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Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for low epitope coverage allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas in some cases (e.g., orchard grass, Alternaria, cypress, and Russian thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen-derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of specific immunotherapy treatment or varying disease severity (asthma or rhinitis).

Rhesus and pigtail macaques have proven to be valuable animal models for several important human diseases, including HIV, where they exhibit similar pathology and disease progression. Because rhesus macaques have been extensively characterized in terms of their major histocompatibility complex (MHC) class I alleles, their demand has soared, making them increasingly difficult to obtain for research purposes. This problem has been exacerbated by a continued export ban in place since 1978. Pigtail macaques represent a potential alternative animal model. However, because their MHC class I alleles have not been characterized in detail, their use has been hindered. To address this, in the present study, we have characterized the peptide binding specificity of the pigtail macaque class I allele Mane-A1*082:01 (formerly known as Mane A*0301), representative of the second most common MHC class I antigen detected across several cohorts. The motif was defined on the basis of binding studies utilizing purified MHC protein and panels of single amino acid substitution analog peptides, as well as sequences of peptide ligands eluted from Mane- A1*082:01. Based on these analyses, Mane-A1*082:01 was found to recognize a motif with H in position 2 and the aromatic residues F and Y, or the hydrophobic/aliphatic residue M, at the C-terminus. Finally, analysis of the binding of a combinatorial peptide library allowed the generation of a detailed quantitative motif that proved effective in the prediction of a set of high-affinity binders derived from chimeric SIV/HIV, an important model virus for studying HIV infection in humans.

Of the two rhesus macaque subspecies used for AIDS studies, the Simian immunodeficiency virus-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection, providing both insight into pathogenesis and a system for testing novel vaccines. Despite the Chinese rhesus macaque potentially being a more relevant model for AIDS outcomes than the Indian rhesus macaque, the Chinese-origin rhesus macaques have not been well-characterized for their major histocompatibility complex (MHC) composition and function, reducing their greater utilization. In this study, we characterized a total of 50 unique Chinese rhesus macaques from several varying origins for their entire MHC class I allele composition and identified a total of 58 unique complete MHC class I sequences. Only nine of the sequences had been associated with Indian rhesus macaques, and 28/58 (48.3%) of the sequences identified were novel. From all MHC alleles detected, we prioritized Mamu-A1*02201 for functional characterization based on its higher frequency of expression. Upon the development of MHC/peptide binding assays and definition of its associated motif, we revealed that this allele shares peptide binding characteristics with the HLA-B7 supertype, the most frequent supertype in human populations. These studies provide the first functional characterization of an MHC class I molecule in the context of Chinese rhesus macaques and the first instance of HLA-B7 analogy for rhesus macaques. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0450-3) contains supplementary material, which is available to authorized users.

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.

