protein and transcript - expression analyses of several Plasmodium species with the aim to identify candidate genes for the generation of novel GAPs. With a lack of liver stage expression data for human malaria parasites, we used data available for liver stage development of Plasmodium yoelii, a rodent malaria model, to identify proteins expressed in the liver stage but absent from blood stage parasites. An orthology-based search was then employed to identify orthologous proteins in the human malaria parasite Plasmodium falciparum resulting in a total of 310 genes expressed in the liver stage but lacking evidence of protein expression in blood stage parasites. Among these 310 possible GAP candidates, we further studied Plasmodium liver stage proteins by phyletic distribution and functional domain analyses and shortlisted twenty GAP-candidates; these are: fabB/F, fabI, arp, 3 genes encoding subunits of the PDH complex, dnaJ, urm1, rS5, ancp, mcp, arh, gk, lisp2, valS, palm, and four conserved Plasmodium proteins of unknown function. Parasites lacking one or several of these genes might yield new attenuated malaria parasites for experimental vaccination studies.

  19. Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

    Institute of Scientific and Technical Information of China (English)

    王恒樑; 冯尔玲; 林云; 廖翔; 金明; 黄留玉; 苏国富; 黄翠芬

    2002-01-01

    In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.

  20. Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling.

    Directory of Open Access Journals (Sweden)

    Fahad N Almajhdi

    Full Text Available Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16 is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.

  1. What Is Known about the Immune Response Induced by Plasmodium vivax Malaria Vaccine Candidates?

    Science.gov (United States)

    López, Carolina; Yepes-Pérez, Yoelis; Hincapié-Escobar, Natalia; Díaz-Arévalo, Diana; Patarroyo, Manuel A.

    2017-01-01

    Malaria caused by Plasmodium vivax continues being one of the most important infectious diseases around the world; P. vivax is the second most prevalent species and has the greatest geographic distribution. Developing an effective antimalarial vaccine is considered a relevant control strategy in the search for means of preventing the disease. Studying parasite-expressed proteins, which are essential in host cell invasion, has led to identifying the regions recognized by individuals who are naturally exposed to infection. Furthermore, immunogenicity studies have revealed that such regions can trigger a robust immune response that can inhibit sporozoite (hepatic stage) or merozoite (erythrocyte stage) invasion of a host cell and induce protection. This review provides a synthesis of the most important studies to date concerning the antigenicity and immunogenicity of both synthetic peptide and recombinant protein candidates for a vaccine against malaria produced by P. vivax. PMID:28243235

  2. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone.

    Science.gov (United States)

    Osorio, Jorge E; Wallace, Derek; Stinchcomb, Dan T

    2016-01-01

    Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever.

  3. Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites.

    Science.gov (United States)

    Spiegel, Holger; Schinkel, Helga; Kastilan, Robin; Dahm, Pia; Boes, Alexander; Scheuermayer, Matthias; Chudobová, Ivana; Maskus, Dominika; Fendel, Rolf; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-04-01

    We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.

  4. Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice.

    Science.gov (United States)

    Bannantine, John P; Everman, Jamie L; Rose, Sasha J; Babrak, Lmar; Katani, Robab; Barletta, Raúl G; Talaat, Adel M; Gröhn, Yrjö T; Chang, Yung-Fu; Kapur, Vivek; Bermudez, Luiz E

    2014-01-01

    Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne's disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.

  5. Conservation analysis of dengue virus T-cell epitope-based vaccine candidates using peptide block entropy

    Directory of Open Access Journals (Sweden)

    Lars Ronn Olsen

    2011-12-01

    Full Text Available Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches to assembling broadly covering sets of peptides are commonly based on assembling highly conserved epitopes. Peptide block entropy analysis is a novel approach to assembling sets of broadly covering antigens. Since T-cell epitopes are recognized as peptides rather than individual residues, this method is based on calculating the information content of blocks of peptides from a multiple sequence alignment of homologous proteins rather than individual residues. The block entropy analysis provides broad coverage by variant inclusion, since high frequency may not be the sole determinant of the immunogenic potential of a predicted MHC class I binder. We applied block entropy analysis method to the proteomes of the four serotypes of dengue virus and found 1,551 blocks of 9-mer peptides, which covered all available sequences with five or fewer unique peptides. In contrast, the benchmark study by Khan et al. (2008, resulted in 165 9-mers being determined as conserved. Many of the blocks are located consecutively in the proteins, so connecting these blocks resulted in 78 conserved regions which can be covered with 457 subunit peptides. Of the 1551 blocks of 9-mer peptides, 110 blocks consisted of peptides all predicted to bind to MHC with similar affinity and the same HLA restriction. In total, we identified a pool of 333 peptides as T-cell epitope candidates. This set could form the basis for a broadly neutralizing dengue virus vaccine. The peptide block entropy analysis approach significantly increases the number of conserved peptide regions in comparison to traditional conservation analysis of individual residues. We determined 457 subunit peptides with the capacity to encompass the diversity of all sequenced DENV strains.

  6. Comparative genomic analysis of Brucella abortus vaccine strain 104M reveals a set of candidate genes associated with its virulence attenuation.

    Science.gov (United States)

    Yu, Dong; Hui, Yiming; Zai, Xiaodong; Xu, Junjie; Liang, Long; Wang, Bingxiang; Yue, Junjie; Li, Shanhu

    2015-01-01

    The Brucella abortus strain 104M, a spontaneously attenuated strain, has been used as a vaccine strain in humans against brucellosis for 6 decades in China. Despite many studies, the molecular mechanisms that cause the attenuation are still unclear. Here, we determined the whole-genome sequence of 104M and conducted a comprehensive comparative analysis against the whole genome sequences of the virulent strain, A13334, and other reference strains. This analysis revealed a highly similar genome structure between 104M and A13334. The further comparative genomic analysis between 104M and A13334 revealed a set of genes missing in 104M. Some of these genes were identified to be directly or indirectly associated with virulence. Similarly, a set of mutations in the virulence-related genes was also identified, which may be related to virulence alteration. This study provides a set of candidate genes associated with virulence attenuation in B.abortus vaccine strain 104M.

  7. Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases.

    Science.gov (United States)

    Perez-Casal, Jose; Potter, Andrew A

    2016-02-17

    The enzyme glyceraldehyde-3-P-dehydrogenase (GAPDH) has been identified as having other properties in addition to its key role in glycolysis. The ability of GAPDH to bind to numerous extracellular matrices, modulation of host-immune responses, a role in virulence and surface location has prompted numerous investigators to postulate that GAPDH may be a good vaccine candidate for protection against numerous pathogens. Although immune responses against GAPDH have been described for many microorganisms, vaccines containing GAPDH have been successfully tested in few cases including those against the trematode-Schistosoma mansoni, the helminth-Enchinococcus multilocularis; the nematode filaria- Litomosoides sigmodontis; fish pathogens such as Aeromonas spp., Vibrio spp., Edwarsiella spp., and Streptococcus iniae; and environmental streptococci, namely, Streptococcus uberis and Streptococcus dysgalactiae. Before GAPDH-based vaccines are considered viable options for protection against numerous pathogens, we need to take into account the homology between the host and pathogen GAPDH proteins to prevent potential autoimmune reactions, thus protective GAPDH epitopes unique to the pathogen protein must be identified.

  8. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein.

    Science.gov (United States)

    Chen, Edwin; Salinas, Nichole D; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D; Gross, Michael L; Adams, John H; Tolia, Niraj Harish

    2016-05-31

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifs in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. The identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.

  9. Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

    Science.gov (United States)

    Fernández, Sonsire; Año, Gemma; Castaño, Jorge; Pino, Yadira; Uribarri, Evangelina; Riverón, Luis A; Cedré, Bárbara; Valmaseda, Tania; Falero, Gustavo; Pérez, José L; Infante, Juan F; García, Luis G; Solís, Rosa L; Sierra, Gustavo; Talavera, Arturo

    2013-01-01

    A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine.

  10. Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

    Directory of Open Access Journals (Sweden)

    Felipe Romero-Saavedra

    Full Text Available Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens.We extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72% between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm, and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm. These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens.Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier

  11. 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

    2016-04-07

    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.

  12. Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets.

    Directory of Open Access Journals (Sweden)

    Amorsolo L Suguitan

    2006-09-01

    Full Text Available BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA and a wild-type (wt N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2, were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3 that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.

  13. Conservation analysis of dengue virust-cell epitope-based vaccine candidates using peptide block entropy

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Zhang, Guang Lan; Keskin, Derin B.;

    2011-01-01

    Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches are based on combinations of highly conserved T-cell epitopes. Peptide block entropy analysis is a novel approach for assembling sets...... residues. The block entropy analysis provides broad coverage of variant antigens. We applied the block entropy analysis method to the proteomes of the four serotypes of dengue virus (DENV) and found 1,551 blocks of 9-mer peptides, which cover 99% of available sequences with five or fewer unique peptides...

  14. Early Transcriptional Signatures of the Immune Response to a Live Attenuated Tetravalent Dengue Vaccine Candidate in Non-human Primates.

    Directory of Open Access Journals (Sweden)

    Fiona R Strouts

    2016-05-01

    Full Text Available The development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection.In this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV, suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30.These results suggest that early transcriptional responses may be predictive of development of adaptive immunity to TDV

  15. Early Transcriptional Signatures of the Immune Response to a Live Attenuated Tetravalent Dengue Vaccine Candidate in Non-human Primates

    Science.gov (United States)

    Strouts, Fiona R.; Popper, Stephen J.; Partidos, Charalambos D.; Stinchcomb, Dan T.; Osorio, Jorge E.; Relman, David A.

    2016-01-01

    Background The development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection. Methodology/Principal Findings In this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV), suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN) response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal) vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30. Conclusions/Significance These results suggest that early transcriptional responses may be

  16. Draft Genome Sequence of Lactobacillus reuteri Strain CRL 1098, an Interesting Candidate for Functional Food Development

    Science.gov (United States)

    Torres, Andrea C.; Suárez, Nadia E.; Font, Graciela; Saavedra, Lucila

    2016-01-01

    We report here the draft genome sequence of Lactobacillus reuteri strain CRL 1098. This strain represents an interesting candidate for functional food development because of its proven probiotic properties. The draft genome sequence is composed of 1,969,471 bp assembled into 45 contigs and an average G+C content of 38.8%. PMID:27563038

  17. Analysis of the coverage capacity of the StreptInCor candidate vaccine against Streptococcus pyogenes.

    Science.gov (United States)

    De Amicis, Karine M; Freschi de Barros, Samar; Alencar, Raquel E; Postól, Edilberto; Martins, Carlo de Oliveira; Arcuri, Helen Andrade; Goulart, Cibelly; Kalil, Jorge; Guilherme, Luiza

    2014-07-07

    Streptococcus pyogenes is responsible for infections as pharyngitis, sepsis, necrotizing fasciitis and streptococcal toxic shock syndrome. The M protein is the major bacterial antigen and consists of both polymorphic N-terminal portion and a conserved region. In the present study, we analyzed the in vitro ability of StreptInCor a C-terminal candidate vaccine against S. pyogenes to induce antibodies to neutralize/opsonize the most common S. pyogenes strains in Sao Paulo by examining the recognition by sera from StreptInCor immunized mice. We also evaluated the presence of cross-reactive antibodies against human heart valve tissue. Anti-StreptInCor antibodies were able to neutralize/opsonize at least 5 strains, showing that immunization with StreptInCor is effective against several S. pyogenes strains and can prevent infection and subsequent sequelae without causing autoimmune reactions.

  18. Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine.

    Science.gov (United States)

    Yang, Yanling; Wang, Lin; Yin, Jigang; Wang, Xinglong; Cheng, Shipeng; Lang, Xulong; Wang, Xiuran; Qu, Hailong; Sun, Chunhui; Wang, Jinglong; Zhang, Rui

    2011-10-01

    In order to screen immunogenic candidate antigens for the development of a brucellosis subunit vaccine, an immunoproteomic assay was used to identify immunogenic proteins from Brucella melitensis 16 M soluble proteins. In this study, a total of 56 immunodominant proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two proteins of interest, riboflavin synthase alpha chain (RS-α) and Loraine synthase (LS-2), which are both involved in riboflavin synthesis, were detected by two-dimensional immunoblots using antisera obtained from Brucella-infected human and goats. LS-2, however, is an already well-known vaccine candidate. Therefore, we focussed our studies on the novel vaccine candidate RS-α. B. melitensis RS-α and LS-2 were then expressed in Escherichia coli as fusion proteins with His tag. The humoral and cellular immune responses to the recombinant (r)RS-α was characterized. In response to in vitro stimulation by rRS-α, splenocytes from mice vaccinated with rRS-α were able to produce γ-interferon (IFN-γ) and interleukin (IL)-2 but not interleukin (IL)-4 and interleukin (IL)-10. Furthermore, rRS-α or rLS-2-vaccinated mice were partially protected against B. melitensis infection. Our results suggested that we have developed a high-throughout, accurate, rapid and highly efficient method for the identification of candidate antigens by a combination of immunoproteomics with immunisation and bacterial challenge and rRs-α could be a useful candidate for the development of subunit vaccines against B. melitensis.

  19. Construction and preliminary immunobiological characterization of a novel, non-reverting, intranasal live attenuated whooping cough vaccine candidate.

    Science.gov (United States)

    Cornford-Nairns, Renee; Daggard, Grant; Mukkur, Trilochan

    2012-06-01

    We describe the construction and immunobiological properties of a novel whooping cough vaccine candidate, in which the aroQ gene, encoding 3-dehydroquinase, was deleted by insertional inactivation using the kanamycin resistance gene cassette and allelic exchange using a Bordetella suicide vector. The aroQ B. pertussis mutant required supplementation of media to grow but failed to grow on an unsupplemented medium. The aroQ B. pertussis mutant was undetectable in the trachea and lungs of mice at days 6 and 12 post-infection, respectively. Antigen-specific antibody isotypes IgG1 and IgG2a, were produced, and cell-mediated immunity [CMI], using interleukin-2 and interferon-gamma as indirect indicators, was induced in mice vaccinated with the aroQ B. pertussis vaccine candidate, which were substantially enhanced upon second exposure to virulent B. pertussis. Interleukin- 12 was also produced in the aroQ B. pertussis-vaccinated mice. On the other hand, neither IgG2a nor CMI-indicator cytokines were produced in DTaP-vaccinated mice, although the CMI-indicator cytokines became detectable post-challenge with virulent B. pertussis. Intranasal immunization with one dose of the aroQ B. pertussis mutant protected vaccinated mice against an intranasal challenge infection, with no pathogen being detected in the lungs of immunized mice by day 7 post-challenge. B. pertussis aroQ thus constitutes a safe, non-reverting, metabolite-deficient vaccine candidate that induces both humoral and cellmediated immune responses with potential for use as a single-dose vaccine in adolescents and adults, in the first instance, with a view to disrupting the transmission cycle of whooping cough to infants and the community.

  20. Identification and evaluation of vaccine candidate antigens from the poultry red mite (Dermanyssus gallinae).

    Science.gov (United States)

    Bartley, Kathryn; Wright, Harry W; Huntley, John F; Manson, Erin D T; Inglis, Neil F; McLean, Kevin; Nath, Mintu; Bartley, Yvonne; Nisbet, Alasdair J

    2015-11-01

    An aqueous extract of the haematophagous poultry ectoparasite, Dermanyssus gallinae, was subfractionated using anion exchange chromatography. Six of these subfractions were used to immunise hens and the blood from these hens was fed, in vitro, to poultry red mites. Mite mortality following these feeds was indicative of protective antigens in two of the subfractions, with the risks of mites dying being 3.1 and 3.7 times higher than in the control group (P<0.001). A combination of two-dimensional immunoblotting and immunoaffinity chromatography, using IgY from hens immunised with these subfractions, was used in concert with proteomic analyses to identify the strongest immunogenic proteins in each of these subfractions. Ten of the immunoreactive proteins were selected for assessment as vaccine candidates using the following criteria: intensity of immune recognition; likelihood of exposure of the antigen to the antibodies in a blood meal; proposed function and known vaccine potential of orthologous molecules. Recombinant versions of each of these 10 proteins were produced in Escherichia coli and were used to immunise hens. Subsequent in vitro feeding of mites on blood from these birds indicated that immunisation with Deg-SRP-1 (serpin), Deg-VIT-1 (vitellogenin), Deg-HGP-1 (hemelipoglycoprotein) or Deg-PUF-1 (a protein of unknown function) resulted in significantly increased risk of mite death (1.7-2.8times higher than in mites fed blood from control hens immunised with adjuvant only, P<0.001). The potential for using these antigens in a recombinant vaccine is discussed.

  1. Satisfactory safety and immunogenicity of MSP3 malaria vaccine candidate in Tanzanian children aged 12–24 months

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    Segeja Method D

    2009-07-01

    Full Text Available Abstract Background Development and deployment of an effective malaria vaccine would complement existing malaria control measures. A blood stage malaria vaccine candidate, Merozoite Surface Protein-3 (MSP3, produced as a long synthetic peptide, has been shown to be safe in non-immune and semi-immune adults. A phase Ib dose-escalating study was conducted to assess the vaccine's safety and immunogenicity in children aged 12 to 24 months in Korogwe, Tanzania (ClinicalTrials.gov number: NCT00469651. Methods This was a double-blind, randomized, controlled, dose escalation phase Ib trial, in which children were given one of two different doses of the MSP3 antigen (15 μg or 30 μg or a control vaccine (Engerix B. Children were randomly allocated either to the MSP3 candidate malaria vaccine or the control vaccine administered at a schedule of 0, 1, and 2 months. Immunization with lower and higher doses was staggered for safety reasons starting with the lower dose. The primary endpoint was safety and reactogenicity within 28 days post-vaccination. Blood samples were obtained at different time points to measure immunological responses. Results are presented up to 84 days post-vaccination. Results A total of 45 children were enrolled, 15 in each of the two MSP3 dose groups and 15 in the Engerix B group. There were no important differences in reactogenicity between the two MSP3 groups and Engerix B. Grade 3 adverse events were infrequent; only five were detected throughout the study, all of which were transient and resolved without sequelae. No serious adverse event reported was considered to be related to MSP3 vaccine. Both MSP3 dose regimens elicited strong cytophilic IgG responses (subclasses IgG1 and IgG3, the isotypes involved in the monocyte-dependant mechanism of Plasmodium falciparum parasite-killing. The titers reached are similar to those from African adults having reached a state of premunition. Furthermore, vaccination induced seroconversion in

  2. Efficacy of a recombinant Rift Valley fever virus MP-12 with NSm deletion as a vaccine candidate in sheep.

    Science.gov (United States)

    Weingartl, Hana M; Nfon, Charles K; Zhang, Shunzhen; Marszal, Peter; Wilson, William C; Morrill, John C; Bettinger, George E; Peters, Clarence J

    2014-04-25

    Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family and Phlebovirus genus, causes RVF, a disease of ruminants and man, endemic in Sub-Saharan African countries. However, outbreaks in Yemen and Saudi Arabia demonstrate the ability for RVFV to spread into virgin territory and thus the need exists to develop safe and efficacious vaccines that can be used outside the endemic zones. Commercial RVFV vaccines are available but have limitations that prevent their use in disease-free countries. Consequently, there are ongoing efforts to develop and/or improve RVFV vaccines with global acceptability. In this study a previously developed MP-12-derived vaccine candidate with a large deletion of the NSm gene in the pre Gn region of the M segment (arMP-12-ΔNSm21/384) developed by T. Ikegami, that was already shown to be safe in pregnant sheep causing neither abortion nor fetal malformation was further evaluated. This vaccine was tested for protection of sheep from viremia and fever following challenge with virulent RVFV ZH501 strain. A single vaccination with arMP-12-ΔNSm21/384 fully protected sheep when challenged four weeks post vaccination, thereby demonstrating that this vaccine is efficacious in protecting these animals from RVFV infection.

  3. Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice

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    John P Bannantine

    2014-07-01

    Full Text Available Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP, which results in serious economic losses worldwide in farmed livestock such as cattle, sheep and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne’s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321 and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370 was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.

  4. A New Approach for Designing a Potentially Vaccine Candidate against Urinary Tract Infection by Using Protein Display on Lactobacillus

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    Gholamreza Goudarzi

    2015-10-01

    Full Text Available Background: The prevalence of Urinary Tract Infection (UTI is really high in the world. Escherichia coli is a major agent of UTI. One of the strategies for decreasing UTI infections is vaccine development. As the attachment is a really important stage in colonization and infection, at- tachment inhibition has an applied strategy.  FimH protein is a major factor during bacterial colonization in urinary tract and could be used as a vaccine. Thus, it was considered in this research as a candidate anti- gen.Methods: The sequences of fimH and acmA genes were used for de- signing a synthetic gene. It was cloned to pET23a expression vector and transformed  to E. coli (DE3 Origami.  To confirm the expression  of recombinant  protein,  SDS-PAGE  and western  blotting  methods  were used.  Subsequently,  recombinant  protein  was  purified.  On  the  other hand, Lactobacillus reuteri was cultured and mixed with FimH / AcmA recombinant  protein. The rate of protein localization  on lactobacillus surface was assessed using ELISA method.Results: It was showed that the recombinant protein was expressed inE. coli (DE3 Origami and purified by affinity chromatography. More- over, this protein could be localized on lactobacillus surface by 5 days. Conclusion:  In current study,  a fusion recombinant  protein was pre- pared and displayed on L. reuteri surface. This strain could be used for animal  experiment  as  a  competitor  against  Uropathogenic   E.  coli (UPEC. Using manipulated probiotics strains instead of antibiotic ther- apy could decrease the antibiotic consumption  and reduce multi-drug resistant strains.

  5. Immunoscreening of Plasmodium falciparum proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate

    Science.gov (United States)

    Morita, Masayuki; Takashima, Eizo; Ito, Daisuke; Miura, Kazutoyo; Thongkukiatkul, Amporn; Diouf, Ababacar; Fairhurst, Rick M.; Diakite, Mahamadou; Long, Carole A.; Torii, Motomi; Tsuboi, Takafumi

    2017-01-01

    The number of malaria vaccine candidates in preclinical and clinical development is limited. To identify novel blood-stage malaria vaccine candidates, we constructed a library of 1,827P. falciparum proteins prepared using the wheat germ cell-free system (WGCFS). Also, a high-throughput AlphaScreen procedure was developed to measure antibody reactivity to the recombinant products. Purified IgGs from residents in malaria endemic areas have shown functional activity against blood-stage parasites as judged by an in vitro parasite Growth Inhibition Assay (GIA). Therefore, we evaluated the GIA activity of 51 plasma samples prepared from Malian adults living in a malaria endemic area against the WGCFS library. Using the AlphaScreen-based immunoreactivity measurements, antibody reactivity against 3 proteins was positively associated with GIA activity. Since anti-LSA3-C responses showed the strongest correlation with GIA activity, this protein was investigated further. Anti-LSA3-C-specific antibody purified from Malian adult plasmas showed GIA activity, and expression of LSA3 in blood-stage parasites was confirmed by western blotting. Taken together, we identified LSA3 as a novel blood-stage vaccine candidate, and we propose that this system will be useful for future vaccine candidate discovery. PMID:28378857

  6. Linear synthesis and immunological properties of a fully synthetic vaccine candidate containing a sialylated MUC1 glycopeptide

    NARCIS (Netherlands)

    Thompson, Pamela; Lakshminarayanan, Vani; Supekar, Nitin T.; Bradley, Judy M.; Cohen, Peter A.; Wolfert, Margreet A.; Gendler, Sandra J.; Boons, Geert Jan

    2015-01-01

    A strategy for the linear synthesis of a sialylated glycolipopeptide cancer vaccine candidate has been developed using a strategically designed sialyl-Tn building block and microwave-assisted solid-phase peptide synthesis. The glycolipopeptide elicited potent humoral and cellular immune responses. T

  7. Naturally acquired antibody responses to recombinant Pfs230 and Pfs48/45 transmission blocking vaccine candidates

    DEFF Research Database (Denmark)

    Jones, Sophie; Grignard, Lynn; Nebie, Issa;

    2015-01-01

    OBJECTIVES: Pfs48/45 and Pfs230 are Plasmodium falciparum sexual stage proteins and promising malaria transmission-blocking vaccine candidates. Antibody responses against these proteins may be naturally acquired and target antigens may be under selective pressure. This has consequences for the fu...

  8. Antigenic community between Schistosoma mansoni and Biomphalaria glabrata: on the search of candidate antigens for vaccines

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    N Chacón

    2002-10-01

    Full Text Available We have previously confirmed the presence of common antigens between Schistosoma mansoni and its vector, Biomphalaria glabrata. Cross-reactive antigens may be important as possible candidates for vaccine and diagnosis of schistosomiasis. Sera from outbred mice immunized with a soluble Biomphalaria glabrata antigen (SBgA of non-infected B. glabrata snails recognized molecules of SBgA itself and S. mansoni AWA by Western blot. Recognition of several molecules of the SBgA were inhibited by pre-incubation with AWA (16, 30, 36, 60 and 155 kDa. The only specific molecule of AWA, inhibited by SBgA, was a 120 kDa protein. In order to determine which epitopes of SBgA were glycoproteins, the antigen was treated with sodium metaperiodate and compared with non-treated antigen. Molecules of 140, 60 and 24 kDa in the SBgA appear to be glycoproteins. Possible protective effects of the SBgA were evaluated immunizing outbred mice in two different experiments using Freund's Adjuvant. In the first one (12 mice/group, we obtained a significant level of protection (46% in the total worm load, with a high variability in worm recovery. In the second experiment (22 mice/group, no significant protection was observed, neither in worm load nor in egg production per female. Our results suggest that SBgA constitutes a rich source of candidate antigens for diagnosis and prophylactic studies.

  9. African Green Monkeys Recapitulate the Clinical Experience with Replication of Live Attenuated Pandemic Influenza Virus Vaccine Candidates

    Science.gov (United States)

    Matsuoka, Yumiko; Suguitan, Amorsolo; Orandle, Marlene; Paskel, Myeisha; Boonnak, Kobporn; Gardner, Donald J.; Feldmann, Friederike; Feldmann, Heinz; Marino, Michael; Jin, Hong; Kemble, George

    2014-01-01

    ABSTRACT Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. IMPORTANCE Ferrets and mice are commonly used for preclinical evaluation of influenza

  10. Development of a human live attenuated West Nile infectious DNA vaccine: conceptual design of the vaccine candidate.

    Science.gov (United States)

    Yamshchikov, Vladimir

    2015-10-01

    West Nile virus has become an important epidemiological problem attracting significant attention of health authorities, mass media, and the public. Although there are promising advancements toward addressing the vaccine need, the perspectives of the commercial availability of the vaccine remain uncertain. To a large extent this is due to lack of a sustained interest for further commercial development of the vaccines already undergoing the preclinical and clinical development, and a predicted insignificant cost effectiveness of mass vaccination. There is a need for a safe, efficacious and cost effective vaccine, which can improve the feasibility of a targeted vaccination program. In the present report, we summarize the background, the rationale, and the choice of the development pathway that we selected for the design of a live attenuated human West Nile vaccine in a novel infectious DNA format.

  11. Immunological characterization of recombinantWuchereria bancrofti cuticular collagen (COL-4) as putative vaccine candidate for human lymphatic filariasis

    Institute of Scientific and Technical Information of China (English)

    Chakkaravarthy Arunkumar; Pandurangan Pandiaraja; P. R. Prince; Perumal Kaliraj

    2014-01-01

    Objective:To elucidate immunoprophylactic potential of recombinantWuchereria bancrofti(W. bancrofti) cuticular collagen(COL-4) inBALB/c mice and filarial clinical samples.Methods:col-4 gene wasPCR amplified fromW. bancroftiL3 cDNA library and cloned in pRSETB vector. RecombinantCOL-4 was over expressed in salt inducible system and was purified by nickel affinity chromatography.Humoral and cellular responses were measured byELISA and peripheral blood mononuclear cells(PBMC) of various filarial clinical samples respectively using purified recombinantCOL-4 antigen.Then the protective immune responses ofCOL-4 immunizedBALB/c mice were characterized.Results:Sequence analysis ofCOL-4 with human host proteins reveals lack of homology.The recombinantCOL-4 was found to be at15 kDa fusion protein.The affinity purifiedCOL-4 showed significant reactivity with putatively immune sera and in a similar fashion it demonstrated marked proliferation inPBMC samples.Immunization studies in experimental filarial host(mice) elicited significant titers with protective antibody isotype profile(IgM and IgG).Cellular immune responses were also significant in terms of splenocytes proliferation assay on mice samples.Conclusions:Our immunological findings in experimental host suggestTh2 mediated immune response.Hence, we propose thatW. bancroftiCOL-4 could be an efficacious vaccine candidate against lymphatic filariasis.

  12. Protection of mice against Staphylococcus aureus infection by a recombinant protein ClfA-IsdB-Hlg as a vaccine candidate.

    Science.gov (United States)

    Delfani, Somayeh; Mohabati Mobarez, Ashraf; Imani Fooladi, Abbas Ali; Amani, Jafar; Emaneini, Mohammad

    2016-02-01

    Staphylococcus aureus is one of the most important causes of nosocomial infections. An effective vaccine to prevent S. aureus infections is urgently required due to the dramatic increase in the number of antibiotic-resistant strains. In this report, we evaluated a newly recombinant protein composed of selected antigenic regions of clumping factor A (ClfA), iron surface determinant B (IsdB) and gamma hemolysin B (HlgB) of S. aureus and sequence coding for hydrophobic linkers between three domains. The recombinant gene was constructed in pET-28a (+) and expressed in Escherichia coli BL21. In addition, sequence coding for a His(6)-tag was added followed by a hybrid procedure of nickel chelate protein purification. Immunization of BALB/c mice with the recombinant protein ClfA-IsdB-Hlg evoked antigen-specific antibodies that could opsonize S. aureus cells, enhancing in vitro phagocytosis by macrophages. Vaccination with the recombinant protein also reduced the bacterial load recovered from mice spleen samples and increased survival following the intraperitoneal challenge with pathogenic S. aureus compared to the control mice. Our results showed that the recombinant protein ClfA-IsdB-Hlg is a promising vaccine candidate for the prevention of S. aureus bacteremia infections.

  13. Production of H5N1 influenza virus matrix protein 2 ectodomain protein bodies in tobacco plants and in insect cells as a candidate universal influenza vaccine

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    Sandiswa Mbewana

    2015-12-01

    Full Text Available The spread of influenza A viruses is partially controlled and prevented by vaccination. The matrix protein 2 ectodomain (M2e is the most conserved sequence in influenza A viruses, and is therefore a good potential target for a vaccine to protect against multiple virus subtypes. We explored the feasibility of a M2e-based universal influenza A vaccine candidate based on the highly pathogenic avian influenza A virus, H5N1. A synthetic M2e gene was human and plant codon optimised and fused in-frame with a sequence encoding the N-terminal proline-rich domain (Zera® of the γ-zein protein of maize. Zera®M2e was expressed transiently in Nicotiana benthamiana and Sf21 baculovirus / insect cell expression systems, and Zera®M2e protein bodies (PBs were successfully produced in both expression systems. The plant-produced Zera®M2e PBs were purified and injected into Balb/c mice. Western blot analysis using insect cell-produced Zera®M2e PBs and multiple tandem M2e sequences (5xM2e fused with the avian influenza H5N1 transmembrane and cytosolic tail (5xM2e_tHA confirmed the presence of M2e-specific antibodies in immunised mice sera. The immunogenicity of the Zera®M2e indicates that our plant-produced protein has potential as an inexpensive universal influenza A vaccine.

  14. Neisseria meningitidis antigen NMB0088: sequence variability, protein topology and vaccine potential.

    Science.gov (United States)

    Sardiñas, Gretel; Yero, Daniel; Climent, Yanet; Caballero, Evelin; Cobas, Karem; Niebla, Olivia

    2009-02-01

    The significance of Neisseria meningitidis serogroup B membrane proteins as vaccine candidates is continually growing. Here, we studied different aspects of antigen NMB0088, a protein that is abundant in outer-membrane vesicle preparations and is thought to be a surface protein. The gene encoding protein NMB0088 was sequenced in a panel of 34 different meningococcal strains with clinical and epidemiological relevance. After this analysis, four variants of NMB0088 were identified; the variability was confined to three specific segments, designated VR1, VR2 and VR3. Secondary structure predictions, refined with alignment analysis and homology modelling using FadL of Escherichia coli, revealed that almost all the variable regions were located in extracellular loop domains. In addition, the NMB0088 antigen was expressed in E. coli and a procedure for obtaining purified recombinant NMB0088 is described. The humoral immune response elicited in BALB/c mice was measured by ELISA and Western blotting, while the functional activity of these antibodies was determined in a serum bactericidal assay and an animal protection model. After immunization in mice, the recombinant protein was capable of inducing a protective response when it was administered inserted into liposomes. According to our results, the recombinant NMB0088 protein may represent a novel antigen for a vaccine against meningococcal disease. However, results from the variability study should be considered for designing a cross-protective formulation in future studies.

  15. A cell wall protein-based vaccine candidate induce protective immune response against Sporothrix schenckii infection.

    Science.gov (United States)

    Portuondo, Deivys Leandro; Batista-Duharte, Alexander; Ferreira, Lucas Souza; Martínez, Damiana Téllez; Polesi, Marisa Campos; Duarte, Roberta Aparecida; de Paula E Silva, Ana Carolina Alves; Marcos, Caroline Maria; Almeida, Ana Marisa Fusco de; Carlos, Iracilda Zeppone

    2016-02-01

    Sporotrichosis is a subcutaneous mycosis caused by several closely related thermo-dimorphic fungi of the Sporothrix schenckii species complex, affecting humans and other mammals. In the last few years, new strategies have been proposed for controlling sporotrichosis owning to concerns about its growing incidence in humans, cats, and dogs in Brazil, as well as the toxicity and limited efficacy of conventional antifungal drugs. In this study, we assessed the immunogenicity and protective properties of two aluminum hydroxide (AH)-adsorbed S. schenckii cell wall protein (ssCWP)-based vaccine formulations in a mouse model of systemic S. schenckii infection. Fractioning by SDS-PAGE revealed nine protein bands, two of which were functionally characterized: a 44kDa peptide hydrolase and a 47kDa enolase, which was predicted to be an adhesin. Sera from immunized mice recognized the 47kDa enolase and another unidentified 71kDa protein, whereas serum from S. schenckii-infected mice recognized both these proteins plus another unidentified 9.4kDa protein. Furthermore, opsonization with the anti-ssCWP sera led to markedly increased phagocytosis and was able to strongly inhibit the fungus' adhesion to fibroblasts. Immunization with the higher-dose AH-adjuvanted formulation led to increased ex vivo release of IL-12, IFN-γ, IL-4, and IL-17, whereas only IL-12 and IFN-γ were induced by the higher-dose non-adjuvanted formulation. Lastly, passive transference of the higher-dose AH-adjuvanted formulation's anti-ssCWP serum was able to afford in vivo protection in a subsequent challenge with S. schenckii, becoming a viable vaccine candidate for further testing.

  16. Development of live attenuated Bordetella pertussis strains expressing the universal influenza vaccine candidate M2e.

    Science.gov (United States)

    Li, Rui; Lim, Annabelle; Ow, Stephanie T L; Phoon, Meng Chee; Locht, Camille; Chow, Vincent T; Alonso, Sylvie

    2011-07-26

    The attenuated Bordetella pertussis BPZE1 vaccine strain represents an attractive platform for the delivery of heterologous vaccine candidates via the nasal route. The filamentous hemagglutinin (FHA) has been used to secrete or expose the foreign antigens at the bacterial surface. In this study, one, two and three copies of the Cys-containing ectodomain of matrix protein 2 (M2e) from influenza A virus were genetically fused to full length FHA and expressed in BPZE1. The secretion efficacy of the FHA-(M2e)(1,2,3) chimera in the extracellular milieu and the ability of the recombinant bacteria to colonize the mouse lungs inversely correlated with the number of M2e copies fused to FHA. Nevertheless FHA-(M2e)(3)-producing bacteria (BPLR3) triggered the highest systemic anti-M2e antibody response upon nasal administration to BALB/c mice. Nasal immunization with BPLR3 bacteria resulted in a significant reduction in the viral loads upon challenge with H1N1/PR8 influenza A virus, but did not improve the survival rate compared to BPZE1-immunized mice. Furthermore, since previous work reported that disulfide bond formation in Cys-containing passenger antigens affects the secretion efficacy of the FHA chimera, the dsbA gene encoding a periplasmic disulfide isomerase was deleted in the FHA-(M2e)(3)-producing strain. Despite improving significantly the secretion efficacy of the FHA-(M2e)(3) chimera, the dsbA deletion did not result in higher anti-M2e antibody titers in mice, due to impaired bacterial fitness and colonization ability.

