Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.     

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.     

Minimal T-cell-stimulatory sequences and spectrum of HLA restriction of immunodominant CD4+ T-cell epitopes within hepatitis C virus NS3 and NS4 proteins

The hepatitis C virus (HCV)-specific CD4+ T-cell response against nonstructural proteins is strongly associated with successful viral clearance during acute hepatitis C. To further develop these observations into peptide-based vaccines and clinical immunomonitoring tools like HLA class II tetramers, a detailed characterization of immunodominant CD4+ T-cell epitopes is required. We studied peripheral blood mononuclear cells from 20 patients with acute hepatitis C using 83 overlapping 20-mer peptides covering the NS3 helicase and NS4. Eight peptides were recognized by > or = 40% of patients, and specific CD4+ T-cell clones were obtained for seven of these and three additional, subdominant epitopes. Mapping of minimal stimulatory sequences defined epitopes of 8 to 13 amino acids in length, but optimal T-cell stimulation was observed with 10- to 15-mers. While some epitopes were presented by different HLA molecules, others were presented by only a single HLA class II molecule, which has implications for patient selection in clinical trials of peptide-based immunotherapies. In conclusion, using two different approaches we identified and characterized a set of CD4+ T-cell epitopes in the HCV NS3-NS4 region which are immunodominant in patients achieving transient or persistent viral control. This information allows the construction of a valuable panel of HCV-specific HLA class II tetramers for further study of CD4+ T-cell responses in chronic hepatitis C. The finding of immunodominant epitopes with very constrained HLA restriction has implications for patient selection in clinical trials of peptide-based immunotherapies.     

T-cell epitopes in type 1 diabetes autoantigen tyrosine phosphatase IA-2: potential for mimicry with rotavirus and other environmental agents.

The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes. The dominant epitope, VIVMLTPLVEDGVKQC (aa 805-820), which elicited the highest T-cell responses in all at-risk relatives, has 56% identity and 100% similarity over 9 amino acids (aa) with a sequence in VP7, a major immunogenic protein of human rotavirus. Both peptides bind to HLA-DR4(*0401) and are deduced to present identical aa to the T-cell receptor. The contiguous sequence of VP7 has 75% identity and 92% similarity over 12 aa with a known T-cell epitope in glutamic acid decarboxylase (GAD), another autoantigen in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity over 8-14 aa to sequences in Dengue, cytomegalovirus, measles, hepatitis C, and canine distemper viruses, and the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 aa to herpes, rhino-, hanta- and flaviviruses. Two others are 80-82% similar over 10-11 aa to sequences in milk, wheat, and bean proteins. Further studies should now be carried out to directly test the hypothesis that T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and (for rotavirus) GAD.     

Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.     

Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.     

Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets

Background

Influenza A viruses generate an extreme genetic diversity through point mutation and gene segment exchange, resulting in many new strains that emerge from the animal reservoirs, among which was the recent highly pathogenic H5N1 virus. This genetic diversity also endows these viruses with a dynamic adaptability to their habitats, one result being the rapid selection of genomic variants that resist the immune responses of infected hosts. With the possibility of an influenza A pandemic, a critical need is a vaccine that will recognize and protect against any influenza A pathogen. One feasible approach is a vaccine containing conserved immunogenic protein sequences that represent the genotypic diversity of all current and future avian and human influenza viruses as an alternative to current vaccines that address only the known circulating virus strains.

Methodology/Principal Findings

Methodologies for large-scale analysis of the evolutionary variability of the influenza A virus proteins recorded in public databases were developed and used to elucidate the amino acid sequence diversity and conservation of 36,343 sequences of the 11 viral proteins of the recorded virus isolates of the past 30 years. Technologies were also applied to identify the conserved amino acid sequences from isolates of the past decade, and to evaluate the predicted human lymphocyte antigen (HLA) supertype-restricted class I and II T-cell epitopes of the conserved sequences. Fifty-five (55) sequences of 9 or more amino acids of the polymerases (PB2, PB1, and PA), nucleoprotein (NP), and matrix 1 (M1) proteins were completely conserved in at least 80%, many in 95 to 100%, of the avian and human influenza A virus isolates despite the marked evolutionary variability of the viruses. Almost all (50) of these conserved sequences contained putative supertype HLA class I or class II epitopes as predicted by 4 peptide-HLA binding algorithms. Additionally, data of the Immune Epitope Database (IEDB) include 29 experimentally identified HLA class I and II T-cell epitopes present in 14 of the conserved sequences.

