Discovery Of Vaccine Candidates Research Articles

Characterization of Flu MOSAIC nanoparticle vaccine candidate using high performance size-exclusion chromatography to support vaccine process development

This report describes an application of analytical high performance size exclusion chromatography with UV and Fluorescent detection (HPSEC-UV/FLR) method that enabled a bridging from research vaccine candidate discovery (His-tagged model) to clinical product development (Non-His-tagged molecules). HPSEC measurement can accurately determine the total trimer-to-pentamer molar ratio by either titration evaluation during the nanoparticle being assembled or dissociation during a well-formed nanoparticle being dis-assembled. Through experimental design with small sample consumptions, HPSEC can provide a quick determination on the nanoparticle assembling efficiency which can therefore guide the buffer optimization for an assembly, from His-tagged model nanoparticle, to non-His-tagged clinical development product. HPSEC has also discovered a difference in assembling efficiencies for various strains of HAx-dn5B with Pentamer-dn5A components, and different efficiencies for monovalent assembly vs. multivalent assembly. The present study demonstrates HPSEC as a pivotal tool to support the Flu Mosaic nanoparticle vaccine development from research to clinical production.

An insight into differential protein abundance throughout Leishmania donovani promastigote growth and differentiation

Leishmania donovani causes anthroponotic visceral leishmaniasis, responsible for about 50,000 annual deaths worldwide. Current therapies have considerable side effects. Drug resistance has been reported and no vaccine is available nowadays. The development of undifferentiated promastigotes in the sand fly vector’s gut leads to the promastigote form that is highly infective to the mammalian host. Fully differentiated promastigotes play a crucial role in the initial stages of mammalian host infection before internalization in the host phagocytic cell. Therefore, the study of protein levels in the promastigote stage is relevant for disease control, and proteomics analysis is an ideal source of vaccine candidate discovery. This study aims to get insight into the protein levels during the differentiation process of promastigotes by 2DE-MALDI-TOF/TOF. This partial proteome analysis has led to the identification of 75 proteins increased in at least one of the L. donovani promastigote differentiation and growth phases. This study has revealed the differential abundance of said proteins during growth and differentiation. According to previous studies, some are directly involved in parasite survival or are immunostimulatory. The parasite survival–related proteins are ascorbate peroxidase; cystathionine β synthase; an elongation factor 1β paralog; elongation factor 2; endoribonuclease L-PSP; an iron superoxide dismutase paralog; GDP-mannose pyrophosphorylase; several heat shock proteins—HSP70, HSP83-17, mHSP70-rel, HSP110; methylthioadenosine phosphorylase; two thiol-dependent reductase 1 paralogs; transitional endoplasmic reticulum ATPase; and the AhpC thioredoxin paralog. The confirmed immunostimulatory proteins are the heat shock proteins, enolase, and protein kinase C receptor analog. The potential immunostimulatory molecules according to findings in patogenic bacteria are fructose-1,6-diphophate aldolase, dihydrolipoamide acetyltransferase, isocitrate dehydrogenase, pyruvate dehydrogenase E1α and E1β subunits, and triosephosphate isomerase. These proteins may become disease control candidates through future intra-vector control methods or vaccines.

A new mass spectral library for high-coverage and reproducible analysis of the Plasmodium falciparum-infected red blood cell proteome.

Background Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry–based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility.ResultsHere, we present a comprehensive P. falciparum–infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites.ConclusionA reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

The Relevance of Bioinformatics Applications in the Discovery of Vaccine Candidates and Potential Drugs for COVID-19 Treatment.

The application of bioinformatics to vaccine research and drug discovery has never been so essential in the fight against infectious diseases. The greatest combat of the 21st century against a debilitating disease agent SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus discovered in Wuhan, China, December 2019, has piqued an unprecedented usage of bioinformatics tools in deciphering the molecular characterizations of infectious pathogens. With the viral genome data of SARS-COV-2 been made available barely weeks after the reported outbreak, bioinformatics platforms have become an all-time critical tool to gain time in the fight against the disease pandemic. Before the outbreak, different platforms have been developed to explore antigenic epitopes, predict peptide-protein docking and antibody structures, and simulate antigen-antibody reactions and lots more. However, the advent of the pandemic witnessed an upsurge in the application of these pipelines with the development of newer ones such as the Coronavirus Explorer in the development of efficacious vaccines, drug repurposing, and/or discovery. In this review, we have explored the various pipelines available for use, their relevance, and limitations in the timely development of useful therapeutic candidates from genomic data knowledge to clinical therapy.