Chronic hepatitis B virus (HBV) infection, which occurs in 1 to 5% of adult infections and up to 30% of pediatric infections, is characterized by high levels of viral replication averaging a daily production of about 1011 viral particles, hepatic inflammation, necrosis, and ultimately liver failure (36). An estimated 350 million individuals are classified as chronically HBV infected, with the highest concentrations of infection occurring in large parts of Asia and Africa (23). Chronic HBV can be treated with nucleoside analogues that inhibit polymerase activity. Lamivudine was the first licensed polymerase inhibitor, and it results in significant suppression of HBV DNA levels. However, this response, similar to the loss of hepatitis B virus e antigen, is often not sustained upon discontinuation of treatment (11, 28). The emergence of viral resistance in 15 to 20% of treated patients per year clearly pinpoints the limitations of this treatment. Newer drugs such as adefovir dipivoxil, entecavir, and telbivudine can result in less resistance, increased suppression of DNA levels, or in somewhat higher levels of hepatitis B virus e antigen loss. Real long-term treatment data with these drugs are, however, limited, and it is unclear how well these responses would be sustained if therapy were withdrawn. Higher levels of sustained response after the cessation of therapy have been documented for treatment with alpha interferon (IFN-α) and even more so for its pegylated form. This higher efficacy may be related to the fact that interferon has not only an antiviral but also an immunomodulatory function. However, drug cost and toxicities often restrict the use of IFN (11, 28). Thus, the development of supplemental therapies, especially those aiming at an improved immune response, remain a priority, particularly as a combination of lamivudine and IFN failed to improve sustained response rates (19). In acute HBV infection, cellular immune responses are well characterized and temporally associated with the clearance of infection. The HBV-specific cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) responses present during and immediately following resolution of acute infections are readily detected and often broadly specific, targeting epitopes from numerous viral gene products (13, 17). Chronic HBV infection is, however, only rarely resolved by the immune system. When this occurs, viral clearance is associated with increased CTL activity and increased levels of alanine transaminase (ALT), referred to as ALT flares, caused by the destruction of infected hepatocytes by the immune system (40). Viral clearance also can be induced in a significant proportion (10 to 15%) of individuals receiving IFN-α, and, similar to the effect of spontaneous clearance, the effect is correlated with increased CTL and HTL responses. Antiviral effects can be mediated through the action of cytokines, such as IFN-γ, which depress viral replication or inactivate virus particles without killing HBV-infected cells (15, 16). Similarly, in persistent HBV infection, the presence of HBV-specific CTL is associated with lower viral loads, independent from liver damage, further confirming the noncytolytic antiviral activity of CTL (4, 29). Successful vaccine immunotherapy in chronically infected individuals is expected to be characterized by similar CTL and HTL responses. However, in the presence of active HBV replication, the immune system of chronically infected individuals no longer responds vigorously to viral epitopes. The high levels of HBV particles and HBV proteins in the circulation are thought to be responsible for this immune suppression (12, 43). This effect can be so pronounced that it develops into a generalized imbalance of HTL type 1 and 2 responses (Th1/Th2), which manifests as general peripheral tolerance (42). The rare spontaneous resolution of chronic HBV infection and the immune responses observed during IFN-α or lamivudine treatment, in which viral replication is significantly reduced, indicates that deletion of HBV-specific CTL precursors does not occur and that the disease state of immune system tolerance is reversible (7, 8). So far, therapeutic vaccine candidates (6, 20, 25, 38, 44) for HBV have focused on the use of HBsAg and core proteins or parts thereof. However, cellular immune responses to these antigens probably are most prone to suppression, tolerance, or other dysfunctions, since these proteins circulate in very high levels in the infected host (21, 41). This was effectively demonstrated by a single core epitope lipopeptide vaccine candidate, which successfully induced CTL responses in healthy volunteers but failed to do so in chronic HBV patients (24, 25). Even under effective antiviral treatment with polymerase inhibitors or IFN-α, the levels of circulating HBsAg remain very high, possibly pointing to an explanation of why combinations of lamivudine and therapeutic vaccines based on HBsAg so far have failed to show a benefit (44). On the other hand, the polymerase antigen so far has not been as thoroughly investigated as therapeutic vaccine candidate, although the immune system may be more likely to respond to this antigen, which is produced only in minute amounts compared to production of the HBsAg and core antigen. Importantly, polymerase-specific CTL and HTL responses can be readily detected in persons resolving HBV infection (33). The induction of broadly specific CTL and HTL responses against various HBV antigens including the polymerase thus would be a new approach to improve the efficacy of a candidate therapeutic vaccine. Such a vaccine, designed for use in the general population, needs to be based on numerous epitopes restricted by multiple HLA types in order to provide acceptable levels of population coverage. The use of synthetic peptides to deliver large numbers of CTL and HTL epitopes is not feasible, but the technology to design and synthesize genes encoding multiple epitopes for use in DNA plasmid vaccines and in viral vectors is well suited (26, 27). The utility of using genetic constructs, such as DNA vaccines, to deliver multiple epitopes was demonstrated through the development of a vaccine product designed primarily for use with human immunodeficiency virus (HIV)-infected patients (56). This vaccine, which is designated EP HIV-1090, was successfully evaluated for safety and immunogenicity in two phase-1 clinical trials. This first-generation product proved to be safe at the dose levels tested, but immunogenicity was limited. The combined use of DNA vaccines with viral vectors in a heterologous prime-boost sequential immunization format has proved useful for augmenting response levels and rates in some clinical studies. Thus, it was decided that the combined use of DNA and modified vaccinia Ankara (MVA)-vectored vaccines encoding CTL and HTL epitopes warranted investigation. This report describes the design, synthesis, and characterization of a multiepitope-based HBV candidate therapeutic vaccine composed of a plasmid DNA (pDNA) construct (INX102-3697) and a recombinant MVA viral vector (INX102-0557) that each contain a single gene encoding 30 HBV-derived CTL epitopes, 16 HBV-derived HTL epitopes, and the universal pan-DR epitope (PADRE) HTL epitope. These two products are designed for sequential administration using a heterologous prime-boost immunization format to maximize the cellular immune responses induced.

Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8+T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC42-50, GLVGLVTFL; GPC60-68, SLYKGVYEL; and GPC441-449, YLISIFLHL) that displayed high-affinity binding (≤98 nM) to HLA-A*0201, induced CD8+T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC42-50or GPC60-68were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.

We have analyzed byex vivoELISPOT the anti-vaccinia cytotoxic T lymphocyte responses of peripheral blood mononuclear cells from humans vaccinated with Dryvax vaccine. More than 6,000 peptides from 258 putative vaccinia ORFs predicted to bind the common molecules of the HLA A1, A2, A3, A24, B7, and B44 supertypes were screened with peripheral blood mononuclear cells of 31 vaccinees. A total of 48 epitopes derived from 35 different vaccinia antigens were identified, some of which (B8R, D1R, D5R, C10L, C19L, C7L, F12, and O1L) were recognized by multiple donors and contain multiple epitopes recognized in the context of different HLA types. The antigens recognized tend to be >100 residues in length and are expressed predominantly in the early phases of infection, although some late antigens were also recognized. Viral genome regulation and virulence factor were recognized most frequently, whereas few structural proteins were immunogenic. Finally, most epitopes were highly conserved among vaccinia virus Western Reserve, variola major and modified vaccinia Ankara, supporting their potential use in vaccine and diagnostic applications.

