Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics.

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

Identification and selection of immunodominant B and T cell epitopes for dengue multi-epitope-based vaccine.

Dengue virus (DENV) comprises four serotypes (DENV1-4) which cause 390 million global infections with 500,000 hospitalizations and 25,000 fatalities annually. Currently, the only FDA approved DENV vaccine is the chimeric live-attenuated vaccine, Dengvaxia®, which is based on the yellow fever virus (YFV) genome that carries the prM and E genes of the respective DENV 1, 2, 3, and 4 serotypes. However, it has lower efficacies against serotypes DENV1 (51%) and DENV2 (34%) when compared with DENV3 (75%) and DENV4 (77%). The absence of T cell epitopes from non-structural (NS) and capsid (C) proteins of the yellow fever vaccine strain might have prevented Dengvaxia® to elicit robust cellular immune responses, as CD8+ T cell epitopes are mainly localized in the NS3 and NS5 regions. Multi-epitope-based peptide vaccines carrying CD4+, CD8+ T cell and B cell epitopes represent a novel approach to generate specific immune responses. Therefore, assessing and selecting epitopes that can induce robust B and T cell responses is a prerequisite for constructing an efficient multi-epitope peptide vaccine. Potent B and T cell epitopes can be identified by utilizing immunoinformatic analysis, but the immunogenicity of the epitopes have to be experimentally validated. In this review, we presented T cell epitopes that have been predicted by bioinformatic approaches as well as recent experimental validations of CD4+ and CD8+ T cell epitopes by ex-vivo stimulation of PBMCs with specific peptides. Immunoproteomic analysis could be utilized to uncover HLA-specific epitopes presented by DENV-infected cells. Based on various approaches, immunodominant epitopes capable of inducing strong immune responses could be selected and incorporated to form a universally applicable multi-epitope-based peptide dengue vaccine.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Identification of Immunodominant HIV-1 Epitopes Presented by HLA-C*12:02, a Protective Allele, Using an Immunopeptidomics Approach.

Despite the fact that the cell surface expression level of HLA-C on both uninfected and HIV-infected cells is lower than those of HLA-A and -B, increasing evidence suggests an important role for HLA-C and HLA-C-restricted CD8+ T cell responses in determining the efficiency of viral control in HIV-1-infected individuals. Nonetheless, HLA-C-restricted T cell responses are much less well studied than HLA-A/B-restricted ones, and relatively few optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C alleles have been defined. Recent improvements in the sensitivity of mass spectrometry (MS)-based approaches for profiling the immunopeptidome present an opportunity for epitope discovery on a large scale. Here, we employed an MS-based immunopeptidomic strategy to characterize HIV-1 peptides presented by a protective allele, HLA-C*12:02. We identified a total of 10,799 unique 8- to 12-mer peptides, including 15 HIV-1 peptides. The latter included 2 previously reported immunodominant HIV-1 epitopes, and analysis of T cell responses to the other HIV-1 peptides detected revealed an additional immunodominant epitope. These findings illustrate the utility of MS-based approaches for epitope definition and emphasize the capacity of HLA-C to present immunodominant T cell epitopes in HIV-infected individuals, indicating the importance of further evaluation of HLA-C-restricted responses to identify novel targets for HIV-1 prophylactic and therapeutic strategies.IMPORTANCE Mass spectrometry (MS)-based approaches are increasingly being employed for large-scale identification of HLA-bound peptides derived from pathogens, but only very limited profiling of the HIV-1 immunopeptidome has been conducted to date. Notably, a growing body of evidence has recently begun to indicate a protective role for HLA-C in HIV-1 infection, which may suggest that despite the fact that levels of HLA-C expression on both uninfected and HIV-1-infected cells are lower than those of HLA-A/B, HLA-C still presents epitopes to CD8+ T cells effectively. To explore this, we analyzed HLA-C*12:02-restricted HIV-1 peptides presented on HIV-1-infected cells expressing only HLA-C*12:02 (a protective allele) using liquid chromatography-tandem MS (LC-MS/MS). We identified a number of novel HLA-C*12:02-bound HIV-1 peptides and showed that although the majority of them did not elicit T cell responses during natural infection in a Japanese cohort, they included three immunodominant epitopes, emphasizing the contribution of HLA-C to epitope presentation on HIV-infected cells.