  17. Exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in Vibrio cholerae targeted by Piper betel derived compounds.

    Directory of Open Access Journals (Sweden)

    Debmalya Barh

    Full Text Available Vibrio cholerae is the causal organism of the cholera epidemic, which is mostly prevalent in developing and underdeveloped countries. However, incidences of cholera in developed countries are also alarming. Because of the emergence of new drug-resistant strains, even though several generic drugs and vaccines have been developed over time, Vibrio infections remain a global health problem that appeals for the development of novel drugs and vaccines against the pathogen. Here, applying comparative proteomic and reverse vaccinology approaches to the exoproteome and secretome of the pathogen, we have identified three candidate targets (ompU, uppP and yajC for most of the pathogenic Vibrio strains. Two targets (uppP and yajC are novel to Vibrio, and two targets (uppP and ompU can be used to develop both drugs and vaccines (dual targets against broad spectrum Vibrio serotypes. Using our novel computational approach, we have identified three peptide vaccine candidates that have high potential to induce both B- and T-cell-mediated immune responses from our identified two dual targets. These two targets were modeled and subjected to virtual screening against natural compounds derived from Piper betel. Seven compounds were identified first time from Piper betel to be highly effective to render the function of these targets to identify them as emerging potential drugs against Vibrio. Our preliminary validation suggests that these identified peptide vaccines and betel compounds are highly effective against Vibrio cholerae. Currently we are exhaustively validating these targets, candidate peptide vaccines, and betel derived lead compounds against a number of Vibrio species.

  18. Immunogenicity of a polyvalent HIV-1 candidate vaccine based on fourteen wild type gp120 proteins in golden hamsters

    Directory of Open Access Journals (Sweden)

    Ghorbani Masoud

    2006-10-01

    Full Text Available Abstract Background One of the major obstacles in the design of an effective vaccine against HIV-1 is the hypervariability of the HIV-1 envelope glycoprotein. Most HIV-1 vaccine candidates have utilized envelope glycoprotein from a single virus isolate, but to date, none of them elicited broadly reactive humoral immunity. Herein, we hypothesised that a cocktail of HIV-1 gp120 proteins containing multiple epitopes may increase the breadth of immune responses against HIV-1. We compared and evaluated the immunogenicity of HIV-1 vaccines containing either gp120 protein alone or in combinations of four or fourteen gp120s from different primary HIV-1 isolates in immunized hamsters. Results We amplified and characterized 14 different gp120s from primary subtype B isolates with both syncytium and non-syncytium inducing properties, and expressed the proteins in Chinese Hamster Ovary (CHO cell lines. Purified proteins were used either alone or in combinations of four or fourteen different gp120s to vaccinate golden hamsters. The polyvalent vaccine showed higher antibody titers to HIV-1 subtype B isolates MN and SF162 compared to the groups that received one or four gp120 proteins. However, the polyvalent vaccine was not able to show higher neutralizing antibody responses against HIV-1 primary isolates. Interestingly, the polyvalent vaccine group had the highest proliferative immune responses and showed a substantial proportion of cross-subtype CD4 reactivity to HIV-1 subtypes B, C, and A/E Conclusion Although the polyvalent approach achieved only a modest increase in the breadth of humoral and cellular immunity, the qualitative change in the vaccine (14 vs. 1 gp120 resulted in a quantitative improvement in vaccine-induced immunity.

  19. Comparison of the safety and efficacy of a new live Salmonella Gallinarum vaccine candidate, JOL916, with the SG9R vaccine in chickens.

    Science.gov (United States)

    Matsuda, Kiku; Chaudhari, Atul A; Lee, John Hwa

    2011-09-01

    We evaluated a recently developed live vaccine candidate for fowl typhoid (FT)-JOL916, a lon/cpxR mutant of Salmonella Gallinarum (SG)-by comparing its safety and efficacy with that of the well-known rough mutant strain SG9R vaccine in 6-wk-old Hy-Line hens. Forty-five chickens were divided into three groups of 15 chickens each. The chickens were then intramuscularly inoculated with 2 x 10(7) colony-forming units (CFUs) of JOL916 (JOL916 group), 2 x 10(7) CFUs of SG9R (SG9R group), or phosphate-buffered saline (control group). After vaccination, no clinical symptoms were observed in any of the groups. No differences in body weight increase were detected among the three groups postvaccination. A cellular immune response was observed at 2 wk postvaccination (wpv) in the JOL916 group with the peripheral lymphocyte proliferation assay, whereas no response was detected in the SG9R group. Elevation of SG antigen-specific plasma immunoglobulin was observed 2 and 3 wpv in the JOL916 and SG9R vaccine groups, respectively. After virulent challenge on day 25 postvaccination, 0, 1, and 15 chickens in the JOL916 group, SG9R group, and control group, respectively, died by 12 days postchallenge; the death rate of the SG9R vaccine group was statistically similar to that of the JOL916 group. Postmortem examination revealed that the JOL916 vaccine offered more efficient protection than the SG9R vaccine, with significantly decreased hepatic necrotic foci scores, splenic enlargement scores, necrotic foci scores, and recovery of the challenge strain from the spleen. Vaccination with JOL916 appears to be safe and offers better protection than SG9R against FT in chickens.

  20. Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA

    DEFF Research Database (Denmark)

    Avril, Marion; Kulasekara, Bridget R; Gose, Severin O;

    2008-01-01

    Da) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three......Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 k...

  1. Identification by genomic immunization of a pool of DNA vaccine candidates that confer protective immunity in mice against Neisseria meningitidis serogroup B.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Pérez, Yusleydis; Fariñas, Mildrey; Cobas, Karem; Diaz, Daiyana; Solis, Rosa L; Acosta, Armando; Brookes, Charlotte; Taylor, Stephen; Gorringe, Andrew

    2007-07-09

    We have shown previously that expression library immunization is viable alternative approach to induce protective immunity against Neisseria meningitidis serogroup B. In this study we report that few rounds of library screening allow identification of protective pools of defined antigens. A previously reported protective meningococcal library (L8, with 600 clones) was screened and two sub-libraries of 95 clones each were selected based on the induction of bactericidal and protective antibodies in BALB/c mice. After sequence analysis of each clone within these sub-libraries, we identified a pool of 20 individual antigens that induced protective immune responses in mice against N. meningitidis infection, and the observed protection was associated with the induction of bactericidal antibodies. Our studies demonstrate for the first time that ELI combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in a meningococcal vaccine.

  2. Novel Plasmodium falciparum malaria vaccines: evidence-based searching for variant surface antigens as candidates for vaccination against pregnancy-associated malaria

    DEFF Research Database (Denmark)

    Staalsoe, Trine; Jensen, Anja T R; Theander, Thor G;

    2002-01-01

    to statistically significant co-variation with protection rather than on demonstration of causal relationships. We have studied the relationship between variant surface antigen-specific antibodies and clinical protection from Plasmodium falciparum malaria in general, and from pregnancy-associated malaria (PAM......) in particular, to provide robust evidence of a causal link between the two in order to allow efficient and evidence-based identification of candidate antigens for malaria vaccine development....

  3. Functional Exposed Amino Acids of OSPA as a Candidate for Lyme disease Vaccine

    Directory of Open Access Journals (Sweden)

    Negin Kafee

    2016-06-01

    Full Text Available Lyme disease is caused by the bacterium Borreliaburgdorferi and is transmitted to humans through the bite of infected black legged ticks. Typical symptoms include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. If left untreated, infection can spread to joints, the heart, and the nervous system. Lyme disease is diagnosed based on symptoms, physical findings (e.g., rash, and the possibility of exposure to infected ticks. The protective role of antibodies to B. burgdorferi has been explored in animal models of Lyme disease. Findings indicated that antibodies against OspA were protective, making this antigen a likely candidate for a vaccine. Early clinical trials demonstrated that recombinant OspA was immunogenic and well tolerated, even in subjects with a history of Lyme disease. The present study was designed to in silico resolving the major obstacles in the control or in prevention of lyme diseases. We exploited bioinformatic tools to better understanding and characterizing the OspAstructure and select appropriate regions as effectiveB cell epitops.

  4. Puf mediates translation repression of transmission-blocking vaccine candidates in malaria parasites.

    Directory of Open Access Journals (Sweden)

    Jun Miao

    Full Text Available Translational control of gene expression plays an essential role in development. In malaria parasites, translational regulation is critical during the development of specialized transition stages between the vertebrate host and mosquito vector. Here we show that a Pumilio/FBF (Puf family RNA-binding protein, PfPuf2, is required for the translation repression of a number of transcripts in gametocytes including two genes encoding the transmission-blocking vaccine candidates Pfs25 and Pfs28. Whereas studies to date support a paradigm of Puf-mediated translation regulation through 3' untranslated regions (UTRs of target mRNAs, this study, for the first time, identifies a functional Puf-binding element (PBE in the 5'UTR of pfs25. We provide both in vitro and in vivo evidence to demonstrate that PfPuf2 binds to the PBEs in pfs25 and pfs28 to mediate translation repression. This finding provides a renewed view of Pufs as versatile translation regulators and sheds light on their functions in the development of lower branches of eukaryotes.

  5. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine.

    Science.gov (United States)

    Nawaz-Tipu, Hamid

    2015-10-01

    The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1) protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA) class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine's candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Three alleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens more prevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of 50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

  6. Targeted Sequencing Reveals Large-Scale Sequence Polymorphism in Maize Candidate Genes for Biomass Production and Composition.

    Directory of Open Access Journals (Sweden)

    Moses M Muraya

    Full Text Available A major goal of maize genomic research is to identify sequence polymorphisms responsible for phenotypic variation in traits of economic importance. Large-scale detection of sequence variation is critical for linking genes, or genomic regions, to phenotypes. However, due to its size and complexity, it remains expensive to generate whole genome sequences of sufficient coverage for divergent maize lines, even with access to next generation sequencing (NGS technology. Because methods involving reduction of genome complexity, such as genotyping-by-sequencing (GBS, assess only a limited fraction of sequence variation, targeted sequencing of selected genomic loci offers an attractive alternative. We therefore designed a sequence capture assay to target 29 Mb genomic regions and surveyed a total of 4,648 genes possibly affecting biomass production in 21 diverse inbred maize lines (7 flints, 14 dents. Captured and enriched genomic DNA was sequenced using the 454 NGS platform to 19.6-fold average depth coverage, and a broad evaluation of read alignment and variant calling methods was performed to select optimal procedures for variant discovery. Sequence alignment with the B73 reference and de novo assembly identified 383,145 putative single nucleotide polymorphisms (SNPs, of which 42,685 were non-synonymous alterations and 7,139 caused frameshifts. Presence/absence variation (PAV of genes was also detected. We found that substantial sequence variation exists among genomic regions targeted in this study, which was particularly evident within coding regions. This diversification has the potential to broaden functional diversity and generate phenotypic variation that may lead to new adaptations and the modification of important agronomic traits. Further, annotated SNPs identified here will serve as useful genetic tools and as candidates in searches for phenotype-altering DNA variation. In summary, we demonstrated that sequencing of captured DNA is a powerful

  7. Live Attenuated Human Salmonella Vaccine Candidates: tracking the pathogen in natural infection and stimulation of host immunity

    Science.gov (United States)

    Galen, James E.; Buskirk, Amanda D.; Tennant, Sharon M.; Pasetti, Marcela F.

    2016-01-01

    Salmonellosis, caused by members of the genus Salmonella, is responsible for considerable global morbidity and mortality, in both animals and humans. In this review, we will discuss the pathogenesis of S. Typhi and S. Typhimurium, focusing on human Salmonella infections. We will trace the path of Salmonella through the body, including host entry sites, tissues and organs affected, and mechanisms involved in both pathogenesis and stimulation of host immunity. Careful consideration of the natural progression of disease provides an important context in which attenuated live oral vaccines can be rationally designed and developed. With this in mind, we will describe a series of attenuated live oral vaccines that have been successfully tested in clinical trials and demonstrated to be both safe and highly immunogenic. The attenuation strategies summarized in this review offer important insights into further development of attenuated vaccines against other Salmonella for which live oral candidates are currently unavailable. PMID:27809955

  8. Live Attenuated Human Salmonella Vaccine Candidates: Tracking the Pathogen in Natural Infection and Stimulation of Host Immunity.

    Science.gov (United States)

    Galen, James E; Buskirk, Amanda D; Tennant, Sharon M; Pasetti, Marcela F

    2016-11-01

    Salmonellosis, caused by members of the genus Salmonella, is responsible for considerable global morbidity and mortality in both animals and humans. In this review, we will discuss the pathogenesis of Salmonella enterica serovar Typhi and Salmonella enterica serovar Typhimurium, focusing on human Salmonella infections. We will trace the path of Salmonella through the body, including host entry sites, tissues and organs affected, and mechanisms involved in both pathogenesis and stimulation of host immunity. Careful consideration of the natural progression of disease provides an important context in which attenuated live oral vaccines can be rationally designed and developed. With this in mind, we will describe a series of attenuated live oral vaccines that have been successfully tested in clinical trials and demonstrated to be both safe and highly immunogenic. The attenuation strategies summarized in this review offer important insights into further development of attenuated vaccines against other Salmonella for which live oral candidates are currently unavailable.

  9. A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Amy R Noe

    Full Text Available The circumsporozoite protein (CSP of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA and immunofluorescence assay (IFA, as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime

  10. Recombinant vesicular stomatitis virus-based dengue-2 vaccine candidate induces humoral response and protects mice against lethal infection.

    Science.gov (United States)

    Lauretti, Flavio; Chattopadhyay, Anasuya; de Oliveira França, Rafael Freitas; Castro-Jorge, Luiza; Rose, John; Fonseca, Benedito A L da

    2016-09-01

    Dengue is the most important arbovirus disease throughout the world and it is responsible for more than 500,000 dengue hemorrhagic cases and 22,000 deaths every year. One vaccine was recently licensed for human use in Brazil, Mexico and Philippines and although at least seven candidates have been in clinical trials the results of the most developed CYD vaccine have demonstrated immunization problems, such as uneven protection and interference between serotypes. We constructed a vaccine candidate based on vesicular stomatitis virus (VSV) expression of pre-membrane (prM) and envelope (E) proteins of dengue-2 virus (DENV-2) and tested it in mice to evaluate immunogenicity and protection against DENV-2 infection. VSV has been successfully used as vaccine vectors for several viruses to induce strong humoral and cellular immune responses. The VSV-DENV-2 recombinant was constructed by inserting the DENV-2 structural proteins into a VSV plasmid DNA for recombinant VSV-DENV-2 recovery. Infectious recombinant VSV viruses were plaque purified and prM and E expression were confirmed by immunofluorescence and radiolabeling of proteins of infected cells. Forty Balb/C mice were inoculated through subcutaneous (s.c.) route with VSV-DENV-2 vaccine in a two doses schedule 15 d apart and 29 d after first inoculation, sera were collected and the mice were challenged with 50 lethal doses (LD50) of a neurovirulent DENV-2. The VSV-DENV-2 induced anti-DENV-2 antibodies and protected animals in the challenge experiment comparable to DENV-2 immunization control group. We conclude that VSV is a promising platform to test as a DENV vaccine and perhaps against others Flaviviridae.

  11. Generation and preclinical evaluation of a DENV-1/2 prM+E chimeric live attenuated vaccine candidate with enhanced prM cleavage.

    Science.gov (United States)

    Keelapang, Poonsook; Nitatpattana, Narong; Suphatrakul, Amporn; Punyahathaikul, Surat; Sriburi, Rungtawan; Pulmanausahakul, Rojjanaporn; Pichyangkul, Sathit; Malasit, Prida; Yoksan, Sutee; Sittisombut, Nopporn

    2013-10-17

    In the absence of a vaccine or sustainable vector control measures, illnesses caused by dengue virus infection remain an important public health problem in many tropical countries. During the export of dengue virus particles, furin-mediated cleavage of the prM envelope protein is usually incomplete, thus generating a mixture of immature, partially mature and mature extracellular particles. Variations in the arrangement and conformation of the envelope proteins among these particles may be associated with their different roles in shaping the antibody response. In an attempt to improve upon live, attenuated dengue vaccine approaches, a mutant chimeric virus, with enhanced prM cleavage, was generated by introducing a cleavage-enhancing substitution into a chimeric DENV-1/2 virus genome, encoding the prM+E sequence of a recent DENV-1 isolate under an attenuated DENV-2 genetic background. A modest increase in virus specific infectivity observed in the mutant chimeric virus affected neither the attenuation phenotype, when assessed in the suckling mouse neurovirulence model, nor multiplication in mosquitoes. The two chimeric viruses induced similar levels of anti-DENV-1 neutralizing antibody response in mice and rhesus macaques, but more efficient control of viremia during viral challenge was observed in macaques immunized with the mutant chimeric virus. These results indicate that the DENV-1/2 chimeric virus, with enhanced prM cleavage, could be useful as an alternative live, attenuated vaccine candidate for further tests in humans.

  12. First assessment of classical swine fever marker vaccine candidate CP7_E2alf for oral immunization of wild boar under field conditions.

    Science.gov (United States)

    Feliziani, Francesco; Blome, Sandra; Petrini, Stefano; Giammarioli, Monica; Iscaro, Carmen; Severi, Giulio; Convito, Luca; Pietschmann, Jana; Beer, Martin; De Mia, Gian Mario

    2014-04-11

    Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7_E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7_E2alf bait vaccines in two areas called 'faunistic-hunting farms' in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3-35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future.

  13. Evaluation of Salmonella enterica serovar Enteritidis pathogenicity island-1 proteins as vaccine candidates against S. Enteritidis challenge in chickens.

    Science.gov (United States)

    Desin, Taseen S; Wisner, Amanda L S; Lam, Po-King S; Berberov, Emil; Mickael, Claudia S; Potter, Andrew A; Köster, Wolfgang

    2011-03-24

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of gastrointestinal disease in humans worldwide, which mainly results from the consumption of contaminated poultry meat and eggs. Vaccination of chickens is an important strategy to lower the prevalence of Salmonella in poultry flocks. The S. Enteritidis type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island-1 (SPI-1) is an important virulence factor that plays a role in invasion and systemic spread in chickens. In this manuscript, we evaluated the efficacy of SPI-1 proteins as vaccine candidates for protection against S. Enteritidis oral challenge. Our results demonstrate for the first time that SPI-1 T3SS proteins elicit antigen specific IgG antibody responses in chickens. In one study we show that vaccination with the aforementioned proteins reduces the levels of S. Enteritidis in the liver, but not in the spleen and cecal contents of chickens. However, a second study shows that vaccination of hens with SPI-1 proteins using a seeder model of infection does not affect the levels of S. Enteritidis in the cecal contents or internal organs of progeny obtained from these hens. Hence, the SPI-1 proteins, in conjunction with other proteins, may form important components of subunit vaccines used for protection against colonization by S. Enteritidis in poultry.

  14. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

    Directory of Open Access Journals (Sweden)

    Ho-Hsien Lee

    2014-09-01

    Full Text Available CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB and the membrane-proximal region of gp41 (MPR, the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1, and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

  15. Immunogenicity and in vitro Protective Efficacy of a Recombinant Multistage Plasmodium falciparum Candidate Vaccine

    Science.gov (United States)

    Shi, Ya Ping; Hasnain, Seyed E.; Sacci, John B.; Holloway, Brian P.; Fujioka, Hisashi; Kumar, Nirbhay; Wohlhueter, Robert; Hoffman, Stephen L.; Collins, William E.; Lal, Altaf A.

    1999-02-01

    Compared with a single-stage antigen-based vaccine, a multistage and multivalent Plasmodium falciparum vaccine would be more efficacious by inducing "multiple layers" of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specific P. falciparum antigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages of P. falciparum. In vitro assays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponent P. falciparum vaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.

  16. Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: an uptake and processing study in the midgut of Penaeus monodon

    NARCIS (Netherlands)

    Kulkarni, V.; Rombout, J.H.W.M.; Singh, I.S.B.; Sudheer, N.S.; Vlak, J.M.; Caipang, C.M.A.; Brinchmann, M.; Kiron, V.

    2013-01-01

    Several oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral deliver

  17. Vaccination with a Streptococcus pneumoniae trivalent recombinant PcpA, PhtD and PlyD1 protein vaccine candidate protects against lethal pneumonia in an infant murine model.

    Science.gov (United States)

    Verhoeven, David; Xu, Qingfu; Pichichero, Michael E

    2014-05-30

    Streptococcus pneumoniae infections continue to cause significant worldwide morbidity and mortality despite the availability of efficacious serotype-dependent vaccines. The need to incorporate emergent strains expressing additional serotypes into pneumococcal polysaccharide conjugate vaccines has led to an identified need for a pneumococcal protein-based vaccine effective against a broad scope of serotypes. A vaccine consisting of several conserved proteins with different functions during pathogenesis would be preferred. Here, we investigated the efficacy of a trivalent recombinant protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad D (PhtD), and genetically detoxified pneumolysin (PlyD1) in an infant mouse model. We found the trivalent vaccine conferred protection from lethal pneumonia challenges using serotypes 6A and 3. The observed protection with trivalent PcpA, PhtD, and PlyD1 vaccine in infant mice supports the ongoing study of this candidate vaccine in human infant clinical trials.

  18. Molecular characterisation and nucleotide sequence analysis of canine parvovirus strains in vaccines in India

    Directory of Open Access Journals (Sweden)

    Sukdeb Nandi

    2010-03-01

    Full Text Available Canine parvovirus 2 (CPV‑2 is one of the most important viruses that causes haemorrhagic gastroenteritis and myocarditis of dogs worldwide. The picture has been complicated further due to the emergence of new mutants of CPV, namely: CPV‑2a, CPV‑2b and CPV‑2c. In this study, the molecular characterisation of strains present in the CPV vaccines available on the Indian market was performed using polymerase chain reaction and DNA sequencing. The VP1/VP2 genes of two vaccine strains and a field strain (Bhopal were sequenced and the nucleotide and the deduced amino acid sequences were compared. The results indicated that the isolate belonged to CPV type 2b and the strains in the vaccines belonged to type CPV‑2. From the study, it is inferred that the CPV strain used in commercially available vaccine preparation differed from the strains present in CPV infection in dogs in India

  19. Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency.

    Science.gov (United States)

    Farrag, Mohamed A; Amer, Haitham M; Öhlschläger, Peter; Hamad, Maaweya E; Almajhdi, Fahad N

    2017-03-08

    The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8(+) T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

  20. Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

    Directory of Open Access Journals (Sweden)

    Liudmila A Stepanova

    Full Text Available Matrix 2 protein ectodomain (M2e is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek. Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1 and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1 and A/Chicken/Kurgan/05/05 RG (H5N1 to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2 and avian influenza virus (H5N1. Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.

  1. Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

    Directory of Open Access Journals (Sweden)

    Yan Mylene L

    2011-08-01

    Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

  2. Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen.

    Science.gov (United States)

    Arumugam, Thangavelu U; Takeo, Satoru; Yamasaki, Tsutomu; Thonkukiatkul, Amporn; Miura, Kazutoyo; Otsuki, Hitoshi; Zhou, Hong; Long, Carole A; Sattabongkot, Jetsumon; Thompson, Jennifer; Wilson, Danny W; Beeson, James G; Healer, Julie; Crabb, Brendan S; Cowman, Alan F; Torii, Motomi; Tsuboi, Takafumi

    2011-11-01

    One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

  3. Sequencing and characterization of Varicella-Zoster virus vaccine strain SuduVax

    Directory of Open Access Journals (Sweden)

    Kim Jong

    2011-12-01

    Full Text Available Abstract Background Varicella-zoster virus (VZV causes chickenpox in children and shingles in older people. Currently, live attenuated vaccines based on the Oka strain are available worldwide. In Korea, an attenuated VZV vaccine has been developed from a Korean isolate and has been commercially available since 1994. Despite this long history of use, the mechanism for the attenuation of the vaccine strain is still elusive. We attempted to understand the molecular basis of attenuation mechanism by full genome sequencing and comparative genomic analyses of the Korean vaccine strain SuduVax. Results SuduVax was found to contain a genome that was 124,759 bp and possessed 74 open reading frames (ORFs. SuduVax was genetically most close to Oka strains and these Korean-Japanese strains formed a strong clade in phylogenetic trees. SuduVax, similar to the Oka vaccine strains, underwent T- > C substitution at the stop codon of ORF0, resulting in a read-through mutation to code for an extended form of ORF0 protein. SuduVax also shared certain deletion and insertion mutations in ORFs 17, 29, 56 and 60 with Oka vaccine strains and some clinical strains. Conclusions The Korean VZV vaccine strain SuduVax is genetically similar to the Oka vaccine strains. Further comparative genomic and bioinformatics analyses will help to elucidate the molecular basis of the attenuation of the VZV vaccine strains.

  4. Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates.

    Science.gov (United States)

    Ortega-Villaizan, M; Martinez-Lopez, A; Garcia-Valtanen, P; Chico, V; Perez, L; Coll, J M; Estepa, A

    2012-09-07

    DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.

  5. CFP10: mFcγ2 as a novel tuberculosis vaccine candidate increases immune response in mouse

    Science.gov (United States)

    Baghani, Ali Asghar; Soleimanpour, Saman; Farsiani, Hadi; Mosavat, Arman; Yousefi, Masoud; Meshkat, Zahra; Rezaee, Seyed Abdolrahim; Jamehdar, Saeid Amel; Eydgahi, Mohammad Reza Akbari; Sadeghian, Hamid; Ghazvini, Kiarash

    2017-01-01

    Objective(s): Despite treatment with antibiotics and vaccination with BCG, tuberculosis (TB) is still considered as one of the most important public health problems in the world. Therefore, designing and producing a more effective vaccine against TB seems urgently. In this study, immunogenicity of a fusion protein which consisting or comprising CFP-10 from Mycobacterium tuberculosis and the Fc-domain of mouse IgG2a was evaluated as a novel subunit vaccine candidate against TB. Materials and Methods: The genetic constructs were cloned in pPICZαA expression vector and recombinant vectors (pPICZαA-CFP-10: Fcγ2a and pPICZαA-CFP-10:His) were transformed into Pichia pastoris. To evaluate the expression of recombinant proteins, SDS-PAGE and immunoblotting were used. The immunogenicity of recombinant proteins, with and without BCG were assessed in BALB/c mice and specific cytokines against recombinant proteins (IFN-γ, IL-12, IL-4, IL-17 and TGF-β) were evaluated. Results: The levels of IFN-γ and IL-12 in mice that received recombinant proteins was higher than the control groups (BCG and PBS). Thus, both recombinant proteins (CFP-10:Fcγ2a and CFP-10:His) could excite good response in Th1-cells. The Fc-tagged protein had a stronger Th1 response with low levels of IL-4, as compared to CFP-10:His. However, the highest level of Th1 response was observed in groups that were vaccinated with BCG (prime) and then received recombinant protein CFP-10: Fcγ2a (booster). Conclusion: The results demonstrated that binding mice Fc-domain to CFP-10 protein can increase the immunogenicity of the subunit vaccine. Further studies, might be able to design and produce a new generation of subunit vaccines based on the Fc-fused immunogen. PMID:28293387

  6. Comparison of serological and sequence-based methods for typing feline calcivirus isolates from vaccine failures.

    Science.gov (United States)

    Radford, A D; Dawson, S; Wharmby, C; Ryvar, R; Gaskell, R M

    2000-01-29

    Feline calicivirus (FCV) can be typed by exploiting antigenic differences between isolates or, more recently, by the sequence analysis of a hypervariable region of the virus's capsid gene. These two methods were used to characterise FCV isolates from 20 vaccine failures which occurred after the use of a commercial, live-attenuated vaccine. Using virus neutralisation, the isolates showed a spectrum of relatedness to the vaccine; depending on the criterion adopted for identity, 10 to 40 per cent of them appeared to be similar to the vaccine virus. Using sequence analysis, the isolates fell into one of two categories; 20 per cent had a similar sequence to the vaccine (0-67 to 2-67 per cent distant), and the remainder had a dissimilar sequence (21-3 to 36-0 per cent distant). Sequence analysis identified one cat that appeared to be infected with two distinct FCVs. The serological and sequence-based typing methods gave the same result in 80 to 95 per cent of individual cases, depending on the criterion adopted for serological identity. It is suggested that molecular typing is a more definitive method for characterising the relatedness of FCV isolates.

  7. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine

    Directory of Open Access Journals (Sweden)

    Hamid Nawaz-Tipu

    2015-10-01

    Full Text Available The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1 protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine’s candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Threealleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens moreprevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

  8. Malaria: deploying a candidate vaccine (RTS,S/AS02A) for an old scourge of humankind.

    Science.gov (United States)

    Alonso, Pedro L

    2006-06-01

    Malaria is an infectious disease caused by the protist Plasmodium spp. and it currently kills more than one million people annually. The burden of malaria is concentrated in sub-Saharan Africa, India, and Southeast Asia. The parasite's resistance to commonly used anti-malarial drugs has worsened the situation in the poorest countries. The World Health Organization (WHO) estimates that more than 100 countries suffer from endemic malaria episodes. In addition to numerous control measures and treatments, several vaccines are at different research stages and trials. We have assayed RTS,S/AS02A, a pre-erythrocytic candidate vaccine that has shown promising protection levels in phase IIb trials in Mozambique. The vaccine is directed against the sporozoite form of the parasite, which is injected by the mosquito Anopheles spp. The vaccine induces a strong antibody response and stimulates Th1 cells-a subset of helper T cells that participates in cell-mediated immunity. Recent interest by international funding agencies has provided new inputs into initiatives and programs to fight malaria, which, under normal welfare and adequate social development conditions, is a curable disease.

  9. Generation of Recombinant Equine Influenza Vaccine Candidate RgH3N1 Virus by Reverse Genetics

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yun; LIU Ming; YU Kang-zhen; Webster Robert

    2005-01-01

    The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate 7 + 1 reassortants that contain seven genes from a high-yield virus A/Puerto Rico/8/34[A/PR/8/34](H1N1) and the HA gene from the circulating strains. By using this DNA-based cotransfection technique, we generated 7 + 1 reassortants rgH3N1 which had the antigenic determinants of influenza virus A/Songbird/HongKong/102/00[SB/HK/01](H3N8) and 7 other genes from A/PR/8/34. The hemagglutinin of A/Songbird/HongKong/102/00 is 96.3% homologous to that of A/Equine/Jilin/98[Eq/J1/89] (H3N8). The resulting virus rgH3N1 grows to high HA titers in chicken embryonated eggs, allowing vaccine preparation in unconcentrated allantoic fluid. The rgH3N1 is stable after multiple passages in embryonated eggs. The reassortant rgH3N1 virus could be used as vaccine candidate to reduce the reemergence of equine influenza outbreaks.

  10. 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...

  11. Safety and immunogenicity of the malaria candidate vaccines FP9 CS and MVA CS in adult Gambian men.

    Science.gov (United States)

    Imoukhuede, E B; Berthoud, T; Milligan, P; Bojang, K; Ismaili, J; Keating, S; Nwakanma, D; Keita, S; Njie, F; Sowe, M; Todryk, S; Laidlaw, S M; Skinner, M A; Lang, T; Gilbert, S; Greenwood, B M; Hill, A V S

    2006-10-30

    We assessed the safety and immunogenicity of prime-boost vectors encoding the Plasmodium falciparum circumsporozoite (CS) protein expressed either in the attenuated fowl-pox virus (FP9) or modified vaccinia virus Ankara (MVA). Thirty-two adult Gambians in groups of four to eight received one, two or three doses of FP9 CS and/or MVA CS. No serious adverse event was observed following vaccination. The most immunogenic regimen was two doses of FP9 followed by a single dose of MVA 4 weeks later (an average of 1000 IFN-gamma spot forming units/million PBMCs). This level of effector T-cell responses appears higher than that seen in previously reported studies of CS-based candidate malaria vaccines.

  12. Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens.

    Directory of Open Access Journals (Sweden)

    Hyesun Jang

    Full Text Available Influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1-truncated mutants are attractive candidates for avian live attenuated influenza vaccine (LAIV development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens.

  13. Development of influenza A(H7N9) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs

    Science.gov (United States)

    Ridenour, Callie; Johnson, Adam; Winne, Emily; Hossain, Jaber; Mateu-Petit, Guaniri; Balish, Amanda; Santana, Wanda; Kim, Taejoong; Davis, Charles; Cox, Nancy J; Barr, John R; Donis, Ruben O; Villanueva, Julie; Williams, Tracie L; Chen, Li-Mei

    2015-01-01

    Background The emergence of avian influenza A(H7N9) virus in poultry causing zoonotic human infections was reported on March 31, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was initiated without delay. Candidate vaccine viruses were derived by reverse genetics using the internal genes of A/Puerto/Rico/8/34 (PR8). The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation. Methods Two CVVs derived by reverse genetics were serially passaged in embryonated eggs to improve the hemagglutinin (HA) antigen yield. The total viral protein and HA antigen yields of six egg-passaged CVVs were determined by the BCA assay and isotope dilution mass spectrometry (IDMS) analysis, respectively. CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera. Results Improvement of total viral protein yield was observed for the six egg-passaged CVVs; HA quantification by IDMS indicated approximately a twofold increase in yield of several egg-passaged viruses as compared to that of the parental CVV. Several different amino acid substitutions were identified in the HA of all viruses after serial passage. However, HI tests indicated that the antigenic properties of two CVVs remained unchanged. Conclusions If influenza A(H7N9) viruses were to acquire sustained human-to-human transmissibility, the improved HA yield of the egg-passaged CVVs generated in this study could expedite vaccine manufacturing for pandemic mitigation. PMID:25962412

  14. Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants.

    Science.gov (United States)

    Voepel, Nadja; Boes, Alexander; Edgue, Güven; Beiss, Veronique; Kapelski, Stephanie; Reimann, Andreas; Schillberg, Stefan; Pradel, Gabriele; Fendel, Rolf; Scheuermayer, Matthias; Spiegel, Holger; Fischer, Rainer

    2014-11-01

    Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80°C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP_TSR, PfTRAP_TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails.

  15. Toxoplasma gondii Elongation Factor 1-Alpha (TgEF-1α) Is a Novel Vaccine Candidate Antigen against Toxoplasmosis

    Science.gov (United States)

    Wang, Shuai; Zhang, Zhenchao; Wang, Yujian; Gadahi, Javaid A.; Xu, Lixin; Yan, Ruofeng; Song, Xiaokai; Li, Xiangrui

    2017-01-01

    Toxoplasma gondii (T. gondii) is an obligate intracellular parasite which can infect almost all warm-blood animals, leading to toxoplasmosis. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, the recombinant T. gondii elongation factor 1-alpha (rTgEF-1α) was successfully expressed in in Escherichia coli. Passive immunization of mice with anti-rTgEF-1α polyclonal antibody following challenge with a lethal dose of tachyzoites significantly increased the survival time compared with PBS control group. The survival time of mice challenged with tachyzoites pretreated with anti-rTgEF-1α PcAb also was significantly increased. Invasion of tachyzoites into mouse macrophages was significantly inhibited in the anti-rTgEF-1α PcAb pretreated group. Mice vaccinated with rTgEF-1α induced a high level of specific anti-T. gondii antibodies and production of IFN-gamma, interleukin-4. The expression levels of MHC-I and MHC-II molecules as well as the percentages of CD4+ and CD8+ T cells in mice vaccinated with rTgEF-1α was significantly increased, respectively (P < 0.05), compared with all the controls. Immunization with rTgEF-1α significantly (P < 0.05) prolonged survival time (14.53 ± 1.72 days) after challenge infection with the virulent T. gondii RH strain. These results indicate that T. gondii EF-1α plays an essential role in mediating host cell invasion by the parasite and, as such, could be a candidate vaccine antigen against toxoplasmosis.