At the functional level, the majority of human leukocyte antigen (HLA) class I MHC variants can be classified into about ten different major groups, or supertypes, characterized by overlapping peptide binding motifs and repertoires. Previous studies have detailed the peptide binding specificity of the HLA A2, A3, B7, and B44 supertypes, and predicted, on the basis of MHC pocket structures, known motifs, or the sequence of T cell epitopes, the existence of the HLA A1 and A24 supertypes. Direct experimental validation of the A1 and A24 supertypes, however, has been lacking. In the current study, the peptide-binding repertoires and main anchor specificities of several common HLA A molecules (A*0101, A*2301, A*2402, A*2601, A*2902, and A*3002) predicted to be members of the A1 or A24 supertypes were analyzed and defined using single amino acid substituted peptides and a large peptide library. Based on the present findings, the A1 supertype includes A*0101, A*2601, A*2902, and A*3002, whereas the A24 supertype includes A*2301 and A*2402. Interestingly, A*2902 is associated with a motif and peptide binding repertoire that overlaps significantly with those of all of the A1- and A24-supertype molecules studied, representing-to our knowledge-the first report of significant cross-reactivity among molecules belonging to different supertypes.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-γ ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context ofPlasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuatedP. falciparumsporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterizedP. falciparum-derived protein antigens. The data suggest that immune responses toPlasmodiumare dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens.

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4+ T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4+ T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.

Assays to measure the binding capacity of peptides for HLA-DQA1*0501/B*0201 (DQ2.3) and DQA1*0301/B*0302 (DQ3.2) were developed using solubilized MHC molecules purified from EBV-transformed cell lines. These quantitative assays, based on the principle of the inhibition of binding of a high-affinity radiolabeled ligand, were validated by examining the binding capacity of known DQ-restricted epitopes or ligands. The availability of these assays allowed an investigation of patterns of cross-reactivity between different DQ molecules and with various common DR molecules. DQ2.3 and DQ3.2 were found to have significantly overlapping peptide binding repertoires. Specifically, of 13 peptides that bound either DQ2.3 or DQ3.2, nine (69.2%) bound both. The molecular basis of this high degree of cross-reactivity was further investigated with panels of single substitution analogs of the thyroid peroxidase 632–645Y epitope. It was found that DQ2.3 and DQ3.2 bind the same ligands by using similar anchor residues but different registers. These data suggest that in analogy to what was previously described for HLA-DR molecules, HLA-DQ supertypes characterized by largely overlapping binding repertoires can be defined. In light of the known linkage of both HLA-DQ2.3 and -DQ3.2 with insulin-dependent diabetes mellitus and celiac disease, these results might have important implications for understanding HLA class II autoimmune disease associations.

While most immunotherapies for cancer have focused on eliciting specific CD8+ cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4+ T cell help may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4+ helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase. This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4+ tumor-infiltrating lymphocyte line. Here, we present the crystal structures of HLA-DR1 in complex with both wild-type and mutant TPI peptide antigens, the first structures of tumor peptide antigen/MHC class II complexes recognized by CD4+ T cells to be reported. These structures show that very minor changes in the binding surface for T cell receptor correspond to the dramatic differences in T cell stimulation. Defining the structural basis by which CD4+ T cell help is invoked in an anti-tumor immune response will likely aid the design of more effective cancer immunotherapies.

Certain peptide analogs that carry substitutions at residues other than the main major histocompatibility complex anchors and are surprisingly much more antigenic than wild-type peptide (heteroclitic analogs). To date, it was unknown how frequently wild-type epitopes could be modified to obtain heteroclitic activity. In this study, we analyzed a large panel of analogs of two different human histocompatibility leukocyte antigen (HLA)-A2.1–restricted epitopes and found that heteroclitic analogs were associated with higher magnitude responses and increased (up to 107-fold) sensitivity to antigen, and corresponded to conservative or semiconservative substitutions at odd-numbered positions in the middle of the peptide (positions 3, 5, or 7). These findings were validated by performing additional immunogenicity studies in murine and human systems with four additional epitopes. The biological relevance of heteroclitic analogs was underlined when predicted analogs of the p53.261 epitope was shown to induce cytotoxic T lymphocytes (CTLs) that recognize low concentrations of peptide (high avidity) in vivo and demonstrate in vitro antitumor recognition. Finally, in vitro immunization of human peripheral blood mononuclear cells with two heteroclitic analogs resulted in recruitment of more numerous CTLs which were associated with increased antigen sensitivity. In conclusion, heteroclitic analogs were identified in each of the six cases studied and structural features were defined which allow identification of such analogs. The strong CTL immunity elicited by heteroclitic epitopes suggest that they could be of significant value in vaccination against tolerant or weakly immunogenic tumor-associated and viral antigens.

