Field Application of a Subunit Vaccine against an Enteric Protozoan Disease

Background

Coccidiosis is a major global veterinary health problem in intensively reared chickens. It is caused by apicomplexan parasites of the genus Eimeria.

Principal Findings

A subunit vaccine composed of purified antigens from the gametocytes of Eimeria maxima was used to stimulate the production and transfer of maternal antibodies between breeding hens and their hatchlings. The vaccine was injected into hens twice before they began laying eggs. Immunization had no adverse affects on egg laying or health of the hens and resulted in high antibody levels throughout the life of the hens. Progeny of immunized hens excreted significantly less oocysts of various species of Eimeria in their faeces than chicks from unvaccinated hens. Furthermore, the offspring of vaccinated hens developed stronger natural immunity to Eimeria, so that they were resistant to challenge infection even at 8 weeks of age, well after all maternal antibodies had left their circulation. Field trials were conducted in South Africa, Brazil and Thailand, involving at least 1 million progeny of vaccinated hens and at least 1 million positive control birds (raised on feed containing anticoccidial drugs or immunized with a live vaccine) in each country. Additionally, trials were carried out in Israel involving 60 million progeny of vaccinated hens and 112 million positive control birds. There were no significant differences in growth rate, feed conversion ratios or mortality in the offspring of vaccinated hens compared with the positive control chickens in any of these countries regardless of different management practices, different breeds of chickens or climate.

Conclusions

These results demonstrate that a vaccine composed of antigens purified from the gametocytes of Eimeria can be used safely and effectively to prevent the deleterious effects of coccidiosis. It is the first subunit vaccine against any protozoan parasite to be successfully applied on a commercial scale.     

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth

Background

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/Principal Finding

The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/Significance

Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.     

Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery

Background

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood.

Methods and Findings

In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1) that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences.

Conclusion

We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division.     

Immunological Interactions between 2 Common Pathogens,Th1-Inducing Protozoan Toxoplasma gondii and the Th2-Inducing Helminth Fasciola hepatica

Background

The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-γ, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Principal Findings

Regardless, of whether F. hepatica infection preceded or succeeded T. gondii infection, there was little impact on the production of the Th1 cytokines IL-12, IFN-γ or on the development of classically-activated macrophages induced by T. gondii. By contrast, the production of helminth-specific Th2 cytokines, such as IL-4 and IL-5, was suppressed by infection with T. gondii. Additionally, the recruitment and alternative activation of macrophages by F. hepatica was blocked or reversed by subsequent infection with T. gondii. The clinical symptoms of toxoplasmosis and the survival rate of infected mice were not significantly altered by the helminth.

Conclusions

Despite previous studies showing that F. hepatica suppressed the classical activation of macrophages and the Th1-driven responses of mice to bystander microbial infection, as well as reduced their ability to reject these, here we found that the potent immune responses to T. gondii were capable of suppressing the responses to helminth infection. Clearly, the outcome of particular infections in polyparasitoses depends on the means and potency by which each pathogen controls the immune response.     

Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV

Background

Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection.

Results

We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism.

Conclusions

Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-39) contains supplementary material, which is available to authorized users.     

The genome of Eimeria falciformis - reduction and specialization in a single host apicomplexan parasite

Background

The phylum Apicomplexa comprises important unicellular human parasites such as Toxoplasma and Plasmodium. Eimeria is the largest and most diverse genus of apicomplexan parasites and some species of the genus are the causative agent of coccidiosis, a disease economically devastating in poultry. We report a complete genome sequence of the mouse parasite Eimeria falciformis. We assembled and annotated the genome sequence to study host-parasite interactions in this understudied genus in a model organism host.

Results

The genome of E. falciformis is 44 Mb in size and contains 5,879 predicted protein coding genes. Comparative analysis of E. falciformis with Toxoplasma gondii shows an emergence and diversification of gene families associated with motility and invasion mainly at the level of the Coccidia. Many rhoptry kinases, among them important virulence factors in T. gondii, are absent from the E. falciformis genome. Surface antigens are divergent between Eimeria species. Comparisons with T. gondii showed differences between genes involved in metabolism, N-glycan and GPI-anchor synthesis. E. falciformis possesses a reduced set of transmembrane transporters and we suggest an altered mode of iron uptake in the genus Eimeria.