  16. Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss

    DEFF Research Database (Denmark)

    Marana, Moonika Haahr; Jørgensen, Louise von Gersdorff; Skov, Jakob;

    2017-01-01

    Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish...... rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain...... A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i...

  17. Immunostimulation by Synthetic Lipopeptide-Based Vaccine Candidates: Structure-Activity Relationships

    OpenAIRE

    Zaman, Mehfuz; Toth, Istvan

    2013-01-01

    Peptide-based vaccines offer several advantages over conventional whole organism or protein approaches by offering improved purity and specificity in inducing immune response. However, peptides alone are generally non-immunogenic. Concerns remain about the toxicity of adjuvants which are critical for immunogenicity of synthetic peptides. The use of lipopeptides in peptide vaccines is currently under intensive investigation because potent immune responses can be generated without the use of ad...

  18. Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

    Directory of Open Access Journals (Sweden)

    Fresno Manuel

    2011-07-01

    Full Text Available Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II, to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus, infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

  19. Genome sequencing and analysis of BCG vaccine strains.

    Directory of Open Access Journals (Sweden)

    Wen Zhang

    Full Text Available BACKGROUND: Although the Bacillus Calmette-Guérin (BCG vaccine against tuberculosis (TB has been available for more than 75 years, one third of the world's population is still infected with Mycobacterium tuberculosis and approximately 2 million people die of TB every year. To reduce this immense TB burden, a clearer understanding of the functional genes underlying the action of BCG and the development of new vaccines are urgently needed. METHODS AND FINDINGS: Comparative genomic analysis of 19 M. tuberculosis complex strains showed that BCG strains underwent repeated human manipulation, had higher region of deletion rates than those of natural M. tuberculosis strains, and lost several essential components such as T-cell epitopes. A total of 188 BCG strain T-cell epitopes were lost to various degrees. The non-virulent BCG Tokyo strain, which has the largest number of T-cell epitopes (359, lost 124. Here we propose that BCG strain protection variability results from different epitopes. This study is the first to present BCG as a model organism for genetics research. BCG strains have a very well-documented history and now detailed genome information. Genome comparison revealed the selection process of BCG strains under human manipulation (1908-1966. CONCLUSIONS: Our results revealed the cause of BCG vaccine strain protection variability at the genome level and supported the hypothesis that the restoration of lost BCG Tokyo epitopes is a useful future vaccine development strategy. Furthermore, these detailed BCG vaccine genome investigation results will be useful in microbial genetics, microbial engineering and other research fields.

  20. Medical sequencing of candidate genes for nonsyndromic cleft lip and palate.

    Directory of Open Access Journals (Sweden)

    Alexandre R Vieira

    2005-12-01

    Full Text Available Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father. The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Etude du Polymorphisme Humain (CEPH diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate. Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.

  1. Medical Sequencing of Candidate Genes for Nonsyndromic Cleft Lip and Palate.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father. The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Etude du Polymorphisme Humain (CEPH diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate. Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.

  2. Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51.

    Directory of Open Access Journals (Sweden)

    Yimin Wu

    Full Text Available BACKGROUND: Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion. METHODOLOGY/PRINCIPAL FINDINGS: The trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005-April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 microg of Pfs25/ISA 51, 5 microg of Pvs25/ISA 51, or 20 microg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51. Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity. CONCLUSION/SIGNIFICANCE: It is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum. TRIAL REGISTRATION: ClinicalTrials.gov NCT00295581.

  3. In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

    Science.gov (United States)

    Rodrigues-da-Silva, Rodrigo Nunes; Martins da Silva, João Hermínio; Singh, Balwan; Jiang, Jianlin; Meyer, Esmeralda V. S.; Santos, Fátima; Banic, Dalma Maria; Moreno, Alberto; Galinski, Mary R.; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa

    2016-01-01

    Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and

  4. Comparison of a live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit with a commercial vaccine for efficacy of protection against internal egg contamination by Salmonella in hens.

    Science.gov (United States)

    Nandre, Rahul M; Eo, Seong Kug; Park, Sang Youel; Lee, John Hwa

    2015-07-01

    This study compared a new live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit (SE-LTB) with a commercial Salmonella Enteritidis (SE) vaccine for efficacy of protection against SE infection in laying hens. Chickens were divided into 3 groups of 20 each. Group A chickens were inoculated orally with phosphate-buffered saline and served as controls, group B chickens were inoculated orally with the vaccine candidate, and group C chickens were inoculated intramuscularly with a commercial vaccine, the primary inoculation in groups B and C being at 10 wk of age and the booster at 16 wk. Groups B and C showed significantly higher titers of plasma immunoglobulin G, intestinal secretory immunoglobulin A, and egg yolk immunoglobulin Y antibodies compared with the control group, and both vaccinated groups showed a significantly elevated cellular immune response. After virulent challenge, group B had significantly lower production of thin-shelled and/or malformed eggs and a significantly lower rate of SE contamination of eggs compared with the control group. Furthermore, the challenge strain was detected significantly less in all of the examined organs of group B compared with the control group. Group C had lower gross lesion scores only in the spleen and had lower bacterial counts only in the spleen, ceca, and ovary. These findings indicate that vaccination with the SE-LTB vaccine candidate can efficiently reduce internal egg and internal organ contamination by Salmonella and has advantages over the commercial vaccine.

  5. The candidate TB vaccine, MVA85A, induces highly durable Th1 responses.

    Directory of Open Access Journals (Sweden)

    Michele Tameris

    Full Text Available BACKGROUND: Vaccination against tuberculosis (TB should provide long-term protective immunity against Mycobacterium tuberculosis (M.tb. The current TB vaccine, Bacille Calmette-Guerin (BCG, protects against disseminated childhood TB, but protection against lung TB in adolescents and adults is variable and mostly poor. One potential reason for the limited durability of protection may be waning of immunity through gradual attrition of BCG-induced T cells. We determined if a MVA85A viral-vector boost could enhance the durability of mycobacteria-specific T cell responses above those induced by BCG alone. METHODS: We describe a long-term follow-up study of persons previously vaccinated with MVA85A. We performed a medical history and clinical examination, a tuberculin skin test and measured vaccine-specific T cell responses in persons previously enrolled as adults, adolescents, children or infants into three different Phase II trials, between 2005 and 2011. RESULTS: Of 252 potential participants, 183 (72.6% consented and completed the study visit. Vaccine-induced Ag85A-specific CD4+ T cell responses were remarkably persistent in healthy, HIV-uninfected adults, adolescents, children and infants, up to 6 years after MVA85A vaccination. Specific CD4+ T cells expressed surface markers consistent with either CD45RA-CCR7+ central memory or CD45RA-CCR7- effector memory T cells. Similarly durable Ag85A-specific CD4+ T cell responses were detected in HIV-infected persons who were on successful antiretroviral therapy when MVA85A was administered. By contrast, Ag85A-specific CD4+ T cell frequencies in untreated MVA85A-vaccinated HIV-infected persons were mostly undetectable 3-5 years after vaccination. CONCLUSION: MVA85A induces remarkably durable T cell responses in immunocompetent persons. However, results from a recent phase IIb trial of MVA85A, conducted in infants from the same geographic area and study population, showed no vaccine efficacy, suggesting

  6. Complete Genome Sequence of the Bacterium Aalborg_AAW-1, Representing a Novel Family within the Candidate Phylum SR1

    DEFF Research Database (Denmark)

    Dueholm, Morten Simonsen; Albertsen, Mads; Stokholm-Bjerregaard, Mikkel;

    2015-01-01

    Here, we present the complete genome sequence of the candidate phylum SR1 bacterium Aalborg_AAW-1. Its 16S rRNA gene is only 85.5% similar to that of the closest relative, RAAC1_SR1, and the genome of Aalborg_AAW-1 consequently represents the first of a novel family within the candidate phylum SR1....

  7. Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss

    Science.gov (United States)

    Jørgensen, Louise von Gersdorff; Skov, Jakob; Chettri, Jiwan Kumar; Holm Mattsson, Andreas; Dalsgaard, Inger; Kania, Per Walter; Buchmann, Kurt

    2017-01-01

    Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17–30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival. PMID:28182704

  8. Immunostimulation by synthetic lipopeptide based vaccine candidates: structure-activity relationships.

    Directory of Open Access Journals (Sweden)

    Mehfuz eZaman

    2013-10-01

    Full Text Available Peptide based vaccines offer several advantages over conventional whole organism or protein approaches by offering improved purity and specificity in inducing immune response. However, peptides alone are generally non-immunogenic. Concerns remain about the toxicity of adjuvants which are critical for immunogenicity of synthetic peptides. The use of lipopeptides in peptide vaccines is currently under intensive investigation because potent immune responses can be generated without the use of adjuvant (thus are self-adjuvanting. Several lipopeptides derived from microbial origin, and their synthetic versions or simpler fatty acid moieties impart this self-adjuvanting activity by signalling via Toll-like receptor 2 (TLR2. Engagement of this innate immune receptor on antigen-presenting cell leads to the initiation and development of potent immune responses. Therefore optimization of lipopeptides to enhance TLR2-mediated activation is a promising strategy for vaccine development. Considerable structure-activity relationships that determine TLR2 binding and consequent stimulation of innate immune responses have been investigated for a range of lipopeptides. In this review we address the development of lipopeptide vaccines, mechanism of TLR2 recognition, and immune activation. An overview is provided of the best studied lipopeptide vaccine systems.

  9. An alphavirus replicon-derived candidate vaccine against Rift Valley fever virus.

    Science.gov (United States)

    Heise, M T; Whitmore, A; Thompson, J; Parsons, M; Grobbelaar, A A; Kemp, A; Paweska, J T; Madric, K; White, L J; Swanepoel, R; Burt, F J

    2009-09-01

    Rift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus (genus Phlebovirus) associated with severe disease in livestock and fatal encephalitis or haemorrhagic fever in a proportion of infected humans. Although live attenuated and inactivated vaccines have been used in livestock, and on a limited scale in humans, there is a need for improved anti-RVFV vaccines. Towards this goal, Sindbis virus replicon vectors expressing the RVFV Gn and Gc glycoproteins, as well as the non-structural nsM protein, were constructed and evaluated for their ability to induce protective immune responses against RVFV. These replicon vectors were shown to produce the RVFV glycoproteins to high levels in vitro and to induce systemic anti-RVFV antibody responses in immunized mice, as determined by RVFV-specific ELISA, fluorescent antibody tests, and demonstration of a neutralizing antibody response. Replicon vaccination also provided 100% protection against lethal RVFV challenge by either the intraperitoneal or intranasal route. Furthermore, preliminary results indicate that the replicon vectors elicit RVFV-specific neutralizing antibody responses in vaccinated sheep. These results suggest that alphavirus-based replicon vectors can induce protective immunity against RVFV, and that this approach merits further investigation into its potential utility as a RVFV vaccine.

  10. Identification of novel and candidate miRNAs in rice by high throughput sequencing

    Directory of Open Access Journals (Sweden)

    Zhang Weixiong

    2008-02-01

    Full Text Available Abstract Background Small RNA-guided gene silencing at the transcriptional and post-transcriptional levels has emerged as an important mode of gene regulation in plants and animals. Thus far, conventional sequencing of small RNA libraries from rice led to the identification of most of the conserved miRNAs. Deep sequencing of small RNA libraries is an effective approach to uncover rare and lineage- and/or species-specific microRNAs (miRNAs in any organism. Results In order to identify new miRNAs and possibly abiotic-stress regulated small RNAs in rice, three small RNA libraries were constructed from control rice seedlings and seedlings exposed to drought or salt stress, and then subjected to pyrosequencing. A total of 58,781, 43,003 and 80,990 unique genome-matching small RNAs were obtained from the control, drought and salt stress libraries, respectively. Sequence analysis confirmed the expression of most of the conserved miRNAs in rice. Importantly, 23 new miRNAs mostly each derived from a unique locus in rice genome were identified. Six of the new miRNAs are conserved in other monocots. Additionally, we identified 40 candidate miRNAs. Allowing not more than 3 mis-matches between a miRNA and its target mRNA, we predicted 20 targets for 9 of the new miRNAs. Conclusion Deep sequencing proved to be an effective strategy that allowed the discovery of 23 low-abundance new miRNAs and 40 candidate miRNAs in rice.

  11. Phase 1 safety and immunogenicity evaluation of ADVAX, a multigenic, DNA-based clade C/B' HIV-1 candidate vaccine.

    Directory of Open Access Journals (Sweden)

    Sandhya Vasan

    Full Text Available BACKGROUND: We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADVAX or placebo was administered intramuscularly at months 0, 1 and 3 to 45 healthy volunteers not at high risk for HIV-1. Three dosage levels [0.2 mg (low, 1.0 mg (mid, and 4.0 mg (high] were tested. Twelve volunteers in each dosage group were assigned to receive ADVAX and three to receive placebo in a double-blind design. Subjects were followed for local and systemic reactogenicity, adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. ADVAX was safe and well-tolerated, with no vaccine-related serious adverse events. Local and systemic reactogenicity events were reported by 64% and 42% of vaccine recipients, respectively. The majority of events were mild. The IFNgamma ELISpot response rates to any HIV antigen were 0/9 (0% in the placebo group, 3/12 (25% in the low-dosage group, 4/12 (33% in the mid-dosage group, and 2/12 (17% in the high-dosage group. Overall, responses were generally transient and occurred to each gene product, although volunteers responded to single antigens only. Binding antibodies to gp120 were not detected in any volunteers, and HIV seroconversion did not occur. CONCLUSIONS/SIGNIFICANCE: ADVAX delivered intramuscularly is safe, well-tolerated, and elicits modest but transient cellular immune responses. TRIAL REGISTRATION: Clinicaltrials.gov NCT00249106.

  12. Rising Cellular Immune Response after Injection of pVax/iutA: A Genetic DNA Cassette as Candidate Vaccine against Urinary Tract Infection

    Directory of Open Access Journals (Sweden)

    Ronak BAKHTIARI

    2016-08-01

    Full Text Available Background: Uropathogenic Escherichia coli (UPEC are major bacterial agent of Urinary Tract Infection (UTI. This infection is more prevalent among women because approximately half of all women will experience a UTI in their life-time and near a quarter of them will have a recurrent infection within 6–12 months. IutA protein has a major role during UPEC pathogenesis and consequently infection. Therefore, the aim of current study was assessment of IutA protein roles as a potential candidate antigen based for vaccine designing.Methods: This survey was conducted during 2014-2015 at the University of Tehran, Iran. Chromosomal DNA extracted from E. coli 35218 and iutA gene amplified by PCR. The amplicon cloned to pVax.1 eukaryotic expression vector and recombinant vector confirmed by sequencing. The iutA gene expression in genetic cassette of pVax/iutA was evaluated in COS7 cell line by RT-PCR. Then, injected to mouse model, which divided to three groups: injected with pVax vector, PBS and pVax/iutA cassette respectively in two stages (d 1 and 14. One week after the second injection, bleeding from immunized mouse was performed and IFN-gamma was measured.Results: The mice immunized with pVax/iutA showed increased interferon-γ responses significantly higher than two non-immunized groups (P<0.05.Conclusion: Cellular immune response has a main protective role against UTI. Raising this kind of immune response is important to preventing of recurrent infection. Moreover, the current DNA cassette will be valuable for more trying to prepare a new vaccine against UTI. Keywords: Genetic vaccination, Uropathogenic escherichia coli, IutA

  13. High level expression, purification and characterization of recombinant CCR5 as a vaccine candidate against HIV.

    Science.gov (United States)

    Wu, Kongtian; Xue, Xiaochang; Li, Meng; Qin, Xin; Zhang, Cun; Li, Weina; Hao, Qiang; Wang, Zenglu; Liu, Qian; Zhang, Wei; Zhang, Yingqi

    2013-06-01

    Cysteine-cysteine chemokine receptor type 5 (CCR5) is an important co-receptor for human immunodeficiency virus (HIV) infection and CCR5 neutralizing agents have proven efficient in patients suffering from HIV infection. Here, we expressed and purified various CCR5 vaccines named rCCR5, PADRE-rCCR5, GST-C1 and GST-C2 composed of different epitopes of CCR5. Results showed that vaccines containing multiple epitopes (rCCR5 and PADRE-rCCR5) induced stronger immune responses than single-epitope ones (GST-C1 and GST-C2). In addition, the elicited antibodies can specifically bind CCR5(+) U937 but not CCR5(-) Wish cells. These results demonstrate that the CCR5 vaccines are useful for further research, especially for the in vitro preclinical evaluation of their potential as biological CCR5 neutralizing agents.

  14. Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

    Directory of Open Access Journals (Sweden)

    Gilles Riveau

    1998-01-01

    Full Text Available For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

  15. A novel sequence-based antigenic distance measure for H1N1, with application to vaccine effectiveness and the selection of vaccine strains

    Science.gov (United States)

    Pan, Keyao; Subieta, Krystina C.; Deem, Michael W.

    2011-01-01

    H1N1 influenza causes substantial seasonal illness and was the subtype of the 2009 influenza pandemic. Precise measures of antigenic distance between the vaccine and circulating virus strains help researchers design influenza vaccines with high vaccine effectiveness. We here introduce a sequence-based method to predict vaccine effectiveness in humans. Historical epidemiological data show that this sequence-based method is as predictive of vaccine effectiveness as hemagglutination inhibition assay data from ferret animal model studies. Interestingly, the expected vaccine effectiveness is greater against H1N1 than H3N2, suggesting a stronger immune response against H1N1 than H3N2. The evolution rate of hemagglutinin in H1N1 is also shown to be greater than that in H3N2, presumably due to greater immune selection pressure. PMID:21123189

  16. Evaluation of Borrelia burgdorferi BbHtrA Protease as a Vaccine Candidate for Lyme Borreliosis in Mice.

    Directory of Open Access Journals (Sweden)

    Amy J Ullmann

    Full Text Available Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA(S226A. Although inoculation with either BbHtrA or BbHtrA(S226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.

  17. Comparative evaluation of two vaccine candidates against experimental leishmaniasis due to Leishmania major infection in four inbred mouse strains.

    Science.gov (United States)

    Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

    2009-11-01

    Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

  18. Comparative Evaluation of Two Vaccine Candidates against Experimental Leishmaniasis Due to Leishmania major Infection in Four Inbred Mouse Strains▿

    Science.gov (United States)

    Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

    2009-01-01

    Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials. PMID:19726616

  19. Stellar Parameters of Main Sequence Turn-off Star Candidates Observed with the LAMOST and Kepler

    CERN Document Server

    Wu, Yaqian; Zhang, Xianfei; Li, Tanda; Bi, Shaolan; Liu, Xiaowei; Fu, Jianning; Huang, Yang; Tian, Zhijia; Liu, Kang; Ge, Zhishuai; He, Xin; Zhang, Jinghua

    2016-01-01

    Main sequence turn-off (MSTO) stars have advantages as indicators of Galactic evolution since their ages could be robustly estimated from atmospheric parameters. Hundreds of thousands of MSTO stars have been selected from the LAMOST Galactic sur- vey to study the evolution of the Galaxy, and it is vital to derive accurate stellar parameters. In this work, we select 150 MSTO star candidates from the MSTO stars sample of Xiang that have asteroseismic parameters and determine accurate stellar parameters for these stars combing the asteroseismic parameters deduced from the Kepler photometry and atmospheric parameters deduced from the LAMOST spectra.With this sample, we examine the age deter- mination as well as the contamination rate of the MSTO stars sample. A comparison of age between this work and Xiang shows a mean difference of 0.53 Gyr (7%) and a dispersion of 2.71 Gyr (28%). The results show that 79 of the candidates are MSTO stars, while the others are contaminations from either main sequence or sub-giant...

  20. Phase I randomised clinical trial of an HIV-1(CN54, clade C, trimeric envelope vaccine candidate delivered vaginally.

    Directory of Open Access Journals (Sweden)

    David J Lewis

    Full Text Available UNLABELLED: We conducted a phase 1 double-blind randomised controlled trial (RCT of a HIV-1 envelope protein (CN54 gp140 candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18-45 years were entered into the trial, the first receiving open-label active product. Subsequently, 16 women were randomised to receive 9 doses of 100 µg of gp140 in 3 ml of a Carbopol 974P based gel, 5 were randomised to placebo solution in the same gel, delivered vaginally via an applicator. Participants delivered the vaccine three times a week over three weeks during one menstrual cycle, and were followed up for two further months. There were no serious adverse events, and the vaccine was well tolerated. No sustained systemic or local IgG, IgA, or T cell responses to the gp140 were detected following vaginal immunisations. Repeated vaginal immunisation with a HIV-1 envelope protein alone formulated in Carbopol gel was safe, but did not induce local or systemic immune responses in healthy women. TRIAL REGISTRATION: ClinicalTrials.gov NCT00637962.

  1. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

    Directory of Open Access Journals (Sweden)

    Jessica B Hostetler

    2015-12-01

    Full Text Available A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC, and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins

  2. Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate

    Science.gov (United States)

    2016-01-01

    Purpose Salmonella enterica serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid. Materials and Methods The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization. Results Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4+ and CD8+ T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4+ and CD8+ T-cell population and IL-6 cytokine in mRNA levels. Conclusion Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses. PMID:27489805

  3. Genome-Wide Analysis of Evolutionary Markers of Human Influenza A(H1N1)pdm09 and A(H3N2) Viruses May Guide Selection of Vaccine Strain Candidates.

    Science.gov (United States)

    Belanov, Sergei S; Bychkov, Dmitrii; Benner, Christian; Ripatti, Samuli; Ojala, Teija; Kankainen, Matti; Kai Lee, Hong; Wei-Tze Tang, Julian; Kainov, Denis E

    2015-11-27

    Here we analyzed whole-genome sequences of 3,969 influenza A(H1N1)pdm09 and 4,774 A(H3N2) strains that circulated during 2009-2015 in the world. The analysis revealed changes at 481 and 533 amino acid sites in proteins of influenza A(H1N1)pdm09 and A(H3N2) strains, respectively. Many of these changes were introduced as a result of random drift. However, there were 61 and 68 changes that were present in relatively large number of A(H1N1)pdm09 and A(H3N2) strains, respectively, that circulated during relatively long time. We named these amino acid substitutions evolutionary markers, as they seemed to contain valuable information regarding the viral evolution. Interestingly, influenza A(H1N1)pdm09 and A(H3N2) viruses acquired non-overlapping sets of evolutionary markers. We next analyzed these characteristic markers in vaccine strains recommended by the World Health Organization for the past five years. Our analysis revealed that vaccine strains carried only few evolutionary markers at antigenic sites of viral hemagglutinin (HA) and neuraminidase (NA). The absence of these markers at antigenic sites could affect the recognition of HA and NA by human antibodies generated in response to vaccinations. This could, in part, explain moderate efficacy of influenza vaccines during 2009-2014. Finally, we identified influenza A(H1N1)pdm09 and A(H3N2) strains, which contain all the evolutionary markers of influenza A strains circulated in 2015, and which could be used as vaccine candidates for the 2015/2016 season. Thus, genome-wide analysis of evolutionary markers of influenza A(H1N1)pdm09 and A(H3N2) viruses may guide selection of vaccine strain candidates.

  4. Assessment of vaccine candidates for persons aged 50 and older : a review

    NARCIS (Netherlands)

    Eilers, Renske; Krabbe, Paul F. M.; van Essen, Ted G. A.; Suijkerbuijk, Anita; van Lier, Alies; de Melker, Hester E.

    2013-01-01

    Background: The increasing life expectancy in most European countries has resulted in growth of the population 50 and older. This population is more susceptible to infectious diseases because of immunosenescence, comorbidity and general frailty. Thus, to promote healthy aging, vaccination against va

  5. Targeted modifications of foot-and-mouth disease virus; towards improved vaccine candidates

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette

    these into susceptible cells it is possible to rescue specifically altered FMDVs. We have used this approach to generate modified viruses that have particular properties; these studies can assist in the development of improved and safer vaccines to protect against FMDV. For example, we have made changes to the leader (L...

  6. Anti-cattle tick vaccines: Many candidate antigens, but will a commercially viable product emerge?

    Science.gov (United States)

    This is an invited paper from the editor-in-chief of International Journal for Parasitology who requested a Current Opinion manuscript to discuss the status of anti-cattle tick vaccine research. Arguably the world's most significant arthropod pest of cattle, control of the cattle tick, Rhipicephalus...

  7. Bo-lysin: A Potential Candidate as a biomarker of Protection after Vaccination against Tuberculosis

    Science.gov (United States)

    Tuberculosis (TB) is still a major health problem worldwide. A Th1 type response with release of IFN {gamma}https://webmail.utmb.edu/math/gamma.gif and cytotoxic granules such as granulysin and perforin, play a major role in the disease. Measurements of protection after TB vaccination include IFN {...

  8. A randomized controlled phase Ib trial of the malaria vaccine candidate GMZ2 in African children.

    Directory of Open Access Journals (Sweden)

    Sabine Bélard

    Full Text Available BACKGROUND: GMZ2 is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3 and glutamate rich protein (GLURP that mediates an immune response against the blood stage of the parasite. Two previous phase I clinical trials, one in naïve European adults and one in malaria-exposed Gabonese adults showed that GMZ2 was well tolerated and immunogenic. Here, we present data on safety and immunogenicity of GMZ2 in one to five year old Gabonese children, a target population for future malaria vaccine efficacy trials. METHODOLOGY/PRINCIPAL FINDINGS: Thirty children one to five years of age were randomized to receive three doses of either 30 µg or 100 µg of GMZ2, or rabies vaccine. GMZ2, adjuvanted in aluminum hydroxide, was administered on Days 0, 28 and 56. All participants received a full course of their respective vaccination and were followed up for one year. Both 30 µg and 100 µg GMZ2 vaccine doses were well tolerated and induced antibodies and memory B-cells against GMZ2 as well as its antigenic constituents MSP3 and GLURP. After three doses of vaccine, the geometric mean concentration of antibodies to GMZ2 was 19-fold (95%CI: 11,34 higher in the 30 µg GMZ2 group than in the rabies vaccine controls, and 16-fold (7,36 higher in the 100 µg GMZ2 group than the rabies group. Geometric mean concentration of antibodies to MSP3 was 2.7-fold (1.6,4.6 higher in the 30 µg group than in the rabies group and 3.8-fold (1.5,9.6 higher in the 100 µg group. Memory B-cells against GMZ2 developed in both GMZ2 vaccinated groups. CONCLUSIONS/SIGNIFICANCE: Both 30 µg as well as 100 µg intramuscular GMZ2 are immunogenic, well tolerated, and safe in young, malaria-exposed Gabonese children. This result confirms previous findings in naïve and malaria-exposed adults and supports further clinical development of GMZ2. TRIAL REGISTRATION: ClinicalTrials.gov NCT00703066.

  9. Non-toxic perfringolysin O and α-toxin derivatives as potential vaccine candidates against bovine necrohaemorrhagic enteritis.

    Science.gov (United States)

    Verherstraeten, S; Goossens, E; Valgaeren, B; Pardon, B; Timbermont, L; Haesebrouck, F; Ducatelle, R; Deprez, P; Van Immerseel, F

    2016-11-01

    Bovine necrohaemorrhagic enteritis is a fatal Clostridium perfringens type A-induced disease that is characterised by sudden death. Recently the involvement of perfringolysin O and α-toxin in the development of necrohaemorrhagic lesions in the gut of calves was suggested, and thus derivatives of these toxins are potentially suitable as vaccine antigens. In the current study, the perfringolysin O derivative PFO(L491D), alone or in combination with α-toxin derivative GST-cpa247-370, was evaluated as possible vaccine candidate, using in vitro assays. PFO(L491D) showed no haemolytic effect on horse red blood cells and no cytotoxic effect on bovine endothelial cells. Furthermore, calves immunised with PFO(L491D) raised antibodies against perfringolysin O that could inhibit the perfringolysin O-associated haemolytic activity on horse red blood cells. Antisera from calves immunised with PFO(L491D) had a significantly higher neutralising capacity against the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells than serum from control calves (P O and α-toxin and consequently inhibited both the perfringolysin O-associated haemolytic activity and the α-toxin-associated lecithinase activity in vitro. Additionally, the neutralising ability of these antisera on the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells was significantly higher than that from calves immunised with PFO(L491D) (P O derivative PFO(L491D) is an immunogenic antigen that can potentially be used to produce vaccine against bovine necrohaemorrhagic enteritis. Including α-toxin derivative GST-cpa247-370 has an additional protective effect and therefore vaccination of calves with a combination of both antigens seems even more promising.

  10. Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Qiu Chunhui

    2012-08-01

    Full Text Available Abstract Background Thyroid hormones (TH modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs. The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHRβ and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. Methods The full-length cDNA sequence of SjTHRβ, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated. Results The SjTHRβ cDNA was cloned, verified by 5’ and 3’ Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3’end, suggesting the transcript is full-length. SjTHRβ is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHRβ was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRβ-DBD and ligand binding domain (SjTHRβ-LBD were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRβ (rSjTHRβ. Western blotting revealed that anti-rSjTHRβ-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA analysis showed that rSjTHRβ-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRβ-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs

  11. Antigen-Specific lgA B Memory Cell Responses to Shigella Antigens Elicited in Volunteers Immunized with Live Attenuated Shigella flexneri 2a Oral Vaccine Candidates

    Science.gov (United States)

    2011-01-01

    cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates J.K. Simona,b... Shigella ;. B cell memory; Immunoglobulin lgA; Mucosal immunity Abstract We studied the induction of antigen-specific lgA memory B cells (BM) in...volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 107 , 108 or 109 CFU of S. flexneri 2a with

  12. Immunogenicity of a Candidate DNA Vaccine Based on the prM/E Genes of a Dengue Type 2 Virus Cosmopolitan Genotype Strain.

    Science.gov (United States)

    Putri, Dwi Hilda; Sudiro, Tjahjani Mirawati; Yunita, Rina; Jaya, Ungke Anton; Dewi, Beti Ernawati; Sjatha, Fithriyah; Konishi, Eiji; Hotta, Hak; Sudarmono, Pratiwi

    2015-01-01

    The development of a dengue virus vaccine is a major priority in efforts to control the diseases. Several researchers are currently using the Asian 1 and Asian 2 genotypes as vaccine candidates for dengue type 2 virus (DENV-2). However, in this study, we constructed a recombinant plasmid-based prM/E gene, from a DENV-2 Cosmopolitan genotype strain as a dengue DNA vaccine candidate. The protein expression of the recombinant plasmid in CHO cells was analyzed using an enzyme-linked immunosorbent assay, western blotting, and sucrose gradient sedimentation. After being used to immunize ddY mice three times at doses of 25 or 100 μg, the DNA vaccine induced humoral immune responses. There was no difference in the neutralizing antibody titer (focus reduction neutralization test 50% value) of mice immunized with 25 and 100 μg DNA vaccine doses. When challenged with 3 × 10(5) FFU DENV-2, immunized mice could raise anamnestic neutralizing antibody responses, which were observed at day 4 and day 8 post-challenge. Analysis of immunogenicity using BALB/c mice showed that their antibody neutralization titers were lower than those of ddY mice. In addition, the antibodies produced after immunization and challenge could also neutralize a DENV-2 Asian 2 genotype (New Guinea C) strain. Therefore, the DENV-2 Cosmopolitan genotype may be a DENV-2 vaccine candidate.

  13. Construction and Evaluation of a Polyvalent Genetically Engineered Vaccine Candidate for VEE.

    Science.gov (United States)

    1997-01-01

    subcutaneous inoculation of attenuated VEE induces mucosal immunity may broaden our understanding of the mucosal immune system and the interaction between...genotype and route for induction of mucosal immunity . Serum and saliva samples were collected from some of the mice inoculated in. with V3526 three...engineered vaccine strains. REFERENCES Charles, P.C., K.W. Brown, N.L. Davis, M.K. Hart and R.E. Johnston. 1997. Mucosal immunity induced by parenteral

  14. Characterization and evaluation of a Sarcoptes scabiei allergen as a candidate vaccine

    OpenAIRE

    Zhang Runhui; Jise Quwu; Zheng Wanpeng; Ren Yongjun; Nong Xiang; Wu Xuhang; Gu Xiaobin; Wang Shuxian; Peng Xuerong; Lai Songjia; Yang Guangyou

    2012-01-01

    Abstract Background Sarcoptic mange caused by the mite Sarcoptes scabiei is a worldwide disease affecting both humans and animals. Here we report the molecular characterization and evaluation of a recombinant S. scabiei tropomyosin (SsTm) protein in a vaccination trial in rabbits. Methods The full-length cDNA was cloned in a bacterial pET vector, and the recombinant protein was expressed in BL21 (DE3) cells and purified. Using specific rabbit antiserum, tropomyosin was localized immunohistoch...

  15. Vacuna contra la fiebre hemorrágica argentina Candid#1 producida en la Argentina: Inmunogenicidad y seguridad Candid#1 vaccine against Argentine Hemorrhagic Fever produced in Argentina: Immunogenicity and safety

    Directory of Open Access Journals (Sweden)

    Delia A. Enria

    2010-06-01

    Full Text Available Se realizó un estudio clínico en 946 voluntarios humanos sanos, donde se comparó la vacuna Candid#1 producida en Argentina con la elaborada en EE.UU., que había sido utilizada en estudios previos. Como objetivo primario se evaluó la equivalencia en la eficacia utilizando como marcador subrogante a la inmunogenicidad medida por detección de anticuerpos neutralizantes. Como objetivo secundario se evaluó la equivalencia en inocuidad comparando las tasas de reacciones adversas. Ambas vacunas mostraron una tasa equivalente de inmunogenicidad ligeramente superior al 95.5%, que es la eficacia estimada para Candid #1 en estudios previos. No se observaron eventos adversos graves relacionados con la vacuna. Los eventos adversos generales considerados relacionados fueron de escasa significación clínica y de resolución espontánea o con tratamiento sintomático; se presentaron en los receptores de ambas vacunas en tasas equivalentes (29.9% para la vacuna fabricada en la Argentina y 35.0% para la fabricada en EE.UU., e incluyeron: cefalea, decaimiento, mialgias, plaquetopenia leve (A clinical study in 946 human volunteers was done to compare Candid #1 vaccine manufactured in Argentina with the vaccine produced in USA that had been previously used. The efficacy was evaluated using immunogenicity measured by the detection of neutralizing antibodies as a subrogate marker. Safety was evaluated comparing the rate of adverse events. Both vaccines showed a comparable rate of seroconversion, slighty higher than the efficacy estimated from previous studies (95.5%. There were no severe adverse events related to the vaccines. The general events considered related to the vaccines were not clinically relevant and disappeared either spontaneously or with symptomatic treatment. Similar rates of adverse events (29.9% for the Argentine vaccine and 35.0% for the USA vaccine were found for both vaccines. These included: headache, weakness, myalgias, mild low blood

  16. Protectome analysis: a new selective bioinformatics tool for bacterial vaccine candidate discovery.

    Science.gov (United States)

    Altindis, Emrah; Cozzi, Roberta; Di Palo, Benedetta; Necchi, Francesca; Mishra, Ravi P; Fontana, Maria Rita; Soriani, Marco; Bagnoli, Fabio; Maione, Domenico; Grandi, Guido; Liberatori, Sabrina

    2015-02-01

    New generation vaccines are in demand to include only the key antigens sufficient to confer protective immunity among the plethora of pathogen molecules. In the last decade, large-scale genomics-based technologies have emerged. Among them, the Reverse Vaccinology approach was successfully applied to the development of an innovative vaccine against Neisseria meningitidis serogroup B, now available on the market with the commercial name BEXSERO® (Novartis Vaccines). The limiting step of such approaches is the number of antigens to be tested in in vivo models. Several laboratories have been trying to refine the original approach in order to get to the identification of the relevant antigens straight from the genome. Here we report a new bioinformatics tool that moves a first step in this direction. The tool has been developed by identifying structural/functional features recurring in known bacterial protective antigens, the so called "Protectome space," and using such "protective signatures" for protective antigen discovery. In particular, we applied this new approach to Staphylococcus aureus and Group B Streptococcus and we show that not only already known protective antigens were re-discovered, but also two new protective antigens were identified.