Proteomics, Bioinformatics and Structure-Function Antigen Mining For Gonorrhea Vaccines.

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the serious health consequences combined with the prevalence and the dire possibility of untreatable gonorrhea. Reverse vaccinology, which includes genome and proteome mining, has proven successful in the discovery of vaccine candidates against many pathogenic bacteria. Here, we describe proteomic applications including comprehensive, quantitative proteomic platforms and immunoproteomics coupled with broad-ranging bioinformatics that have been applied for antigen mining to develop gonorrhea vaccine(s). We further focus on outlining the vaccine candidate decision tree, describe the structure-function of novel proteome-derived antigens as well as ways to gain insights into their roles in the cell envelope, and underscore new lessons learned about the fascinating biology of Neisseria gonorrhoeae.

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate

BackgroundTechnical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated.ResultsDetailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity.ConclusionsThe MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Vaccine candidate discovery for the next generation of malaria vaccines.

SummaryAlthough epidemiological observations, IgG passive transfer studies and experimental infections in humans all support the feasibility of developing highly effective malaria vaccines, the precise antigens that induce protective immunity remain uncertain. Here, we review the methodologies applied to vaccine candidate discovery for Plasmodium falciparum malaria from the pre‐ to post‐genomic era. Probing of genomic and cDNA libraries with antibodies of defined specificities or functional activity predominated the former, whereas reverse vaccinology encompassing high throughput in silico analyses of genomic, transcriptomic or proteomic parasite data sets is the mainstay of the latter. Antibody‐guided vaccine design spanned both eras but currently benefits from technological advances facilitating high‐throughput screening and downstream applications. We make the case that although we have exponentially increased our ability to identify numerous potential vaccine candidates in a relatively short space of time, a significant bottleneck remains in their validation and prioritization for evaluation in clinical trials. Longitudinal cohort studies provide supportive evidence but results are often conflicting between studies. Demonstration of antigen‐specific antibody function is valuable but the relative importance of one mechanism over another with regards to protection remains undetermined. Animal models offer useful insights but may not accurately reflect human disease. Challenge studies in humans are preferable but prohibitively expensive. In the absence of reliable correlates of protection, suitable animal models or a better understanding of the mechanisms underlying protective immunity in humans, vaccine candidate discovery per se may not be sufficient to provide the paradigm shift necessary to develop the next generation of highly effective subunit malaria vaccines.

Purification of Biotinylated Cell Surface Proteins from <em>Rhipicephalus microplus</em> Epithelial Gut Cells

Rhipicephalus microplus – the cattle tick – is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells.

Rhipicephalus microplus - the cattle tick - is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

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

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.

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform-isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry-to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.

Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression.

Vacceed: a high-throughput in silico vaccine candidate discovery pipeline for eukaryotic pathogens based on reverse vaccinology.

Summary: We present Vacceed, a highly configurable and scalable framework designed to automate the process of high-throughput in silico vaccine candidate discovery for eukaryotic pathogens. Given thousands of protein sequences from the target pathogen as input, the main output is a ranked list of protein candidates determined by a set of machine learning algorithms. Vacceed has the potential to save time and money by reducing the number of false candidates allocated for laboratory validation. Vacceed, if required, can also predict protein sequences from the pathogen’s genome.Availability and implementation: Vacceed is tested on Linux and can be freely downloaded from https://github.com/sgoodswe/vacceed/releases (includes a worked example with sample data). Vacceed User Guide can be obtained from https://github.com/sgoodswe/vacceed.Contact: John.Ellis@uts.edu.auSupplementary information: Supplementary data are available at Bioinformatics online.

