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 Cross-Reactive Norovirus CD4+ T Cell Epitopes

Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4⁺ T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans.Norovirus infection is characterized by the induction of both humoral and cellular immune responses. Humoral immunity in humans following norovirus infection has been described in detail for a limited number of norovirus strains (8, 10, 12, 17, 18, 29). Humans mount specific antibody responses to the infecting strain, which bear complex patterns of unique and cross-reactive, yet undefined, epitopes to other strains within or across genogroups (23, 29). Short-term immunity following homologous norovirus challenge has been documented, but long-term immunity remains controversial (16, 25). Furthermore, no studies to date have demonstrated cross-protection following heterologous norovirus challenge (30). While some susceptible individuals can become reinfected with multiple norovirus strains throughout their lifetimes, the mechanism of short-term protection and the impact of previous exposures on susceptibility to reinfection remain largely unknown.The role of T cells in controlling norovirus infection also remains largely undefined. A single comprehensive study detailing immune responses in genogroup II Snow Mountain virus-infected individuals revealed that CD4⁺ TH1 cells can be stimulated by virus-like particles (VLPs) to secrete gamma interferon (IFN-γ) and interleukin-2 (IL-2) (17). Furthermore, heterologous stimulation from VLPs derived from different norovirus strains within but not across genogroups also induced significant IFN-γ secretion compared to that for uninfected individuals (17). A follow-up study with genogroup I Norwalk virus (NV)-infected individuals confirmed high T cell cross-reactivity within a genogroup as measured by IFN-γ secretion (18). Further, vaccination of humans with VLPs also results in short-term IFN-γ production (27).Because norovirus infection studies in humans are confounded by previous exposure histories, the use of inbred mice maintained in pathogen-free environments allows for the study of norovirus immune responses in a naive background. While mice cannot be infected with human norovirus strains, VLP vaccines expressing norovirus structural proteins induce immune responses that can be measured and studied (14, 20). Mice immunized orally or intranasally with VLP vaccines in the presence of adjuvant similarly induced CD4⁺ IFN-γ responses in Peyer''s patches and spleen (22, 26). Induction of CD8⁺ T cells and secretion of the TH2 cytokine IL-4 were separately noted; however, it is unclear if these responses were influenced by VLPs or the coadministered vaccine adjuvants (22, 26). Further, coadministration of alphavirus adjuvant particles with multivalent norovirus VLP vaccine, including or excluding mouse norovirus (MNV) VLPs, resulted in significantly reduced MNV loads following MNV challenge (21). Multivalent VLP vaccines induced robust receptor-blocking antibody responses to heterologous human strains not included in the vaccine composition (20, 21). Moreover, natural infection with MNV supports a role for T cell immunity in viral clearance and protection (5).To advance our understanding of the scope of the cellular immune response within and between strains, we immunized mice with Venezuelan equine encephalitis (VEE) virus replicon particles (VRPs) expressing norovirus VLPs derived from the Norwalk virus (GI.1-1968) (1) or Farmington Hills virus (FH) (GII.4-2002) (19) strains and analyzed splenocytes for cytokine secretion, epitope identification, and heterologous stimulation. The data presented here indicate that the major capsid proteins of genogroup I and II noroviruses contain robust T cell epitopes that cross-react with related strains in the mouse yet also occur within regions of known variation, especially among the GII.4 noroviruses.     

CD8+ T Cells Use TRAIL To Restrict West Nile Virus Pathogenesis by Controlling Infection in Neurons

Previous studies of mice have demonstrated that an orchestrated sequence of innate and adaptive immune responses is required to control West Nile virus (WNV) infection in peripheral and central nervous system (CNS) tissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also known as CD253) has been reported to inhibit infection with dengue virus, a closely related flavivirus, in cell culture. To determine the physiological function of TRAIL in the context of flavivirus infection, we compared the pathogenesis of WNV in wild-type and TRAIL(-/-) mice. Mice lacking TRAIL showed increased vulnerability and death after subcutaneous WNV infection. Although no difference in viral burden was detected in peripheral tissues, greater viral infection was detected in the brain and spinal cord at late times after infection, and this was associated with delayed viral clearance in the few surviving TRAIL(-/-) mice. While priming of adaptive B and T cell responses and trafficking of immune and antigen-specific cells to the brain were undistinguishable from those in normal mice, in TRAIL(-/-) mice, CD8(+) T cells showed qualitative defects in the ability to clear WNV infection. Adoptive transfer of WNV-primed wild-type but not TRAIL(-/-) CD8(+) T cells to recipient CD8(-/-) mice efficiently limited infection in the brain and spinal cord, and analogous results were obtained when wild-type or TRAIL(-/-) CD8(+) T cells were added to WNV-infected primary cortical neuron cultures ex vivo. Collectively, our results suggest that TRAIL produced by CD8(+) T cells contributes to disease resolution by helping to clear WNV infection from neurons in the central nervous system.     