Forty-two wild-type and analogue peptides derived from p53, carcinoembryonic Ag, Her2/neu, and MAGE2/3 were screened for their capacity to induce CTLs, in vitro, capable of recognizing tumor target lines. All the peptides bound HLA-A*0201 and two or more additional A2 supertype alleles with an IC50 of 500 nM or less. A total of 20 of 22 wild-type and 9 of 12 single amino acid substitution analogues were found to be immunogenic in primary in vitro CTL induction assays, using normal PBMCs and GM-CSF/IL-4-induced dendritic cells. These results suggest that peripheral T cell tolerance does not prevent, in this system, induction of CTL responses against tumor-associated Ag peptides, and confirm that an HLA class I affinity of 500 nM or less is associated with CTL epitope immunogenicity. CTLs generated by 13 of 20 of the wild-type epitopes, 6 of 9 of the single, and 2 of 5 of the double substitution analogues tested recognized epitopes generated by endogenous processing of tumor-associated Ags and expressed by HLA-matched cancer cell lines. Further analysis revealed that recognition of naturally processed Ag was correlated with high HLA-A2.1-binding affinity (IC50 = 200 nM or less; p = 0.008), suggesting that high binding affinity epitopes are frequently generated and can be recognized as a result of natural Ag processing. These results have implications for the development of cancer vaccines, in particular, and for the process of epitope selection in general.

Here, we report the identification of a novel CD8+cytotoxic T-lymphocyte epitope on thePlasmodium falciparumcircumsporozoite protein (3D7; amino acids 310 to 319 [EPSDKHIKEY]) that is restricted by HLA-A*01 and is recognized by human volunteers immunized with irradiatedP. falciparumsporozoites. HLA-A*01 is the second most common HLA allele among Caucasians.

The ‘classical’ myelin basic protein (MBP) exons belong to a much larger unit, termed the ‘Golli-MBP’ gene. Here we have examined the T-cell determinant structure of the Golli protein region in the BALB/c mouse. Golli p10-24, which was shown to have the strongest affinity for I-A d , could not induce T-cell activation. Paradoxically, the poorer binding, overlapping p5–19 was effective at inducing T-cell proliferation. Thus, immunogenicity is not necessarily related to the MHC-binding affinity of self-peptides. In addition, MBP: p151–168-specific T cell clones responded only poorly to J37, a Golli-MBP protein, while MBP: 59–76-specific clones responded well to J37.

Studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. To define antigens and epitopes ofMycobacterium tuberculosis(MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A*0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA30–38), RNA polymerase β-subunit (RpoB127–135), and a putative phosphate transport system permease protein A-1 (PstA175–83). In addition, CD8+ T cell lines specific for three peptides (ThyA30–38, PstA175–83, and 85B15–23) were generated from peripheral blood mononuclear cells of normal HLA-A*0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-γ and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against the pathogen.

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-γ responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8.7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

Previous studies indicated that thePlasmodium yoeliicircumsporozoite protein (PyCSP) 57–70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58–67 sequence contains an H-2dbinding motif, which binds purified Kdmolecules in vitro with low affinity (3,267 nM) and encodes an H-2d-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57–70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59–67 specific, H-2drestricted, and CD8+T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8+- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines.

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87–99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87–99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.

The inverse relationship between peripheral blood CTL responsiveness to multiple hepatitis C virus (HCV) epitopes and viral titer in patients with persistent HCV infection suggests that enhancement of the CTL response might result in viral clearance. Since several HLA-A2-restricted HCV CTL epitopes are already known, we aimed to identify CTL epitopes restricted by other HLA types in an effort to expand the epitope repertoire available for T cell-mediated therapeutic vaccine development. Scanning of 14 different HCV genome sequences for the presence of conserved peptides containing the HLA-A3 and -B7 motifs revealed 9- to 10-mer peptides that were synthesized and assayed for binding to HLA-A3, -B7 supertype molecules. Peptides with good HLA-binding affinities and cross-reactivities with at least three of five most common molecules of each supertype were tested for the ability to stimulate a memory CTL response in the peripheral blood from selected HCV-infected patients and normal seronegative donors in vitro. We identified eight HLA-A3 supertype-restricted CTL epitopes and one HLA-B7 supertype-restricted CTL epitope that were recognized by infected patients but not by healthy seronegative donors. HLA class I serotyping of 158 chronically infected patients revealed that 80% expressed one or more of HLA molecules belong to either the A2, A3, or B7 supertypes. In conclusion, the epitopes, herein identified combined with previously defined HLA-A2-restricted CTL epitopes, should be useful for the design of an ethnically unbiased, therapeutic CTL vaccine for the treatment of patients with chronic HCV infection.

T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).