Conclusions/Significance

This study of all reported influenza A virus protein sequences, avian and human, has identified 55 highly conserved sequences, most of which are predicted to have immune relevance as T-cell epitopes. This is a necessary first step in the design and analysis of a polyepitope, pan-influenza A vaccine. In addition to the application described herein, these technologies can be applied to other pathogens and to other therapeutic modalities designed to attack DNA, RNA, or protein sequences critical to pathogen function.     

Characterization of the West Nile virus protease substrate specificity and inhibitors

West Nile virus (WNV), a mosquito-borne member of Flaviviridae, is a human pathogen causing widespread disease for which there is no vaccine or chemotherapy. The two-component viral serine protease consists of a heterodimeric complex between the hydrophilic domain of the cofactor, NS2B (NS2BH) and the protease domain (NS3-pro). The protease is essential for polyprotein processing followed by assembly of viral replicase and genome replication. Therefore, the protease is an excellent target for development of antiviral therapeutics. Here, we report the expression in Escherichia coli, purification, and characterization of biochemical and kinetic properties of the WNV protease. Furthermore, we show that the WNV and the dengue virus type 2 (DENV-2) proteases are inhibited by aprotinin with inhibitor constants of 0.16 and 0.026 microM, respectively. Molecular modeling of the WNV protease/aprotinin complex, based on the known crystal structures of the WNV NS2BH-N3pro and aprotinin, suggest a potentially strong interaction between the P2 Lys and the protease activator peptide, NS2BH. This conclusion based on molecular modeling is in agreement with our data of a higher k(cat)/Km value with the substrate, Boc-Gly-Lys-Arg-MCA than the Boc-Gly-Arg-Arg-MCA and is also consistent with the results of an earlier study that were based on substrate-based inhibitor peptides.     

Analysis of Promiscuous T cell Epitopes for Vaccine Development Against West Nile Virus Using Bioinformatics Approaches

West Nile virus (WNV) is a major pathogenic flavivirus which causes human neuro-invasive disease, worldwide. Still successful vaccine and therapeutic treatment against WNV infection is not available, which demands the development of more potential WNV vaccines. The present study used immunoinformatics methods viz. Matrix and Artificial Neural Network (ANN) based algorithm to identify the promiscuous and conserve T cell epitopes from entire WNV proteome followed by structure based analysis of identified epitopes. The epitopes were also taken for TAP binding analysis and epitope conservancy analysis. Among 89 identified epitopes, eight epitopes showed high potential and conserve nature but two epitopes viz. capsid ⁴⁰FVLALLAFF⁴⁸ and NS2B ⁹LMFAIVGGL¹⁷ were found most promiscuous and having high population coverage in comparison of other identified epitopes and known antigenic positive control epitopes. Further, Autodock 4.2 and NAMD–VMD molecular dynamics simulation were used for docking and molecular dynamics simulation respectively, to validate epitope and allele complex stability. The 3D structure models were generated for epitopes and corresponding HLA allele by Pepstr and Modeller 9.10, respectively. Epitope FVLALLAFF-B*3501 allele and epitope LMFAIVGGL-B*5101 HLA allele complexes have shown best energy minimization and stable complexes during simulation. The study also showed the optimum binding epitopes FVLALLAFF and LMFAIVGGL with cTAP1 (PDB ID: 1JJ7) cavity, as revealed by Autodock 4.2, concluding favored passage through the ER membrane from cytosol to the ER lumen during cytosolic processing. The docking experiment of epitopes FVLALLAFF, LMFAIVGGL with cTAP1 very well show 1 H-bond state with a binding energy of ?1.62 and ?0.23 kcal/mol, respectively. These results show a smooth pass through of the epitope across the channel of cTAP1 via being weakly bonded and released into the ER lumen through the cavity of cTAP1. Overall, identified peptides have potential application in the development of short peptide based vaccines and diagnostic agents for West Nile virus.     