  17. Immunological evaluation of an alginate-based conjugate as a vaccine candidate against Pseudomonas aeruginosa.

    Science.gov (United States)

    Farjah, Ali; Owlia, Parviz; Siadat, Seyed Davar; Mousavi, Seyed Fazlollah; Ardestani, Mehdi Shafiee; Mohammadpour, Hashem Khorsand

    2015-02-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections, is usually resistant to antimicrobial agents, and is the leading cause of morbidity and premature mortality in patients with cystic fibrosis (CF). Mucoid strains of P. aeruginosa produce a virulence factor known as alginate. Developing a strategy to raise opsonic antibodies against alginate could be promising for the treatment of P. aeruginosa infection in CF patients. Conjugation of alginate to a carrier protein is a good method for increasing the immunogenicity of alginate. We conjugated alginate to the outer membrane vesicle (OMV) of Neisseria meningitidis serogroup B, which is a safe carrier protein, and evaluated its efficacy in mice. To evaluate the immune response, total IgG, IgG1, IgG2a, and IgG2b titers were analyzed. Immunization of mice with the alginate-OMV conjugate raised the levels of opsonic antibodies, and the vaccinated mice were protected when challenged intranasally with P. aeruginosa. Further studies showed that the conjugated vaccine could eliminate P. aeruginosa from the lungs of infected mice. This study supports the proposal that immunization of mice with an alginate-OMV conjugate vaccine could be safe and protective against P. aeruginosa infection.

  18. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  19. Candidate gene analysis and exome sequencing confirm LBX1 as a susceptibility gene for idiopathic scoliosis

    DEFF Research Database (Denmark)

    Grauers, Anna; Wang, Jingwen; Einarsdottir, Elisabet;

    2015-01-01

    BACKGROUND CONTEXT: Idiopathic scoliosis is a spinal deformity affecting approximately 3% of otherwise healthy children or adolescents. The etiology is still largely unknown but has an important genetic component. Genome-wide association studies have identified a number of common genetic variants...... that are significantly associated with idiopathic scoliosis in Asian and Caucasian populations, rs11190870 close to the LBX1 gene being the most replicated finding. PURPOSE: The aim of the present study was to investigate the genetics of idiopathic scoliosis in a Scandinavian cohort by performing a candidate gene study...... of four variants previously shown to be associated with idiopathic scoliosis and exome sequencing of idiopathic scoliosis patients with a severe phenotype to identify possible novel scoliosis risk variants. STUDY DESIGN: This was a case control study. PATIENT SAMPLE: A total of 1,739 patients...

  20. Whole-genome sequencing reveals the effect of vaccination on the evolution of Bordetella pertussis.

    Science.gov (United States)

    Xu, Yinghua; Liu, Bin; Gröndahl-Yli-Hannuksila, Kirsi; Tan, Yajun; Feng, Lu; Kallonen, Teemu; Wang, Lichan; Peng, Ding; He, Qiushui; Wang, Lei; Zhang, Shumin

    2015-08-18

    Herd immunity can potentially induce a change of circulating viruses. However, it remains largely unknown that how bacterial pathogens adapt to vaccination. In this study, Bordetella pertussis, the causative agent of whooping cough, was selected as an example to explore possible effect of vaccination on the bacterial pathogen. We sequenced and analysed the complete genomes of 40 B. pertussis strains from Finland and China, as well as 11 previously sequenced strains from the Netherlands, where different vaccination strategies have been used over the past 50 years. The results showed that the molecular clock moved at different rates in these countries and in distinct periods, which suggested that evolution of the B. pertussis population was closely associated with the country vaccination coverage. Comparative whole-genome analyses indicated that evolution in this human-restricted pathogen was mainly characterised by ongoing genetic shift and gene loss. Furthermore, 116 SNPs were specifically detected in currently circulating ptxP3-containing strains. The finding might explain the successful emergence of this lineage and its spread worldwide. Collectively, our results suggest that the immune pressure of vaccination is one major driving force for the evolution of B. pertussis, which facilitates further exploration of the pathogenicity of B. pertussis.

  1. Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine.

    Directory of Open Access Journals (Sweden)

    Sandhya Vasan

    Full Text Available BACKGROUND: We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1x10(7 (low, 5x10(7 (mid, or 2.5x10(8 pfu (high] volunteers were randomized in a 3:1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNgamma ELISpot response rate to any HIV antigen was 0/12 (0% in the placebo group, 3/12 (25% in the low dosage group, 6/12 (50% in the mid dosage group, and 8/13 (62% in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%, 8/13 (62%, 6/12 (50% and 10/13 (77% in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement

  2. Phase I safety and immunogenicity evaluation of MVA-CMDR, a multigenic, recombinant modified vaccinia Ankara-HIV-1 vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Jeffrey R Currier

    Full Text Available BACKGROUND: We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand. METHODOLOGY/PRINCIPAL FINDINGS: MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7 or 10(8 pfu or intradermally (ID; 10(6 or 10(7 pfu at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2. Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6 PBMC at 10(8 pfu IM, but high in response rate (70% (51Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8 pfu IM; (ii predominantly HIV Env-specific CD4(+ T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route; (iv dose- and route-dependent with 10(8 pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8 pfu IM. CONCLUSIONS/SIGNIFICANCE: MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and

  3. Plasmodium falciparum CS protein - prime malaria vaccine candidate: definition of the human CTL domain and analysis of its variation

    Directory of Open Access Journals (Sweden)

    Denise L. Doolan

    1992-01-01

    Full Text Available Studies in mice have shown that immunity to malaria sporozoites is mediated primarily by citotoxic T lymphocytes (CTL specific for epitopes within the circumsporozoite (CS protein. Humans, had never been shown to generate CTL against any malaria or other parasite protein. The design of a sub-unit vaccine for humans ralies on the epitopes recognized by CTL being identified and polymorphisms therein being defined. We have developed a novel technique using an entire series of overlapping synthetic peptides to define the epitopes of the Plasmodium falciparum CS protein recognized by human CTL and have analyzed the sequence variation of the protein with respect to the identified CTL epitopic domain. We have demonstrated that some humans can indeed generate CTL. against the P. falciparum CS protein. Furthermore, the extent of variation observed for the CTL recognition domain is finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways can be found to increase immune responsiveness, then a vaccine designed to stimulate CS protein-specific CTL activity may prevent malaria.

  4. Vaccinations

    Science.gov (United States)

    ... vaccinated? For many years, a set of annual vaccinations was considered normal and necessary for dogs and ... to protect for a full year. Consequently, one vaccination schedule will not work well for all pets. ...

  5. Multiparametric analyses of human PBMCs loaded ex vivo with a candidate idiotype vaccine for HCV-related lymphoproliferative disorders.

    Directory of Open Access Journals (Sweden)

    Annacarmen Petrizzo

    Full Text Available Hepatitis C virus (HCV has been identified as one of the major risk factors for type II mixed cryoglobulinemia (MC, during the clinical evolution of chronic hepatitis, which may lead to development of B cell non-Hodgkin's lymphoma (NHL. We have previously shown that the candidate idiotype vaccine, based on the IGKV3-20 light chain protein, is able to induce activation and maturation of circulating antigen presenting cells (APCs in both HCV-positive and HCV-negative healthy control subjects, with production of Th2-type cytokines. Here, the effect of the recombinant IGKV3-20 protein on human peripheral blood mononuclear cells (PBMCs from HCV-positive subjects, with known blood levels of cryoglobulins, is shown via gene expression profiling analysis combined to multiparameter flow cytometry and multiplex analyses of cytokines.

  6. Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate

    Institute of Scientific and Technical Information of China (English)

    AN Wen-qi; LIU Xiu-fan; WANG Xi-liang; YANG Peng-hui; DUAN Yue-qiang; LUO De-yan; TANG Chong; JIA Wei-hong; XING Li; SHI Xin-fu; ZHANG Yu-jing

    2009-01-01

    Background H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics. Methods In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (te), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A). Results In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found. Conclusion The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.

  7. A randomized controlled Phase Ib trial of the malaria vaccine candidate GMZ2 in African children

    DEFF Research Database (Denmark)

    Bélard, Sabine; Issifou, Saadou; Hounkpatin, Aurore B

    2011-01-01

    GMZ2 is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP) that mediates an immune response against the blood stage of the parasite. Two previous phase I clinical trials, one in naïve European adults and one in malaria-exposed Gabonese ...... adults showed that GMZ2 was well tolerated and immunogenic. Here, we present data on safety and immunogenicity of GMZ2 in one to five year old Gabonese children, a target population for future malaria vaccine efficacy trials....

  8. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

    Directory of Open Access Journals (Sweden)

    Gabriel Gomez

    Full Text Available Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

  9. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

    Science.gov (United States)

    Gomez, Gabriel; Pei, Jianwu; Mwangi, Waithaka; Adams, L Garry; Rice-Ficht, Allison; Ficht, Thomas A

    2013-01-01

    Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.

  10. High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Fangye; Zhou, Jian; Ma, Lei; Song, Shaohui; Zhang, Xinwen; Li, Weidong; Jiang, Shude [No. 5, Department of Bioproducts, Institute of Medical Biology, Chinese Academy of Medical Science and Pecking Union Medical College, Jiaoling Avenue 935, Kunming, Yunnan Province 650102, People' s Republic of China (China); Wang, Yue, E-mail: euy-tokyo@umin.ac.jp [National Institute for Viral Disease Control and Prevention, China Center for Disease Control and Prevention, Yingxin Lane 100, Xicheng District, Beijing 100052, People' s Republic of China (China); Liao, Guoyang, E-mail: liaogy@21cn.com [No. 5, Department of Bioproducts, Institute of Medical Biology, Chinese Academy of Medical Science and Pecking Union Medical College, Jiaoling Avenue 935, Kunming, Yunnan Province 650102, People' s Republic of China (China)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Vero cell-based HPAI H5N1 vaccine with stable high yield. Black-Right-Pointing-Pointer Stable high yield derived from the YNVa H3N2 backbone. Black-Right-Pointing-Pointer H5N1/YNVa has a similar safety and immunogenicity to H5N1delta. -- Abstract: Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.

  11. Broad blockade antibody responses in human volunteers after immunization with a multivalent norovirus VLP candidate vaccine: immunological analyses from a phase I clinical trial.

    Directory of Open Access Journals (Sweden)

    Lisa C Lindesmith

    2015-03-01

    Full Text Available Human noroviruses (NoVs are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP candidate vaccine in human volunteers.Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4 were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential

  12. Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates

    OpenAIRE

    Tao, Tao; Skiadopoulos, Mario H.; Davoodi, Fatemeh; Riggs, Jeffrey M.; Collins, Peter L.; Murphy, Brian R

    2000-01-01

    We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoul...

  13. Rising Cellular Immune Response after Injection of pVax/iutA: A Genetic DNA Cassette as Candidate Vaccine against Urinary Tract Infection

    Science.gov (United States)

    BAKHTIARI, Ronak; AHMADIAN, Shahin; FALLAH MEHRABADI, Jalil

    2016-01-01

    Background: Uropathogenic Escherichia coli (UPEC) are major bacterial agent of Urinary Tract Infection (UTI). This infection is more prevalent among women because approximately half of all women will experience a UTI in their life-time and near a quarter of them will have a recurrent infection within 6–12 months. IutA protein has a major role during UPEC pathogenesis and consequently infection. Therefore, the aim of current study was assessment of IutA protein roles as a potential candidate antigen based for vaccine designing. Methods: This survey was conducted during 2014–2015 at the University of Tehran, Iran. Chromosomal DNA extracted from E. coli 35218 and iutA gene amplified by PCR. The amplicon cloned to pVax.1 eukaryotic expression vector and recombinant vector confirmed by sequencing. The iutA gene expression in genetic cassette of pVax/iutA was evaluated in COS7 cell line by RT-PCR. Then, injected to mouse model, which divided to three groups: injected with pVax vector, PBS and pVax/iutA cassette respectively in two stages (d 1 and 14). One week after the second injection, bleeding from immunized mouse was performed and IFN-gamma was measured. Results: The mice immunized with pVax/iutA showed increased interferon-γ responses significantly higher than two non-immunized groups (P<0.05). Conclusion: Cellular immune response has a main protective role against UTI. Raising this kind of immune response is important to preventing of recurrent infection. Moreover, the current DNA cassette will be valuable for more trying to prepare a new vaccine against UTI. PMID:27516995

  14. Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

    Directory of Open Access Journals (Sweden)

    Xiangyang Min

    2016-01-01

    Full Text Available Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold and IL-10 (2-fold in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

  15. A New Approach for Designing A Potentially Vaccine Candidate against Urinary Tract Infection by Using Protein Display on Lacto-bacillus Surface

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    Jalil Fallah Mehrabadi

    2013-07-01

    Full Text Available Background: The prevalence of Urinary Tract Infection (UTI is really high in the world. Escherichia coli is a major agent of UTI. One of the strategies for decreasing UTI infections is vaccine development. As the attachment is a really important stage in colonization and infection, at­tachment inhibition has an applied strategy. FimH protein is a major factor during bacterial colonization in urinary tract and could be used as a vaccine. Thus, it was considered in this research as a candidate antigen. Methods: The sequences of fimH and acmA genes were used for designing a synthetic gene. It was cloned to pET23a expression vector and transformed to E. coli (DE3 Origami. To confirm the expression of recombinant protein, SDS-PAGE and western blotting methods were used. Subsequently, recombinant protein was purified. On the other hand, Lactobacillus reuteri was cultured and mixed with FimH / AcmA recombinant protein. The rate of protein localization on lactobacillus surface was assessed using ELISA method. Results: It was showed that the recombinant protein was expressed in E. coli (DE3 Origami and purified by affinity chromatography. Moreover, this protein could be localized on lactobacillus surface by 5 days. Conclusion: In current study, a fusion recombinant protein was pre­pared and displayed on L. reuteri surface. This strain could be used for animal experiment as a competitor against Uropathogenic E. coli (UPEC. Using manipulated probiotics strains instead of antibiotic ther­apy could decrease the antibiotic consumption and reduce multi-drug resistant strains.

  16. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

    DEFF Research Database (Denmark)

    Tamborrini, Marco; Stoffel, Sabine A; Westerfeld, Nicole;

    2011-01-01

    In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs) have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant...... fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP....

  17. Safety of the malaria vaccine candidate, RTS,S/AS01E in 5 to 17 month old Kenyan and Tanzanian Children

    DEFF Research Database (Denmark)

    Lusingu, John; Olotu, Ally; Leach, Amanda

    2010-01-01

    The malaria vaccine candidate, RTS,S/AS01(E), showed promising protective efficacy in a trial of Kenyan and Tanzanian children aged 5 to 17 months. Here we report on the vaccine's safety and tolerability. The experimental design was a Phase 2b, two-centre, double-blind (observer- and participant...... taken at baseline, 3, 10 and 14 months after dose 1. A total of 894 children received RTS,S/AS01(E) or rabies vaccine between March and August 2007. Overall, children vaccinated with RTS,S/AS01(E) had fewer SAEs (51/447) than children in the control group (88/447). One SAE episode in a RTS,S/AS01(E...... reported unsolicited AE. Fever was the most frequently observed solicited AE and was recorded after 11% of RTS,S/AS01(E) doses compared to 31% of doses of rabies vaccine. The candidate vaccine RTS,S/AS01(E) showed an acceptable safety profile in children living in a malaria-endemic area in East Africa...

  18. Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis

    Directory of Open Access Journals (Sweden)

    Ratna eGurung

    2014-07-01

    Full Text Available Control of Johne’s disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP in ruminants using commercially available vaccine reduces production losses, mortality, faecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johne’s disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE (ISA 50V2, 61VG, 71VG and 201VG adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate.

  19. Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis

    Science.gov (United States)

    Gurung, Ratna B.; Purdie, Auriol C.; Whittington, Richard J.; Begg, Douglas J.

    2014-01-01

    Control of Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP) in ruminants using commercially available vaccine reduces production losses, mortality, fecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johne's disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE™ (ISA 50V2, 61VG, 71VG, and 201VG) adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ) release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE™ ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate. PMID:25077074

  20. Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis.

    Science.gov (United States)

    Gurung, Ratna B; Purdie, Auriol C; Whittington, Richard J; Begg, Douglas J

    2014-01-01

    Control of Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP) in ruminants using commercially available vaccine reduces production losses, mortality, fecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johne's disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE™ (ISA 50V2, 61VG, 71VG, and 201VG) adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ) release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE™ ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate.

  1. An adjuvanted, tetravalent dengue virus purified inactivated vaccine candidate induces long-lasting and protective antibody responses against dengue challenge in rhesus macaques.

    Science.gov (United States)

    Fernandez, Stefan; Thomas, Stephen J; De La Barrera, Rafael; Im-Erbsin, Rawiwan; Jarman, Richard G; Baras, Benoît; Toussaint, Jean-François; Mossman, Sally; Innis, Bruce L; Schmidt, Alexander; Malice, Marie-Pierre; Festraets, Pascale; Warter, Lucile; Putnak, J Robert; Eckels, Kenneth H

    2015-04-01

    The immunogenicity and protective efficacy of a candidate tetravalent dengue virus purified inactivated vaccine (TDENV PIV) formulated with alum or an Adjuvant System (AS01, AS03 tested at three different dose levels, or AS04) was evaluated in a 0, 1-month vaccination schedule in rhesus macaques. One month after dose 2, all adjuvanted formulations elicited robust and persisting neutralizing antibody titers against all four dengue virus serotypes. Most of the formulations tested prevented viremia after challenge, with the dengue serotype 1 and 2 virus strains administered at 40 and 32 weeks post-dose 2, respectively. This study shows that inactivated dengue vaccines, when formulated with alum or an Adjuvant System, are candidates for further development.

  2. Immunomodulatory molecules of Fasciola hepatica: candidates for both vaccine and immunotherapeutic development.

    Science.gov (United States)

    Dalton, John P; Robinson, Mark W; Mulcahy, Grace; O'Neill, Sandra M; Donnelly, Sheila

    2013-08-01

    The liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle), a global disease that is also an important infection of humans. As soon as the parasite invades the gut wall its interaction with various host immune cells (e.g. dendritic cells, macrophages and mast cells) is complex. The parasite secretes a myriad of molecules that direct the immune response towards a favourable non-protective Th2-mediate/regulatory environment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1), are under development as the first generation of fluke vaccines. However, this peptidase and other molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treat immune-related conditions in humans and animals.

  3. Immunogenicity of a recombinant Rift Valley fever MP-12-NSm deletion vaccine candidate in calves.

    Science.gov (United States)

    Morrill, John C; Laughlin, Richard C; Lokugamage, Nandadeva; Wu, Jing; Pugh, Roberta; Kanani, Pooja; Adams, L Garry; Makino, Shinji; Peters, C J

    2013-10-09

    The safety and immunogenicity of an authentic recombinant (ar) of the live, attenuated MP-12 Rift Valley fever (RVF) vaccine virus with a large deletion of the NSm gene in the pre-Gn region of the M RNA segment (arMP-12ΔNSm21/384) was tested in 4-6 month old Bos taurus calves. Phase I of this study evaluated the neutralizing antibody response, measured by 80% plaque reduction neutralization (PRNT80), and clinical response of calves to doses of 1 × 10(1) through 1 × 10(7) plaque forming units (PFU) administered subcutaneously (s.c.). Phase II evaluated the clinical and neutralizing antibody response of calves inoculated s.c. or intramuscularly (i.m.) with 1 × 10(3), 1 × 10(4) or 1 × 10(5)PFU of arMP-12ΔNSm21/384. No significant adverse clinical events were observed in the animals in these studies. Of all specimens tested, only one vaccine viral isolate was recovered and that virus retained the introduced deletion. In the Phase I study, there was no statistically significant difference in the PRNT80 response between the dosage groups though the difference in IgG response between the 1 × 10(1)PFU group and the 1 × 10(5)PFU group was statistically significant (pvaccine with the 1 × 10(1)PFU dose group showing the least response. The Phase II study also showed no statistically significant difference in PRNT80 response between the dosage groups though the difference in RVFV-specific IgG values was significantly increased (pvaccine.

  4. Yersinia pestis with regulated delayed attenuation as a vaccine candidate to induce protective immunity against plague.

    Science.gov (United States)

    Sun, Wei; Roland, Kenneth L; Kuang, Xiaoying; Branger, Christine G; Curtiss, Roy

    2010-03-01

    Two mutant strains of Yersinia pestis KIM5+, a Deltacrp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P(BAD) crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD(50)s) of the Deltacrp and araC P(BAD) crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5+, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 x 10(7) CFU of the Deltacrp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 x 10(4) CFU of the araC P(BAD) crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.

  5. Comparative full-length sequence analysis of Marek's disease virus vaccine strain 814.

    Science.gov (United States)

    Zhang, Feng; Liu, Chang-Jun; Zhang, Yan-Ping; Li, Zhi-Jie; Liu, Ai-Ling; Yan, Fu-Hai; Cong, Feng; Cheng, Yun

    2012-01-01

    The complete DNA sequence of Marek's disease virus (MDV) serotype 1 vaccine strain 814 was determined. It consisted of 172,541 bp, with an overall gene organization identical to that of the MDV-1 type strains. Comparative genomic analysis of vaccine strains (814 and CVI988) and other strains (CU-2, Md5, and Md11) showed that 814 was most similar to CVI988. Several unique insertions, deletions, and substitutions were identified in strain 814. Of note, a 177-bp insertion in the overlapping genes encoding the Meq, RLORF6, and 23-kDa proteins of strain 814 was identified, and a 69-bp deletion was also located in the origin of replication site (Ori) in the gene encoding RLORF12. Compared to the CVI988 vaccine strain, a deletion of 510 bp was identified in the UL36 gene. These analyses identified key mutations in the 814 strain and the vaccine strain that could be exploited for future MDV vaccine design.

  6. MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate.

    Science.gov (United States)

    Cloward, Jason M; Shafer, William M

    2013-01-01

    The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes.

  7. A randomized trial assessing the safety and immunogenicity of AS01 and AS02 adjuvanted RTS,S malaria vaccine candidates in children in Gabon.

    Directory of Open Access Journals (Sweden)

    Bertrand Lell

    Full Text Available BACKGROUND: The malaria vaccine candidate antigen RTS,S includes parts of the pre-erythrocytic stage circumsporozoite protein fused to the Hepatitis B surface antigen. Two Adjuvant Systems are in development for this vaccine, an oil-in water emulsion--based formulation (AS02 and a formulation based on liposomes (AS01. METHODS & PRINCIPAL FINDINGS: In this Phase II, double-blind study (NCT00307021, 180 healthy Gabonese children aged 18 months to 4 years were randomized to receive either RTS,S/AS01(E or RTS,S/AS02(D, on a 0-1-2 month vaccination schedule. The children were followed-up daily for six days after each vaccination and monthly for 14 months. Blood samples were collected at 4 time-points. Both vaccines were well tolerated. Safety parameters were distributed similarly between the two groups. Both vaccines elicited a strong specific immune response after Doses 2 and 3 with a ratio of anti-CS GMT titers (AS02(D/AS01(E of 0.88 (95% CI: 0.68-1.15 post-Dose 3. After Doses 2 and 3 of experimental vaccines, anti-CS and anti-HBs antibody GMTs were higher in children who had been previously vaccinated with at least one dose of hepatitis B vaccine compared to those not previously vaccinated. CONCLUSIONS: RTS,S/AS01(E proved similarly as well tolerated and immunogenic as RTS,S/AS02(D, completing an essential step in the age de-escalation process within the RTS,S clinical development plan. TRIAL REGISTRATION: ClinicalTrials.gov. NCT00307021.

  8. Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46 and Pol amino acid sequences for vaccine design

    Directory of Open Access Journals (Sweden)

    Aline Cristina Mota-Miranda

    2007-09-01

    Full Text Available This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15 and Pol (n = 43 nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59 using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

  9. Safety of the malaria vaccine candidate, RTS,S/AS01E in 5 to 17 month old Kenyan and Tanzanian Children.

    Directory of Open Access Journals (Sweden)

    John Lusingu

    Full Text Available The malaria vaccine candidate, RTS,S/AS01(E, showed promising protective efficacy in a trial of Kenyan and Tanzanian children aged 5 to 17 months. Here we report on the vaccine's safety and tolerability. The experimental design was a Phase 2b, two-centre, double-blind (observer- and participant-blind, randomised (1∶1 ratio controlled trial. Three doses of study or control (rabies vaccines were administered intramuscularly at 1 month intervals. Solicited adverse events (AEs were collected for 7 days after each vaccination. There was surveillance and reporting for unsolicited adverse events for 30 days after each vaccination. Serious adverse events (SAEs were recorded throughout the study period which lasted for 14 months after dose 1 in Korogwe, Tanzania and an average of 18 months post-dose 1 in Kilifi, Kenya. Blood samples for safety monitoring of haematological, renal and hepatic functions were taken at baseline, 3, 10 and 14 months after dose 1. A total of 894 children received RTS,S/AS01(E or rabies vaccine between March and August 2007. Overall, children vaccinated with RTS,S/AS01(E had fewer SAEs (51/447 than children in the control group (88/447. One SAE episode in a RTS,S/AS01(E recipient and nine episodes among eight rabies vaccine recipients met the criteria for severe malaria. Unsolicited AEs were reported in 78% of subjects in the RTS,S/AS01(E group and 74% of subjects in the rabies vaccine group. In both vaccine groups, gastroenteritis and pneumonia were the most frequently reported unsolicited AE. Fever was the most frequently observed solicited AE and was recorded after 11% of RTS,S/AS01(E doses compared to 31% of doses of rabies vaccine. The candidate vaccine RTS,S/AS01(E showed an acceptable safety profile in children living in a malaria-endemic area in East Africa. More data on the safety of RTS,S/AS01(E will become available from the Phase 3 programme.

  10. Candidate sequence variants and fetal hemoglobin in children with sickle cell disease treated with hydroxyurea.

    Directory of Open Access Journals (Sweden)

    Nancy S Green

    Full Text Available BACKGROUND: Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin. METHODOLOGY/PRINCIPAL FINDINGS: In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively, with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels. CONCLUSIONS/SIGNIFICANCE: These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease.

  11. Extraction of Sequence Conservation Features for the Prioritization of Candidate Single Amino Acid Polymorphisms

    Directory of Open Access Journals (Sweden)

    Jiaxin Wu

    2011-03-01

    Full Text Available Although remarkable success has been achieved by genome-wide association (GWA studies over the past few years, genetic variants discovered in GWA studies can typically account for only a small fraction of heritability of most common diseases. As such, the identification of multiple rare variants that are associated with complex diseases has been receiving more and more attentions. However, most of the recently developed statistical approaches for detecting association of rare variants with diseases require the selection of functional variants before the successive analysis, making an effective bioinformatics method for filtering out non-relevant rare variants indispensible. In this paper, we focus on a specific type of genetic variants called single amino acid polymorphisms (SAAPs. We propose to prioritize candidate SAAPs for a specific disease according to their association scores that are calculated using a guilt-by-association model with a set of features derived from protein sequences. We validate the proposed approach in a systematic way and demonstrate that the proposed model is powerful in distinguishing disease-associated SAAPs for the specific disease of interest.

  12. Generation of a safe Salmonella Gallinarum vaccine candidate that secretes an adjuvant protein with immunogenicity and protective efficacy against fowl typhoid.

    Science.gov (United States)

    Nandre, R M; Lee, J H

    2014-01-01

    We constructed a live, attenuated Salmonella Gallinarum (SG) that secretes heat-labile enterotoxin B subunit protein (LTB), and evaluated this strain as a new vaccine candidate by assessing its safety, immunogenicity and protective efficacy against fowl typhoid. An asd(+) p15A ori low-copy plasmid containing eltB encoding LTB was transformed into a ΔlonΔcpxRΔasd SG (JOL967) to construct the candidate, JOL1355. In Experiments 1 and 2, birds were orally immunized with JOL1355 at 4 weeks of age, while control birds were inoculated with sterile phosphate-buffered saline. In Experiment 2, the birds of both groups were orally challenged with a virulent SG at 8 weeks of age. In Experiment 1, examination for safety revealed that the immunized group did not show any bacterial counts of the vaccine candidate in the liver and spleen. Birds immunized with the vaccine candidate showed a significant increase in systemic IgG and mucosal secretory IgA levels in Experiment 2. In addition, the lymphocyte proliferation response and the numbers of CD3(+)CD4(+) and CD3(+)CD8(+) T cells were also significantly elevated in the immunized group, which indicated that the candidate also induced cellular immune responses. In the protection assay, efficient protection with only 16% mortality in the immunized group was observed against challenge compared with 76% mortality in the control group. These results indicate that the live, attenuated SG secreting LTB can be a safe vaccine candidate. In addition, it can induce humoral and cellular immune responses and can efficiently reduce mortality of birds exposed to fowl typhoid.

  13. Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display

    Directory of Open Access Journals (Sweden)

    Nini Guo

    2015-08-01

    Research in Context: We used a large number of randomly produced small proteins (“peptides” to identify peptides containing specific protein sequences that bind efficiently to an antibody that can prevent hepatitis C virus infection in cell culture. After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody. This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines.

  14. DENGUE VACCINES.

    Science.gov (United States)

    Thisyakorn, Usa; Thisyakorn, Chule

    2015-01-01

    The uniqueness of the dengue viruses (DENVs) and the spectrum of disease resulting from infection have made dengue vaccine development difficult. Several vaccine candidates are currently being evaluated in clinical studies. The candidate currently at the most advanced clinical development stage, a live-attenuated tetravalent vaccine based on the chimeric yellow fever-dengue virus (CYD-TDV), has progressed to Phase 3 efficacy studies. Several other live-attenuated vaccines, as well as subunit, DNA, and purified inactivated vaccine candidates are at earlier stages of clinical development. Additional technological approaches, such as virus-vectored and Virus-Like Particles (VLP)-based vaccines are under evaluation in preclinical studies.

  15. EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Hamid Sedighian Rad

    2013-09-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS. Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium.A divalent recombinant EspA-Intimin (EI protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+ vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI.Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group.The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.

  16. Strain-transcending immune response generated by chimeras of the malaria vaccine candidate merozoite surface protein 2

    Science.gov (United States)

    Krishnarjuna, Bankala; Andrew, Dean; MacRaild, Christopher A.; Morales, Rodrigo A. V.; Beeson, James G.; Anders, Robin F.; Richards, Jack S.; Norton, Raymond S.

    2016-01-01

    MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum. Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro. We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum. PMID:26865062

  17. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing

    Science.gov (United States)

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G.

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  18. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael;

    2011-01-01

    consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

  19. In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity.

    Science.gov (United States)

    Nilsson, Ola B; Adedoyin, Justus; Rhyner, Claudio; Neimert-Andersson, Theresa; Grundström, Jeanette; Berndt, Kurt D; Crameri, Reto; Grönlund, Hans

    2011-01-01

    Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

  20. Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

    Directory of Open Access Journals (Sweden)

    Murphy Brian R

    2004-10-01

    Full Text Available Abstract Background A dengue virus type 2 (DEN-2 Tonga/74 isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30 in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. Methods A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2 and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice, in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. Results The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. Conclusions The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

  1. A non-allergenic Ole e 1-like protein from birch pollen as a tool to design hypoallergenic vaccine candidates.

    Science.gov (United States)

    Marazuela, Eva G; Hajek, Roswitha; Villalba, Mayte; Barber, Domingo; Breiteneder, Heimo; Rodríguez, Rosalía; Batanero, Eva

    2012-02-01

    Recombinant DNA technology offers several approaches to convert allergens into hypoallergenic derivatives that can represent the basis of novel, safer and more effective forms of allergy vaccines. In this context, we used a new strategy for the design of a hypoallergenic derivative of Ole e 1, the main allergen of olive pollen. By screening a cDNA library from birch pollen, the clone BB18, encoding the birch counterpart of Ole e 1, was identified. In this study, BB18 has been produce in Pichia pastoris as a recombinant protein and immunologically characterized. The well-established non-allergenic properties of BB18 were used to generate a genetic variant of Ole e 1, named OB(55-58), by site-direct mutagenesis of four residues (E(55)V(56)G(57)Y(58)) in an IgE/IgG epitope of Ole e 1 by the corresponding ones in BB18 (SDSE). OB(55-58) was expressed in P. pastoris, purified to homogeneity and analyzed for IgE-reactivity by means of ELISA using sera from olive pollen allergic patients and rat basophil activation assay. T cell reactivity was assayed in a mouse model of Ole e 1 sensitization. The mutant OB(55-58) exhibited an impaired IgE reactivity, but not affected T cell reactivity, compared to wild type rOle e 1. This study emphasizes the usefulness of BB18 as a tool for epitope mapping and for engineering hypoallergenic derivatives of Ole e 1 as vaccine candidates for allergy prevention and treatment.

