Analysis of anchor residues in a naturally processed HLA-DR53 ligand

 The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. Received: 29 April 1996 / Revised: 16 June 1996     

Tyrosinase family proteins are antigens specific to Vogt-Koyanagi-Harada disease

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease, and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins.     

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

 HLA-B^*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B^*3501-restricted antigens is the characterization of peptide-HLA-B^*3501 interactions. In this study, peptide-HLA-B^*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B^*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B^*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B^*3501 binding cleft. Received: 29 May 1996 / Revised: 5 August 1996     

Identification of immunogenic epitopes of GAD 65 presented by A g7 in non-obese diabetic mice

 The autoantigen glutamic acid decarboxylase 65 (GAD 65) is believed to be an important target antigen in insulin-dependent diabetes mellitus (IDDM), since an age-related spontaneous breakdown in tolerance is observed, and cell-mediated and autoantibody immune responses have been reported in humans and NOD mice. We sought to identify immunogenic epitopes of GAD 65 which are presented to T cells by the type I diabetes susceptibility allele (A ^g7 ), using overlapping 15-mer synthetic peptides spanning the entire sequence of this protein. Four epitopes (p206 – 220, p221 – 235, p286 – 300, p571 – 585) were identified by screening a panel of T-cell hybridomas generated from GAD 65-immunized NOD mice. These immunogenic epitopes are unrelated to the previously described T-cell epitopes of GAD 65 reported in NOD mice. Of the GAD 65 amino acid sequence, 206 – 220 and 221 – 235 are the two most dominant T-cell epitopes identified in this study. Sixty-three percent and 25% of GAD 65-responding T cell hybridomas react to p206 – 220 and p221 – 235, respectively. The remaining two peptides (p286 – 300, p571 – 585) are less dominant T-cell responses. The identification of the whole spectrum of GAD 65 A^g7 epitopes should further the investigation of the role of this autoantigen in the pathogenesis of IDDM.     

Importance of GAD65 peptides and I-Ag7 in the development of insulitis in nonobese diabetic mice

 Insulin-dependent diabetes mellitus (IDDM) develops in nonobese diabetic (NOD) mice through the destruction of the B cells in pancreatic Langerhans islets by islet autoantigen-specific T cells. The islet autoantigen glutamic acid decarboxylase 65 (GAD65) is thought to be a major target autoantigen in IDDM. In the present report, we established GAD65-specific T-cell clones using overlapping peptides that cover the amino acid sequences of mouse GAD65. T-cell epitopes of GAD65 were characterized by proliferation and binding assays using various analogue peptides and wild-type or mutant I-A^g7 transfectants. The efficacy of the peptide vaccine in IDDM was determined by administering T-cell epitope peptides to NOD mice and evaluating the histopathology of their insulitis. We obtained two types of T-cell clone, one specific for peptide p316–335 and another specific for p531–545 of GAD65. The p531–545 site has already been identified, but we report the p316–335 site for the first time. T-cell clones recognized those peptides in the wild-type I-A^g7 but not in the mutant I-A^g7 in which the serine at position 57 of the β-chain was replaced by an aspartic acid. Both the p316–335 and p531–545 peptides bound weakly to I-A^g7. Some peptides with amino acid substitutions had antagonistic activity, and administration of a large amount of wild-type peptide reduced the severity of insulitis in NOD mice. Our results suggest that peptide vaccine therapy may be useful in autoimmune diseases, including IDDM. Received: 19 July 1999 / Revised: 4 January 2000     

A possible association between basic amino acids of position 13 of DRB1 chains and autoimmune hepatitis

Fifty-one patients with autoimmune hepatitis have been studied for HLA association by conventional serology and also by modified polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) genotyping.HLA-DR4 was significantly associated with autoimmmune hepatitis (46 of 51 patients, 90.2%). DNA typing of the DRB1 gene for 43 DR4-positive patients by using the PCR-RFLP technique revealed that of 43 patients, 33 had DRB1 ^* 0405 (Dw15), five had DRB1 ^* 0406 (DwKT2), four had DRB1 ^* 0403 (Dw13a), two had DRB1 ^* 0401 (Dw4), two of 43 had DRB1 ^* 0407 (Dw13b) and one had DRB1 ^* 0408 (Dw14b). Thus, there was no significant difference in Dw frequencies between DR4-positive patients and DR4-positive healthy subjects. These findings suggest that the DR4-specific sequence (Val 11 and His 13 at amino acid positions 11 and 13, respectively), but not particular Dw-associated DR4 sequence, in the first domain of the DRB1 chain contributes to susceptibility to autoimmune hepatitis among Japanese. Interestingly, all five of the DR4-negative patients had the DR2 specificity (DRB1 ¹⁵⁰² or 1601). Taken together, these results imply that the basic amino acids at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), are most important for determining the predisposition to autoimmune hepatitis. Address correspondence and offprint requests to: M. Ota.     