Bioinformatics tools for identifying class I-restricted epitopes

The lack of simple methods to identify relevant T-cell epitopes, the high mutation rate of many pathogens, and restriction of T-cell response to epitopes due to human lymphocyte antigen (HLA) polymorphism have significantly hindered the development of cytotoxic T-lymphocyte (CTL) epitope-based or "epitope-driven" vaccines. Previously, CTL epitopes were mapped using large arrays of overlapping synthetic peptides. The large number of protein sequences available for mapping is now making this method prohibitively expensive and time-consuming. Bioinformatics tools such as EpiMatrix and Conservatrix, which search for unique or multi-HLA-restricted (promiscuous) T-cell epitopes and identify epitopes that are conserved across variant strains of the same pathogen, accelerate epitope mapping. These tools offer a significant advantage over other methods of epitope selection because high-throughput screening can be performed in silico, followed by confirmatory studies in vitro. CTL epitopes discovered using these tools might be used to develop novel vaccines and therapeutics for the prevention and treatment of infectious diseases such as human immunodeficiency virus, hepatitis C, tuberculosis, and some cancers.     

Antiviral peptides targeting the west nile virus envelope protein

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.     

Heat Shock Protein-derived peptides as a potential immuno-regulatory vaccine in allergy. "In silico" analysis

Background: Allergic and autoimmune diseases are forms of altered immune responses directed respectively against exogenous and endogenous antigens. A growing body of experimental evidence exists to support a com-mon pathogenetic link between allergy and autoimmunity. Identification of shared antigens might thus be usefully exploited in the therapy of both conditions. The allergy to cat allergens is very common and fre-quently associated with asthma. Sequences of the main cat allergen Fel d1, containing overlapping T cell epitopes that bind to HLA molecules, have been used as an effective allergen-specific peptide vaccine. It has been observed that human proteins identified as cross-reactive auto-antigens with structural homology with environmental allergens often belong to evolutionarily conserved proteins.Heat shock proteins (HSPs) are among the most conserved proteins and some of them have been recog-nized as antigens in autoimmune diseases and also as the main proteins of environmental allergens. We performed an extensive “in silico” analysis to test the hypothesis that the human HSPs Grp94, Grp78, HSP90, HSP71 and HSP60 could show similarity with both Fel d1 and other common allergens, thus serv-ing as a source of peptides in a broad-spectrum allergen-specific immuno-therapy.Results: An extensive structural similarity was observed between Fel d1 and sequences of HSP71, Grp78, HSP90 and Grp94. These HSP sequences contained T-cell epitopes with binding capacity to HLA alleles better than that displayed by corresponding similar Fel d1-derived peptides employed so far as therapeutic vac-cines. A significant similarity was also found between these HSP sequences and other aeroallergens, not shared by Fel d1-derived peptides. HLA alleles involved in binding HSP sequences were mostly those as-sociated with an increased predisposition to both allergy and autoimmune diseases.Conclusion: Results support the possibility that HSP-derived immuno-dominant epitopes are exploitable as therapeutic peptides in allergies other than cat allergy.     

A systematic bioinformatics approach for selection of epitope-based vaccine targets

Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with dengue virus as a model system. A combination of publicly available bioinformatics algorithms and computational tools are used to screen and select antigen sequences as potential T-cell epitopes of supertype human leukocyte antigen (HLA) alleles. The selected sequences are tested for biological function by their activation of T-cells of HLA transgenic mice and of pathogen infected subjects. This approach provides an experimental basis for the design of pathogen specific, T-cell epitope-based vaccines that are targeted to majority of the genetic variants of the pathogen, and are effective for a broad range of differences in human leukocyte antigens among the global human population.     