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

K88 and F18 fimbrial enterotoxigenic Escherichia coli (ETEC) are the major causes of post-weaning diarrhea (PWD) in pigs. A vaccine that induces broad immunity to prevent K88 and F18 fimbrial ETEC bacterial attachment and colonization in pig small intestines and to neutralize enterotoxin enterotoxicity would be effective for PWD. Structure-based multiepitope-fusion-antigen (MEFA) technology using a backbone immunogen to present neutralizing epitopes of representing virulence factors capacitates development of broadly protective ETEC vaccines. Neutralizing epitopes have been identified from K88 fimbrial adhesin (FaeG) and enterotoxins but not F18 fimbrial adhesin. In this study, we in silico identified immunodominant epitopes from F18ac fimbrial subunit FedF which plays a critical role in F18 fimbrial adherence, genetically fused each epitope to a carrier, examined immunogenicity of each epitope fusion, and determined epitope-derived antibodies neutralizing activities against F18 fimbrial adherence. Data showed that seven immune-dominant epitopes were identified from FedF subunit. Fused to heterologous human ETEC adhesin subunit CfaB, epitope fusions induced anti-F18 antibodies in subcutaneously immunized mice. Moreover, antibodies derived from each fusion significantly blocked adherence of a F18-fimbrial E. coli bacteria to pig intestinal cell line IPEC-J2. While all seven epitopes exhibited neutralizing activity, results from this study identified FedF epitopes #3 (IPSSSGTLTCQAGT) and #7 (QPDATGSWYD) the most effective for antibodies against F18 fimbrial adherence, and suggested their future application in PWD vaccine development.

Determination of immunodominant scaffolds of Com1 and OmpH antigens of Coxiella burnetii.

Today, there is an increasing emphasis on recombinant vaccines to eliminate the side effects of conventional vaccines such as whole-cell bacteria. Query fever is an emerging disease that causes irreparable complications for both humans and domestic animals. The cause of this disease is Coxiella burnetii, a gram-negative intracellular bacteria. In order to determine the most immunodominant epitopes of Com1 and OmpH antigens of C. burnetii, the most reliable bioinformatics tools with high rates of citation in predicting B cell and T cell epitopes were used. Finally, by comparing the results of all servers, the best overlapped epitopes with the highest antigenicity among different servers were selected. In this regard, epitopes in 18-27and 67-82 amino acids residues were introduced for MHCI and MHCII of T cell, respectively, whereas epitope in 16-25 amino acids residues was introduced for B cell of OmpH antigen. The epitopes in the range of 193-202, 100-108 and 215-223 amino acid residues were preferred for MHCI class of T cell, MHCII class of T cell and B cell of Com1 antigen, respectively. For each antigen, some empirical common epitopic regions were introduced, which included both T and B cells epitopes, 53-65 and 102-111 amino acid residues of OmpH antigen as well as 38-54 range of the amino acid of Com1 antigen. All the predicted epitopes were selected based on their high antigenicity scores and number of non-digestive enzymes. To optimize the application of reported epitopes, various orders of epitopes were arranged in three categories of B cell, T cell and common T and B cells epitopes for each antigen. Then, the best immunodominant scaffolds for each antigen were proposed in these categories. The results demonstrated that the scaffold arranged based on B cell epitopes had the highest antigenicity in both antigens.

Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence.

Preclinical development of HIvax: Human survivin highly immunogenic vaccines.

Our previous work involved the development of a recombinant fowlpox virus encoding survivin (FP-surv) vaccine that was evaluated for efficacy in mesothelioma mouse models. Results showed that FP-surv vaccination generated significant immune responses, which led to delayed tumor growth and improved animal survival. We have extended those previous findings in the current study, which involves the pre-clinical development of an optimized version of FP-surv designed for human immunization (HIvax). Survivin-derived peptides for the most common haplotypes in the human population were identified and their immunogenicity confirmed in co-culture experiments using dendritic cells and T cells isolated from healthy donors. Peptides confirmed to induce CD8(+) and CD4(+) T cells activation in humans were then included in 2 transgenes optimized for presentation of processed peptides on MHC-I (HIvax1) and MHC-II (HIvax2). Fowlpox vectors expressing the HIvax transgenes were then generated and their efficacy was evaluated with subsequent co-culture experiments to measure interferon-γ and granzyme B secretion. In these experiments, both antigen specific CD4(+) and CD8(+) T cells were activated by HIvax vaccines with resultant cytotoxic activity against survivin-overexpressing mesothelioma cancer cells. These results provide a rationale for clinical testing of HIvax1 and HIvax2 vaccines in patients with survivin-expressing cancers.

Development of a POC test for TB based on multiple immunodominant epitopes of M. tuberculosis specific cell-wall proteins.

The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings.

Identification of novel B cell epitopes within Toxoplasma gondii GRA1.

Newly synthesized epitopes are one of the most promising antigens for the development of diagnostic kits and peptide vaccines. Very little is known about the B cell epitopes on GRA1 of Toxoplasma gondii, which are recognized by the humoral immune response in pigs. In this study, epitopes derived from GRA1 of T. gondii were identified using synthetic peptide techniques and bioinformatics. Three (PG10, PG13 and PG18) out of the eighteen peptides tested were recognized by all pig sera from different time points after infection, and the other peptides were recognized by select sera from various time points after infection. Our data indicate that many regions of GRA1, and in particular, the regions represented by the peptides PG10, PG13 and PG18, are involved in the pig antibody response. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope-based marker vaccines and diagnostic tools for toxoplasmosis.