CD8+ T Cell Recognition of Cryptic Epitopes Is a Ubiquitous Feature of AIDS Virus Infection

Vaccines designed to elicit AIDS virus-specific CD8⁺ T cells should engender broad responses. Emerging data indicate that alternate reading frames (ARFs) of both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encode CD8⁺ T cell epitopes, termed cryptic epitopes. Here, we show that SIV-specific CD8⁺ T cells from SIV-infected rhesus macaques target 14 epitopes in eight ARFs during SIV infection. Animals recognized up to five epitopes, totaling nearly one-quarter of the anti-SIV responses. The epitopes were targeted by high-frequency responses as early as 2 weeks postinfection and in the chronic phase. Hence, previously overlooked ARF-encoded epitopes could be important components of AIDS vaccines.CD8⁺ T cells control AIDS virus replication (5, 9, 17, 21); however, their role in prophylactic AIDS vaccines is topic for debate. CD8⁺ T cells recognize infected cells by the presence of virus-derived peptides bound to major histocompatibility complex class I (MHC-I) molecules on the cell surface. The nine defined human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) proteins have long been thought to be the sole sources of virus-derived, MHC-I bound epitopes because researchers assume the classical viral protein annotations to represent the totality of the viral translation products despite increasing evidence to the contrary. Our laboratory and others have shown that MHC-I-bound epitopes can be derived from translation of viral alternate reading frames (ARFs), termed cryptic epitopes (2, 4, 6, 10, 15, 16). Collectively, these data indicate that cryptic CD8⁺ T cell responses might be more common, and more important, than previously appreciated.Rhesus macaques infected with a molecularly cloned strain of SIV offer several important advantages for studying specific CD8⁺ T cell responses (22). Since the exact sequence of the inoculum is known, it is possible to track precisely the CD8⁺ T cell responses against all possible viral ARF translations. We used a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay to screen SIVmac239-infected rhesus macaques in both the acute and chronic stages of infection for T cell responses against an overlapping peptide set (15-mers, overlapping by 11) spanning the entire potential ARF-encoded proteome in the “sense” direction. Altogether, we defined eight novel MHC-I epitope-containing translation products putatively ranging in length from 32 to 71 amino acids, each containing from one to five epitopes. We found that, in some animals, the cryptic epitope-directed response can be a dominant component of the total antiviral response, comprising nearly a quarter of the total response. Together, our data indicate that translation and immune recognition of viral ARFs are common features of AIDS virus infection.     

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.     

Role of CD8+ T cells in control of West Nile virus infection

Infection with West Nile virus (WNV) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. Although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. Previously, we demonstrated that components of humoral immunity, particularly immunoglobulin M (IgM) and IgG, have critical roles in preventing dissemination of WNV infection to the central nervous system. In this study, we addressed the function of CD8(+) T cells in controlling WNV infection. Mice that lacked CD8(+) T cells or classical class Ia major histocompatibility complex (MHC) antigens had higher central nervous system viral burdens and increased mortality rates after infection with a low-passage-number WNV isolate. In contrast, an absence of CD8(+) T cells had no effect on the qualitative or quantitative antibody response and did not alter the kinetics or magnitude of viremia. In the subset of CD8(+)-T-cell-deficient mice that survived initial WNV challenge, infectious virus was recovered from central nervous system compartments for several weeks. Primary or memory CD8(+) T cells that were generated in vivo efficiently killed target cells that displayed WNV antigens in a class I MHC-restricted manner. Collectively, our experiments suggest that, while specific antibody is responsible for terminating viremia, CD8(+) T cells have an important function in clearing infection from tissues and preventing viral persistence.     

CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis

C57BL/6J mice infected intravenously with the Sarafend strain of West Nile virus (WNV) develop a characteristic central nervous system (CNS) disease, including an acute inflammatory reaction. Dose response studies indicate two distinct kinetics of mortality. At high doses of infection (10(8) PFU), direct infection of the brain occurred within 24 h, resulting in 100% mortality with a 6-day mean survival time (MST), and there was minimal destruction of neural tissue. A low dose (10(3) PFU) of infection resulted in 27% mortality (MST, 11 days), and virus could be detected in the CNS 7 days postinfection (p.i.). Virus was present in the hypogastric lymph nodes and spleens at days 4 to 7 p.i. Histology of the brains revealed neuronal degeneration and inflammation within leptomeninges and brain parenchyma. Inflammatory cell infiltration was detectable in brains from day 4 p.i. onward in the high-dose group and from day 7 p.i. in the low-dose group, with the severity of infiltration increasing over time. The cellular infiltrates in brain consisted predominantly of CD8(+), but not CD4(+), T cells. CD8(+) T cells in the brain and the spleen expressed the activation markers CD69 early and expressed CD25 at later time points. CD8(+) T-cell-deficient mice infected with 10(3) PFU of WNV showed increased mortalities but prolonged MST and early infection of the CNS compared to wild-type mice. Using high doses of virus in CD8-deficient mice leads to increased survival. These results provide evidence that CD8(+) T cells are involved in both recovery and immunopathology in WNV infection.     

Pattern Recognition Receptor MDA5 Modulates CD8+ T Cell-Dependent Clearance of West Nile Virus from the Central Nervous System

Many viruses induce type I interferon responses by activating cytoplasmic RNA sensors, including the RIG-I-like receptors (RLRs). Although two members of the RLR family, RIG-I and MDA5, have been implicated in host control of virus infection, the relative role of each RLR in restricting pathogenesis in vivo remains unclear. Recent studies have demonstrated that MAVS, the adaptor central to RLR signaling, is required to trigger innate immune defenses and program adaptive immune responses, which together restrict West Nile virus (WNV) infection in vivo. In this study, we examined the specific contribution of MDA5 in controlling WNV in animals. MDA5^−/− mice exhibited enhanced susceptibility, as characterized by reduced survival and elevated viral burden in the central nervous system (CNS) at late times after infection, even though small effects on systemic type I interferon response or viral replication were observed in peripheral tissues. Intracranial inoculation studies and infection experiments with primary neurons ex vivo revealed that an absence of MDA5 did not impact viral infection in neurons directly. Rather, subtle defects were observed in CNS-specific CD8⁺ T cells in MDA5^−/− mice. Adoptive transfer into recipient MDA5^+/+ mice established that a non-cell-autonomous deficiency of MDA5 was associated with functional defects in CD8⁺ T cells, which resulted in a failure to clear WNV efficiently from CNS tissues. Our studies suggest that MDA5 in the immune priming environment shapes optimal CD8⁺ T cell activation and subsequent clearance of WNV from the CNS.     

CD8+ T cells require perforin to clear West Nile virus from infected neurons

Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.     

计算机辅助T细胞表位鉴定

免疫相关抗原T细胞表位的鉴定与筛选是疫苗开发的关键之一。目前,基于对天然MHC配体或合成肽与MHC分子结合特性的分析,若干计算机算法已被用于MHCI／Ⅱ类分子限制性T细胞表位的预测。而且,基于对蛋白酶体裂解片段的分析,开发了预测蛋白酶体酶切位点的算法。为进一步证实预测的表位肽在体有效性及免疫原性,若干实验技术也被相继开发和应用。同时,若干方法也被用于检测活化T细胞针对表达特定抗原靶细胞的免疫识别和T细胞活化后所分泌的细胞因子的产生。该综述了计算机辅助设计在表位筛选中的应用。     

Establishment of HLA-DR4 Transgenic Mice for the Identification of CD4+ T Cell Epitopes of Tumor-Associated Antigens

Reports have shown that activation of tumor-specific CD4⁺ helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E^d, where I-E^d α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E^d has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA1_55-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms'' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A_494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA1_55-78 and KIF20A_494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1_191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.     

Induction of a Cross-Reactive CD8+ T Cell Response following Foot-and-Mouth Disease Virus Vaccination