One approach to development of specific cancer immunotherapy relies on the induction of cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens (TAA). Induction of TAA-specific CTL could be used towards the eradication of established tumors, or to prevent their dissemination or recurrence after primary treatment. The present study identifies a set of CTL epitopes from TAA frequently found on solid epithelial tumors such as breast, lung and gastro-intestinal tumors. Specifically, HLA-A2.1 binding peptides from the MAGE2, MAGE3, HER-2/neu and CEA antigens were tested for their capacity to elicit in vitro anti-tumor CTL using lymphocytes from normal volunteers and autologous dendritic cells as antigen-presenting cells. A total of 6 new epitopes (MAGE2[10(157)], MAGE3[9(112)], CEA[9(691)], CEA[9(24)], HER2[9(435)] and HER2[9(5)]) were identified which were capable of specifically recognizing tumor cell lines lines expressing HLA-A2.1 and the corresponding TAA. In one case (CEA[9(24)]), induction of vigorous anti-tumor CTL responses required epitope engineering to increase HLA-A2.1 binding affinity. Finally, most of the newly identified epitopes (5 out of 6) were found to be highly crossreactive with other common HLA alleles of the A2 supertype (A2.2, A2.3, A2.6 and A6802), thus demonstrating their potential in providing broad and non-ethnically biased population coverage. The results are discussed in the context of the development of multi-epitope-based therapies with broad applicability for patients suffering from commonly found tumors.

Quantitative A*0207 peptide binding assays have been developed utilizing HLA transfected cells and affinity purified molecules. By using a panel of single substitution analog peptides, it was demonstrated that A*0207 binds peptides with main anchor specificity at position 2 and the C-terminus similar to A*0201. Previous data indicating that A*0207 (but not A*0201) also requires the presence of D or P in position 3 of its peptide ligands was confirmed by the analysis of additional single substituted analogs. Finally, by analyzing the A*0201 and A*0207 binding capacities of panels of unrelated synthetic peptides, it was found that 8/15 (53.3%) A*0201 binders with D or P in position 3 bound A*0207, while only 5/72 (6.9%) A*0201 binders without D or P in position 3 also bound A*0207. Together, these data indicate that although A*0207 may be included amongst A2 supertype alleles, its peptide binding repertoire is largely limited to a subset of that bound by A*0201.

Binding of peptides to MHC class I molecules is a prerequisite for their recognition by cytotoxic T cells. Consequently, identification of peptides that will bind to a given MHC molecule must constitute a central part of any algorithm for prediction of T-cell antigenic peptides based on the amino acid sequence of the protein. Binding motifs, defined by anchor positions only, have proven to be insufficient to ensure binding, suggesting that other positions along the peptide sequence also affect peptide-MHC interaction. The second phase of prediction schemes therefore take into account the effect of all positions along the peptide sequence, and are based on position-dependent-coefficients that are used in the calculation of a peptide score. These coefficients can be extracted from a large ensemble of binding sequences that were tested experimentally, or derived from structural considerations, as in the algorithm developed by us recently. This algorithm uses the coordinates of solved complexes to evaluate the interactions of peptide amino acids with MHC contact residues, and results in a peptide score that reflects its binding energy. Here we present our analysis for peptide binding to four MHC alleles (HLA-A2, HLA-A68, HLA-B27 and H-2Kb), and compare the predictions of the algorithm to experimental binding data. The algorithm performs successfully in predicting peptide binding to MHC molecules with hydrophobic binding pockets but not when MHC molecules with hydrophilic, charged pockets are considered. For MHC molecules with hydrophobic pockets it is demonstrated how the algorithm succeeds in distinguishing binding from non-binding peptides, and in high ranking of immunogenic peptides within all overlapping same-length peptides spanning their respective protein sequences. The latter property of the algorithm makes it a useful tool in the rational design of peptide vaccines aimed at T-cell immunity.

The strength of the cytotoxic T lymphocyte (CTL) response is believed to influence the final outcome of hepatitis B virus (HBV) infection. Among the different CTL epitopes so far identified, the sequence 18-27 of the HBV nucleocapsid antigen is widely recognized by CTL of HLA-A2-positive patients with acute self-limited HBV infection, and represents the main component of a peptide-based therapeutic vaccine aimed at stimulating the antiviral CTL response in patients with chronic hepatitis B. In the present study, we further analyzed the features of this important HBV region by the following: 1) defining the contribution of individual residues of the epitope to the interaction with the T-cell receptor (TCR) and with the HLA-A0201 molecule; 2) assessing the antigenicity of this viral region in the context of the different HLA-A2 subtypes; and 3) testing whether this sequence can stimulate not only HLA-class I but also HLA class II restricted T-cell responses. A clear hierarchy was observed in the ability of individual residues to act as TCR or HLA binding sites. Furthermore, the sequence HBc18-27 was able to be recognized by specific CTL when presented in the context of different HLA-A2 subtypes. Finally, this HBV region was also found to stimulate HLA class II restricted T-cell responses. These data further increase the potential coverage and efficacy of therapeutic vaccines based on the HBc18-27 sequence.