  2. In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

    Science.gov (United States)

    Nilsson, Ola B.; Adedoyin, Justus; Rhyner, Claudio; Neimert-Andersson, Theresa; Grundström, Jeanette; Berndt, Kurt D.; Crameri, Reto; Grönlund, Hans

    2011-01-01

    Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients. PMID:21931754

  3. Gene deleted live attenuated Leishmania vaccine candidates against visceral leishmaniasis elicit pro-inflammatory cytokines response in human PBMCs

    Science.gov (United States)

    Avishek, Kumar; Kaushal, Himanshu; Gannavaram, Sreenivas; Dey, Ranadhir; Selvapandiyan, Angamuthu; Ramesh, V.; Negi, Narender Singh; Dubey, Uma S.; Nakhasi, Hira L.; Salotra, Poonam

    2016-01-01

    Currently no effective vaccine is available for human visceral leishmaniasis(VL) caused by Leishmania donovani. Previously, we showed that centrin1 and p27gene deleted live attenuated Leishmania parasites (LdCen1−/− and Ldp27−/−) are safe, immunogenic and protective in animal models. Here, to assess the correlates of protection, we evaluated immune responses induced by LdCen1−/− and Ldp27−/− in human blood samples obtained from healthy, healed VL (HVL), post kala-azar dermal leishmaniasis(PKDL) and VL subjects. Both parasites infected human macrophages, as effectively as the wild type parasites. Further, LdCen1−/− and Ldp27−/− strongly stimulated production of pro-inflammatory cytokines including, IL-12, IFN-γ, TNF-α, IL-2, IL-6 and IL-17 in the PBMCs obtained from individuals with a prior exposure to Leishmania (HVL and PKDL). There was no significant stimulation of anti-inflammatory cytokines (IL-4 and IL-10). Induction of Th1 biased immune responses was supported by a remarkable increase in IFN-γ secreting CD4+ and CD8+ T cells and IL-17 secreting CD4+ cells in PBMCs from HVL cases with no increase in IL-10 secreting T cells. Hence, LdCen1−/− and Ldp27−/− are promising as live vaccine candidates against VL since they elicit strong protective immune response in human PBMCs from HVL, similar to the wild type parasite infection, mimicking a naturally acquired protection following cure. PMID:27624408

  4. In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity.

    Directory of Open Access Journals (Sweden)

    Ola B Nilsson

    Full Text Available Allergy and asthma to cat (Felis domesticus affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

  5. Heterologous protection elicited by candidate monomeric recombinant HIV-1 gp120 vaccine in the absence of cross neutralising antibodies in a macaque model

    Directory of Open Access Journals (Sweden)

    Page Mark

    2012-07-01

    Full Text Available Abstract Background Current data suggest that an efficacious human immunodeficiency virus type 1 (HIV-1 vaccine should elicit both adaptive humoral and cell mediated immune responses. Such a vaccine will also need to protect against infection from a range of heterologous viral variants. Here we have developed a simian-human immunodeficiency virus (SHIV based model in cynomolgus macaques to investigate the breadth of protection conferred by HIV-1W61D recombinant gp120 vaccination against SHIVsbg and SHIVSF33 challenge, and to identify correlates of protection. Results High titres of anti-envelope antibodies were detected in all vaccinees. The antibodies reacted with both the homologous HIV-1W61D and heterologous HIV-1IIIB envelope rgp120 which has an identical sequence to the SHIVsbg challenge virus. Significant titres of virus neutralising antibodies were detected against SHIVW61D expressing an envelope homologous with the vaccine, but only limited cross neutralisation against SHIVsbg, SHIV-4 and SHIVSF33 was observed. Protection against SHIVsbg infection was observed in vaccinated animals but none was observed against SHIVSF33 challenge. Transfer of immune sera from vaccinated macaques to naive recipients did not confer protection against SHIVsbg challenge. In a follow-up study, T cell proliferative responses detected after immunisation with the same vaccine against a single peptide present in the second conserved region 2 of HIV-1 W61D and HIV-1 IIIB gp120, but not SF33 gp120. Conclusions Following extended vaccination with a HIV-1 rgp120 vaccine, protection was observed against heterologous virus challenge with SHIVsbg, but not SHIVSF33. Protection did not correlate with serological responses generated by vaccination, but might be associated with T cell proliferative responses against an epitope in the second constant region of HIV-1 gp120. Broader protection may be obtained with recombinant HIV-1 envelope based vaccines formulated with

  6. Emerging Vaccine Informatics

    Directory of Open Access Journals (Sweden)

    Yongqun He

    2010-01-01

    Full Text Available Vaccine informatics is an emerging research area that focuses on development and applications of bioinformatics methods that can be used to facilitate every aspect of the preclinical, clinical, and postlicensure vaccine enterprises. Many immunoinformatics algorithms and resources have been developed to predict T- and B-cell immune epitopes for epitope vaccine development and protective immunity analysis. Vaccine protein candidates are predictable in silico from genome sequences using reverse vaccinology. Systematic transcriptomics and proteomics gene expression analyses facilitate rational vaccine design and identification of gene responses that are correlates of protection in vivo. Mathematical simulations have been used to model host-pathogen interactions and improve vaccine production and vaccination protocols. Computational methods have also been used for development of immunization registries or immunization information systems, assessment of vaccine safety and efficacy, and immunization modeling. Computational literature mining and databases effectively process, mine, and store large amounts of vaccine literature and data. Vaccine Ontology (VO has been initiated to integrate various vaccine data and support automated reasoning.

  7. Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

    Science.gov (United States)

    Kashino, S S; Abeijon, C; Qin, L; Kanunfre, K A; Kubrusly, F S; Silva, F O; Costa, D L; Campos, D; Costa, C H N; Raw, I; Campos-Neto, A

    2012-07-01

    Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

  8. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    Science.gov (United States)

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

  9. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis

    Science.gov (United States)

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. PMID:26197085

  10. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Mehdi Shahbazi

    Full Text Available Canine Visceral Leishmaniasis (CVL is a major veterinary and public health problem caused by Leishmania infantum (L. infantum in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

  11. B cells from knock-in mice expressing broadly neutralizing HIV antibody b12 carry an innocuous B cell receptor responsive to HIV vaccine candidates.

    Science.gov (United States)

    Ota, Takayuki; Doyle-Cooper, Colleen; Cooper, Anthony B; Doores, Katherine J; Aoki-Ota, Miyo; Le, Khoa; Schief, William R; Wyatt, Richard T; Burton, Dennis R; Nemazee, David

    2013-09-15

    Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic ("knock-in") mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol. 191: 3186-3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.

  12. Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2010-10-01

    virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles. Conclusions These results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.

  13. Heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein.

    Science.gov (United States)

    Beiss, Veronique; Spiegel, Holger; Boes, Alexander; Kapelski, Stephanie; Scheuermayer, Matthias; Edgue, Gueven; Sack, Markus; Fendel, Rolf; Reimann, Andreas; Schillberg, Stefan; Pradel, Gabriele; Fischer, Rainer

    2015-07-01

    Malaria is a vector-borne disease affecting more than two million people and accounting for more than 600,000 deaths each year, especially in developing countries. The most serious form of malaria is caused by Plasmodium falciparum. The complex life cycle of this parasite, involving pre-erythrocytic, asexual and sexual stages, makes vaccine development cumbersome but also offers a broad spectrum of vaccine candidates targeting exactly those stages. Vaccines targeting the sexual stage of P. falciparum are called transmission-blocking vaccines (TBVs). They do not confer protection for the vaccinated individual but aim to reduce or prevent the transmission of the parasite within a population and are therefore regarded as an essential tool in the fight against the disease. Malaria predominantly affects large populations in developing countries, so TBVs need to be produced in large quantities at low cost. Combining the advantages of eukaryotic expression with a virtually unlimited upscaling potential and a good product safety profile, plant-based expression systems represent a suitable alternative for the production of TBVs. We report here the high level (300 μg/g fresh leaf weight (FLW)) transient expression in Nicotiana benthamiana leaves of an effective TBV candidate based on a fusion protein F0 comprising Pfs25 and the C0-domain of Pfs230, and the implementation of a simple and cost-effective heat treatment step for purification that yields intact recombinant protein at >90% purity with a recovery rate of >70%. The immunization of mice clearly showed that antibodies raised against plant-derived F0 completely blocked the formation of oocysts in a malaria transmission-blocking assay (TBA) making F0 an interesting TBV candidate or a component of a multi-stage malaria vaccine cocktail.

  14. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  15. 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.

  16. Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models

    Directory of Open Access Journals (Sweden)

    Tania Rivera-Hernandez

    2016-06-01

    Full Text Available Group A Streptococcus (GAS is an important human pathogen responsible for both superficial infections and invasive diseases. Autoimmune sequelae may occur upon repeated infection. For this reason, development of a vaccine against GAS represents a major challenge, since certain GAS components may trigger autoimmunity. We formulated three combination vaccines containing the following: (i streptolysin O (SLO, interleukin 8 (IL-8 protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP], group A streptococcal C5a peptidase (SCPA, arginine deiminase (ADI, and trigger factor (TF; (ii the conserved M-protein-derived J8 peptide conjugated to ADI; and (iii group A carbohydrate lacking the N-acetylglucosamine side chain conjugated to ADI. We compared these combination vaccines to a “gold standard” for immunogenicity, full-length M1 protein. Vaccines were adjuvanted with alum, and mice were immunized on days 0, 21, and 28. On day 42, mice were challenged via cutaneous or subcutaneous routes. High-titer antigen-specific antibody responses with bactericidal activity were detected in mouse serum samples for all vaccine candidates. In comparison with sham-immunized mice, all vaccines afforded protection against cutaneous challenge. However, only full-length M1 protein provided protection in the subcutaneous invasive disease model.

  17. Safety and immunogenicity of the malaria vaccine candidate GMZ2 in malaria-exposed, adult individuals from Lambaréné, Gabon.

    Science.gov (United States)

    Mordmüller, Benjamin; Szywon, Katja; Greutelaers, Benedikt; Esen, Meral; Mewono, Ludovic; Treut, Carolin; Mürbeth, Raymund E; Chilengi, Roma; Noor, Ramadhani; Kilama, Wen L; Imoukhuede, Egeruan Babatunde; Imbault, Nathalie; Leroy, Odile; Theisen, Michael; Jepsen, Søren; Milligan, Paul; Fendel, Rolf; Kremsner, Peter G; Issifou, Saadou

    2010-09-24

    Malaria is still one of the major public health threats in sub-Saharan Africa. An effective vaccine could be a sustainable control measure that can be integrated into existing health infrastructures. The malaria vaccine candidate GMZ2 is a recombinant fusion protein of conserved parts of Plasmodium falciparum Glutamate Rich Protein and Merozoite Surface Protein 3 adjuvanted with aluminium hydroxide. GMZ2 is immunogenic and well tolerated in malaria-naive adults from Germany. To assess safety and immunogenicity in malaria-exposed individuals, 40 adults from Lambaréné, Gabon were randomly assigned to receive either 100 μg GMZ2 or a rabies control vaccine three times in monthly intervals. Both vaccines were well tolerated. One month after a full course of vaccination, GMZ2-vaccinated individuals had 1.4-fold (95% confidence interval: [1.1, 1.7]) higher baseline-corrected anti-GMZ2 antibody levels and more GMZ2-specific memory B-cells compared to the rabies group (p=0.039), despite a high prevalence of GMZ2-specific immune reactivity due to previous intense exposure to P. falciparum.

  18. "In vitro systems to characterize the immune response to HIV-1 and HIV-1 vaccine candidates", NIAID Workshop Report, Bethesda, August 4, 2010.

    Science.gov (United States)

    Malaspina, Angela; Rinaldo, Charles R; Sekaly, Rafick P; Flores, Jorge; D'Souza, Patricia M

    2011-06-24

    Although clinical trials are the ultimate way to prove vaccine safety and efficacy, the complexity, cost and time required to develop a product to enter human trials demand a serious, long-term investment. Lack of knowledge on immune correlates of protection from HIV infections makes investments in HIV vaccine research significantly risky. Preclinical testing of HIV vaccines is routinely carried out in non-human primate models however these studies have a significant cost and their predictive value is still questionable. The potential value of screening new HIV-1 vaccine candidates on human cells and tissues via high throughput in vitro systems that allow rapid, cost-effective and accurate predictions of in vivo immune responses would be enormous. A one-day workshop was convened by Division of AIDS, National Institutes of Health on August 4, 2010 to address the benefits and challenges of assessing HIV-1 vaccine responses in alternative ways. Consideration was given to the use of various in vitro model systems, human mucosal tissue explants and humanized mouse models as ways to predict immunogenicity and efficacy of HIV-1 vaccines early in the development process, and support decisions on whether a product may be worthy of moving into non-human primates or human trials. This report summarizes the outcome of the workshop.

  19. An In Silico Identification of Common Putative Vaccine Candidates against Treponema pallidum: A Reverse Vaccinology and Subtractive Genomics Based Approach

    Science.gov (United States)

    Kumar Jaiswal, Arun; Tiwari, Sandeep; Jamal, Syed Babar; Barh, Debmalya; Azevedo, Vasco; Soares, Siomar C.

    2017-01-01

    Sexually transmitted infections (STIs) are caused by a wide variety of bacteria, viruses, and parasites that are transmitted from one person to another primarily by vaginal, anal, or oral sexual contact. Syphilis is a serious disease caused by a sexually transmitted infection. Syphilis is caused by the bacterium Treponema pallidum subspecies pallidum. Treponema pallidum (T. pallidum) is a motile, gram-negative spirochete, which can be transmitted both sexually and from mother to child, and can invade virtually any organ or structure in the human body. The current worldwide prevalence of syphilis emphasizes the need for continued preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine targets and putative drugs against syphilis disease using reverse vaccinology and subtractive genomics. We compared 13 strains of T. pallidum using T. pallidum Nichols as the reference genome. Using an in silicoapproach, four pathogenic islands were detected in the genome of T. pallidum Nichols. We identified 15 putative antigenic proteins and sixdrug targets through reverse vaccinology and subtractive genomics, respectively, which can be used as candidate therapeutic targets in the future. PMID:28216574

  20. Plant-based production of recombinant Plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate.

    Science.gov (United States)

    Feller, Tatjana; Thom, Pascal; Koch, Natalie; Spiegel, Holger; Addai-Mensah, Otchere; Fischer, Rainer; Reimann, Andreas; Pradel, Gabriele; Fendel, Rolf; Schillberg, Stefan; Scheuermayer, Matthias; Schinkel, Helga

    2013-01-01

    Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1∶11.000 and 1∶39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using αPf38 antibodies demonstrated strong inhibition (≥60%) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by αPf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.

  1. Plant-based production of recombinant Plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Tatjana Feller

    Full Text Available Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38 using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1∶11.000 and 1∶39.000, respectively. In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using αPf38 antibodies demonstrated strong inhibition (≥60% of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by αPf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.

  2. A multi-stage malaria vaccine candidate targeting both transmission and asexual parasite life-cycle stages

    DEFF Research Database (Denmark)

    Theisen, Michael; Roeffen, Will; Singh, Susheel K;

    2014-01-01

    Effective control and eventual eradication of malaria drives the imperative need for clinical development of a malaria vaccine. Asexual parasite forms are responsible for clinical disease and death while apathogenic gametocytes are responsible for transmission from man to mosquito. Vaccines...

  3. Efficacy of marker vaccine candidate CP7 E2alf in piglets with maternally derived C-strain antibodies

    DEFF Research Database (Denmark)

    Rangelova, Desislava Yordanova; Nielsen, Jens; Strandbygaard, Bertel

    2012-01-01

    virus (CSFV) strain “Koslov”. CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could...... intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever...... to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8 weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4 weeks before farrowing with one dose of C-strain vaccine....

  4. Vaccination status and sequence of vaccinations as risk factors for hospitalisation among outpatients in a high mortality country

    DEFF Research Database (Denmark)

    Biai, Sidu; Rodrigues, Amabelia; Nielsen, Jens

    2011-01-01

    Most developing countries are implementing the WHO immunisation programme. Although vaccines reach most children, many modifications of the recommended schedule are observed in practice. We investigated the association between vaccination status and risk of hospitalisation in Guinea-Bissau....

  5. Construction and characterization of bivalent vaccine candidate expressing HspA and Mr18000 OMP from Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    Zheng Jiang; Ai-Long Huang; Xiao-Hong Tao; Pi-Long Wang

    2003-01-01

    AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with Mr18 000 and heat shock protein A (HspA) from Helicobacter pylori(H.pylori)in E.coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection.METHODS: The target gene of HspA was amplified from H.pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn Ⅰ, BamH Ⅰ simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp18, digested by restrictive endonuclease enzyme Hind Ⅲ and BamH Ⅰ simultaneously. pET32a(+)/HspA and Omp18 were recovered from 1% agarose gel by gel kit, and ligated with T4 ligase by BarmH Ⅰ digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp18 was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot.RESULTS: Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 %and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (Mr) of the expressed product was Mr 51 000,Mr of protein expressed by pET32a (+) was about Mr 20 000, and soluble expression product accounted for 18.96 % of total bacterial protein.After purification with Ni+2-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients′ serum infected with H. pylori and anti-Omp18 monoclone, suggesting that this protein had good antigenicity.CONCLUSION: The gene coding for H. pylori Mr18 000OMP and

  6. Pulmonary immunity and durable protection induced by the ID93/GLA-SE vaccine candidate against the hyper-virulent Korean Beijing Mycobacterium tuberculosis strain K.

    Science.gov (United States)

    Cha, Seung Bin; Kim, Woo Sik; Kim, Jong-Seok; Kim, Hongmin; Kwon, Kee Woong; Han, Seung Jung; Cho, Sang-Nae; Coler, Rhea N; Reed, Steven G; Shin, Sung Jae

    2016-04-27

    The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4(+) T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFN-γ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8(+) central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent.

  7. A live attenuated H7N7 candidate vaccine virus induces neutralizing antibody that confers protection from challenge in mice, ferrets and monkeys

    Science.gov (United States)

    A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of HP A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (...

  8. Evaluation of the Efficacy, Potential for Vector Transmission, and Duration of Immunity of MP-12, an Attenuated Rift Valley Fever Virus Vaccine Candidate, in Sheep

    Science.gov (United States)

    2015-08-01

    and L (24–26), thus significantly reducing the risk of reversion to viru - lence that might otherwise arise from a recombination event with other viruses ...Transmission, and Duration of Immunity of MP-12, an Attenuated Rift Valley Fever Virus Vaccine Candidate, in Sheep 5a. CONTRACT NUMBER 5b. GRANT...Immunol. 2015; 22(8):930-937 14. ABSTRACT Rift Valley fever virus (RVFV) causes serious disease in ruminants and humans in Africa. In North America

  9. Construction of an American mink Bacterial Artificial Chromosome (BAC library and sequencing candidate genes important for the fur industry

    Directory of Open Access Journals (Sweden)

    Christensen Knud

    2011-07-01

    Full Text Available Abstract Background Bacterial artificial chromosome (BAC libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. Results Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison. The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs, representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes. Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome. Conclusions The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the

  10. A Phase I study evaluating the safety and immunogenicity of MVA85A, a candidate TB vaccine, in HIV-infected adults.

    Science.gov (United States)

    Minassian, Angela M; Rowland, Rosalind; Beveridge, Natalie E R; Poulton, Ian D; Satti, Iman; Harris, Stephanie; Poyntz, Hazel; Hamill, Matthew; Griffiths, Kristin; Sander, Clare R; Ambrozak, David R; Price, David A; Hill, Brenna J; Casazza, Joseph P; Douek, Daniel C; Koup, Richard A; Roederer, Mario; Winston, Alan; Ross, Jonathan; Sherrard, Jackie; Rooney, Guy; Williams, Nicola; Lawrie, Alison M; Fletcher, Helen A; Pathan, Ansar A; McShane, Helen

    2011-01-01

    Objectives Control of the tuberculosis (TB) epidemic is a global health priority and one that is likely to be achieved only through vaccination. The critical overlap with the HIV epidemic requires any effective TB vaccine regimen to be safe in individuals who are infected with HIV. The objectives of this clinical trial were to evaluate the safety and immunogenicity of a leading candidate TB vaccine, MVA85A, in healthy, HIV-infected adults. Design This was an open-label Phase I trial, performed in 20 healthy HIV-infected, antiretroviral-naïve subjects. Two different doses of MVA85A were each evaluated as a single immunisation in 10 subjects, with 24 weeks of follow-up. The safety of MVA85A was assessed by clinical and laboratory markers, including regular CD4 counts and HIV RNA load measurements. Vaccine immunogenicity was assessed by ex vivo interferon γ (IFN-γ) ELISpot assays and flow-cytometric analysis. Results MVA85A was safe in subjects with HIV infection, with an adverse-event profile comparable with historical data from previous trials in HIV-uninfected subjects. There were no clinically significant vaccine-related changes in CD4 count or HIV RNA load in any subjects, and no evidence from qPCR analyses to indicate that MVA85A vaccination leads to widespread preferential infection of vaccine-induced CD4 T cell populations. Both doses of MVA85A induced an antigen-specific IFN-γ response that was durable for 24 weeks, although of a lesser magnitude compared with historical data from HIV-uninfected subjects. The functional quality of the vaccine-induced T cell response in HIV-infected subjects was remarkably comparable with that observed in healthy HIV-uninfected controls, but less durable. Conclusion MVA85A is safe and immunogenic in healthy adults infected with HIV. Further safety and efficacy evaluation of this candidate vaccine in TB- and HIV-endemic areas is merited.

  11. A phase I randomized clinical trial of candidate human immunodeficiency virus type 1 vaccine MVA.HIVA administered to Gambian infants.

    Directory of Open Access Journals (Sweden)

    Muhammed O Afolabi

    Full Text Available BACKGROUND: A vaccine to decrease transmission of human immunodeficiency virus type 1 (HIV-1 during breast-feeding would complement efforts to eliminate infant HIV-1 infection by antiretroviral therapy. Relative to adults, infants have distinct immune development, potentially high-risk of transmission when exposed to HIV-1 and rapid progression to AIDS when infected. To date, there have been only three published HIV-1 vaccine trials in infants. TRIAL DESIGN: We conducted a randomized phase I clinical trial PedVacc 001 assessing the feasibility, safety and immunogenicity of a single dose of candidate vaccine MVA.HIVA administered intramuscularly to 20-week-old infants born to HIV-1-negative mothers in The Gambia. METHODS: Infants were followed to 9 months of age with assessment of safety, immunogenicity and interference with Expanded Program on Immunization (EPI vaccines. The trial is the first stage of developing more complex prime-boost vaccination strategies against breast milk transmission of HIV-1. RESULTS: From March to October 2010, 48 infants (24 vaccine and 24 no-treatment were enrolled with 100% retention. The MVA.HIVA vaccine was safe with no difference in adverse events between vaccinees and untreated infants. Two vaccine recipients (9% and no controls had positive ex vivo interferon-γ ELISPOT assay responses. Antibody levels elicited to the EPI vaccines, which included diphtheria, tetanus, whole-cell pertussis, hepatitis B virus, Haemophilus influenzae type b and oral poliovirus, reached protective levels for the vast majority and were similar between the two arms. CONCLUSIONS: A single low-dose of MVA.HIVA administered to 20-week-old infants in The Gambia was found to be safe and without interference with the induction of protective antibody levels by EPI vaccines, but did not alone induce sufficient HIV-1-specific responses. These data support the use of MVA carrying other transgenes as a boosting vector within more complex prime

  12. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    Science.gov (United States)

    Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

    2013-01-01

    Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  13. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    Directory of Open Access Journals (Sweden)

    Qicheng Zhang

    Full Text Available Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1 viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1 and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs. ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  14. Comparison of microarray-predicted closest genomes to sequencing for poliovirus vaccine strain similarity and influenza A phylogeny.

    Science.gov (United States)

    Maurer-Stroh, Sebastian; Lee, Charlie W H; Patel, Champa; Lucero, Marilla; Nohynek, Hanna; Sung, Wing-Kin; Murad, Chrysanti; Ma, Jianmin; Hibberd, Martin L; Wong, Christopher W; Simões, Eric A F

    2016-03-01

    We evaluate sequence data from the PathChip high-density hybridization array for epidemiological interpretation of detected pathogens. For influenza A, we derive similar relative outbreak clustering in phylogenetic trees from PathChip-derived compared to classical Sanger-derived sequences. For a positive polio detection, recent infection could be excluded based on vaccine strain similarity.

  15. CASCAD : a database of annotated candidate single nucleotide polymorphisms associated with expressed sequences

    NARCIS (Netherlands)

    Guryev, Victor; Berezikov, Eugene; Cuppen, Edwin

    2005-01-01

    BACKGROUND: With the recent progress made in large-scale genome sequencing projects a vast amount of novel data is becoming available. A comparative sequence analysis, exploiting sequence information from various resources, can be used to uncover hidden information, such as genetic variation. Althou

  16. Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis.

    Science.gov (United States)

    Cheng, Weiqiang; Curti, Elena; Rezende, Wanderson C; Kwityn, Clifford; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan; Joshi, Sangeeta B; Volkin, David B; Hotez, Peter J; Middaugh, C Russell; Bottazzi, Maria Elena

    2013-11-01

    A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0-8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

  17. Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar enteritidis as a potential vaccine candidate against homologous challenge in chickens.

    Science.gov (United States)

    Okamura, Masashi; Matsumoto, Wakako; Seike, Fumio; Tanaka, Yuuya; Teratani, Chie; Tozuka, Maki; Kashimoto, Takashige; Takehara, Kazuaki; Nakamura, Masayuki; Yoshikawa, Yasuhiro

    2012-06-01

    FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.

  18. Phase I Safety and Immunogenicity Study of a Candidate Meningococcal Disease Vaccine Based on Neisseria lactamica Outer Membrane Vesicles▿

    Science.gov (United States)

    Gorringe, Andrew R.; Taylor, Stephen; Brookes, Charlotte; Matheson, Mary; Finney, Michelle; Kerr, Moyra; Hudson, Michael; Findlow, Jamie; Borrow, Ray; Andrews, Nick; Kafatos, George; Evans, Cariad M.; Read, Robert C.

    2009-01-01

    Natural immunity to meningococcal disease in young children is associated epidemiologically with carriage of commensal Neisseria species, including Neisseria lactamica. We have previously demonstrated that outer membrane vesicles (OMVs) from N. lactamica provide protection against lethal challenge in a mouse model of meningococcal septicemia. We evaluated the safety and immunogenicity of an N. lactamica OMV vaccine in a phase I placebo-controlled, double-blinded clinical trial. Ninety-seven healthy young adult male volunteers were randomized to receive three doses of either an OMV vaccine or an Alhydrogel control. Subsequently, some subjects who had received the OMV vaccine also received a fourth dose of OMV vaccine, 6 months after the third dose. Injection site reactions were more frequent in the OMV-receiving group, but all reactions were mild or moderate in intensity. The OMV vaccine was immunogenic, eliciting rises in titers of immunoglobulin G (IgG) against the vaccine OMVs, together with a significant booster response, as determined by an enzyme-linked immunosorbent assay. Additionally, the vaccine induced modest cross-reactive immunity to six diverse strains of serogroup B Neisseria meningitidis, including IgG against meningococcal OMVs, serum bactericidal antibodies, and opsonophagocytic activity. The percentages of subjects showing ≥4-fold rises in bactericidal antibody titer obtained were similar to those previously reported for the Norwegian meningococcal OMV vaccine against the same heterologous meningococcal strain panel. In conclusion, this N. lactamica OMV vaccine is safe and induces a weak but broad humoral immune response to N. meningitidis. PMID:19553555

  19. Phase I safety and immunogenicity study of a candidate meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles.

    Science.gov (United States)

    Gorringe, Andrew R; Taylor, Stephen; Brookes, Charlotte; Matheson, Mary; Finney, Michelle; Kerr, Moyra; Hudson, Michael; Findlow, Jamie; Borrow, Ray; Andrews, Nick; Kafatos, George; Evans, Cariad M; Read, Robert C

    2009-08-01

    Natural immunity to meningococcal disease in young children is associated epidemiologically with carriage of commensal Neisseria species, including Neisseria lactamica. We have previously demonstrated that outer membrane vesicles (OMVs) from N. lactamica provide protection against lethal challenge in a mouse model of meningococcal septicemia. We evaluated the safety and immunogenicity of an N. lactamica OMV vaccine in a phase I placebo-controlled, double-blinded clinical trial. Ninety-seven healthy young adult male volunteers were randomized to receive three doses of either an OMV vaccine or an Alhydrogel control. Subsequently, some subjects who had received the OMV vaccine also received a fourth dose of OMV vaccine, 6 months after the third dose. Injection site reactions were more frequent in the OMV-receiving group, but all reactions were mild or moderate in intensity. The OMV vaccine was immunogenic, eliciting rises in titers of immunoglobulin G (IgG) against the vaccine OMVs, together with a significant booster response, as determined by an enzyme-linked immunosorbent assay. Additionally, the vaccine induced modest cross-reactive immunity to six diverse strains of serogroup B Neisseria meningitidis, including IgG against meningococcal OMVs, serum bactericidal antibodies, and opsonophagocytic activity. The percentages of subjects showing > or =4-fold rises in bactericidal antibody titer obtained were similar to those previously reported for the Norwegian meningococcal OMV vaccine against the same heterologous meningococcal strain panel. In conclusion, this N. lactamica OMV vaccine is safe and induces a weak but broad humoral immune response to N. meningitidis.

  20. Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum

    Directory of Open Access Journals (Sweden)

    Vahid NASIRI

    2016-10-01

    Full Text Available Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development. 

  1. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    Science.gov (United States)

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  2. Targeted sequencing of 351 candidate genes for epileptic encephalopathy in a large cohort of patients

    DEFF Research Database (Denmark)

    de Kovel, Carolien G F; Brilstra, Eva H; van Kempen, Marjan J A;

    2016-01-01

    BACKGROUND: Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation. METHODS: To increase the number of validated EE...

  3. Molecular and phylogenetic analysis of the partial tams1 gene sequence of a vaccine strain of Theileria annulata

    Directory of Open Access Journals (Sweden)

    Majid Esmaelizad

    2011-12-01

    Full Text Available The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata and is highly variable. In this study, the partial nucleotide (nt sequence of the Tams1 (522 nt gene of Iranian vaccine strain (Vaccine-ir68 recovered from an outbreak of disease in Iran was determined and compared with the corresponding sequences of eighteen previously published Tams1 genes. According to sequencing result, a novel amino acid substitution at the Tams1 region (K→Q was found exclusively in isolate Vaccine-ir68. Genetic distance values, estimated from the sequence data, revealed striking sequence homology (approximately 99% between Vaccine-ir68 isolate and Tunisian isolates, showing that they were same isolates of T. annulata which were spread in these areas. The phylogenetic tree constructed based on the sequence alignment of 19 Tams1 coding regions was distinctly divided into five lineages. There might be some unknown tick carrier birds immigrating to the different geographical regions. These birds have an effective role to distribute the T. annulata species in North Africa, Palestine and Iran.

  4. CD8+ T-cell Responses in Flavivirus-Naive Individuals Following Immunization with a Live-Attenuated Tetravalent Dengue Vaccine Candidate.

    Science.gov (United States)

    Chu, Haiyan; George, Sarah L; Stinchcomb, Dan T; Osorio, Jorge E; Partidos, Charalambos D

    2015-11-15

    We are developing a live-attenuated tetravalent dengue vaccine (TDV) candidate based on an attenuated dengue 2 virus (TDV-2) and 3 chimeric viruses containing the premembrane and envelope genes of dengue viruses (DENVs) -1, -3, and -4 expressed in the context of the attenuated TDV-2 genome (TDV-1, TDV-3, and TDV-4, respectively). In this study, we analyzed and characterized the CD8(+) T-cell response in flavivirus-naive human volunteers vaccinated with 2 doses of TDV 90 days apart via the subcutaneous or intradermal routes. Using peptide arrays and intracellular cytokine staining, we demonstrated that TDV elicits CD8(+) T cells targeting the nonstructural NS1, NS3, and NS5 proteins of TDV-2. The cells were characterized by the production of interferon-γ, tumor necrosis factor-α, and to a lesser extent interleukin-2. Responses were highest on day 90 after the first dose and were still detectable on 180 days after the second dose. In addition, CD8(+) T cells were multifunctional, producing ≥2 cytokines simultaneously, and cross-reactive to NS proteins of the other 3 DENV serotypes. Overall, these findings describe the capacity of our candidate dengue vaccine to elicit cellular immune responses and support the further evaluation of T-cell responses in samples from future TDV clinical trials.

  5. Effect of the pre-erythrocytic candidate malaria vaccine RTS,S/AS01E on blood stage immunity in young children

    DEFF Research Database (Denmark)

    Bejon, Philip; Cook, Jackie; Bergmann-Leitner, Elke

    2011-01-01

    -linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. Results. Antibody......(See the article by Greenhouse et al, on pages 19-26.) Background. RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. Methods. We measured, by enzyme...... concentrations to AMA-1, EBA-175, and MSP-1(42) decreased with age during the first year of life, then increased to 32 months of age. Anti-MSP-3 antibody concentrations gradually increased, and GIA gradually decreased up to 32 months. Vaccination with RTS,S/AS01(E) resulted in modest reductions in AMA-1, EBA-175...

  6. Salmonella Enteritidis with double deletion in phoPfliC--a potential live Salmonella vaccine candidate with novel characteristics for use in chickens.

    Science.gov (United States)

    Methner, Ulrich; Barrow, Paul A; Berndt, Angela; Rychlik, Ivan

    2011-04-12

    Salmonella Enteritidis mutants with deletions in phoP, fliC or phoPfliC were tested for their virulence and their ability to induce parameters of the innate and adaptive immunity in addition to their potential for serological differentiation between vaccinated, non-vaccinated and infected chickens. The double phoPfliC deletion mutant was sufficiently attenuated but not diminished in its capability to inhibit the caecal colonisation and systemic invasion of homologous Salmonella Enteritidis shortly after administration of the vaccine strain to very young chicks. Immunisation with the attenuated ΔphoPfliC mutant resulted in protective effects which were only slightly and insignificantly lower than after "immunisation" with a Salmonella wild-type strain, indicating the capability to induce an intense adaptive immune response and protection against Salmonella exposure in older chickens. The deletion in fliC enabled the effective the differentiation between immunised and infected chickens using a commercially available ELISA kit. The double phoPfliC deletion mutant of Salmonella Enteritidis might be a potential and promising live Salmonella vaccine candidate with novel characteristics for use in poultry.

  7. The Malaria Vaccine Candidate GMZ2 Elicits Functional Antibodies in Individuals From Malaria Endemic and Non-Endemic Areas

    DEFF Research Database (Denmark)

    Jepsen, Micha Phill Grønholm; Jogdand, Prajakta S; Singh, Susheel K

    2013-01-01

    against Plasmodium falciparum. Results. We showed that the maximum level of immunoglobulin G (IgG) antibodies obtained by GMZ2 vaccination is independent of ethnicity, time under malaria-exposure, and vaccine dose and that GMZ2 elicits high levels of functionally active IgG antibodies. Both, malaria......-naive adults and malaria-exposed preschool children elicit vaccine-specific antibodies with broad inhibitory activity against geographically diverse P. falciparum isolates. Peptide-mapping studies of IgG subclass responses identified IgG3 against a peptide derived from MSP3 as the strongest predictor...

  8. Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

    Directory of Open Access Journals (Sweden)

    Chaudhari Atul A

    2012-05-01

    Full Text Available Abstract In order to develop a novel, safe and immunogenic fowl typhoid (FT vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene E expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control, group B (orally immunized, group C (subcutaneously immunized and group D (intramuscularly immunized. The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi. Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4+ and CD8+ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine

  9. Targeted sequencing of 351 candidate genes for epileptic encephalopathy in a large cohort of patients

    DEFF Research Database (Denmark)

    de Kovel, Carolien G F; Brilstra, Eva H; van Kempen, Marjan J A

    2016-01-01

    BACKGROUND: Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation. METHODS: To increase the number of validated EE......, intellectual disability gene can lead to phenotypes that get classified as EE in the clinic. We confirmed existing literature reports that de novo loss-of-function HNRNPUmutations lead to severe developmental delay and febrile seizures in the first year of life....

  10. Optimized Blanching Reduces the Host Cell Protein Content and Substantially Enhances the Recovery and Stability of Two Plant-Derived Malaria Vaccine Candidates.

    Science.gov (United States)

    Menzel, Stephan; Holland, Tanja; Boes, Alexander; Spiegel, Holger; Bolzenius, Johanna; Fischer, Rainer; Buyel, Johannes F

    2016-01-01

    Plants provide an advantageous expression platform for biopharmaceutical proteins because of their low pathogen burden and potential for inexpensive, large-scale production. However, the purification of target proteins can be challenging due to issues with extraction, the removal of host cell proteins (HCPs), and low expression levels. The heat treatment of crude extracts can reduce the quantity of HCPs by precipitation thus increasing the purity of the target protein and streamlining downstream purification. In the overall context of downstream process (DSP) development for plant-derived malaria vaccine candidates, we applied a design-of-experiments approach to enhance HCP precipitation from Nicotiana benthamiana extracts generated after transient expression, using temperatures in the 20-80°C range, pH values of 3.0-8.0 and incubation times of 0-60 min. We also investigated the recovery of two protein-based malaria vaccine candidates under these conditions and determined their stability in the heat-treated extract while it was maintained at room temperature for 24 h. The heat precipitation of HCPs was also carried out by blanching intact plants in water or buffer prior to extraction in a blender. Our data show that all the heat precipitation methods reduced the amount of HCP in the crude plant extracts by more than 80%, simplifying the subsequent DSP steps. Furthermore, when the heat treatment was performed at 80°C rather than 65°C, both malaria vaccine candidates were more stable after extraction and the recovery of both proteins increased by more than 30%.