Conclusions

Reduced diversity of genes required for host-parasite interaction and transmembrane transport allow hypotheses on host adaptation and specialization of a single host parasite. The E. falciformis genome sequence sheds light on the evolution of the Coccidia and helps to identify determinants of host-parasite interaction critical for drug and vaccine development.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-696) contains supplementary material, which is available to authorized users.     

Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii

Background

Toxoplasma gondii is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromized hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the T. gondii proteome.

Methodology/Principal Findings

We have explored the proteome of T. gondii tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the T. gondii proteome. The proteomics survey identified 609 proteins that are unique to Toxoplasma as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%.

Conclusions/Significance

This study not only provides the largest proteomics exploration of the T. gondii proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes.     

Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptide-microarray

Background

Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals.

Methodology

A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100).

Findings

The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II–III, type I–III or type I–II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers.

Conclusions

Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution.     

High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection

Background

Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.

Methods

In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.

Findings

A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.

Conclusions

These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.     

Serotyping of Toxoplasma gondii in Cats (Felis domesticus) Reveals Predominance of Type II Infections in Germany

Background

Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany.

Methodology

To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7).

Findings

Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III.

Conclusions

Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.     

A virosomal malaria peptide vaccine elicits a long-lasting sporozoite-inhibitory antibody response in a phase 1a clinical trial

Objectives

Peptides delivered on the surface of influenza virosomes have been shown to induce solid humoral immune responses in experimental animals. High titers of peptide-specific antibodies were also induced in a phase 1a clinical trial in volunteers immunized with virosomal formulations of two peptides derived from the circumsporozoite protein (CSP) and the apical membrane antigen 1 (AMA-1) of Plasmodium falciparum. The main objective of this study was to perform a detailed immunological and functional analysis of the CSP-specific antibodies elicited in this phase 1a trial.

Methodology/Principal Findings

46 healthy malaria-naïve adults were immunized with virosomal formulations of two peptide-phosphatidylethanolamine conjugates, one derived from the NANP repeat region of P. falciparum CSP (designated UK-39) the other from P. falciparum AMA-1 (designated AMA49-C1). The two antigens were delivered in two different concentrations, alone and in combination. One group was immunized with empty virosomes as control. In this report we show a detailed analysis of the antibody response against UK-39. Three vaccinations with a 10 µg dose of UK-39 induced high titers of sporozoite-binding antibodies in all volunteers. This IgG response was affinity maturated and long-lived. Co-administration of UK-39 and AMA49-C1 loaded virosomes did not interfere with the immunogenicity of UK-39. Purified total IgG from UK-39 immunized volunteers inhibited sporozoite migration and invasion of hepatocytes in vitro. Sporozoite inhibition closely correlated with titers measured in immunogenicity assays.

Conclusions

Virosomal delivery of a short, conformationally constrained peptide derived from P. falciparum CSP induced a long-lived parasite-inhibitory antibody response in humans. Combination with a second virosomally-formulated peptide derived from P. falciparum AMA-1 did not interfere with the immunogenicity of either peptide, demonstrating the potential of influenza virosomes as a versatile, human-compatible antigen delivery platform for the development of multivalent subunit vaccines.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00400101","term_id":"NCT00400101"}}NCT00400101     

Serological Surveillance Development for Tropical Infectious Diseases Using Simultaneous Microsphere-Based Multiplex Assays and Finite Mixture Models

Background

A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya.

Methods

We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized.

Findings

Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively.

Interpretation

A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.     

A novel system for the efficient generation of antibodies following immunization of unique knockout mouse strains

Background

We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen.