We recently described human leukocyte antigen (HLA) A2, A3 and B7 supertypes, characterized by largely overlapping peptide-binding specificities and represented in a high percentage of different populations. Here, we identified 17 Plasmodium falciparum peptides capable of binding these supertypes and assessed antigenicity in both vaccinated and naturally exposed populations. Positive cytotoxic T lymphocyte recall and cytokine (interferon-γ and tumor necrosis factor α) responses were detected for all peptides; all were recognized in the context of more than one HLA class I molecule; and at least 12 of the 17 were recognized in the context of all HLA alleles studied. These data validate the concept of HLA supertypes at the biological level, show that highly degenerate peptides are almost always recognized as epitopes, and demonstrate the feasibility of developing a universally effective vaccine by focusing on a limited number of peptide specificities.

Chinese rhesus macaques are of particular interest in simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) research as these animals have prolonged kinetics of disease progression to acquired immunodeficiency syndrome (AIDS), compared to their Indian counterparts, suggesting that they may be a better model for HIV. Nevertheless, the specific mechanism(s) accounting for these kinetics remains unclear. The study of major histocompatibility complex (MHC) molecules, including their MHC/peptide-binding motifs, provides valuable information for measuring cellular immune responses and deciphering outcomes of infection and vaccine efficacy. In this study, we have provided detailed characterization of six prevalent Chinese rhesus macaque MHC class I alleles, yielding a combined phenotypic frequency of 29 %. The peptide-binding specificity of two of these alleles, Mamu-A2*01:02 and Mamu-B*010:01, as well as the previously characterized allele Mamu-B*003:01 (and Indian rhesus Mamu-B*003:01), was found to be analogous to that of alleles in the HLA-B27 supertype family. Specific alleles in the HLA-B27 supertype family, including HLA-B*27:05, have been associated with long-term nonprogression to AIDS in humans. All six alleles characterized in the present study were found to have specificities analogous to HLA supertype alleles. These data contribute to the concept that Chinese rhesus macaque MHC immunogenetics is more similar to HLA than their Indian rhesus macaque counterparts and thereby warrants further studies to decipher the role of these alleles in the context of SIV infection.

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.

Radio frequency (RF) signals provide a near ubiquitous energy source due to the large number of TV, radio, cellular, and Wi-Fi transmitters throughout our urban environments. While the traditional use of RF signals is for data transfer, it is possible to harvest, convert, and store the energy in these signals for use in a variety of applications. In general, the energy harvesting process includes four stages: excitation of current in an antenna by an incident electromagnetic wave, recti?cation of the resulting power at the antenna output, conversion of this power to optimal voltage and current levels, and ?nally, energy storage in a capacitor or battery for later use. This chapter describes a sensor node that is powered by UHF television broadcast signals. The sensor node transmits the measured data wirelessly over a short distance using a IEEE 802.15.4 radio. Typically broadcast television signals are considered to be sources of information, but this chapter demonstrates that these ubiquitous ambient signals can be used as a power source to do non-trivial work.

We have focused on conserved regions of the hepatitis C Virus (HCV) genome to identify viral peptides that contain HLA class I binding motifs and bind with high affinity to the corresponding purified HLA molecules. Accordingly, we have identified 31 candidate epitopes in the HCV that have the potential to be recognized by either HLA-A1, A2.1-, A3, A11- or A24-restricted cytotoxic T lymphocytes (CTL). Twelve conserved peptides that bind HLA-A2.1 with high or intermediate affinity were tested for immunogenicity in vitro in human primary CTL cultures and in vivo by direct immunization of HLA-A2.1/Kb transgenic mice. Six HLA-A2.1-restricted CTL epitopes were immunogenic in both systems. At least three of these peptide epitopes were endogenously processed and presented for CTL recognition. Overall, these data illustrate the value of this approach for the development of virus-specific, peptide-based vaccines.

The interaction of the TCR with the immunogenic peptide bound to MHC class II molecules leads to the activation of CD4 + T helper cells. T helper cells have been divided into two subsets, Th1 and Th2, on the basis of the cytokines secreted by them. Th1 cells which secrete IL-2, IFN-y and tumor necrosis factor-p" induce delayed-type hypersensitivity, while Th2 cells secreting IL-4, IL-5, IL-6 and IL-10 induce humoral immune response. However, the mechanism of selective activation of Th1 and Th2 cells in response to different antigens is not fully understood. In this study we examined the selective activation of Th1 and Th2 cells in response to strongly immunogenic synthetic peptides EYK(EYA)3, abbreviated as K3; EYK(EYA)4, abbreviated as K4; and EYKEYAAYA(EYA)2, abbreviated as K1A2. These peptides are recognized by H-2 d T cells in the context of I-A d, and are strongly cross-reactive in both T cell response and antibody response. The peptide K1A2 has very high affinity for I-Ad while K3 has a much lower affinity. K4 has affinity intermediate between K1A2 and K3. The peptide K1A2 induced Th1 and K3 induced Th2 cells in BALB/c mice as suggested by their cytokine profiles. K4 induced both Th1- and Th2-type cytokines. This was also confirmed by the analysis of peptide-specific antibody responses. K3 induced primarily lgG1 response, whereas K4 induced both lgG1 and lgG2a responses in vivo. There was a shift toward a Th1-type cytokine profile when K3-primed T cells were challenged with K1A2 in vitro but K1A2-primed cells did not show any shift when challenged with K3. Immunization with higher doses of K3 shifted the response towards Th1 type, while immunization with lower doses of K1A2 did not shift the response toward Th2. We conclude that cells primed with high-affinity peptide are committed to differentiate into Th1 irrespective of the priming dose and affinity of challenge antigen. On the other hand, the differentiation of cells primed with low-affinity peptide depends upon the dose of immunization and binding affinity of the challenge antigen for MHC.