  11. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

    Directory of Open Access Journals (Sweden)

    Tamborrini Marco

    2011-12-01

    Full Text Available Abstract Background In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP. Methods The highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds. Results Intramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins. Conclusion A virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal

  12. Ensayo preclínico de la vacuna candid #1 producida en Argentina contra la Fiebre Hemorrágica Argentina Preclinical assay of Candid #1 vaccine against Argentine Hemorrhagic Fever made in Argentina

    Directory of Open Access Journals (Sweden)

    Ana M. Ambrosio

    2005-08-01

    Full Text Available Se comparó en cobayos la seguridad, inmunogenicidad y eficacia protectora de un lote de vacuna Candid #1(C#1 fabricada en Estados Unidos de América (EE.UU. y distintos lotes de la misma vacuna fabricados en Argentina (Arg. El lote TSI 5-1-92 (EE.UU. y los lotes Exp Nº 3, 7A y 8A (Arg fueron inoculados (0.5 ml, IM en cobayos de 250-400 g. Para cada ensayo diez animales recibieron solución fisiológica y sirvieron como control. Todos fueron desafiados con la cepa patógena P23790 de virus Junin. Se registró: a temperatura rectal, b peso corporal, c presencia de anticuerpos neutralizantes (AcNT pre y post-vacunación, d respuesta al desafío. Todos los animales vacunados desarrollaron AcNT anti virus Junin (rango = 40- 81920 y sobrevivieron al desafío. En cada grupo control 8/10 animales murieron (día 23.3 ± 5.4 post-desafío. Los cobayos resultaron idénticamente protegidos de una descarga letal de virus Junin por la vacuna importada y los diferentes lotes de C#1 producidos en Argentina.Candid #1 vaccine against Argentine Hemorrhagic Fever produced in USA versus lots of the same vaccine made in Argentina were compared in guinea pigs regarding safety, immunogenicity and protective efficacy against a challenge with pathogenic Junin virus. Lots Nº Exp 3, 7A and 8A of Argentine origin as well as lot TSI 5-1-92 from USA were inoculated in guinea pigs of 250-400 g in two consecutive assays. Ten animals inoculated with saline performed as normal controls in each experiment. Parameters studied were: a temperature; b body weight; c neutralizing antibodies to Junin virus; d response to viral challenge. Animals gained weight and remained normothermic up to the challenge. Guinea pigs that received Candid #1 from any manufacturer elicited neutralizing antibodies to Junin virus (titles from 40 to 81920 and survived to challenge whilst 8/10 animals died in each control group. Data presented demonstrated that Candid #1 vaccines from USA or Argentine

  13. 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

    2015-01-09

    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.

  14. Recombinant Leishmania tarentolae expressing the A2 virulence gene as a novel candidate vaccine against visceral leishmaniasis.

    Science.gov (United States)

    Mizbani, Amir; Taheri, Tahereh; Zahedifard, Farnaz; Taslimi, Yasaman; Azizi, Hiva; Azadmanesh, Kayhan; Papadopoulou, Barbara; Rafati, Sima

    2009-12-10

    Visceral leishmaniasis is the most severe form of leishmaniasis. To date, there is no effective vaccine against this disease. Many antigens have been examined so far as protein- or DNA-based vaccines, but none of them conferred complete long-term protection. The use of live attenuated vaccines has recently emerged as a promising vaccination strategy. In this study, we stably expressed the Leishmania donovani A2 antigen in Leishmania tarentolae, a non-pathogenic member of the genus Leishmania, and evaluated its protective efficacy as a live vaccine against L. infantum challenge. Our results show that a single intraperitoneal administration of the A2-recombinant L. tarentolae strain protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-gamma production prior and after challenge. This is accompanied by reduced levels of IL-5 production after challenge, leading to a potent Th1 immune response. In contrast, intravenous injection elicited a Th2 type response, characterized by higher levels of IL-5 and high humoral immune response, resulting in a less efficient protection. All together, these results indicate the promise of A2-expressing L. tarentolae as a safe live vaccine against visceral leishmaniasis.

  15. Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1 vaccine candidates containing stabilized mutations in the P/C and L genes

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    Skiadopoulos Mario H

    2007-07-01

    Full Text Available Abstract Background Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1 mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts attenuating (att mutations primarily in the P/C gene, namely CR84GHNT553A (two point mutations used together as a set and CΔ170 (a short deletion mutation, and two ts att mutations in the L gene, namely LY942A (a point mutation, and LΔ1710–11 (a short deletion, the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs. Results The rHPIV1 mutant bearing the novel LΔ1710–11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates were highly ts, with shut-off temperatures of 38°C and 35°C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G/Δ170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host. Conclusion The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.

  16. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael

    2011-01-01

    consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library......BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...... report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each...

  17. Implication of respiratory syncytial virus (RSV) F transgene sequence heterogeneity observed in Phase 1 evaluation of MEDI-534, a live attenuated parainfluenza type 3 vectored RSV vaccine.

    Science.gov (United States)

    Yang, Chin-Fen; Wang, C Kathy; Malkin, Elissa; Schickli, Jeanne H; Shambaugh, Cindy; Zuo, Fengrong; Galinski, Mark S; Dubovsky, Filip; Tang, Roderick S

    2013-06-10

    MEDI-534 is the first live vectored RSV vaccine candidate to be evaluated in seronegative children. It consists of the bovine parainfluenza virus type 3 (PIV3) genome with substituted human PIV3 F and HN glycoproteins engineered to express RSV F protein. A Phase 1 study of 49 healthy RSV and PIV3 seronegative children 6 to <24 months of age demonstrated an acceptable safety profile at the following doses: 10(4), 10(5) and 10(6)TCID50. After 3 doses of MEDI-534 at 10(6)TCID50, administered at 0, 2 and 4 month intervals, 100% of subjects seroresponded to PIV3, whereas only 50% seroresponded to RSV. To investigate the discordance in seroresponse rates, the RSV F transgene and its flanking non-coding nucleotides were sequenced from shed virus recovered from the nasal washes of 24 MEDI-534-vaccinated children. Eleven out of 24 samples contained no nucleotide changes in the analyzed region. The other 13 samples contained mixtures of variant subpopulations. Fifty-five percent exhibited changes in the transcription termination poly A gene sequences of the upstream bPIV3N gene while 21% had variant subpopulations in the RSV F open reading frame that resulted in pre-mature stop codons. Both types of changes are expected to reduce RSV F expression. Evaluation of the administered vaccine by dual immunofluorescence staining showed ~2.5% variants with low or no RSV F expression while single nucleotide primer extension detected ~1% variation at nucleotide 2045 that resulted in a pre-mature translational termination at codon 85. An association between shedding of variants and lower RSV F serological response was observed but it was not possible to establish a definitive clinical significance due to the small number of subjects in this study.

  18. DNA sequence analysis of the Hind III M fragment from Chinese vaccine strain of vaccinia virus.

    Science.gov (United States)

    Liu, V J; Jin, Q; Jin, D Y; Hou, Y D

    1989-01-01

    The complete DNA sequence of the Hind III M fragment of vaccinia virus (VV) Tian Tan strain genome was determined by the dideoxynucleotide chain termination method. Three open reading frames (ORFs) were identified in the complementary strand of the sequence, comprised of 2218bp. Among them, ORF K1 initiates its transcription at -45 of the Hind III K fragment. The deduced peptide encoded by K1 contains 284 amino acids with a calculated molecular weight of 32.48 KDa. Its sequence is homologous to the host range protein of VV Copenhagen strain; the variation is only 2.46% at the amino acid level. ORF M2 could encode a peptide of 21.94 KDa with 196 amino acids. This gene was shown to be homologous to that of the 23 KDa peptide of herpes simplex virus type I. A non-coding region of 204bp located between K1 and M2 is rich in palindromic structures. ORF M1 extends its 3' terminus into the Hind III N fragment. Within the M fragment, M1 can only encode 212 amino acids. The major part of ORF M1 is very similar to the M portion of a possible alpha-amanitin resistance gene isolated from VV-WR strain. This work provides a molecular foundation in the construction of a new insertion vector for the preparation of a recombinant vaccinia virus to be used as a polyvalent live vaccine.

  19. Candidate genes revealed by a genome scan for mosquito resistance to a bacterial insecticide: sequence and gene expression variations

    Directory of Open Access Journals (Sweden)

    David Jean-Philippe

    2009-11-01

    Full Text Available Abstract Background Genome scans are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation, but on their own, they are often helpless at identifying the specific gene(s or mutation(s targeted by selection. This shortcoming is hopefully bound to disappear in the near future, thanks to the wealth of new genomic resources that are currently being developed for many species. In this article, we provide a foretaste of this exciting new era by conducting a genome scan in the mosquito Aedes aegypti with the aim to look for candidate genes involved in resistance to Bacillus thuringiensis subsp. israelensis (Bti insecticidal toxins. Results The genome of a Bti-resistant and a Bti-susceptible strains was surveyed using about 500 MITE-based molecular markers, and the loci showing the highest inter-strain genetic differentiation were sequenced and mapped on the Aedes aegypti genome sequence. Several good candidate genes for Bti-resistance were identified in the vicinity of these highly differentiated markers. Two of them, coding for a cadherin and a leucine aminopeptidase, were further examined at the sequence and gene expression levels. In the resistant strain, the cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection (e.g. an excess of high compared to intermediate frequency mutations, as well as a significant under-expression compared to the susceptible strain. Conclusion Both sequence and gene expression analyses agree to suggest a role for positive selection in the evolution of this cadherin gene in the resistant strain. However, it is unlikely that resistance to Bti is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with Bti resistance in Ae. aegypti. Beyond these results, this article illustrates how genome scans can build on the body of new genomic information (here, full

  20. Discovery and functional prioritization of Parkinson's disease candidate genes from large-scale whole exome sequencing

    NARCIS (Netherlands)

    I. Jansen (Iris); Ye, H. (Hui); Heetveld, S. (Sasja); Lechler, M.C. (Marie C.); Michels, H. (Helen); Seinstra, R.I. (Renée I.); Lubbe, S.J. (Steven J.); Drouet, V. (Valérie); S. Lesage (Suzanne); E. Majounie (Elisa); Gibbs, J.R. (J.Raphael); M.A. Nalls (Michael); M. Ryten (Mina); Botia, J.A. (Juan A.); J. Vandrovcova (Jana); J. Simón-Sánchez (Javier); Castillo-Lizardo, M. (Melissa); P. Rizzu (Patrizia); Blauwendraat, C. (Cornelis); Chouhan, A.K. (Amit K.); Li, Y. (Yarong); Yogi, P. (Puja); N. Amin (Najaf); C.M. van Duijn (Cock); Morris, H.R. (Huw R.); Brice, A. (Alexis); A. Singleton (Andrew); David, D.C. (Della C.); Nollen, E.A. (Ellen A.); A. Jain (Ashok); J.M. Shulman; P. Heutink (Peter); D.G. Hernandez (Dena); S. Arepalli (Sampath); J. Brooks (Janet); Price, R. (Ryan); Nicolas, A. (Aude); S. Chong (Sean); M.R. Cookson (Mark); A. Dillman (Allissa); M. Moore (Matt); B.J. Traynor (Bryan); A. Singleton (Andrew); V. Plagnol (Vincent); Nicholas W Wood,; U.-M. Sheerin (Una-Marie); Jose M Bras,; K. Charlesworth (Kate); M. Gardner (Mac); R. Guerreiro (Rita); D. Trabzuni (Danyah); Hardy, J. (John); M. Sharma; M. Saad (Mohamad); Javier Simón-Sánchez,; C. Schulte (Claudia); J.C. Corvol (Jean-Christophe); Dürr, A. (Alexandra); M. Vidailhet (M.); S. Sveinbjörnsdóttir (Sigurlaug); R.A. Barker (Roger); Caroline H Williams-Gray,; Y. Ben-Shlomo; H.W. Berendse (Henk W.); K.D. van Dijk (Karin); D. Berg (Daniela); K. Brockmann; K.D. Wurster (Kathrin); Mätzler, W. (Walter); Gasser, T. (Thomas); M. Martinez (Maria); R.M.A. de Bie (Rob); A. Biffi (Alessandro); D. Velseboer (Daan); B.R. Bloem (Bastiaan); B. Post (Bart); M. Wickremaratchi (Mirdhu); B. van de Warrenburg (Bart); Z. Bochdanovits (Zoltan); M. von Bonin (Malte); H. Pétursson (Hjörvar); O. Riess (Olaf); D.J. Burn (David); Lubbe, S. (Steven); Cooper, J.M. (J Mark); N.H. McNeill (Nathan); Schapira, A. (Anthony); Lungu, C. (Codrin); Chen, H. (Honglei); Dong, J. (Jing); Chinnery, P.F. (Patrick F.); G. Hudson (Gavin); Clarke, C.E. (Carl E.); C. Moorby (Catriona); C. Counsell (Carl); P. Damier (Philippe); J.-F. Dartigues; P. Deloukas (Panagiotis); E. Gray (Emma); T. Edkins (Ted); Hunt, S.E. (Sarah E.); S.C. Potter (Simon); A. Tashakkori-Ghanbaria (Avazeh); G. Deuschl (Günther); D. Lorenz (Delia); D.T. Dexter (David); F. Durif (Frank); J. Evans (Jonathan Mark); Langford, C. (Cordelia); T. Foltynie (Thomas); A.M. Goate (Alison); C. Harris (Clare); J.J. van Hilten (Jacobus); A. Hofman (Albert); J.R. Hollenbeck (John R.); J.L. Holton (Janice); Hu, M. (Michele); X. Huang (Xiaohong); Illig, T. (Thomas); P.V. Jónsson (Pálmi); J.-C. Lambert; S.S. O'Sullivan (Sean); T. Revesz (Tamas); K. Shaw (Karen); A.J. Lees (Andrew); P. Lichtner (Peter); P. Limousin (Patricia); G. Lopez; Escott-Price, V. (Valentina); J. Pearson (Justin); N. Williams (Nigel); E. Mudanohwo (Ese); J.S. Perlmutter (Joel); Pollak, P. (Pierre); F. Rivadeneira Ramirez (Fernando); A.G. Uitterlinden (André); S.J. Sawcer (Stephen); H. Scheffer (Hans); I. Shoulson (Ira); L. Shulman (Lee); Smith, C. (Colin); R. Walker (Robert); C.C.A. Spencer (Chris C.); A. Strange (Amy); H. Stefansson (Hreinn); F. Bettella (Francesco); J-A. Zwart (John-Anker); Stockton, J.D. (Joanna D.); D. Talbot; C.M. Tanner (Carlie); F. Tison (François); S. Winder-Rhodes (Sophie); K.P. Bhatia (Kailash)

    2017-01-01

    textabstractBackground: Whole-exome sequencing (WES) has been successful in identifying genes that cause familial Parkinson's disease (PD). However, until now this approach has not been deployed to study large cohorts of unrelated participants. To discover rare PD susceptibility variants, we perform

  1. Comparative evaluation of the protective efficacy of two formulations of a recombinant Chlamydia abortus subunit candidate vaccine in a mouse model.

    Science.gov (United States)

    Pan, Qing; Pais, Roshan; Ohandjo, Adaugo; He, Cheng; He, Qing; Omosun, Yusuf; Igietseme, J U; Eko, F O

    2015-04-08

    Chlamydia abortus (C. abortus) is the causative agent of ovine enzootic abortion (OEA) and poses a zoonotic risk to pregnant women. Current live attenuated 1B vaccines are efficacious but cause disease in vaccinated animals and inactivated vaccines are only marginally protective. We tested the ability of a new C. abortus subunit vaccine candidate based on the conserved and immunogenic polymorphic membrane protein D (Pmp18D) formulated in CpG1826+FL (Fms-like tyrosine kinase 3 Ligand; Flt3L) or Vibrio cholerae ghosts (VCG) to induce innate and cross protective immunity against genital C. abortus infection. We found that delivery of rPmp18D with VCG was more effective than with CpG+FL in up-regulating the expression of molecules critically involved in T cell activation and differentiation, including MHC II, CD40, CD80, and CD86, activation of TLRs and NLRP3 inflammasome engagement, and secretion of IL-1β and TNF-α but not IL-10 and IL-4. rVCG-Pmp18D-immunized mice elicited more robust antigen-specific IFN-γ, IgA and IgG2c antibody responses compared to CpG+FL-delivered rPmp18D. Based on the number of mice with positive vaginal cultures, length of vaginal shedding, and number of inclusion forming units recovered following challenge with the heterologous C. abortus strain B577, vaccine delivery with VCG induced superior protective immunity than delivery with a combination of CpG1826 and FL, a nasal DC-targeting adjuvant. These results demonstrate that the ability of VCG to enhance protective immunity against genital C. abortus infection is superior to that of CpG+FL adjuvants.

  2. Formalin-inactivated EV71 vaccine candidate induced cross-neutralizing antibody against subgenotypes B1, B4, B5 and C4A in adult volunteers.

    Directory of Open Access Journals (Sweden)

    Ai-Hsiang Chou

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 has caused several epidemics of hand, foot and mouth diseases (HFMD in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16. METHODS: Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses. RESULTS: The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had 4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (∼20% of participants against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8 against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16. CONCLUSION: EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT01268787.

  3. Draft Genome Sequence of the Intermediate Rough Vaccine Strain Brucella abortus S19Δper Mutant

    Science.gov (United States)

    Chaudhuri, Pallab; Goswami T, Tapas K.; Lalsiamthara, Jonathan; Kaur, Gurpreet; Vishnu, Udayakumar S.; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy

    2015-01-01

    Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted. PMID:26564050

  4. Randomized controlled trial of RTS,S/AS02D and RTS,S/AS01E malaria candidate vaccines given according to different schedules in Ghanaian children.

    Directory of Open Access Journals (Sweden)

    Seth Owusu-Agyei

    Full Text Available BACKGROUND: The target delivery channel of RTS,S candidate malaria vaccines in malaria-endemic countries in Africa is the World Health Organisation Expanded Program on Immunization. As an Adjuvant System, age de-escalation and schedule selection step, this study assessed 3 schedules of RTS,S/AS01(E and RTS,S/AS02(D in infants and young children 5-17 months of age in Ghana. METHODOLOGY: A Phase II, partially-blind randomized controlled study (blind to vaccine, not to schedule, of 19 months duration was conducted in two (2 centres in Ghana between August 2006 and May 2008. Subjects were allocated randomly (1:1:1:1:1:1 to one of six study groups at each study site, each defining which vaccine should be given and by which schedule (0,1-, 0,1,2- or 0,1,7-months. For the 0,1,2-month schedule participants received RTS,S/AS01(E or rabies vaccine at one center and RTS,S/AS01(E or RTS,S/AS02(D at the other. For the other schedules at both study sites, they received RTS,S/AS01(E or RTS,S/AS02(D. The primary outcome measure was the occurrence of serious adverse events until 10 months post dose 1. RESULTS: The number of serious adverse events reported across groups was balanced. One child had a simple febrile convulsion, which evolved favourably without sequelae, considered to be related to RTS,S/AS01(E vaccination. Low grade reactions occurred slightly more frequently in recipients of RTS,S/AS than rabies vaccines; grade 3 reactions were infrequent. Less local reactogenicity occurred with RTS,S/AS01(E than RTS,S/AS02(D. Both candidate vaccines were highly immunogenic for anti-circumsporozoite and anti-Hepatitis B Virus surface antigen antibodies. Recipients of RTS,S/AS01(E compared to RTS,S/AS02(D had higher peak anti-circumsporozoite antibody responses for all 3 schedules. Three dose schedules were more immunogenic than 2 dose schedules. Area under the curve analyses for anti-circumsporozoite antibodies were comparable between the 0,1,2- and 0,1,7-month

  5. A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus.

    Directory of Open Access Journals (Sweden)

    Alan G Goodman

    Full Text Available There is a need to develop a universal vaccine against influenza virus infection to avoid developing new formulations of a seasonal vaccine each year. Many of the vaccine strategies for a universal vaccine target strain-conserved influenza virus proteins, such as the matrix, polymerase, and nucleoproteins, rather than the surface hemagglutinin and neuraminidase proteins. In addition, non-disease-causing viral vectors are a popular choice as a delivery system for the influenza virus antigens. As a proof-of-concept, we have designed a novel influenza virus immunogen based on the NP backbone containing human T cell epitopes for M1, NS1, NP, PB1 and PA proteins (referred as NPmix as well as a construct containing the conserved regions of influenza virus neuraminidase (N-terminal and hemagglutinin (C-terminal (referred as NA-HA. DNA vectors and vaccinia virus recombinants expressing NPmix (WR-NP or both NPmix plus NA-HA (WR-flu in the cytosol were tested in a heterologous DNA-prime/vaccinia virus-boost vaccine regimen in mice. We observed an increase in the number of influenza virus-specific IFNγ-secreting splenocytes, composed of populations marked by CD4(+ and CD8(+ T cells producing IFNγ or TNFα. Upon challenge with influenza virus, the vaccinated mice exhibited decreased viral load in the lungs and a delay in mortality. These findings suggest that DNA prime/poxvirus boost with human multi-epitope recombinant influenza virus proteins is a valid approach for a general T-cell vaccine to protect against influenza virus infection.

  6. Identification of Immunodominant Th1-type T cell Epitopes from Schistosoma japonicum 28 kDa Glutathione-S-transferase, a Vaccine Candidate

    Institute of Scientific and Technical Information of China (English)

    Guang-Fu LI; Guan-Ling WU; Yong WANG; Zhao-Song ZHANG; Xin-Jun WANG; Min-Jun JI; Xiang ZHU; Feng LIU; Xiao-Ping CAI; Hai-Wei WU

    2005-01-01

    Th1-type cytokines produced by the stimulation of Th1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection.Schistosoma mansoni 28 kDa glutathione-S-transferase (Sm28GST), a major detoxification enzyme, has been recognized as a vaccine candidate and a phase Ⅱ clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST (Sj28GST) have shown immune protection against the parasite infection. In the present study, six candidate peptides (P1, P2, P3, P4, P7 and P8) from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope (190-211 aa) identified from Sm28GST was selected and named P9.The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c(+)/BL21(DE3) system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6(73-86 aa) generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ and IL-2. Therefore, P6 is a functional Th1-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Th1-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.

  7. Main-sequence progenitor configurations of the NN Ser candidate circumbinary planetary system are dynamically unstable

    CERN Document Server

    Mustill, Alexander J; Villaver, Eva; Veras, Dimitri; Davis, Philip J; Horner, Jonathan; Wittenmyer, Robert A

    2013-01-01

    Recent observations of the NN Serpentis post-common envelope binary system have revealed eclipse timing variations that have been attributed to the presence of two Jovian-mass exoplanets. Under the assumption that these planets are real and survived from the binary's Main Sequence state, we reconstruct initial binaries that give rise to the present NN Ser configuration and test the dynamical stability of the original system. Under standard assumptions about binary evolution, we find that survival of the planets through the entire Main Sequence life-time is very unlikely. Hence, we conclude that the planets are not survivors from before the Common Envelope phase, implying that either they formed recently out of material ejected from the primary, or that the observed signals are of non-planetary origin.

  8. Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC)

    Energy Technology Data Exchange (ETDEWEB)

    Leonhardt, E.A.; Kapp, L.N.; Young, B.R.; Murnane, J.P. (Univ. of California, San Francisco, CA (United States))

    1994-01-01

    A radioresistant cell clone (1B3) was previously isolated after transfection of an ataxia-telangiectasia (AT) group D cell line with a human cosmid library. A cosmid rescued from the integration site in 1B3 contained human DNA from chromosome position 11q23, the same region shown by both genetic linkage and chromosome transfer to contain the genes for AT complementation groups A/B, C, and D. A gene within the cosmid (ATDC) was found to produce mRNAs of different sizes. A cDNA for one of the most abundant mRNAs (3.0 kb) was isolated from a HeLa cell library. In the present study, the authors sequenced the 3.0-kb cDNA and the surrounding intron DNA in the cosmids. They used polymerase chain reaction, with primers in the introns, to confirm the number of exons and to analyze DNA from AT group D cells for mutations within this gene. Although no mutations were found, they do not rule out the possibility that mutations may be present within the regulatory sequences or coding sequences found in other mRNAs specific for this gene. From the sequence analysis, they found that the ATDC gene product is one of a group of proteins that share multiple zinc finger motifs and an adjacent leucine zipper motif. These proteins have been proposed to form homo- or hetero-dimers involved in nucleic acid binding, consistent with the fact that many of these proteins appear to be transcriptional regulatory factors involved in carcinogenesis and/or differentiation. The likelihood that the ATDC gene product is involved in transcriptional regulation could explain the pleiomorphic characteristics of AT, including abnormal cell cycle regulation. 36 refs., 5 figs., 2 tabs.

  9. Comparative analysis of SNP candidates in disparate milk yielding river buffaloes using targeted sequencing

    OpenAIRE

    Menon, Ramesh; Anand B. Patel; Joshi, Chaitanya

    2016-01-01

    River buffalo (Bubalus bubalis) milk plays an important role in economy and nutritious diet in several developing countries. However, reliable milk-yield genomic markers and their functional insights remain unexposed. Here, we have used a target capture sequencing approach in three economically important buffalo breeds namely: Banni, Jafrabadi and Mehsani, belonging to either high or low milk-yield group. Blood samples were collected from the milk-yield/breed balanced group of 12 buffaloes, a...

  10. Evaluation of the immunoprophylactic potential of a killed vaccine candidate in combination with different adjuvants against murine visceral leishmaniasis.

    Science.gov (United States)

    Thakur, Ankita; Kaur, Harpreet; Kaur, Sukhbir

    2015-02-01

    Despite a large number of field trials, till date no prophylactic antileishmanial vaccine exists for human use. Killed antigen formulations offer the advantage of being safe but they have limited immunogenicity. Recent research has documented that efforts to develop effective Leishmania vaccine have been limited due to the lack of an appropriate adjuvant. Addition of adjuvants to vaccines boosts and directs the immunogenicity of antigens. So, the present study was done to evaluate the effectiveness of four adjuvants i.e. alum, saponin, cationic liposomes and monophosphoryl lipid-A in combination with Autoclaved Leishmania donovani (ALD) antigen against murine visceral leishmaniasis (VL). BALB/c mice were immunized thrice with respective vaccine formulation. Two weeks after last booster, challenge infection was given. Mice were sacrificed 15 days after last immunization and on 30, 60 and 90 post infection/challenge days. A considerable protective efficacy was shown by all vaccine formulations. It was evident from significant reduction in parasite load, profound delayed type hypersensitivity responses (DTH), increased IgG2a titres and high levels of Th1 cytokines (IFN-γ, IL-12) as compared to the infected controls. However, level of protection varied with the type of adjuvant used. Maximum protection was achieved with the use of liposome encapsulated ALD antigen and it was closely followed by group immunized with ALD+MPL-A. Significant results were also obtained with ALD+saponin, ALD+alum and ALD antigen (alone) but the protective efficacy was reduced as compared to other immunized groups. The present study reveals greater efficacy of two vaccine formulations i.e. ALD+liposome and ALD+MPL-A against murine VL.

  11. Sequence analysis of candidate genes in two Roma families with severe tooth agenesis

    Directory of Open Access Journals (Sweden)

    Gabriková Dana

    2016-01-01

    Full Text Available Selective tooth agenesis is the most common congenital disorder affecting the formation of dentition in humans. Both its forms (hypodontia and more severe oligodontia can be found either in isolated form and they can be associated with systemic condition (syndromic tooth agenesis. In addition to previously known genes (PAX9, MSX1 and AXIN2 mutations in EDA, EDARADD and WNT10 gene were recently found to be involved in isolated forms of tooth agenesis. The objective of this study was to characterize the phenotype of affected members in two large families of Roma origin segregating severe isolated tooth agenesis with very variable phenotype and to perform mutation analysis of seven genes with aim to find causal mutation. 26 family members were clinically examined and coding regions of seven genes (MSX1, PAX9, AXIN2, EDA, EDAR, EDARADD and WNT10A were sequenced. With exclusion of third molars, average number of missing teeth was 8.2 ± 4.9 in family 1 and 7.1 ± 2.3 in family 2. The most frequently missing teeth were maxillary lateral incisors and first premolars and mandibular central incisors. Sequencing revealed four potentially damaging variants (g.Ala40Gly in MSX1, g.Ala240Pro in PAX9, g.Pro50Ser in AXIN2 and g.Met9Ile in EDARADD; however, none of them was present in all affected family members. Variable phenotype in both families examined in this study is in favour of heterogeneous genetic cause of tooth agenesis in these families: possible interaction of several defected genes, sequence variants in regulatory regions and additional environmental factors is assumed.

  12. Genomic sequence and analysis of a vaccinia virus isolate from a patient with a smallpox vaccine-related complication

    Directory of Open Access Journals (Sweden)

    Damon Inger

    2006-10-01

    Full Text Available Abstract Background Vaccinia virus (VACV-DUKE was isolated from a lesion on a 54 year old female who presented to a doctor at the Duke University Medical Center. She was diagnosed with progressive vaccinia and treated with vaccinia immune globulin. The availability of the VACV-DUKE genome sequence permits a first time genomic comparison of a VACV isolate associated with a smallpox vaccine complication with the sequence of culture-derived clonal isolates of the Dryvax vaccine. Results This study showed that VACV-DUKE is most similar to VACV-ACAM2000 and CLONE3, two VACV clones isolated from the Dryvax® vaccine stock confirming VACV-DUKE as an isolate from Dryvax®. However, VACV-DUKE is unique because it is, to date, the only Dryvax® clone isolated from a patient experiencing a vaccine-associated complication. The 199,960 bp VACV-DUKE genome encodes 225 open reading frames, including 178 intact genes and 47 gene fragments. Between VACV-DUKE and the other Dryvax® isolates, the major genomic differences are in fragmentation of the ankyrin-like, and kelch-like genes, presence of a full-length Interferon-α/β receptor gene, and the absence of a duplication of 12 ORFs in the inverted terminal repeat. Excluding this region, the DNA sequence of VACV-DUKE differs from the other two Dryvax® isolates by less than 0.4%. DNA sequencing also indicated that there was little heterogeneity in the sample, supporting the hypothesis that virus from an individual lesion is clonal in origin despite the fact that the vaccine is a mixed population. Conclusion Virus in lesions that result from progressive vaccinia following vaccination with Dryvax are likely clonal in origin. The genomic sequence of VACV-DUKE is overall very similar to that of Dryvax® cell culture-derived clonal isolates. Furthermore, with the sequences of multiple clones from Dryvax® we can begin to appreciate the diversity of the viral population in the smallpox vaccine.

  13. [Bioinformatics-based Design of Peptide Vaccine Candidates Targeting Spike Protein of MERS-CoV and Immunity analysis in Mice].

    Science.gov (United States)

    Lan, Jiaming; Lu, Shuai; Deng, Yao; Wen, Bo; Chen, Hong; Wang, Wen; Tan, Wenjie

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a novel human coronavirus and posed great threat to public health world wide,which calls for the development of effective and safe vaccine urgently. In the study, peptide epitopes tagrgeting spike antigen were predicted based on bioinformatics methods. Nine polypeptides with high scores were synthesized and linked to keyhole limpet hemocyanin (KLH). Female BALB/C mice were immunized with individual polypeptide-KLH, and the total IgG was detected by ELISA as well as the cellular mediated immunity (CMI) was analyzed using ELIs-pot assay. The results showed that an individual peptide of YVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI could induce the highest level of total IgG as well as CMI (high frequency of IFN-γ secretion) against MERS-CoV antigen in mice. Our study identified a promising peptide vaccine candidate against MERS-CoV and provided an experimental support for bioinformatics-based design of peptide vaccine.

  14. Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A.

    Science.gov (United States)

    Robert, A; Silva, A; Benitez, J A; Rodriguez, B L; Fando, R; Campos, J; Sengupta, D K; Boesman-Finkelstein, M; Finkelstein, R A

    1996-11-01

    The celA gene encoding Clostridium thermocellum endoglucanase A was expressed in Vibrio cholerae on its own promoter and used to tag a candidate El Tor biotype cholera vaccine strain. Colonies of the tagged strain could be unequivocally distinguished by overlaying them with CM-cellulose indicator agar and Congo Red staining. Expression of celA did not affect growth of V. cholerae in vitro and in vivo. The celA gene was inserted in the chromosomal hap locus encoding V. cholerae hemagglutinin/protease, a putative "detachase", to create a hap- mutant that could be identified and scored by its halo of cellulolytic activity. The inactivation of hap had a positive effect on colonization in the infant mice model. The above results indicate that celA is a suitable marker gene for V. cholerae and hap is an appropriate locus for insertion of foreign DNA in vaccine development. Inactivation of hap, by increasing the duration of adherence, might decrease excretion of the resulting vaccine vector strain and thus increase its immunogenicity.

  15. Adjuvant and carrier protein-dependent T-cell priming promotes a robust antibody response against the Plasmodium falciparum Pfs25 vaccine candidate

    Science.gov (United States)

    Radtke, Andrea J.; Anderson, Charles F.; Riteau, Nicolas; Rausch, Kelly; Scaria, Puthupparampil; Kelnhofer, Emily R.; Howard, Randall F.; Sher, Alan; Germain, Ronald N.; Duffy, Patrick

    2017-01-01

    Humoral immune responses have the potential to maintain protective antibody levels for years due to the immunoglobulin-secreting activity of long-lived plasma cells (LLPCs). However, many subunit vaccines under development fail to generate robust LLPC responses, and therefore a variety of strategies are being employed to overcome this limitation, including conjugation to carrier proteins and/or formulation with potent adjuvants. Pfs25, an antigen expressed on malaria zygotes and ookinetes, is a leading transmission blocking vaccine (TBV) candidate for Plasmodium falciparum. Currently, the conjugate vaccine Pfs25-EPA/Alhydrogel is in Phase 1 clinical trials in the USA and Africa. Thus far, it has proven to be safe and immunogenic, but it is expected that a more potent formulation will be required to establish antibody titers that persist for several malaria transmission seasons. We sought to determine the contribution of carrier determinants and adjuvants in promoting high-titer, long-lived antibody responses against Pfs25. We found that both adjuvants and carrier proteins influence the magnitude and capacity of Pfs25-specific humoral responses to remain above a protective level. Furthermore, a liposomal adjuvant with QS21 and a TLR4 agonist (GLA-LSQ) was especially effective at inducing T follicular helper (Tfh) and LLPC responses to Pfs25 when coupled to immunogenic carrier proteins. PMID:28091576

  16. Evaluation of vaccine candidate potential of deltaaroA, deltahtrA and deltaaroAdeltahtrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi in guinea pigs.

    Science.gov (United States)

    Singh, Bhoj Raj; Chandra, Mudit; Hansda, Dhananjoy; Alam, Javed; Babu, Narayanan; Siddiqui, Mehtab Z; Agrawal, Ravi K; Sharma, Gautam

    2013-04-01

    Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), a host adapted Salmonella causes abortions, still births and foal mortality in equids. Though known since more than 100 years, it is still a problem in many of the developing countries including India. There is dearth of really good vaccine affording immunity lasting at least for one full gestation. In search of a potential vaccine candidate, three defined deletion mutants (deltaaroA, deltahtrA and deltaaroAdeltahtrA) of S. Abortusequi were tested in guinea pig model for attenuation, safety, immunogenicity, humoral immune response, protective efficacy and persistence in host. The deltahtrA and deltaaroAdeltahtrA mutants were found to be safe on oral inoculation in doses as high as 4.2 x 10(9) cfu/animal. Also through subcutaneous inoculation deltaaroAdeltahtrA mutant did not induce any abortion in pregnant guinea pigs. All the three mutants did not induce any illness or death in 1-2 week-old baby guinea pigs except deltahtrA mutant which caused mortality on intraperitoneal inoculation. Inoculation with mutants protected against challenge and increased breeding efficiency of guinea pigs. After >4.5 months of mutant inoculation, guinea pigs were protected against abortifacient dose of wild type S. Abortusequi and mother guinea pigs also conferred resistance to their babies to the similar challenge. Early humoral immune response of S. Abortusequi mutants was characteristic. Faecal excretion of deltaaroA and htrA mutants was detected up to 45 days of inoculation in guinea pigs while deltaaroAdeltahtrA mutant could not be detected after 21 days of inoculation. The results indicated that the double deletion mutant (deltaaroAdeltahtrA) was the most effective and safe candidate for vaccination against S. Abortusequi through mucosal route of inoculation.