Repertoire of Theileria equi immunodominant antigens bound by equine antibody.

Theileriosis in horses and cattle is caused by tick-borne Apicomplexa parasites and results in death or life-long infection in their respective hosts. Transmission risk associated with persistent infection severely limits movement of horses and cattle resulting in economic losses. The recent reemergence of Theileria equi infection in U.S. horses demonstrates the continual threat Apicomplexa parasites represent to global animal health. A paucity of data concerning equine immune responses to T. equi, including antigens recognized by antibodies in clinically asymptomatic, persistently infected horses, precludes vaccine development. Therefore, this investigation was initiated to characterize antigens recognized by the equine antibody response to T. equi. This goal was accomplished by defining T. equi merozoite antigens that are recognized by antibodies in horses infected with distinct T. equi isolates. Previously it was shown that equine post-infection serum consistently recognized at least five T. equi merozoite antigens, but their precise identity remained unknown. To determine specificity of antibody target identification, T. equi merozoite antigens were first isolated using equine post-infection serum in affinity chromatography. Proteins recognized by the equine antibodies were then isolated from two-dimensional electrophoresis gels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using the recently available T. equi genome database. Five T. equi antigens were identified and include Equi Merozoite Antigen-2 (EMA-2), EMA-3 and EMA-6, a previously uncharacterized protein annotated as "signal peptide containing protein", and 40S ribosomal protein S12.

Constitutive and inflammatory immunopeptidome of pancreatic ß-cells.

Type 1 diabetes is characterized by the autoimmune destruction of pancreatic ß-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 ß-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant K(d)-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206₋214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by ß-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP206₋214 revealed low-level presentation by K(d) (~25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed K(d)-restricted peptide Janus kinase-1355₋363 (~15,000 copies/cell). We have successfully sequenced peptides from NIT-1 ß-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice.

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (ß), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ∼84 HSV-1 proteins are recognized by CD8(+) T cells, and most (∼80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 ß and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.

Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.

Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS.

Francisella tularensis is a gram-negative intracellular bacterium that is the causative agent of tularemia. Small mammals such as rodents and rabbits, as well as some biting arthropods, serve as the main vectors for environmental reservoirs of F. tularensis. The low infectious dose, ability to aerosolize the organism, and the possibility of generating antibiotic resistant strains make F. tularensis a prime organism for use in bioterrorism. As a result, some strains of F. tularensis have been placed on the CDC category A select agent list. T cell immune responses are thought to be a critical component in protective immunity to this organism. However, investigation into the immune responses to F. tularensis has been hampered by the lack of molecularly defined epitopes. Here we report the identification of a major CD4(+) T cell epitope in C57Bl/6 (B6) mice. The murine model of F. tularensis infection is relevant as mice are a natural host for F. tularensis LVS and exhibit many of the same features of tularemia seen in humans. Using T cell hybridomas derived from B6 mice that had either been inoculated with F. tularensis and allowed to clear the infection or which had been immunized by conventional means using purified recombinant protein in adjuvant, we have identified amino acids 86-99 of the lipoprotein Tul4 (RLQWQAPEGSKCHD) as an immunodominant CD4 T cell epitope in B6 mice. This epitope is a major component of both the acute and memory responses to F. tularensis infection and can constitute as much as 20% of the responding CD4 T cells in an acute infection. Reactive T cells can also effectively enter the long-term memory T cell pool. The identification of this epitope will greatly aid in monitoring the course of F. tularensis infection and will also aid in the development of effective vaccine strategies for F. tularensis.

Expression, purification and immunodetection of a recombinant fragment (residues 179-281) of the G protein from rabies virus ERA strain.

The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGERA179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.

Babesia gibsoni: identification of an immunodominant, interspersed repeat antigen.

In this report, an immunodominant antigen called BgIRA from Babesia gibsoni is identified and described. A highly repetitive antigen was screened from a cDNA library. The genomic BgIRA gene exists as single cope gene and contains 10 introns. BgIRA plays a dominant role in the immune response in dogs infected with B. gibsoni. The specificity and sensitivity of the rBgIRA in an ELISA indicated that this antigen might be useful in a diagnostic test.

Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae.

BACKGROUND: The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. RESULTS: An antigenic sequence corresponding to amino acids 420-457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. CONCLUSION: The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.

Antigen-specific cytotoxic T lymphocytes protect against lethal West Nile virus encephalitis.

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden Markov model analysis was used to identify WNV-encoded peptides that bound the MHC class I proteins K(b) or D(b). Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8(+) T cells from WNV-infected mice induced IFN-gamma production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope- and non-structural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.

Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus.

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.