Major histocompatibility complex class II-associated invariant chain (Ii) provides several important functions that regulate class II expression and function. One of these is the ability to inhibit class II peptide loading early in biosynthesis. This allows for efficient class II folding and egress from the endoplasmic reticulum, and protects the class II peptide binding site from loading with peptides before entry into endosomal compartments. The ability of Ii to interact with class II and interfere with peptide loading has been mapped to Ii exon 3, which encodes amino acids 82-107. This same region of Ii has been described as a nested set of class II-associated Ii peptides (CLIPs) that are transiently associated with class II in normal cells and accumulate in human histocompatibility leukocyte antigen-DM-negative cell lines. Currently it is not clear how CLIP and the CLIP region of Ii blocks peptide binding. CLIP may bind directly to the class II peptide binding site, or may bind elsewhere on class II and modulate class II peptide binding allosterically. In this report, we show that CLIP can interact with many different murine and human class II molecules, but that the affinity of this interaction is controlled by polymorphic residues in the class II chains. Likewise, structural changes in CLIP also modulate class II binding in an allele-dependent manner. Finally, the specificity and kinetics of CLIP binding to class II molecule is similar to antigenic peptide binding to class II. These data indicate that CLIP binds to class II in an analogous fashion as conventional antigenic peptides, suggesting that the CLIP segment of Ii may actually occupy the class II peptide binding site.

Idiosyncratic adverse drug reactions are unpredictable, dose-independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkages between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8 + T cells that required HLA-B*57:01 molecules for their function; however, the mechanism by which abacavir induces this pathologic T-cell response remains unclear. Here we show that abacavir can bind within the F pocket of the peptide-binding groove of HLA-B*57:01, thereby altering its specificity. This provides an explanation for HLA-linked idiosyncratic adverse drug reactions, namely that drugs can alter the repertoire of self-peptides presented to T cells, thus causing the equivalent of an alloreactive T-cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir and that were recognized by T cells of hypersensitive patients. The assays that we have established can be applied to test additional compounds with suspected HLA-linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA-linked hypersensitivities, and guide the development of safer drugs.

Identification of CTL epitopes for tumor-specific responses is important for the development of immunotherapies to treat cancer patients. We have developed a strategy to identify potential CTL epitopes based on screening of sequences of target proteins for presence of specific motifs recognized by the most common HLA-A alleles, and identification of high affinity binding peptides using in vitro quantitative assays. A systematic analysis using the sequence of the product of the tumor-associated MAGE-1 gene has been carried out. All possible peptides of nine and ten residues, containing binding motifs for HLA-A1, -A2.1, A-3.2, -A11 and -A24 were synthesized and tested for binding using a quantitative assay. Out of 237 possible peptide/MHC combinations, 47 cases demonstrated good binding affinity (Kdor = 500 nM). Several peptides were identified as good MHC binders for each one of the five HLA-A alleles studied (five for HLA-A1, 11 for HLA-A2.1, 10 for HLA-A3.2, 16 for HLA-A11 and five for HLA-A24. Furthermore, eight of these peptides were found to bind well to more than one HLA-A allele. These results have important implications for the development of immunotherapeutic vaccines to treat malignant melanoma.

Immunodominant T cell epitopes of myelin basic protein (MBP) may be target antigens for major histocompatibility complex class II-restricted, autoreactive T cells in multiple sclerosis (MS). Since susceptibility to MS is associated with the DR2 haplotype, the binding and presentation of the immunodominant MBP(84-102) peptide by DR2 antigens were examined. The immunodominant MBP(84-102) peptide was found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules of the disease-associated DR2 haplotype. Overlapping but distinct peptide segments were critical for binding to these molecules; hydrophobic residues (Val189 and Phe92) in the MBP(88-95) segment were critical for peptide binding to DRB1*1501 molecules, whereas hydrophobic and charged residues (Phe92, Lys93) in the MBP(89-101/102) sequence contributed to DRB5*0101 binding. The different registers for peptide binding made different peptide side chains available for interaction with the T cell receptor. Although the peptide was bound with high affinity by both DRB1 and DRB5 molecules, only DRB1 (DRB1*1501 and 1602) but not DRB5 molecules served as restriction elements for a panel of T cell clones generated from two MS patients suggesting that the complex of MBP(84-102) and DRB1 molecules is more immunogenic for MBP reactive T cells. The minimal MBP peptide epitope for several T cell clones and the residues important for binding to DRB1*1501 molecules and for T cell stimulation have been defined.