Identification of vaccine antigens using integrated proteomic analyses of surface immunogens from serogroup B Neisseria meningitidis

Meningococcal surface proteins capable of evoking a protective immune response are candidates for inclusion in protein-based vaccines against serogroup B Neisseria meningitidis (NmB). In this study, a 2-dimensional (2-D) gel-based platform integrating surface and immune-proteomics was developed to characterize NmB surface protein antigens. The surface proteome was analyzed by differential 2-D gel electrophoresis following treatment of live bacteria with proteinase K. Alongside, proteins recognized by immune sera from mice challenged with live meningococci were detected using 2-D immunoblots. In combination, seventeen proteins were identified including the well documented antigens PorA, OpcA and factor H-binding protein, previously reported potential antigens and novel potential immunogens. Results were validated for the macrophage infectivity potentiator (MIP), a recently proposed NmB vaccine candidate. MIP-specific antisera bound to meningococci in whole-cell ELISA and facilitated opsonophagocytosis and deposition of complement factors on the surface of meningococcal isolates of different serosubtypes. Cleavage by proteinase K was confirmed in western blots and shown to occur in a fraction of the MIP expressed by meningococci suggesting transient or limited surface exposure. These observations add knowledge for the development of a protein NmB vaccine. The proteomic workflow presented here may be used for the discovery of vaccine candidates against other pathogens. This study presents an integrated proteomic strategy to identify proteins from N. meningitidis with desirable properties (i.e. surface exposure and immunogenicity) for inclusion in subunit vaccines against bacterial meningitis. The effectiveness of the method was demonstrated by the identification of some of the major meningococcal vaccine antigens. Information was also obtained about novel potential immunogens as well as the recently described potential antigen macrophage infectivity potentiator which can be useful for its consideration as a vaccine candidate. Additionally, the proteomic strategy presented in this study provides a generic 2-D gel-based platform for the discovery of vaccine candidates against other bacterial infections.

A case for research consortia to accelerate vaccine candidate discovery: identification of a stable antigenic HIV-1 Clade A Env candidate (P6370)

Abstract IAVI’s NAC provides a multi-disciplinary platform to decipher the interaction of bNAbs with HIV Env and accelerate translation to development of vaccines capable of eliciting potent neutralizing abs. Recent successes highlight the value of a consortium to expedite progress. IAVI initiated Protocol G to identify neutralizing sera from HIV-1 infected patients. Using these samples, a cross-disciplinary team discovered abs with unprecedented potency against HIV. PG9 was isolated from a subject infected with HIV-1 clade A. Consortium members reasoned that Env sequences from the clade A infected donor could lead to immunogen discovery. Using bioinformatics, MRCA sequence was aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database and BG505 was identified with the highest homology. Soluble BG505 retains features of native trimer. While native Env trimer is the target for bnAbs, its instability impedes efforts to develop recombinant Env. Because BG505 retains features of native trimer, it might be optimal for immunogen design. NAC partners report construction of a soluble trimer from BG505 that binds PG9 with high affinity and show it is uniquely stable. Formulations of gp120 and trimers are under analysis for immunogenicity. Challenges remain in development including analysis of structure, immunogenicity, ab responses elicited; and production for human testing. A consortium approach of multidisciplinary experience will be required to expedite advancement of vaccine concepts.

A guide to in silico vaccine discovery for eukaryotic pathogens

In this article, a framework for an in silico pipeline is presented as a guide to high-throughput vaccine candidate discovery for eukaryotic pathogens, such as helminths and protozoa. Eukaryotic pathogens are mostly parasitic and cause some of the most damaging and difficult to treat diseases in humans and livestock. Consequently, these parasitic pathogens have a significant impact on economy and human health. The pipeline is based on the principle of reverse vaccinology and is constructed from freely available bioinformatics programs. There are several successful applications of reverse vaccinology to the discovery of subunit vaccines against prokaryotic pathogens but not yet against eukaryotic pathogens. The overriding aim of the pipeline, which focuses on eukaryotic pathogens, is to generate through computational processes of elimination and evidence gathering a ranked list of proteins based on a scoring system. These proteins are either surface components of the target pathogen or are secreted by the pathogen and are of a type known to be antigenic. No perfect predictive method is yet available; therefore, the highest-scoring proteins from the list require laboratory validation.