Diversity of Natural Self-Derived Ligands Presented by Different HLA Class I Molecules in Transporter Antigen Processing-Deficient Cells

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P₁ position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.     

Sequence uniqueness and sequence variability as modulating factors of human anti-HCV humoral immune response

We recently compared the HCV polyprotein to the human proteome in order to test whether amino acid sequences unique to the virus could represent immunodominant epitopic determinants of the human humoral immune response against HCV. We identified a relatively limited number of HCV fragments with no/low similarity to the human host that represented exclusive HCV motifs. In this study, the peptides corresponding to low/zero similarity sequences were synthesized and assayed with HCV-infected sera. With different patterns, the synthetic HCV peptides corresponding to low/zero similarity sequences were found to be immunoreactive. In particular, the HCV E1 (315-323) HRMAWDMMM, HCV E2/NS1 (547-555) NWFGCTWMN, and HCV NS5 (2638-2646) YDTRCFDST sequences were immunodominant in the HCV-infected cohort under study. These three peptides correspond to sequences that are endowed with low-similarity to the human proteome, are highly conserved among various HCV strains, and have, potentially, a scarce susceptibility to proteolytic attacks. These data may be of help in defining the multiple factors which concur in the modulation of the human immune response against HCV, eventually providing information for the design of effective anti-HCV vaccines.     

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here, we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses, as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals?a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.     

A comprehensive analysis of predicted HLA binding peptides of JE viral proteins specific to north Indian isolates

Japanese encephalitis (JE), a viral disease has significantly increased worldwide especially, in the developing region due to challenges in immunization, vector control and lack of appropriate treatment methods. An effective, yet an expensive heat-killed vaccine is available for the disease. Therefore, the design and development of short peptide vaccine candidate is promising. We used immune-informatics methods to perform a comprehensive analysis of the entire JEV proteome of north Indian isolate to identify the conserved peptides binding known specific HLA alleles among the documented JEV genotypes 1, 2, 3, 4 and 5. The prediction analysis identified 102 class I (using propred I) and 118 class II (using propred) binding peptides at 4% threshold value. These predicted HLA allele binding peptides were further analyzed for potential conserved region using IEDB (an immune epitope database and analysis resource). This analysis shows that 78.81% of class II (in genotype 2) and 76.47% of HLA I (in genotype 3) bound peptides are conserved. The peptides IPIVSVASL, KGAQRLAAL, LAVFLICVL and FRTLFGGMS, VFLICVLTV, are top ranking with potential super antigenic property by binding to all HLA allele members of B7 and DR4 super-types, respectively. This data finds application in the design and development of short peptide vaccine candidates and diagnostic agents for JE following adequate validation and verification.     

Sorting and pooling strategy: a novel tool to map a virus proteome for CD8 T-cell epitopes

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.     

Major histocompatibility complex restriction in tuberculosis susceptibility

More than one mechanism may contribute to disease susceptibility in tuberculosis, viz., major histocompatability complex (MHC) restriction phenomenon, spectrum of immune reactivity/cytokine profile and epidemiology induced anergy. Experiments from our laboratories revealed that (i) human leucocyte antigen D-related allele 2 (HLA DR2) predispose for a more severe form of pulmonary tuberculosis encoding a high responder status, (ii) spectrum of immune reactivity to mycobacteria is ‘innate’, and it is demonstrable in healthy individuals from endemic area, (iii) there is no correlation between the purified protein derivative (PPD) response and peptide responses, (iv) once a person is high responder to P16 and P38 derived peptides (6/22), he/she (whether a patient or control) is a high responder for a wide range of mycobacterial peptides and (v)majority of the T-cell clones generated in vitro, to peptide 16.3 (amino acids 21–40) of 16 kA a mycobacterial antigen, in an HLA DR2 positive healthy individual is HLA DR restricted, permissive and of Th1 phenotype. The results suggested that MHC class II restriction play a role in peptide recognition and the immune response. Nonetheless the outcome and specificity of the immune reactivity and the resultant disease pathogenesis may depend on the promiscuity of peptide recognition and cytokine profiles.