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques’ major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC–peptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0502-8) contains supplementary material, which is available to authorized users.

A direct binding assay specific for IAs molecules has been developed and its immunological relevance validated by examining, for a panel of nine different synthetic peptides, the correlation between their capacity to bind purified IAs and to inhibit IAs-restricted antigen presentation. The IAs assay thus developed has then been used to study the IAs binding affinity of a set of overlapping peptides spanning the entire myelin basic protein (MBP). It was found that the encephalitogenic MBP region corresponds to peptides with high MHC binding affinities. Other regions of the MBP that have not been described as being pathogenic in the context of IAs molecules have also been found to be high IAs binders, suggesting that variables other than MHC affinity are also involved in determining the pathogenic potential of self-derived determinants.

Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and avian H5N1. Our hypothesis is that influenza-derived HLA-class I-restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8(+) T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8(+) T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.

A potential etiology of chronic prostatitis/chronic pelvic pain syndrome is autoimmunity. We determined whether T cells from men with chronic prostatitis/chronic pelvic pain syndrome would recognize peptides derived from the normal self-prostatic proteins prostate specific antigen and prostatic acid phosphatase.CD4 T cells purified from peripheral blood of 31 patients with chronic prostatitis/chronic pelvic pain syndrome and from the buffy coat preparation of 27 normal male blood donors were stimulated in vitro with a panel of immunogenic peptides from prostate specific antigen and prostatic acid phosphatase, and assayed for reactivity with the peptides by interferon-gamma enzyme-linked immunosorbent spot assay. Intermediate resolution HLA typing was done by polymerase chain reaction. Peptides were also tested by binding assay against different class II alleles.Peptide PAP(173-192) was recognized more frequently by CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome than from healthy donors. The recognition of prostate specific antigen peptides was not statistically different when comparing cases to normal male blood donors individually. Peptide reactivity was more common in patients than in normal male blood donors for any prostate specific antigen peptide or any tested peptide. All peptides showed high promiscuity on binding assays. There was no association of cases with any specific HLA class II phenotype at intermediate resolution.CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome have a higher rate of recognizing the self-prostatic proteins prostatic acid phosphatase and prostate specific antigen compared to those from normal male blood donors. Data provide further evidence to support the role of autoimmunity in some men with chronic prostatitis/chronic pelvic pain syndrome.

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. The establishment of an MHC/peptide binding assay, and its subsequent use in determining the MHC binding capacities of peptide ligands, requires sufficient stocks of purified MHC and both labeled and unlabeled peptides. Accordingly, this unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. A support protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a 2-day incubation, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Two methods for separation by size-exclusion gel-filtration chromatography are described, as is data analysis.

CD8+ T-cell responses are central for the resolution of hepatitis C virus (HCV) infection, and viral escape from these CD8+ T-cell responses has been suggested to play a major role in HCV persistence. However, the factors determining the emergence of CD8 escape mutations are not well understood. Here, the first identification of four HLA-A26-restricted CD8+ T-cell epitopes is reported. Of note, two of these four epitopes are located in the NS3/4A and NS5A/5B cleavage sites. The latter epitope is targeted in all (three of three) patients with acute, resolving HCV infection and in a relatively high proportion (four of 14) of patients with chronic HCV infection. Importantly, the epitope corresponding to the NS5A/5B cleavage site is characterized by the complete absence of sequence variations, despite the presence of functional virus-specific CD8+ T cells in our cohort. These results support previous findings that showed defined functional constraints within this region. They also suggest that the absence of viral escape may be determined by viral fitness cost and highlight an attractive target for immunotherapies.

Background T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result. Results To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population. Conclusion We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC50

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome ∼20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8+ T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91–120 and Epo 126–155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.

Previous studies have suggested that murine T cells are tolerant to epitopes derived from germ line variable regions of immunoglobulin (Ig) heavy (VH) or light chains. This has lead to the prediction that germ line VH-region epitopes found in neoplastic B cells cannot be used to provoke an antitumor immune response. To test these assumptions and address the question of how such a vaccine may alter the normal B-cell response, an antibody-forming B-cell hybridoma (1H6) expressing a conserved germ line VH gene with specificity for dextran was generated and used as a tumor model. Using algorithms for predicting major histocompatibility complex (MHC) binding, potential MHC class I and II binding peptides were identified within the 1H6 VH region, synthesized, and tested for MHC binding and immunogenicity. We show that germ line VH peptides, when presented by dendritic cells, are immunogenic in vitro and provoke a tumor-specific protective immune response in vivo. We conclude that (1) it is possible to induce a T-cell response to germ line VH peptides; (2) such peptides can be used to generate a B-cell tumor-specific vaccine; and (3) a vaccine targeting VH peptides expressed by the dominant dextran-specific B-cell clonotype had no effect upon the magnitude of the normal B-cell response to dextran.