Pre-exposure to uninfected sand fly bites protects rhesus monkeys against cutaneous leishmaniasis (94.14)

Abstract Leishmaniasis is a vector-borne disease that distresses millions in the poorest regions of the globe. There are no anti-Leishmania vaccines approved for human use. Mice pre-exposed to uninfected sand fly bites (USFB) are protected against cutaneous leishmaniasis (CL). To test if pre-exposure to USFB protects rhesus monkeys against CL, we first developed a model where monkeys challenged with 50 L. major-infected Phlebotomus duboscqi sand flies developed self-healing cutaneous lesions mimicking human CL. In a following experiment, we exposed animals 3 times, every 21 days, to 20 uninfected P. duboscqi. We found that 70% of the monkeys reacted to USFB(DTH reaction and antibodies). Twenty-one days after the last exposure to USFB naïve, pre-exposed reactive (PE-R) and pre-exposed non-reactive monkeys were challenged with 50 L. major-infected P. duboscqi. We observed a reduction in disease burden and healing time in the PE-R group. This protection correlated with a two log reduction in parasite load and the PE-R animals showed a reduced maximum lesion diameter indicative of diminished disease. Skin biopsies two weeks post-infection showed a higher expression of IFN-γ in the PE-R group compared to naïve monkeys. Altogether, these data show that pre-exposure to uninfected P. duboscqi bites can protect monkeys against L. major infection. Identification of the salivary proteins responsible for the observed protection may lead to the discovery of vaccine candidates against human CL.

The wheat germ cell-free protein synthesis system: A key tool for novel malaria vaccine candidate discovery

Malaria kills more than a million people a year, causes malady in about three hundred million people and poses risk to approximately 40% of the world's population living in malarious countries. This disease is re-emerging mainly due to the development of drug-resistant parasites and insecticide-resistant mosquitoes. Therefore, we are now forced to resort to remedy through vaccination. Until now, not even a single licensed malaria vaccine has been developed despite intensive efforts. Even the efficacy of RTS,S, the most advanced and promising vaccine candidate in the pipeline of malaria vaccine development, was only around 50% based on a number of clinical trials. These facts urge malaria researchers to urgently enrich this pipeline, as much as possible, with potential vaccine candidates. With the availability of malaria genome database, the enrichment of this pipeline is possible if we could now employ an efficient protein expression technology to decode the malaria genomic data, without any codon optimization, into quality recombinant proteins. Then, these synthesized recombinant proteins can be characterized and screened for discovering novel potential vaccine targets. The wheat germ cell-free protein synthesis system will be a promising tool to this end. This review highlights the recent successes in synthesizing quality malaria proteins using this tool.

Developing Antibacterial Vaccines in Genomics and Proteomics Era

Infectious diseases are the greatest cause of global morbidity and mortality. About half of this burden is caused by pathogenic bacteria. One of the most effective ways to prevent infectious diseases is to use vaccines. Limitations in the traditional approaches may be one of the reasons why vaccines are yet not available against some infectious bacterial agents. Recent advancements in high-throughput 'omics' technologies and the availability of complete genome sequences of microbial pathogens and multiple isolates of the same species have dramatically changed the time frame and scope for identifying novel vaccine candidates. At present, 582 microbial genomes have been sequenced and sequencing of 1733 is in progress. There are more than 50 bacterial species for which the genome sequence is available for three or more isolates. Development of tools for in silico analysis has led to the identification of novel virulence genes, metabolic pathways and cell surface proteins that represent potential new targets for anti-microbial drug and vaccine strategies. In this review, we provide an overview of the 'omics'-based techniques that can be used to advance and accelerate the discovery of vaccine candidates against extracellular bacterial pathogens. By citing specific examples, we discuss how high-throughput molecular profiling techniques, such as genomics, transcriptomics and proteomics have contributed to the discovery of novel vaccine candidates. We end by contemplating on the emerging technologies that are likely to have a high impact on the field of vaccinology in the near future.

Multivalent recombinant protein vaccine against coccidioidomycosis.

Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity against Coccidioides posadasii infection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome of C. posadasii and a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed by Escherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population.