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8⁺ T cells in FMDV-vaccinated and -infected cattle. In the current study, we aimed to identify CD8⁺ T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-γ)-producing CD8⁺ T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8⁺ T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN-γ release, proliferation, and target cell killing by αβ CD8⁺ T cells, but not CD4⁺ T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8⁺ T cells from FMDV serotype O-vaccinated A31⁺ cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8⁺ T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes.Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae, genus Aphthovirus. The FMDV particle consists of a positive-strand RNA molecule of approximately 8,500 nucleotides, enclosed within an icosahedral capsid. The genome encodes a unique polyprotein from which four structural proteins (P1A, P1B, P1C, and P1D; also referred to as VP4, VP2, VP3, and VP1, respectively) and nine nonstructural proteins are cleaved by viral proteases (48). FMDV shows a high genetic and antigenic variability, which is reflected in the seven serotypes and multiple subtypes reported so far (13). The virus causes a highly contagious infection in cloven-hoofed animals which is characterized by the formation of vesicles on the mouth, tongue, nose, and feet. In addition, most infected animals develop viremia.The virus elicits a rapid humoral response in both infected and vaccinated animals (26). Virus-specific antibodies protect animals in a serotype-specific manner against reinfection or against infection in the case of vaccination, and protection is generally correlated with high levels of neutralizing antibodies (38). Control of the disease is achieved by vaccination with a chemically inactivated whole-virus vaccine emulsified with adjuvant; however, this provides only short-term, serotype-specific protection (2). The introduction of this vaccine has been very successful in areas of the world where the disease is enzootic. However, one of the major difficulties in implementing vaccination is the inability to distinguish vaccinated animals from infected/recovered animals, which may still be shedding virus. Currently, a number of assays specifically developed for this purpose are being validated (29, 41), and the success of these assays is dependent on the use of purified vaccine antigen. A strategy using replication-deficient adenovirus 5 expressing FMDV antigens has been shown to provide early protection against homologous challenge (39).The identification and characterization of T cell epitopes are important for understanding protective immunity mediated by CD8⁺ and CD4⁺ T lymphocytes. Such T cell responses are pathogen specific and are restricted by major histocompatibility complex (MHC) class I and class II molecules, which present foreign peptides to the immune system (55, 56). The role of cellular immunity in the protection of animals from FMDV is still a matter of some controversy. Specific T cell-mediated antiviral responses have been observed in cattle and swine following either infection or vaccination (3, 7, 24). CD4⁺ T cell responses are suggested to play an important role in protection against FMDV, and published studies demonstrate the presence of FMDV-specific MHC class II-restricted responses in cattle and pigs (22, 24). CD4⁺ epitopes within both P1A and P1D proteins have recently been identified in cattle (23). We have recently reported the presence of FMDV-specific, MHC class I-restricted CD8⁺ T cell responses in cattle following infection or vaccination. Despite these observations, the significance of cell-mediated immune responses in protective immunity to FMDV remains unclear.Cattle MHC (bovine leukocyte antigen [BoLA]) is relatively complex, with variable haplotypes expressing one, two, or three of the six classical class I genes (6, 15). At present, about 60 full-length validated cattle MHC class I cDNA sequences have been identified (www.ebi.ac.uk/ipd/mhc/bola), and the haplotypes commonly found in the Holstein breed are well characterized. We have previously identified amino acid motifs present in peptides binding to BoLA class I alleles N*02101, N*02201, and N*01301 (20). More recently, a number of Theileria parva CD8⁺ T cell epitopes presented through these and additional class I alleles have been described (25). Identification of such epitopes allows detailed analysis of cellular immune responses to vaccination and infection.In the present study, we aimed to identify MHC class I-restricted CD8⁺ T cell epitopes within the FMDV capsid protein. Using a panel of overlapping peptides, we have identified a BoLA A31-restricted epitope that is similar in all FMDV serotypes.     

T Cell Epitope Mapping of the E-Protein of West Nile Virus in BALB/c Mice

West Nile virus (WNV) is a zoonotic virus, which is transmitted by mosquitoes. It is the causative agent of the disease syndrome called West Nile fever. In some human cases, a WNV infection can be associated with severe neurological symptoms. The immune response to WNV is multifactorial and includes both humoral and cellular immunity. T-cell epitope mapping of the WNV envelope (E) protein has been performed in C57BL/6 mice, but not in BALB/c mice. Therefore, we performed in BALB/c mice a T-cell epitope mapping using a series of peptides spanning the WNV envelope (E) protein. To this end, the WNV-E specific T cell repertoire was first expanded by vaccinating BALB/c mice with a DNA vaccine that generates subviral particles that resemble West Nile virus. Furthermore, the WNV structural protein was expressed in Escherichia coli as a series of overlapping 20-mer peptides fused to a carrier-protein. Cytokine-based ELISPOT assays using these purified peptides revealed positive WNV-specific T cell responses to peptides within the different domains of the E-protein.     

Human Mycobacterium tuberculosis CD8 T Cell Antigens/Epitopes Identified by a Proteomic Peptide Library

Identification of CD8⁺ T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8⁺ T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8⁺ T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8⁺ T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ_24–34, B3905-restricted PE9_53–67, and B3514-restricted PE_PGRS42_48–56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8⁺ T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.