  17. Whole-genome sequencing of individuals from a founder population identifies candidate genes for asthma.

    Science.gov (United States)

    Campbell, Catarina D; Mohajeri, Kiana; Malig, Maika; Hormozdiari, Fereydoun; Nelson, Benjamin; Du, Gaixin; Patterson, Kristen M; Eng, Celeste; Torgerson, Dara G; Hu, Donglei; Herman, Catherine; Chong, Jessica X; Ko, Arthur; O'Roak, Brian J; Krumm, Niklas; Vives, Laura; Lee, Choli; Roth, Lindsey A; Rodriguez-Cintron, William; Rodriguez-Santana, Jose; Brigino-Buenaventura, Emerita; Davis, Adam; Meade, Kelley; LeNoir, Michael A; Thyne, Shannon; Jackson, Daniel J; Gern, James E; Lemanske, Robert F; Shendure, Jay; Abney, Mark; Burchard, Esteban G; Ober, Carole; Eichler, Evan E

    2014-01-01

    Asthma is a complex genetic disease caused by a combination of genetic and environmental risk factors. We sought to test classes of genetic variants largely missed by genome-wide association studies (GWAS), including copy number variants (CNVs) and low-frequency variants, by performing whole-genome sequencing (WGS) on 16 individuals from asthma-enriched and asthma-depleted families. The samples were obtained from an extended 13-generation Hutterite pedigree with reduced genetic heterogeneity due to a small founding gene pool and reduced environmental heterogeneity as a result of a communal lifestyle. We sequenced each individual to an average depth of 13-fold, generated a comprehensive catalog of genetic variants, and tested the most severe mutations for association with asthma. We identified and validated 1960 CNVs, 19 nonsense or splice-site single nucleotide variants (SNVs), and 18 insertions or deletions that were out of frame. As follow-up, we performed targeted sequencing of 16 genes in 837 cases and 540 controls of Puerto Rican ancestry and found that controls carry a significantly higher burden of mutations in IL27RA (2.0% of controls; 0.23% of cases; nominal p = 0.004; Bonferroni p = 0.21). We also genotyped 593 CNVs in 1199 Hutterite individuals. We identified a nominally significant association (p = 0.03; Odds ratio (OR) = 3.13) between a 6 kbp deletion in an intron of NEDD4L and increased risk of asthma. We genotyped this deletion in an additional 4787 non-Hutterite individuals (nominal p = 0.056; OR = 1.69). NEDD4L is expressed in bronchial epithelial cells, and conditional knockout of this gene in the lung in mice leads to severe inflammation and mucus accumulation. Our study represents one of the early instances of applying WGS to complex disease with a large environmental component and demonstrates how WGS can identify risk variants, including CNVs and low-frequency variants, largely untested in GWAS.

  18. DNA sequence and haplotype variation in two candidate genes for dilated cardiomyopathy in the turkey Meleagris gallopavo.

    Science.gov (United States)

    Lin, Kuan-chin; Xu, Jun; Kamara, Davida; Geng, Tuoyu; Gyenai, Kwaku; Reed, Kent M; Smith, Edward J

    2007-05-01

    Determining variation in genes is fundamental to understanding their function in the disease state. Cardiac troponin T (cTnT) and phospholamban (PLN) genes have been implicated in dilated cardiomyopathy (DCM) in human and model species. To investigate the role of these 2 candidate genes in DCM in the turkey Meleagris gallopavo, understanding sequence variants and map position distribution is necessary. To this end, a total of 1854 and 1771 bp of cTnT and PLN gene sequences, respectively, were scanned for single nucleotide polymorphisms (SNPs) in a randomly bred population. A total of 15 SNPs was identified in the cTnT and PLN genomic sequences. Nine haplotypes, 5 in cTnT and 4 in PLN, were identified. Observed heterozygosities (0.02-0.39) in the turkey population were low for both genes. Within each gene, 1 SNP corresponding to a restriction enzyme site was identified and used to develop a PCR-restriction fragment length polymorphism (RFLP) genotyping assay. The PLN gene was genetically mapped to turkey chromosome 2, equivalent to Gallus gallus chromosome 3, and cTnT mapped to a turkey microchromosome. Although limited because of the relatively small sample size of 55 birds, the data from this SNP analysis of PLN and cTnT provide a foundation from which to evaluate the function of cTnT and PLN in the turkey. Information about the distribution of the SNPs and haplotypes will facilitate future association and linkage studies.

  19. Rare, Low-Frequency, and Common Variants in the Protein-Coding Sequence of Biological Candidate Genes from GWASs Contribute to Risk of Rheumatoid Arthritis

    NARCIS (Netherlands)

    Diogo, Dorothee; Kurreeman, Fina; Stahl, Eli A.; Liao, Katherine P.; Gupta, Namrata; Greenberg, Jeffrey D.; Rivas, Manuel A.; Hickey, Brendan; Flannick, Jason; Thomson, Brian; Guiducci, Candace; Ripke, Stephan; Adzhubey, Ivan; Barton, Anne; Kremer, Joel M.; Alfredsson, Lars; Sunyaev, Shamil; Martin, Javier; Zhernakova, Alexandra; Bowes, John; Eyre, Steve; Siminovitch, Katherine A.; Gregersen, Peter K.; Worthington, Jane; Klareskog, Lars; Padyukov, Leonid; Raychaudhuri, Soumya; Plenge, Robert M.

    2013-01-01

    The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome

  20. Differential induction of functional IgG using the Plasmodium falciparum placental malaria vaccine candidate VAR2CSA

    DEFF Research Database (Denmark)

    Pinto, Vera V; Ditlev, Sisse B; Jensen, Kamilla E;

    2011-01-01

    and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM...

  1. Generation of growth arrested Leishmania amastigotes: a tool to develop live attenuated vaccine candidates against visceral leishmaniasis.

    Science.gov (United States)

    Selvapandiyan, Angamuthu; Dey, Ranadhir; Gannavaram, Sreenivas; Solanki, Sumit; Salotra, Poonam; Nakhasi, Hira L

    2014-06-30

    Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis.

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

    NARCIS (Netherlands)

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Pijlman, Gorben P.

    2016-01-01

    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

  3. Safety and preliminary immunogenicity of Cuban pneumococcal conjugate vaccine candidate in healthy children: a randomized phase I clinical trial.

    Science.gov (United States)

    Dotres, Carlos P; Puga, Rinaldo; Ricardo, Yariset; Broño, Carmen R; Paredes, Beatriz; Echemendía, Vladimir; Rosell, Sandra; González, Nadezhda; García-Rivera, Dagmar; Valdés, Yury; Goldblatt, David; Vérez-Bencomo, Vicente

    2014-09-15

    A new heptavalent conjugate vaccine (PCV7-TT) is under development in Cuba. PCV7-TT contains 2 μg of serotypes 1, 5, 14, 18C, 19F, 23F and 4 μg of 6B, each one conjugated to tetanus toxoid (TT). This vaccine was designed with the serotypes that cause most invasive pneumococcal diseases (IPD) worldwide. In the present study, we investigated the safety and explored the immunogenicity of PCV7-TT during a controlled, randomized and double blind clinical trial phase I in 4-5-year-old children. PCV7-TT was well tolerated and as safe as Synflorix used as control vaccine. Following a single-dose vaccination, all individual serotypes included in PCV7-TT induced statistically significant increase of IgG GMC and OPA GMT. These are the first clinical results of PCV7-TT in children and they pave the way toward next clinical trials in children and infants. This clinical trial was published in the Cuban Public Register of Clinical Trials with code RPCEC00000173.

  4. Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain

    Directory of Open Access Journals (Sweden)

    I. Valpotic

    2009-12-01

    Full Text Available Colidiarrhea and colienterotoxemia caused by F4+ and/or F18+ enterotoxigenic E. coli (ETEC strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac+ non-ETEC vaccine candidate strain against challenge infection with F4ac+ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs. We also evaluated levamisole as an immune response modifier (IRM and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0 or with levamisole and perorally given F18ac+ non-ETEC strain (at day 0, and challenged with F4ac+ ETEC strain 7 days later. At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3+, CD45RA+, CD45RC+, CD21+, IgA+ and myeloid SWC3+ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3+, CD45RC+ and SWC3+ cells (p<0.05 as compared to those recorded in nontreated control pigs. In the ileum of these pigs we have recorded that only CD21+ cells were significantly increased (p<0.01. The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac+ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.

  5. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

    Science.gov (United States)

    Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

    2012-04-01

    A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.

  6. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

    Science.gov (United States)

    Bernard, Marie-Clotilde; Barban, Véronique; Pradezynski, Fabrine; de Montfort, Aymeric; Ryall, Robert; Caillet, Catherine; Londono-Hayes, Patricia

    2015-01-01

    HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.

  7. Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Marie-Clotilde Bernard

    Full Text Available HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29 has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529 derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a

  8. Testing of four Leishmania vaccine candidates in a mouse model of infection with Leishmania (Viannia) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World.

    Science.gov (United States)

    Salay, G; Dorta, M L; Santos, N M; Mortara, R A; Brodskyn, C; Oliveira, C I; Barbiéri, C L; Rodrigues, M M

    2007-09-01

    We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies.

  9. Testing of Four Leishmania Vaccine Candidates in a Mouse Model of Infection with Leishmania (Viannia) braziliensis, the Main Causative Agent of Cutaneous Leishmaniasis in the New World▿

    Science.gov (United States)

    Salay, G.; Dorta, M. L.; Santos, N. M.; Mortara, R. A.; Brodskyn, C.; Oliveira, C. I.; Barbiéri, C. L.; Rodrigues, M. M.

    2007-01-01

    We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies. PMID:17626159

  10. B-Cells and the Use of Non-Human Primates for Evaluation of HIV Vaccine Candidates.

    Science.gov (United States)

    Demberg, Thorsten; Robert-Guroff, Marjorie

    2015-01-01

    The RV144 clinical trial in Thailand associated vaccine-induced antibodies with protective efficacy, leading to a focus in HIV vaccine research on protective antibody induction. This has necessitated greater understanding of B cell biology in humans as well as non-human primates (NHP), the principle animal model for pre-clinical HIV/SIV vaccine research. This review covers development and maturation of NHP B cells within the framework of current knowledge of human and murine B cells. Identification of many NHP B cell subpopulations is now possible, although consensus is lacking in some cases, and better distinction of some populations is still needed. Elucidation of mechanisms that control germinal center maintenance, selection of B cells into the memory cell pool, and differentiation of B cells into long-lived plasma cells remains critical for improving vaccine design. B cell dysfunction occurs during both HIV and SIV infection. Whether the processes leading to this impairment are identical in humans and NHP is not known. Uncovering the mechanisms involved could lead to improved treatment regimens. The SIV/NHP model effectively mimics HIV infection of people, but key differences between NHP and humans in antibody characteristics such as glycosylation and structure may lead to unexpected outcomes in pre-clinical studies. Important new areas for investigation include the role of B cell cytokines in the immune system and the impact of the microbiome on B cell development and maturation. Enhanced knowledge of B cells in NHP as well as humans should enable improved vaccine design, leading to induction of potent, long-lasting protective antibodies.

  11. Immunogenicity of live attenuated B. pertussis BPZE1 producing the universal influenza vaccine candidate M2e.

    Directory of Open Access Journals (Sweden)

    Hana Kammoun

    Full Text Available BACKGROUND: Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect. CONCLUSIONS/SIGNIFICANCE: Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza.

  12. Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate.

    Science.gov (United States)

    Curti, Elena; Seid, Christopher A; Hudspeth, Elissa; Center, Lori; Rezende, Wanderson; Pollet, Jeroen; Kwityn, Cliff; Hammond, Molly; Matsunami, Rise K; Engler, David A; Hotez, Peter J; Elena Bottazzi, Maria

    2014-01-01

    Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation

  13. RNA deep sequencing reveals novel candidate genes and polymorphisms in boar testis and liver tissues with divergent androstenone levels.

    Directory of Open Access Journals (Sweden)

    Asep Gunawan

    Full Text Available Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one and skatole (3-methylindole. It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq. The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from -4.68 to 2.90 in testis samples and -2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs.

  14. Identification of mesoderm development (mesd) candidate genes by comparative mapping and genome sequence analysis.

    Science.gov (United States)

    Wines, M E; Lee, L; Katari, M S; Zhang, L; DeRossi, C; Shi, Y; Perkins, S; Feldman, M; McCombie, W R; Holdener, B C

    2001-02-15

    The proximal albino deletions identify several functional regions on mouse Chromosome 7 critical for differentiation of mesoderm (mesd), development of the hypothalamus neuroendocrine lineage (nelg), and function of the liver (hsdr1). Using comparative mapping and genomic sequence analysis, we have identified four novel genes and Il16 in the mesd deletion interval. Two of the novel genes, mesdc1 and mesdc2, are located within the mesd critical region defined by BAC transgenic rescue. We have investigated the fetal role of genes located outside the mesd critical region using BAC transgenic complementation of the mesd early embryonic lethality. Using human radiation hybrid mapping and BAC contig construction, we have identified a conserved region of human chromosome 15 homologous to the mesd, nelg, and hsdr1 functional regions. Three human diseases cosegregate with microsatellite markers used in construction of the human BAC/YAC physical map, including autosomal dominant nocturnal frontal lobe epilepsy (ENFL2; also known as ADNFLE), a syndrome of mental retardation, spasticity, and tapetoretinal degeneration (MRST); and a pyogenic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA).

  15. Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Zhang, Xiaodong; Cai, Hui; Niewiesk, Stefan

    2014-01-01

    ABSTRACT The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2′-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2′-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCE Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine

  16. Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus.

    Science.gov (United States)

    Lazo, Laura; Izquierdo, Alienys; Suzarte, Edith; Gil, Lázaro; Valdés, Iris; Marcos, Ernesto; Álvarez, Mayling; Romero, Yaremis; Guzmán, María Guadalupe; Guillén, Gerardo; Hermida Cruz, Lisset

    2014-04-01

    A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non-human primates using this tetravalent formulation in a prime-boost strategy with attenuated viruses are strongly recommended.

  17. The meningococcal vaccine candidate neisserial surface protein A (NspA binds to factor H and enhances meningococcal resistance to complement.

    Directory of Open Access Journals (Sweden)

    Lisa A Lewis

    Full Text Available Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH to fH-binding protein (fHbp is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a approximately 17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA, a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep I chain of lipooligosaccharide (LOS, or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6-7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

  18. Molecular immune responses to aerosol challenge with Francisella tularensis in mice inoculated with live vaccine candidates of varying efficacy.

    Directory of Open Access Journals (Sweden)

    Hua Shen

    Full Text Available BACKGROUND: Francisella tularensis is a facultative intracellular bacterial pathogen and the etiological agent of tularemia. The subspecies F. tularensis tularensis is especially virulent for humans when inhaled and respiratory tularemia is associated with high mortality if not promptly treated. A live vaccine strain (LVS derived from the less virulent holarctica subspecies confers incomplete protection against aerosol challenge with subsp. tularensis. Moreover, correlates of protection have not been established for LVS. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we compare molecular immune responses elicited by LVS and two defined deletion mutants of clinical subsp. tularensis strain, SCHU S4, that confer enhanced protection in a mouse model. BALB/c mice were immunized intradermally then challenged with an aerosol of SCHU S4 six weeks later. Changes in the levels of a selected panel of cytokines and chemokines were examined in the lungs, spleens, and sera of vaccinated and challenged mice. Mostly, increased cytokine and chemokine levels correlated with increased bacterial burden. However, after adjusting for this variable, immunization with either of the two Schu S4 mutants resulted in higher levels of several pulmonary cytokines, versus those resulting after LVS immunization, including IL-17. Moreover, treatment of mice immunized with ΔclpB with anti-IL-17 antibodies post-challenge enhanced lung infection. CONCLUSIONS/SIGNIFICANCE: This is the first report characterizing local and systemic cytokine and chemokine responses in mice immunized with vaccines with different efficacies against aerosol challenge with virulent F. tularensis subsp. tularensis. It shows that increases in the levels of most of these immunomodulators, including those known to be critical for protective immunity, do not superficially correlate with protection unless adjusted for the effects of bacterial burden. Additionally, several cytokines were selectively

  19. Evaluation of Prostatic Acid Phosphatase (PAP) as a Candidate Antigen for the Development of Cancer Vaccines for Prostate Cancer

    Science.gov (United States)

    2001-10-01

    colony-stimulating factor as a vaccine adjuvant elicits both a cellular and humoral response to recombinant human granu - locyte-macrophage colony...cells in recruiting CD8+ CTLs (4) and other inflammatory cells such as macrophages and eosinophils (5). CTLs have generally been believed to be the... eosinophils . Cytotoxic T lymphocytes (CTL) have long been considered to be the primary effector cell population critical for effective antitumor immunity

  20. Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever

    OpenAIRE

    Cashman Kathleen A; Henderson Lee A; Schoepp Randal J; Magliato Susan A; Muncy Ivana J; Boisen Matt L; Geske Frederick J; Grove Jessica N; Branco Luis M; Hensley Lisa E; Garry Robert F

    2010-01-01

    Abstract Background Lassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but...

  1. Complete Genome Sequence of Bordetella pertussis Strain VA-190 Isolated from a Vaccinated 10-Year-Old Patient with Whooping Cough

    Science.gov (United States)

    Eby, Joshua C.; Turner, Lauren; Nguyen, Bryan; Kang, June; Neville, Carly

    2016-01-01

    The number of cases of pertussis has increased in the United States despite vaccination. We present the genome of an isolate of Bordetella pertussis from a vaccinated patient from Virginia. The genome was sequenced by long-read methodology and compared to that of a clinical isolate used for laboratory studies, D420. PMID:27634997

  2. Complete Genome Sequence of Bordetella pertussis Strain VA-190 Isolated from a Vaccinated 10-Year-Old Patient with Whooping Cough.

    Science.gov (United States)

    Eby, Joshua C; Turner, Lauren; Nguyen, Bryan; Kang, June; Neville, Carly; Temple, Louise

    2016-09-15

    The number of cases of pertussis has increased in the United States despite vaccination. We present the genome of an isolate of Bordetella pertussis from a vaccinated patient from Virginia. The genome was sequenced by long-read methodology and compared to that of a clinical isolate used for laboratory studies, D420.

  3. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses.

    Directory of Open Access Journals (Sweden)

    Stuart D Dowall

    Full Text Available Crimean-Congo Haemorrhagic Fever (CCHF is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP. It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge.

  4. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses.

    Science.gov (United States)

    Dowall, Stuart D; Graham, Victoria A; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W; Hewson, Roger

    2016-01-01

    Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge.

  5. Humoral immune response to Plasmodium falciparum vaccine candidate GMZ2 and its components in populations naturally exposed to seasonal malaria in Ethiopia

    DEFF Research Database (Denmark)

    Mamo, Hassen; Esen, Meral; Ajua, Anthony;

    2013-01-01

    for malaria infection microscopically and by the rapid diagnostic test (RDT). Sera were tested by using enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin (Ig) G against P. falciparum blood-stage vaccine candidate GMZ2 and its subunits (Glutamate-rich protein (GLURP-R0), merozoite surface...... protein 3 (MSP3); as well as IgG subclasses against GLURP-R0 and MSP3. RESULTS: Whereas 23(8.6%) blood smear-positive cases for P. falciparum were detected in Boditi, all Shewa Robit study participants had no detectable P. falciparum infection. In both localities, total IgG prevalence and levels to GMZ2...... were significantly higher than the response to the component domains indicating the strong recognition of GMZ2 by antibodies acquired through natural exposure. Total IgG and subclass prevalence and levels were higher in Shewa Robit than Boditi, suggesting difference in the intensity of malaria...

  6. Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate--Tackling the Cocktail Challenge.

    Directory of Open Access Journals (Sweden)

    Alexander Boes

    Full Text Available Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%, blood (up to 90% and sexual parasite stages (100%. Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml, the blood stage (40-60 μg/ml and the sexual stage (1.75 μg/ml. While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy.

  7. Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate--Tackling the Cocktail Challenge.

    Science.gov (United States)

    Boes, Alexander; Spiegel, Holger; Voepel, Nadja; Edgue, Gueven; Beiss, Veronique; Kapelski, Stephanie; Fendel, Rolf; Scheuermayer, Matthias; Pradel, Gabriele; Bolscher, Judith M; Behet, Marije C; Dechering, Koen J; Hermsen, Cornelus C; Sauerwein, Robert W; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-01-01

    Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml), the blood stage (40-60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy.

  8. Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

    Directory of Open Access Journals (Sweden)

    Carmen Elena Gómez

    Full Text Available Based on the partial efficacy of the HIV/AIDS Thai trial (RV144 with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C. MVA-C infection of human monocyte derived dendritic cells (moDCs induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

  9. Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits.

    Science.gov (United States)

    Boes, Alexander; Spiegel, Holger; Edgue, Gueven; Kapelski, Stephanie; Scheuermayer, Matthias; Fendel, Rolf; Remarque, Edmond; Altmann, Friedrich; Maresch, Daniel; Reimann, Andreas; Pradel, Gabriele; Schillberg, Stefan; Fischer, Rainer

    2015-02-01

    One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P. falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2 × 10(6) were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC₅₀ values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.

  10. Development of standardized laboratory methods and quality processes for a phase III study of the RTS, S/AS01 candidate malaria vaccine

    Directory of Open Access Journals (Sweden)

    Carter Terrell

    2011-08-01

    Full Text Available Abstract Background A pivotal phase III study of the RTS,S/AS01 malaria candidate vaccine is ongoing in several research centres across Africa. The development and establishment of quality systems was a requirement for trial conduct to meet international regulatory standards, as well as providing an important capacity strengthening opportunity for study centres. Methods Standardized laboratory methods and quality assurance processes were implemented at each of the study centres, facilitated by funding partners. Results A robust protocol for determination of parasite density based on actual blood cell counts was set up in accordance with World Health Organization recommendations. Automated equipment including haematology and biochemistry analyzers were put in place with standard methods for bedside testing of glycaemia, base excess and lactacidaemia. Facilities for X-rays and basic microbiology testing were also provided or upgraded alongside health care infrastructure in some centres. External quality assurance assessment of all major laboratory methods was established and method qualification by each laboratory demonstrated. The resulting capacity strengthening has ensured laboratory evaluations are conducted locally to the high standards required in clinical trials. Conclusion Major efforts by study centres, together with support from collaborating parties, have allowed standardized methods and robust quality assurance processes to be put in place for the phase III evaluation of the RTS, S/AS01 malaria candidate vaccine. Extensive training programmes, coupled with continuous commitment from research centre staff, have been the key elements behind the successful implementation of quality processes. It is expected these activities will culminate in healthcare benefits for the subjects and communities participating in these trials. Trial registration Clinicaltrials.gov NCT00866619

  11. Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

    Science.gov (United States)

    Fiorentino, Maria; Levine, Myron M; Sztein, Marcelo B; Fasano, Alessio

    2014-01-01

    Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to

  12. Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

    Directory of Open Access Journals (Sweden)

    Maria Fiorentino

    Full Text Available Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa and might contribute (along with enterotoxins to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them

  13. Gut-homing conventional plasmablasts and CD27- plasmablasts elicited after a short time exposure to an oral live attenuated Shigella vaccine candidate in humans

    Directory of Open Access Journals (Sweden)

    Franklin R. Toapanta

    2014-08-01

    Full Text Available Currently, there is no licensed Shigella vaccine; however, various promising live attenuated vaccine candidates have emerged, including CVD1208S (ΔguaBA, Δset, Δsen S. flexneri 2a, which was shown to be safe and immunogenic in Phase 1 clinical trials. Here we report the immune responses elicited in an outpatient Phase 2 clinical trial in which subjects were vaccinated with CVD 1208S. Oral immunization with CVD 1208S elicited high anti-S. flexneri 2a LPS and IpaB antibody responses, as well as an acute plasmablast (PB infiltration in peripheral blood 7 days after immunization. PB sorted based on their expression of homing molecules confirmed that cells expressing integrin α4β7 alone or in combination with CD62L were responsible for antibody production (as measured by ELISpot. Furthermore, using high-color flow-cytometry, on day 7 after immunization, we observed the appearance of conventional PB (CPB, CD19dim CD20- CD27+high CD38+high CD3-, as well as a PB population that did not express CD27 (CD27- PB; pre-plasmablasts. The pattern of individual or simultaneous expression of homing markers (integrin α4β7, CD62L, CXCR3 and CXCR4 suggested that CPB cells homed preferentially to the inflamed gut mucosa. In contrast, ~50% CD27- PB cells appear to home to yet to be identified peripheral lymphoid organs or were in a transition state preceding integrin α4β7 upregulation. In sum, these observations demonstrate that strong immune responses, including distinct PB subsets with the potential to home to the gut and other secondary lymphoid organs, can be elicited after a short time of exposure to a shigella oral

  14. 不可分型流感嗜血杆菌候选疫苗的研究进展%RESEARCH PROGRESS ON NONTYPEABLE HAEMOPHILUS INFLUENZAE VACCINE CANDIDATES

    Institute of Scientific and Technical Information of China (English)

    何多姣; 张彦霞; 贾天军; 张玉妥

    2011-01-01

    不可分型流感嗜血杆菌(nontypeable Hae;ophilus influenzae,NTHi)是多种感染性疾病如急性中耳炎,鼻窦炎,结膜炎,慢性支气管炎的常见病原菌,近年来随着NTHi菌株耐药性的增强,NTHi感染已经成为全球一个较严重的健康问题,因此目前亟待研究一种安全有效的疫苗来预防NTHi的感染.多种NTHi的抗原被作为抗NTHi感染的候选疫苗来研究,该文主要针对近年来的一些研究热点如外膜蛋白P6/P5/P2/P26、Haps蛋白、HMW蛋白(high molecular weight protein,HMW protein)等进行综述,为后续疫苗的研制提供一些依据.%Nontypeable Haemophilus influenzae (NTHi) is a commonly causative pathogen for many infectious diseases, such as acute otitis media, sinusitis, conjunctivitis, chronic bronchitis and so on. With a recent increase in antibiotic resistance of NTHi, infection caused by NTHi has been a serious global health problem, so it is high time to develop a safe and effective vaccine to prevent against NTHi infection. Many antigens of NTHi have been evaluated as vaccine candidates for NTHi, this article will present a conclusion of research highlights such as outer membrane protein P6/P5/P2/P26, Haps protein, HMW protein (high molecular weight protein) for the past few years to offer some foundations for the follow-up vaccine studies.

  15. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes.

    Science.gov (United States)

    Abate, Francesca; Malito, Enrico; Falugi, Fabiana; Margarit Y Ros, Immaculada; Bottomley, Matthew James

    2013-10-01

    Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a=139.2, b=120.4, c=104.3 Å, β=111°.

  16. A plant-produced Pfs25 VLP malaria vaccine candidate induces persistent transmission blocking antibodies against Plasmodium falciparum in immunized mice.

    Directory of Open Access Journals (Sweden)

    R Mark Jones

    Full Text Available Malaria transmission blocking vaccines (TBVs are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs. We engineered VLPs (Pfs25-CP VLP comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.

  17. Tomatine Adjuvantation of Protective Immunity to a Major Pre-erythrocytic Vaccine Candidate of Malaria is Mediated via CD8+ T Cell Release of IFN-γ

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    Karen G. Heal

    2010-01-01

    Full Text Available The glycoalkaloid tomatine, derived from the wild tomato, can act as a powerful adjuvant to elicit an antigen-specific cell-mediated immune response to the circumsporozoite (CS protein, a major pre-erythrocytic stage malaria vaccine candidate antigen. Using a defined MHC-class-I-restricted CS epitope in a Plasmodium berghei rodent model, antigen-specific cytotoxic T lymphocyte activity and IFN-γ secretion ex vivo were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunized mice. Further, through lymphocyte depletion it is demonstrated that antigen-specific IFN-γ is produced exclusively by the CD8+ T cell subset. We conclude that the processing of the P. berghei CS peptide as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells leads to an immune response that is an in vitro correlate of protection against pre-erythrocytic malaria. Further characterization of tomatine as an adjuvant in malaria vaccine development is indicated.

  18. Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia.

    Science.gov (United States)

    Bokori-Brown, Monika; Hall, Charlotte A; Vance, Charlotte; Fernandes da Costa, Sérgio P; Savva, Christos G; Naylor, Claire E; Cole, Ambrose R; Basak, Ajit K; Moss, David S; Titball, Richard W

    2014-05-13

    Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

  19. Omics Approaches for the Study of Adaptive Immunity to Staphylococcus aureus and the Selection of Vaccine Candidates

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    Holtfreter, Silva; Kolata, Julia; Stentzel, Sebastian; Bauerfeind, Stephanie; Schmidt, Frank; Sundaramoorthy, Nandakumar; Bröker, Barbara M.

    2016-01-01

    Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms of protection will be key for the successful prevention or treatment of S. aureus invasion. Omics technologies generate a comprehensive picture of the physiological and pathophysiological processes within cells, tissues, organs, organisms and even populations. This review provides an overview of the contribution of genomics, transcriptomics, proteomics, metabolomics and immunoproteomics to the current understanding of S. aureus‑host interaction, with a focus on the adaptive immune response to the microorganism. While antibody responses during colonization and infection have been analyzed in detail using immunoproteomics, the full potential of omics technologies has not been tapped yet in terms of T-cells. Omics technologies promise to speed up vaccine development by enabling reverse vaccinology approaches. In consequence, omics technologies are powerful tools for deepening our understanding of the “superbug” S. aureus and for improving its control.

  20. Omics Approaches for the Study of Adaptive Immunity to Staphylococcus aureus and the Selection of Vaccine Candidates

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    Silva Holtfreter

    2016-03-01

    Full Text Available Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms of protection will be key for the successful prevention or treatment of S. aureus invasion. Omics technologies generate a comprehensive picture of the physiological and pathophysiological processes within cells, tissues, organs, organisms and even populations. This review provides an overview of the contribution of genomics, transcriptomics, proteomics, metabolomics and immunoproteomics to the current understanding of S. aureus‑host interaction, with a focus on the adaptive immune response to the microorganism. While antibody responses during colonization and infection have been analyzed in detail using immunoproteomics, the full potential of omics technologies has not been tapped yet in terms of T-cells. Omics technologies promise to speed up vaccine development by enabling reverse vaccinology approaches. In consequence, omics technologies are powerful tools for deepening our understanding of the “superbug” S. aureus and for improving its control.

  1. Vaccination with a non-human random sequence amyloid oligomer mimic results in improved cognitive function and reduced plaque deposition and micro hemorrhage in Tg2576 mice

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    Rasool Suhail

    2012-08-01

    Full Text Available Abstract Background It is well established that vaccination of humans and transgenic animals against fibrillar Aβ prevents amyloid accumulation in plaques and preserves cognitive function in transgenic mouse models. However, autoimmune side effects have halted the development of vaccines based on full length human Aβ. Further development of an effective vaccine depends on overcoming these side effects while maintaining an effective immune response. Results We have previously reported that the immune response to amyloid oligomers is largely directed against generic epitopes that are common to amyloid oligomers of many different proteins and independent of a specific amino acid sequence. Here we have examined whether we can exploit this generic immune response to develop a vaccine that targets amyloid oligomers using a non-human random sequence amyloid oligomer. In order to study the effect of vaccination against generic oligomer epitopes, a random sequence oligomer (3A was selected as it forms oligomers that react with the oligomer specific A11 antibody. Oligomer mimics from 3A peptide, Aβ, islet amyloid polypeptide (IAPP, and Aβ fibrils were used to vaccinate Tg2576 mice, which develop a progressive accumulation of plaques and cognitive impairment. Vaccination with the 3A random sequence antigen was just as effective as vaccination with the other antigens in improving cognitive function and reducing total plaque load (Aβ burden in the Tg2576 mouse brains, but was associated with a much lower incidence of micro hemorrhage than Aβ antigens. Conclusion These results shows that the amyloid Aβ sequence is not necessary to produce a protective immune response that specifically targets generic amyloid oligomers. Using a non-human, random sequence antigen may facilitate the development of a vaccine that avoids autoimmune side effects.

  2. Generation of a safety enhanced Salmonella Gallinarum ghost using antibiotic resistance free plasmid and its potential as an effective inactivated vaccine candidate against fowl typhoid.

    Science.gov (United States)

    Jawale, Chetan V; Chaudhari, Atul A; Lee, John Hwa

    2014-02-19

    A safety enhanced Salmonella Gallinarum (SG) ghost was constructed using an antibiotic resistance gene free plasmid and evaluated its potential as fowl typhoid (FT) vaccine candidate. The antibiotic resistance free pYA3342 plasmid possesses aspartate semialdehyde dehydrogenase gene which is complimentary to the deletion of the chromosomal asd gene in the bacterial host. This plasmid was incorporated with a ghost cassette containing the bacteriophage PhiX174 lysis gene E, designated as pJHL101. The plasmid pJHL101 was transformed into a two virulence genes-deleted SG. The SG ghosts with tunnel formation and loss of cytoplasmic contents were observed by scanning electron microscopy and transmission electron microscopy. The cell viability of the culture solution was decreased to 0% at 24h after the induction of gene E expression by an increase in temperature from 37°C to 42°C. The safety and protective efficacy of the SG ghost vaccine was further examined in chickens which were divided into three groups: group A (non-immunized control), group B (orally immunized), and group C (intramuscularly immunized). The birds were immunized at 7d of age. No clinical symptoms associated with FT such as anorexia, depression and greenish diarrhea were observed in the immunized chickens. Upon challenge with a virulent SG strain at 3 week post-immunization, the chickens immunized with the SG ghost via various routes were efficiently protected, as shown by significantly lower mortality and post-mortem lesions in comparison with control group. In addition, all the immunized chickens showed significantly higher antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response along with significantly increased numbers of CD4⁺ and CD8⁺ T lymphocytes. Overall, our results provide a promising approach of generating SG ghosts using the antibiotic resistance free plasmid in order to prepare a non-living bacterial vaccine candidate which could be

  3. Complete Genome Sequence of NC983, a Live Attenuated Strain of Salmonella enterica Serovar Typhimurium

    Science.gov (United States)

    Troxell, Bryan; Fink, Ryan C.; Dickey, Allison N.; Scholl, Elizabeth H.

    2016-01-01

    Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate. PMID:27738027

  4. A 78 kDa host cell invasion protein of Neospora caninum as a potential vaccine candidate.

    Science.gov (United States)

    Lv, Qiang; Xing, Shenyang; Gong, Pengtao; Chang, Le; Bian, Zhengzheng; Wang, Lidong; Zhang, Xichen; Li, Jianhua

    2015-01-01

    Neosporosis is an intracellular protozoan disease caused by Neospora caninum. Until now, there is no effective vaccine to prevent neosporosis. The host cell binding protein has the potential as neosporosis vaccine. In the present study, a T7 phage display library was constructed and screened using Vero cells to obtain host cell binding protein of N. caninum. Two host cell binding proteins, a hypothetical protein of 78 kDa (named as NcP78) homologous to the acylglycerol lipase of Toxoplasma gondii ME49 (XP_002370319.1) and NcGRA7 (known as a dense granules protein that is involved in the invasion of N. caninum to the host cells), were identified. Immune responses induced by recombinant NcP78 and NcGRA7 proteins and their protective efficacies against homologous challenge in BALB/c mice were evaluated respectively. Results showed that recombinant NcP78 and NcGRA7 could elicit both Th1 and Th2 immune responses (with the elevated levels of IgG1 and IgG2a antibody), but predominately a Th2 immune response with a high level of IgG1. The ani-NcP78 and anti-NcGRA7 serum also had inhibitory effects on N. caninum invasion to Vero cells in vitro, which indicated that both NcP78 and NcGRA7 proteins were involved in host cell invasion. Recombinant NcP78 and NcGRA7 could not prolong the survival times and improve the survival rates of dams, but could prolong the survival times and improve the survival rates of offspring significantly. Moreover, the recombinant NcP78 and NcGRA7 could reduce the brain parasite load of dams and offspring. Though these protein vaccines could not effectively alleviate the symptom of abortion, they could increase the number of born offspring significantly, indicating that Nc78 and NcGRA7 recombinant proteins could provide a partial protection against N. caninum infection in mice.

  5. Complete chloroplast genome sequence of an orchid model plant candidate: Erycina pusilla apply in tropical Oncidium breeding.

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    I-Chun Pan

    Full Text Available Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC clones and next-generation sequencing (NGS, the chloroplast (cp genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding.