Methodology/Principal Findings

Jettisoning traditional laborious in silico searching methods to define putative epitopes, we simply immunized available BChE knock-out mice with full-length recombinant BChE protein (having been produced for crystallographic analysis). Immunization with BChE, in practically any form (recombinant human or mouse BChE, BChE purified from human serum, native or denatured), resulted in strong immune responses. Native BChE produced antibodies that favored ELISA and immunostaining detection. Denatured and reduced BChE were more selective for antibodies specific in Western blots. Two especially sensitive monoclonal antibodies were found capable of detecting 0.25 ng of BChE within one min by ELISA. One is specific for human BChE; the other cross-reacts with mouse and rat BChE. Immunization of wild-type mice served as negative controls.

Conclusions/Significance

We examined a simple, fast, and highly efficient strategy to produce antibodies by mining two expanding databases: namely those of knock-out mice and 3D crystallographic protein-structure analysis. We conclude that the immunization of knock-out mice should be a strategy of choice for antibody production.     

Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(pos)CD25(high) T cells for immunotherapy

Background

Regulatory T cell (Treg) based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy.

Methodology/Principal Findings

Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4^posCD25^high Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.

Conclusions/Significance

The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.     

Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program

Background

Large amounts of microarray expression data have been generated for the Apicomplexan parasite Toxoplasma gondii in an effort to identify genes critical for virulence or developmental transitions. However, researchers’ ability to analyze this data is limited by the large number of unannotated genes, including many that appear to be conserved hypothetical proteins restricted to Apicomplexa. Further, differential expression of individual genes is not always informative and often relies on investigators to draw big-picture inferences without the benefit of context. We hypothesized that customization of gene set enrichment analysis (GSEA) to T. gondii would enable us to rigorously test whether groups of genes serving a common biological function are co-regulated during the developmental transition to the latent bradyzoite form.

Results

Using publicly available T. gondii expression microarray data, we created Toxoplasma gene sets related to bradyzoite differentiation, oocyst sporulation, and the cell cycle. We supplemented these with lists of genes derived from community annotation efforts that identified contents of the parasite-specific organelles, rhoptries, micronemes, dense granules, and the apicoplast. Finally, we created gene sets based on metabolic pathways annotated in the KEGG database and Gene Ontology terms associated with gene annotations available at http://www.toxodb.org. These gene sets were used to perform GSEA analysis using two sets of published T. gondii expression data that characterized T. gondii stress response and differentiation to the latent bradyzoite form.

Conclusions

GSEA provides evidence that cell cycle regulation and bradyzoite differentiation are coupled. Δgcn5A mutants unable to induce bradyzoite-associated genes in response to alkaline stress have different patterns of cell cycle and bradyzoite gene expression from stressed wild-type parasites. Extracellular tachyzoites resemble a transitional state that differs in gene expression from both replicating intracellular tachyzoites and in vitro bradyzoites by expressing genes that are enriched in bradyzoites as well as genes that are associated with the G1 phase of the cell cycle. The gene sets we have created are readily modified to reflect ongoing research and will aid researchers’ ability to use a knowledge-based approach to data analysis facilitating the development of new insights into the intricate biology of Toxoplasma gondii.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-515) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> seroprevalence in breeding pigs in Estonia

Background

Toxoplasma gondii is a widespread occurring parasite infecting warm-blooded animals, including pigs and humans. The aims of this study were to estimate the prevalence of anti-T. gondii antibodies and to evaluate risk factors for T. gondii seropositivity in breeding pigs raised in Estonia. Sera from 382 pigs were tested with a commercial direct agglutination test, using a cut-off titer of 40 for seropositivity, for the presence of anti-T. gondii immunoglobulin G antibodies.

Results

Twenty-two (5.8%) of the 382 pigs tested seropositive for T. gondii, and 6 of the 14 herds had at least one seropositive pig. The proportion of seropositive pigs within the herds ranged between 0 and 43%. Gender appeared as a significant factor, with sows having 5.6 times higher odds to be seropositive to T. gondii than boars. Seroprevalence did not increase with age.

Conclusions

Anti-T. gondii antibodies were present in a substantial proportion of breeding pig herds in Estonia. On the other hand, the presence of herds without seropositive pigs illustrates that porcine T. gondii infections can be avoided even in a country where the parasite is endemic and common in several other host species.

     

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

Background

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.

Methods and Findings

We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.

Conclusions

The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.