  6. Pooled Sequencing of Candidate Genes Implicates Rare Variants in the Development of Asthma Following Severe RSV Bronchiolitis in Infancy

    Science.gov (United States)

    Torgerson, Dara G.; Giri, Tusar; Druley, Todd E.; Zheng, Jie; Huntsman, Scott; Seibold, Max A.; Young, Andrew L.; Schweiger, Toni; Yin-Declue, Huiqing; Sajol, Geneline D.; Schechtman, Kenneth B; Hernandez, Ryan D.; Randolph, Adrienne G.; Bacharier, Leonard B.; Castro, Mario

    2015-01-01

    Severe infection with respiratory syncytial virus (RSV) during infancy is strongly associated with the development of asthma. To identify genetic variation that contributes to asthma following severe RSV bronchiolitis during infancy, we sequenced the coding exons of 131 asthma candidate genes in 182 European and African American children with severe RSV bronchiolitis in infancy using anonymous pools for variant discovery, and then directly genotyped a set of 190 nonsynonymous variants. Association testing was performed for physician-diagnosed asthma before the 7th birthday (asthma) using genotypes from 6,500 individuals from the Exome Sequencing Project (ESP) as controls to gain statistical power. In addition, among patients with severe RSV bronchiolitis during infancy, we examined genetic associations with asthma, active asthma, persistent wheeze, and bronchial hyperreactivity (methacholine PC20) at age 6 years. We identified four rare nonsynonymous variants that were significantly associated with asthma following severe RSV bronchiolitis, including single variants in ADRB2, FLG and NCAM1 in European Americans (p = 4.6x10-4, 1.9x10-13 and 5.0x10-5, respectively), and NOS1 in African Americans (p = 2.3x10-11). One of the variants was a highly functional nonsynonymous variant in ADRB2 (rs1800888), which was also nominally associated with asthma (p = 0.027) and active asthma (p = 0.013) among European Americans with severe RSV bronchiolitis without including the ESP. Our results suggest that rare nonsynonymous variants contribute to the development of asthma following severe RSV bronchiolitis in infancy, notably in ADRB2. Additional studies are required to explore the role of rare variants in the etiology of asthma and asthma-related traits following severe RSV bronchiolitis. PMID:26587832

  7. Pooled Sequencing of Candidate Genes Implicates Rare Variants in the Development of Asthma Following Severe RSV Bronchiolitis in Infancy.

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    Dara G Torgerson

    Full Text Available Severe infection with respiratory syncytial virus (RSV during infancy is strongly associated with the development of asthma. To identify genetic variation that contributes to asthma following severe RSV bronchiolitis during infancy, we sequenced the coding exons of 131 asthma candidate genes in 182 European and African American children with severe RSV bronchiolitis in infancy using anonymous pools for variant discovery, and then directly genotyped a set of 190 nonsynonymous variants. Association testing was performed for physician-diagnosed asthma before the 7th birthday (asthma using genotypes from 6,500 individuals from the Exome Sequencing Project (ESP as controls to gain statistical power. In addition, among patients with severe RSV bronchiolitis during infancy, we examined genetic associations with asthma, active asthma, persistent wheeze, and bronchial hyperreactivity (methacholine PC20 at age 6 years. We identified four rare nonsynonymous variants that were significantly associated with asthma following severe RSV bronchiolitis, including single variants in ADRB2, FLG and NCAM1 in European Americans (p = 4.6x10-4, 1.9x10-13 and 5.0x10-5, respectively, and NOS1 in African Americans (p = 2.3x10-11. One of the variants was a highly functional nonsynonymous variant in ADRB2 (rs1800888, which was also nominally associated with asthma (p = 0.027 and active asthma (p = 0.013 among European Americans with severe RSV bronchiolitis without including the ESP. Our results suggest that rare nonsynonymous variants contribute to the development of asthma following severe RSV bronchiolitis in infancy, notably in ADRB2. Additional studies are required to explore the role of rare variants in the etiology of asthma and asthma-related traits following severe RSV bronchiolitis.

  8. Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1.

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    Brigitte M Krenn

    Full Text Available BACKGROUND: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA of highly pathogenic avian influenza viruses (HPAIV was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2. We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4, whereas H5N1 HPAIVs lose infectivity at pH ≤ 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose₅₀. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. CONCLUSION/SIGNIFICANCE: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may

  9. Assessment of severe malaria in a multicenter, phase III, RTS, S/AS01 malaria candidate vaccine trial: case definition, standardization of data collection and patient care

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    Vekemans Johan

    2011-08-01

    Full Text Available Abstract Background An effective malaria vaccine, deployed in conjunction with other malaria interventions, is likely to substantially reduce the malaria burden. Efficacy against severe malaria will be a key driver for decisions on implementation. An initial study of an RTS, S vaccine candidate showed promising efficacy against severe malaria in children in Mozambique. Further evidence of its protective efficacy will be gained in a pivotal, multi-centre, phase III study. This paper describes the case definitions of severe malaria used in this study and the programme for standardized assessment of severe malaria according to the case definition. Methods Case definitions of severe malaria were developed from a literature review and a consensus meeting of expert consultants and the RTS, S Clinical Trial Partnership Committee, in collaboration with the World Health Organization and the Malaria Clinical Trials Alliance. The same groups, with input from an Independent Data Monitoring Committee, developed and implemented a programme for standardized data collection. The case definitions developed reflect the typical presentations of severe malaria in African hospitals. Markers of disease severity were chosen on the basis of their association with poor outcome, occurrence in a significant proportion of cases and on an ability to standardize their measurement across research centres. For the primary case definition, one or more clinical and/or laboratory markers of disease severity have to be present, four major co-morbidities (pneumonia, meningitis, bacteraemia or gastroenteritis with severe dehydration are excluded, and a Plasmodium falciparum parasite density threshold is introduced, in order to maximize the specificity of the case definition. Secondary case definitions allow inclusion of co-morbidities and/or allow for the presence of parasitaemia at any density. The programmatic implementation of standardized case assessment included a clinical

  10. Assessment of Mycobacterium bovis Deleted in p27-p55 Virulence Operon as Candidate Vaccine against Tuberculosis in Animal Models

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    María V. Bianco

    2014-01-01

    Full Text Available A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.

  11. Accelerating Novel Candidate Gene Discovery in Neurogenetic Disorders via Whole-Exome Sequencing of Prescreened Multiplex Consanguineous Families

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    Anas M. Alazami

    2015-01-01

    Full Text Available Our knowledge of disease genes in neurological disorders is incomplete. With the aim of closing this gap, we performed whole-exome sequencing on 143 multiplex consanguineous families in whom known disease genes had been excluded by autozygosity mapping and candidate gene analysis. This prescreening step led to the identification of 69 recessive genes not previously associated with disease, of which 33 are here described (SPDL1, TUBA3E, INO80, NID1, TSEN15, DMBX1, CLHC1, C12orf4, WDR93, ST7, MATN4, SEC24D, PCDHB4, PTPN23, TAF6, TBCK, FAM177A1, KIAA1109, MTSS1L, XIRP1, KCTD3, CHAF1B, ARV1, ISCA2, PTRH2, GEMIN4, MYOCD, PDPR, DPH1, NUP107, TMEM92, EPB41L4A, and FAM120AOS. We also encountered instances in which the phenotype departed significantly from the established clinical presentation of a known disease gene. Overall, a likely causal mutation was identified in >73% of our cases. This study contributes to the global effort toward a full compendium of disease genes affecting brain function.

  12. Accelerating novel candidate gene discovery in neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families.

    Science.gov (United States)

    Alazami, Anas M; Patel, Nisha; Shamseldin, Hanan E; Anazi, Shamsa; Al-Dosari, Mohammed S; Alzahrani, Fatema; Hijazi, Hadia; Alshammari, Muneera; Aldahmesh, Mohammed A; Salih, Mustafa A; Faqeih, Eissa; Alhashem, Amal; Bashiri, Fahad A; Al-Owain, Mohammed; Kentab, Amal Y; Sogaty, Sameera; Al Tala, Saeed; Temsah, Mohamad-Hani; Tulbah, Maha; Aljelaify, Rasha F; Alshahwan, Saad A; Seidahmed, Mohammed Zain; Alhadid, Adnan A; Aldhalaan, Hesham; AlQallaf, Fatema; Kurdi, Wesam; Alfadhel, Majid; Babay, Zainab; Alsogheer, Mohammad; Kaya, Namik; Al-Hassnan, Zuhair N; Abdel-Salam, Ghada M H; Al-Sannaa, Nouriya; Al Mutairi, Fuad; El Khashab, Heba Y; Bohlega, Saeed; Jia, Xiaofei; Nguyen, Henry C; Hammami, Rakad; Adly, Nouran; Mohamed, Jawahir Y; Abdulwahab, Firdous; Ibrahim, Niema; Naim, Ewa A; Al-Younes, Banan; Meyer, Brian F; Hashem, Mais; Shaheen, Ranad; Xiong, Yong; Abouelhoda, Mohamed; Aldeeri, Abdulrahman A; Monies, Dorota M; Alkuraya, Fowzan S

    2015-01-13

    Our knowledge of disease genes in neurological disorders is incomplete. With the aim of closing this gap, we performed whole-exome sequencing on 143 multiplex consanguineous families in whom known disease genes had been excluded by autozygosity mapping and candidate gene analysis. This prescreening step led to the identification of 69 recessive genes not previously associated with disease, of which 33 are here described (SPDL1, TUBA3E, INO80, NID1, TSEN15, DMBX1, CLHC1, C12orf4, WDR93, ST7, MATN4, SEC24D, PCDHB4, PTPN23, TAF6, TBCK, FAM177A1, KIAA1109, MTSS1L, XIRP1, KCTD3, CHAF1B, ARV1, ISCA2, PTRH2, GEMIN4, MYOCD, PDPR, DPH1, NUP107, TMEM92, EPB41L4A, and FAM120AOS). We also encountered instances in which the phenotype departed significantly from the established clinical presentation of a known disease gene. Overall, a likely causal mutation was identified in >73% of our cases. This study contributes to the global effort toward a full compendium of disease genes affecting brain function.

  13. Genome sequence comparison reveals a candidate gene involved in male-hermaphrodite differentiation in papaya (Carica papaya) trees.

    Science.gov (United States)

    Ueno, Hiroki; Urasaki, Naoya; Natsume, Satoshi; Yoshida, Kentaro; Tarora, Kazuhiko; Shudo, Ayano; Terauchi, Ryohei; Matsumura, Hideo

    2015-04-01

    The sex type of papaya (Carica papaya) is determined by the pair of sex chromosomes (XX, female; XY, male; and XY(h), hermaphrodite), in which there is a non-recombining genomic region in the Y and Y(h) chromosomes. This region is presumed to be involved in determination of males and hermaphrodites; it is designated as the male-specific region in the Y chromosome (MSY) and the hermaphrodite-specific region in the Y(h) chromosome (HSY). Here, we identified the genes determining male and hermaphrodite sex types by comparing MSY and HSY genomic sequences. In the MSY and HSY genomic regions, we identified 14,528 nucleotide substitutions and 965 short indels with a large gap and two highly diverged regions. In the predicted genes expressed in flower buds, we found no nucleotide differences leading to amino acid changes between the MSY and HSY. However, we found an HSY-specific transposon insertion in a gene (SVP like) showing a similarity to the Short Vegetative Phase (SVP) gene. Study of SVP-like transcripts revealed that the MSY allele encoded an intact protein, while the HSY allele encoded a truncated protein. Our findings demonstrated that the SVP-like gene is a candidate gene for male-hermaphrodite determination in papaya.

  14. High-purity preparation of HSV-2 vaccine candidate ACAM529 is immunogenic and efficacious in vivo.

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    Sophia T Mundle

    Full Text Available Genital herpes is a sexually transmitted infection (STI caused by herpes simplex virus 2 (HSV-2 and to a lesser extent herpes simplex virus 1 (HSV-1. Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19 without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF. The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.

  15. Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella.

    Science.gov (United States)

    De Benedetto, G; Alfini, R; Cescutti, P; Caboni, M; Lanzilao, L; Necchi, F; Saul, A; MacLennan, C A; Rondini, S; Micoli, F

    2017-01-11

    Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S. Typhimurium and S. Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium ΔtolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications. In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS.

  16. Benefit-of-doubt (BOD) scoring: a sequencing-based method for SNP candidate assessment from high to medium read number data sets.

    Science.gov (United States)

    Sedlazeck, Fritz Joachim; Talloji, Prabhavathi; von Haeseler, Arndt; Bachmair, Andreas

    2013-03-01

    Identification of single nucleotide polymorphisms (SNPs) is a key element in sequence-based genetic analysis. Next generation sequencing offers a cost-effective basis to generate the necessary, large sequence data sets, and bioinformatic methods are being developed to process sequencing machine readouts. We were interested in detection of SNPs in a 350 kb region of an EMS-mutagenized Arabidopsis chromosome 3. The region was selectively analyzed using PCR-generated, overlapping fragments for Solexa sequencing. The ensuing reads provided a high coverage and were processed bioinformatically. In order to assess the SNP candidates obtained with a frequently used alignment program and SNP caller, we developed an additional method that allows the identification of high confidence SNP loci. The method can easily be applied to complete genome sequence data of sufficient coverage.

  17. MRKAd5 HIV-1 Gag/Pol/Nef vaccine-induced T-cell responses inadequately predict distance of breakthrough HIV-1 sequences to the vaccine or viral load.

    Directory of Open Access Journals (Sweden)

    Holly Janes

    Full Text Available BACKGROUND: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. METHODS: Ninety-one male participants (37 placebo and 54 vaccine recipients were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. FINDINGS: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0·04 and greater breadth of post-infection response (median 4.5 vs 2; p = 0·06. Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0·04; Pol p = 0·13; Gag p = 0·89. Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p>0·50. Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4·7 vs 5·1 but the difference was not significant (p = 0·27. Neither was acute viral load associated with distance of the viral sequence to the insert (p>0·30. INTERPRETATION: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect

  18. StreptInCor: a candidate vaccine epitope against S. pyogenes infections induces protection in outbred mice.

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    Edilberto Postol

    Full Text Available Infection with Streptococcus pyogenes (S. pyogenes can result in several diseases, particularly in children. S. pyogenes M protein is the major virulence factor, and certain regions of its N-terminus can trigger autoimmune sequelae such as rheumatic fever in susceptible individuals with untreated group A streptococcal pharyngitis. In a previous study, we utilized a large panel of human peripheral blood cells to define the C-terminal protective epitope StreptInCor (medical identity, which does not induce autoimmune reactions. We recently confirmed the results in HLA-transgenic mice. In the present study, we extended the experimental assays to outbred animals (Swiss mice. Herein, we demonstrate high titers of StreptInCor-specific antibodies, as well as appropriate T-cell immune responses. No cross-reaction to cardiac myosin was detected. Additionally, immunized Swiss mice exhibited 87% survival one month after challenge with S. pyogenes. In conclusion, the data presented herein reinforce previous results in humans and animals and further emphasize that StreptInCor could be an effective and safe vaccine for the prevention of S. pyogenes infections.

  19. Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp.

    Science.gov (United States)

    Maiti, Biswajit; Shetty, Mahesh; Shekar, Malathi; Karunasagar, Iddya; Karunasagar, Indrani

    2011-12-20

    Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His(6) residues was estimated to have a molecular weight of ~38kDa. In Western blot the native protein showed expression at ~36kDa molecular weight which was within the range of major outer membrane proteins (36-44kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.

  20. Comprehensive sieve analysis of breakthrough HIV-1 sequences in the RV144 vaccine efficacy trial.

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    Paul T Edlefsen

    2015-02-01

    Full Text Available The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients. A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or "signatures" and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro. The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021. In particular, site 317 in the third variable loop (V3 overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1 more than did non-signature sites (mean = 0.9 (p < 0.0001, suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine

  1. Leptospirosis vaccines: Past, present, and future

    Directory of Open Access Journals (Sweden)

    Koizumi N

    2005-01-01

    Full Text Available It is well known that Leptospira vaccine prevents the disease. However specificity for serovars limits the efficacy of killed whole cell vaccines. Leptospiral antigens that induce cross-protective immunity to the various serovars are sought as new vaccine candidates. In this paper, we have summarized both past and current findings about leptospiral antigens that are conserved among pathogenic leptospires and that induce protective immunity in animal models. The full-length genome sequences of two Leptospira strains have been published and reverse vaccinology has been used to identify leptospiral vaccine candidates. Although humoral immunity is thought to be dominant in protection from leptospiral infection, a role for cell-mediated immunity is now being explored.

  2. Malaria vaccine based on self-assembling protein nanoparticles.

    Science.gov (United States)

    Burkhard, Peter; Lanar, David E

    2015-01-01

    Despite recent progress with GSK's RTS,S malaria vaccine, there remains a desperate need for an efficient malaria vaccine. We have used a repetitive antigen display technology to display malaria specific B cell and T cell epitopes in an effort to design a vaccine against Plasmodium falciparum malaria. Our protein sequence when assembled into a nanoparticle induces strong, long-lived and protective immune responses against infection with the parasite. We are confident that the clinical trials with our most developed vaccine candidate will show good protection in a controlled human malaria infection trial.

  3. Selection and characterization of vaccine strain for Enterovirus 71 vaccine development.

    Science.gov (United States)

    Chang, Jui-Yuan; Chang, Cheng-Peng; Tsai, Hutchinson Hau-Pong; Lee, Chen-Dou; Lian, Wei-Cheng; Ih-Jen-Su; Sai, I-Hsi; Liu, Chia-Chyi; Chou, Ai-Hsiang; Lu, Ya-Jung; Chen, Ching-Yao; Lee, Pi-Hsiu; Chiang, Jen-Ron; Chong, Pele Choi-Sing

    2012-01-17

    Enterovirus 71 (EV71) has recently emerged as an important neurotropic virus in Asia because effective medications and prophylactic vaccine against EV71 infection are not available. Based on the success of inactivated poliovirus vaccine, the Vero cell-based chemically inactivated EV71 vaccine candidate could be developed. Identification of EV71 vaccine strain which can grow to high titer in Vero cell and induce cross-genotype virus neutralizing antibody responses represents the first step in vaccine development. In this report we describe the characterization and validation of a clinical isolate E59 belonging to B4 sub-genotype based on VP1 genetic analysis. Before selected as the vaccine strain, the genetic stability of E59 in passage had been analyzed based on the nucleotide sequences obtained from the Master Virus Seed, Working Seed banks and the virus harvested from the production lots, and found to be identical to those found in the original isolate. These results indicate that E59 vaccine strain has strong genetic stability in passage. Using this vaccine strain the prototype EV71 vaccine candidate was produced from 20L of Vero cell grown in serum-containing medium. The production processes were investigated, characterized and quantified to establish the potential vaccine manufacturing process including the time for virus harvest, the membrane for diafiltration and concentration, the gel-filtration chromatography for the down-stream virus purification, and the methods for viral inactivation. Finally, the inactivated virion vaccine candidate containing sub-microgram of viral proteins formulated with alum adjuvant was found to induce strong virus neutralizing antibody responses in mice and rabbits. Therefore, these results provide valuable information for cell-based EV71 vaccine development.

  4. Preliminary evaluation of DNA vaccine candidates encoding dengue-2 prM/E and NS1: their immunity and protective efficacy in mice.

    Science.gov (United States)

    Lu, Hui; Xu, Xiao-Feng; Gao, Na; Fan, Dong-Ying; Wang, Juan; An, Jing

    2013-06-01

    Public health is still seriously threatened by dengue virus (DENV) and no vaccine against DENV is yet available for clinical use till now. In this study, DNA vaccine candidates encoding DENV serotype 2 (DENV-2) prM/E (premembrane and envelope proteins) and NS1 (non-structural 1 protein) with or without a gene adjuvant, granulocyte-macrophage colony-stimulating factor (GM-CSF), were evaluated in the aspects of immunity and protective efficacy in mice. We constructed three plasmids, pCAG-prM/E (which only expressed DENV2 prM/E), pCAG-prM/E/NS1 (which only expressed DENV2 prM/E/NS1) and pCAG-DG (which co-expressed DENV2 prM/E/NS1 and GM-CSF). The expressions of the recombined plasmids were analyzed by immuno-staining in Vero cells. Antibody responses and neutralization activity of the sera from the mice were assayed by ELISA and plaque reduction neutralization test after immunization with the plasmids. Immunized BALB/c mice were intracerebrally challenged with DENV2 to evaluate protective efficacy of the plasmids. The recombinant plasmids could be efficiently expressed in Vero cells and induced different levels of specific anti-DENV2 immune responses. The immunized mice were partially protected. The highest survival rate was observed in the pCAG-DG group although the anti-DENV2 titer and neutralization antibody titer were not the highest among the three groups. Our data suggested that pCAG-DG offered better protection against DENV2 infection.

  5. Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

    Directory of Open Access Journals (Sweden)

    P.R. Santos

    2003-11-01

    Full Text Available Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

  6. Mining for Candidate Genes in an Introgression Line by Using RNA Sequencing: The Anthocyanin Overaccumulation Phenotype in Brassica

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    Lulu Xie

    2016-08-01

    Full Text Available Introgression breeding is a widely used method for the genetic improvement of crop plants; however, the mechanism underlying candidate gene flow patterns during hybridization is poorly understood. In this study, we used a powerful pipeline to investigate a Chinese cabbage (Brassica rapa L. ssp. pekinensis introgression line with the anthocyanin overaccumulation phenotype. Our purpose was to analyze the gene flow patterns during hybridization and elucidate the genetic factors responsible for the accumulation of this important pigment compound. We performed RNA-seq analysis by using two pipelines, one with and one without a reference sequence, to obtain transcriptome data. We identified 930 significantly differentially expressed genes (DEGs between the purple-leaf introgression line and B. rapa green cultivar, namely, 389 up-regulated and 541 down-regulated DEGs that mapped to the B. rapa reference genome. Since only one anthocyanin pathway regulatory gene was identified, i.e., Bra037887 (bHLH, we mined unmapped reads, revealing 2,031 de novo assembled unigenes, including c3563g1i2. Phylogenetic analysis suggested that c3563g1i2, which was transferred from the Brassica B genome of the donor parental line Brassica juncea, may represent an R2R3-MYB transcription factor that participates in the ternary transcriptional activation complex responsible for the anthocyanin overaccumulation phenotype of the B. rapa introgression line. We also identified genes involved in cold and light reaction pathways that were highly upregulated in the introgression line, as confirmed using quantitative real-time PCR analysis. The results of this study shed light on the mechanisms underlying the purple leaf trait in Brassica plants and may facilitate the use of introgressive hybridization for many traits of interest.

  7. The Neisseria meningitidis Macrophage Infectivity Potentiator Protein Induces Cross-Strain Serum Bactericidal Activity and Is a Potential Serogroup B Vaccine Candidate

    Science.gov (United States)

    Hung, Miao-Chiu; Salim, Omar; Williams, Jeannette N.; Heckels, John E.; Christodoulides, Myron

    2011-01-01

    A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)3. Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection. PMID:21708989

  8. The Neisseria meningitidis macrophage infectivity potentiator protein induces cross-strain serum bactericidal activity and is a potential serogroup B vaccine candidate.

    Science.gov (United States)

    Hung, Miao-Chiu; Salim, Omar; Williams, Jeannette N; Heckels, John E; Christodoulides, Myron

    2011-09-01

    A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)(3). Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection.

  9. Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice.

    Science.gov (United States)

    Elhaik Goldman, Shirin; Dotan, Shahar; Talias, Amir; Lilo, Amit; Azriel, Shalhevet; Malka, Itay; Portnoi, Maxim; Ohayon, Ariel; Kafka, Daniel; Ellis, Ronald; Elkabets, Moshe; Porgador, Angel; Levin, Ditza; Azhari, Rosa; Swiatlo, Edwin; Ling, Eduard; Feldman, Galia; Tal, Michael; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2016-04-01

    involvement. In addition, rabbit and mouse anti-rFBA antisera significantly protected the mice against a lethal S. pneumoniae challenge in comparison with preimmune sera. Our results emphasize the mixed involvement of the Th1, Th2 and Th17 arms of the immune system in response to immunization with pneumococcal rFBA, a potential vaccine candidate.

  10. Genome-Wide Association Study with Sequence Variants Identifies Candidate Genes for Mastitis Resistance in Dairy Cattle

    DEFF Research Database (Denmark)

    Sahana, Goutam; Guldbrandtsen, Bernt; Bendixen, Christian;

    Effect Predictor (VEP) vers. 2.6 using ENSEMBL vers. 67 databases. Candidate polymorphisms affecting clinical mastitis were selected based on their association with the traits and functional annotations. A strong positional candidate gene for mastitis resistance on chromosome-6 is the NPFFR2 which...

  11. Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

    Science.gov (United States)

    Sircar, Gaurab; Jana, Kuladip; Dasgupta, Angira; Saha, Sudipto; Gupta Bhattacharya, Swati

    2016-08-19

    Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing

  12. Safety and immunogenicity of escalating dosages of a single oral administration of peru-15 pCTB, a candidate live, attenuated vaccine against enterotoxigenic Escherichia coli and Vibrio cholerae.

    Science.gov (United States)

    Chen, Wilbur H; Garza, Jose; Choquette, Monique; Hawkins, Jennifer; Hoeper, Amy; Bernstein, David I; Cohen, Mitchell B

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) organisms are a leading cause of infectious diarrhea in developing countries. A live, attenuated cholera strain that expresses high levels of the nontoxic B subunit of cholera toxin, which might also serve as an ETEC protective antigen, was evaluated for safety, excretion, and immunogenicity in healthy volunteers. We enrolled four inpatient dose-escalation cohorts of 15 to 16 eligible subjects to randomly (3:1) receive a single oral dose of vaccine or placebo (buffer alone), evaluating 1 ×10(7), 1 ×10(8), 1 ×10(9), and 1 ×10(10) CFU of the vaccine. The vaccine was well tolerated, although some subjects experienced moderate diarrhea. The serum Inaba vibriocidal antibody response appeared to display a dose-response relationship with increasing dosages of vaccine, plateauing at the 10(9)-CFU dosage. The serum antitoxin (cholera toxin and heat-labile enterotoxin) antibody seroconversion rate (4-fold increase over baseline) also appeared to display a dose-response relationship. The vaccine strain was excreted in stool cultures, displaying a dose-response relationship. A single oral dose of Peru-15 pCTB at dosages up to 1 ×10(10) CFU was safe and immunogenic in this first-in-human trial. These encouraging data support the ongoing clinical development of this candidate combined cholera and ETEC vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT00654108.).

  13. Liposomal immunostimulatory DNA sequence (ISS-ODN): an efficient parenteral and mucosal adjuvant for influenza and hepatitis B vaccines.

    Science.gov (United States)

    Joseph, Aviva; Louria-Hayon, Igal; Plis-Finarov, Alla; Zeira, Evelyne; Zakay-Rones, Zichria; Raz, Eyal; Hayashi, Tomoko; Takabayashi, Kenji; Barenholz, Yechezkel; Kedar, Eli

    2002-09-10

    Synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory sequences (ISS-ODN, also known as CpG-ODNs) have been shown to display in experimental models potent Th1-biassed immunoadjuvant activity upon parenteral or mucosal co-administration with a variety of antigens. In an attempt to potentiate adjuvant activity, and to reduce dose and number of administrations, ISS-ODN was entrapped (up to 90% efficiency) in large (1.5 microm) multilamellar liposomes using a simple and fast (5 min) procedure. Mice were vaccinated once or twice intramuscularly (i.m.) or intranasally (i.n.) with subunit influenza vaccines (consisting of the viral hemagglutinin and neuraminidase, HN) or with hepatitis B surface antigen particles (HBsAg), either non-encapsulated or liposome-encapsulated, together with free or liposomal ISS-ODN (5-25 microg per dose). At 3-12 weeks post-vaccination, the humoral (systemic, mucosal) and cellular responses and protective immunity were assessed. Vaccine formulations containing liposomal ISS-ODN co-administered with either soluble antigen or liposomal antigen (in the same vesicles or in separate vesicles) were up to 30 times more effective than formulations containing un-encapsulated ISS-ODN in inducing: (a) antigen-specific serum and mucosal IgG2a and IgA antibodies; (b) splenocyte proliferative response, cytotoxic activity and IFNgamma production; (c) a DTH response; and (d) protection against virus challenge. The response was Th1-dominant in the influenza model and a mixed Th1+Th2 response in the hepatitis B model. No adverse reactions were noted. Thus, liposomal encapsulation of ISS-ODN further enhances its inherent adjuvant activity.

  14. The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

    Science.gov (United States)

    Albarracín, Romina M; Becher, Melina Laguía; Farran, Inmaculada; Sander, Valeria A; Corigliano, Mariana G; Yácono, María L; Pariani, Sebastián; López, Edwin Sánchez; Veramendi, Jon; Clemente, Marina

    2015-05-01

    Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.

  15. UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate.

    Science.gov (United States)

    Foucault, Marine; Mayol, Katia; Receveur-Bréchot, Véronique; Bussat, Marie-Claire; Klinguer-Hamour, Christine; Verrier, Bernard; Beck, Alain; Haser, Richard; Gouet, Patrice; Guillon, Christophe

    2010-05-01

    The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function.

  16. Transcriptome Analysis of Human Peripheral Blood Mononuclear Cells Exposed to Lassa Virus and to the Attenuated Mopeia/Lassa Reassortant 29 (ML29), a Vaccine Candidate

    Science.gov (United States)

    Zapata, Juan Carlos; Carrion, Ricardo; Patterson, Jean L.; Crasta, Oswald; Zhang, Yan; Mani, Sachin; Jett, Marti; Poonia, Bhawna; Djavani, Mahmoud; White, David M.; Lukashevich, Igor S.; Salvato, Maria S.

    2013-01-01

    Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease. PMID:24069471

  17. Transcriptome analysis of human peripheral blood mononuclear cells exposed to Lassa virus and to the attenuated Mopeia/Lassa reassortant 29 (ML29, a vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Juan Carlos Zapata

    Full Text Available Lassa virus (LASV is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG, as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

  18. Transcriptome analysis of human peripheral blood mononuclear cells exposed to Lassa virus and to the attenuated Mopeia/Lassa reassortant 29 (ML29), a vaccine candidate.

    Science.gov (United States)

    Zapata, Juan Carlos; Carrion, Ricardo; Patterson, Jean L; Crasta, Oswald; Zhang, Yan; Mani, Sachin; Jett, Marti; Poonia, Bhawna; Djavani, Mahmoud; White, David M; Lukashevich, Igor S; Salvato, Maria S

    2013-01-01

    Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

  19. Disruption of the Putative Vascular Leak Peptide Sequence in the Stabilized Ricin Vaccine Candidate RTA1-33/44-198

    Science.gov (United States)

    2013-01-29

    15]. The crystal structures, PDB 3LC9 and 3MK9, show that only a minor change in a T-cell epitope, conversion of a helix-turn-helix to a helical ...the D75A variant. In total, four triple mutants were constructed. The number of free thiols and apparent Tm were measured as previously described [15...Three of the triple mutant variants (Table 3) had Tm values comparable to RTA1-33/44-198 R48C/T77C or V49C/E99C. Only one variant, RTA1-33/44-198

  20. Genome Sequence of the Soviet/Russian Bacillus anthracis Vaccine Strain 55-VNIIVViM

    Science.gov (United States)

    Kotorashvili, Adam

    2016-01-01

    Bacillus anthracis strain 55-VNIIVViM is a live-attenuated nonencapsulated Soviet/Russian veterinary anthrax vaccine strain. We report here the genome of 55-VNIIVViM and confirm its phylogenetic placement in the global population structure of B. anthracis. PMID:28007853

  1. Phase 1/2a Study of the Malaria Vaccine Candidate Apical Membrane Antigen-1 (AMA-1) Administered in Adjuvant System AS01B or AS02A

    Science.gov (United States)

    2009-04-01

    International Conference on Harmonisation (ICH) guidelines and confirmed the vaccine antigen was stable and potent from date of manufacture through...Maryland, United States of America, 8 Malaria Vaccine Development Program, United States Agency for International Development, Washington D. C., United...into a multi-stage, multi-component vaccine [22]. First, an AMA-1 vaccine must confer significant clinical benefit in either a Phase 2a malaria

  2. Poly(lactic-co-glycolic acid) nanoparticles as candidate DNA vaccine carrier for oral immunization of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus.

    Science.gov (United States)

    Tian, Jiyuan; Yu, Juan

    2011-01-01

    In order to protect DNA vaccine against degradation in alimentary tract of fish, poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating vaccine were prepared using W/O/W emulsification combined with spray drying technique in our laboratory. The characteristics of PLGA nanoparticles were described as follows: (1) shape, spherical; (2) size, 0.05). Pearson's correlation displayed that correlation of immune factors mentioned above (not including serum complement) were all positive for fish vaccinated. The data in this study suggested that PLGA nanoparticles were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by oral approach.

  3. Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

    Directory of Open Access Journals (Sweden)

    Melissa K Boles

    2009-12-01

    Full Text Available An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn, inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

  4. Improving the safety of viral DNA vaccines: development of vectors containing both 5' and 3' homologous regulatory sequences from non-viral origin.

    Science.gov (United States)

    Martinez-Lopez, A; Encinas, P; García-Valtanen, P; Gomez-Casado, E; Coll, J M; Estepa, A

    2013-04-01

    Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

  5. Genetic characterisation of Malawian pneumococci prior to the roll-out of the PCV13 vaccine using a high-throughput whole genome sequencing approach.

    Directory of Open Access Journals (Sweden)

    Dean B Everett

    Full Text Available BACKGROUND: Malawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13 into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction. METHODS: One hundred and twenty seven (127 archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing. RESULTS: The pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds. Several Pneumococcal Molecular Epidemiology Network (PMEN clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching. CONCLUSIONS: High throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.

  6. Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain.

    Science.gov (United States)

    Mitra, Soma; Sinha, Ritam; Mitobe, Jiro; Koley, Hemanta

    2016-04-04

    cost-effective vaccine candidate against shigellosis.

  7. Immunological evaluation of OMV(PagL)+Bap(1-487aa) and AbOmpA(8-346aa)+Bap(1-487aa) as vaccine candidates against Acinetobacter baumannii sepsis infection.

    Science.gov (United States)

    Badmasti, Farzad; Ajdary, Soheila; Bouzari, Saeid; Fooladi, Abbas Ali Imani; Shahcheraghi, Fereshteh; Siadat, Seyed Davar

    2015-10-01

    Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients with sepsis form. The surface exposed virulence proteins and serum resistance factors helping to dissemination of this bacterium to bloodstream are the most promising vaccine candidates against this microorganism. In this project we immunologically evaluated OMV(PagL)+Bap(1-487aa) and AbOmpA (8-346aa)+Bap(1-487aa) as combination forms as well as Bap(1-487aa), AbOmpA(8-346aa) and OMV(PagL) singly, with addition of alum adjuvant as vaccine candidates. The titers of total IgG, IgG1 and IgG2c as well as concentration of IL-4 and IFN-γ and survival rates were measured in a C57BL/6 murine model with disseminated sepsis. The ratio of IgG1/IgG2c and profile of IL-4/IFN-γ in OMV (PagL)+Bap (1-487aa) formulation shows the humoral and cellular immune responses have been induced robustly and have created a full protection against A. baumannii ATCC 19606 and MDR AB-44 strains. We found that the two combination vaccine candidates were protective and induced both Th1 and Th2 responses.

  8. A systems biology perspective on rational design of peptide vaccine against virus infections.

    Science.gov (United States)

    Chen, Jiajia; Wang, Ying; Guo, Deyin; Shen, Bairong

    2012-01-01

    With the recent onset of influenza A (H1N1) pandemic, the need for improved vaccines against virus infections has become an international priority. Strategies for vaccine development have changed over time, from whole-virus to immunogenic proteins and further to antigenic viral peptides. Various algorithms and bioinformatics tools have been developed to predict immunogenic peptide regions in an antigenic protein sequence. Recent advances in next-generation sequencing technologies, as represented by real time DNA