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

HLA antigens and risk for development of pemphigus foliaceus (fogo selvagem) in endemic areas of Brazil

Endemic pemphigus foliaceus (EPF), is an autoimmune disease associated with production of IgG antibodies against epidermal antigens. We have tested 38 patients and 50 control subjects living in endemic areas to investigate whether HLA genes are associated with host factors that determine whether or not exposed individuals will develop this disease. A variant of HLA-DR1, an antigen common in Blacks (DRB1^*0102), was found to be the main susceptibility factor (relative risk=7.3, P<0.0002). Two amino acids, in positions 85 and 86 of DRB1, distinguish DRB1^*0102 from DRB1^*0101. These residues appear to be involved in the formation of a functional epitope that causes T cell recognition and determines disease susceptibility. Moreover, subjects having DQw2 did not develop the disease, while the frequency of DQw2 in controls was 22% (RR=0.04, P<0.006). Thus HLA genes appear to play a crucial role in the response to an environmental factor which in this setting frequently leads to the development of autoimmune disease. An HLA-DQ allele, DQw2, appears to be associated with factors that prevent the development of the disease in exposed individuals.     

Both α and β chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules,with β chain residues being most influential

 We compared the peptide binding specificity of three HLA-DQ molecules; HLA-DQ(α1^*0501, β1^*0201), HLA-DQ(α1^*0201, β1^*0202), and HLA-DQ(α1^*0501, β1^*0301). The first of these molecules confers susceptibility to celiac disease and insulin-dependent diabetes mellitus, while the two latter molecules, which share either the α chain or the nearly identical β chain with HLA-DQ(α1^*0501, β1^*0201), do not predispose to these disorders. The binding of peptides was detected in biochemical binding assays as inhibition of binding of radiolabeled indicator peptides to affinity-purified HLA-DQ molecules. Binding experiments with several peptides demonstrated a clear difference in peptide binding specificity between the three HLA-DQ molecules. Further, single amino acid substitution analyses indicated that the HLA-DQ molecules have different peptide binding motifs. The experimental data were corroborated by computer modelling analysis. Our data suggest that the three HLA-DQ molecules prefer large hydrophobic residues in P1 of peptides with subtle differences in side-chain preferences. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202) both prefer large hydrophobic residues in P9, whereas HLA-DQ(α1^*0501, β1^*0301) prefers much smaller residues in this position. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202), in contrast to HLA-DQ(α1^*0501, β1^*0301), prefer negatively charged residues in P4 and P7. A less prominent P6 pocket also appears to differ between the three HLA-DQ molecules. Our results indicate that polymorphic residues of both the α and the β chain determine the peptide binding specificity of HLA-DQ(α1^*0501, β1^*0201), but that the β chain polymorphisms appears to play the most important role. The information on peptide residues which are advantageous and deleterious for binding to these HLA-DQ molecules may make possible the prediction of characteristic features of peptide that bind to HLA-DQ(α1^*0501, β1^*0201) and precipitate celiac disease. Received: 2 July 1996 / Revised: 7 August 1995     

Induction of determinant spreading and of Th1 responses by in vitro stimulation with HER-2 peptides

Immunization with tumor antigens induces cellular and humoral immune responses. These responses by T cells are specific for defined epitopes (determinants) in the molecule of the immunizing tumor antigen. Extension of such responses to self-antigens requires induction of autoimmunity to the tumor. As with systems of autoimmune disease, expression of T cell autoimmunity is charaterized by diversification of responses from the inducer determinant to other responder (cryptic) determinants. Since similar strategies may be useful for therapy of human cancers, we investigated whether the induction of response to a HER-2 peptide F7 (776–789) induces enhanced reactivity of other HER-2 peptides. We found that stimulation with F7 can expand a response to another epitope F13 (884–899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased interferon γ secretion, and proliferation, but was not observed with another donor who shared HLA-DR14 and HLA-DQ5 with the patient. Since repeated vaccination with the same epitope may lead to a decline of primary cell reactivity caused by apoptosis spreading the response to other epitopes, the tumor antigen may provide an approach for maintaining an inflammatory Th1 response during cancer vaccination. Received: 10 April 2000 / Accepted: 12 July 2000     

Peptide immunisation of HLA-DR–transgenic mice permits the identification of a novel HLA-DRβ1*0101– and HLA-DRβ1*0401–restricted epitope from p53

Because of the central role of CD4⁺ T cells in antitumour immunity, the identification of the MHC class II–restricted peptides to which CD4⁺ T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR–restricted peptides using peptide immunisation of HLA-DR–transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307–319), and then extended to three candidate HLA-DR–restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DR1*0101 (HLA-DR1) and HLA-DR1*0401 (HLA-DR4) molecules. One of these peptides, p53(108–122), consistently induced responses in HLA-DR1– and in HLA-DR4–transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108–122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR–restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II–restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.     

A WT1 protein‐derived,naturally processed 16‐mer peptide,WT1332, is a promiscuous helper peptide for induction of WT1‐specific Th1‐type CD4+ T cells

The Wilms' tumor gene WT1 is overexpressed in various tumors, and the WT1 protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. A WT1 protein‐derived 16‐mer peptide, WT1₃₃₂ (KRYFKLSHLQMHSRKH), which was naturally generated through processing in cells and could elicit Th1‐type CD4⁺ helper T cell responses with an HLA‐DRB1*0405‐restriction has previously been identified by us. In the present study, it has been demonstrated that WT1₃₃₂ can induce WT1₃₃₂‐specific CD4⁺ T cell responses with the restriction of not only HLA‐DRB1*0405 but also HLA‐DRB1*1501, ‐DRB1*1502, or ‐DPB1*0901. These HLA class II‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines produced IFN‐γ but neither IL‐4 nor IL‐10 with WT1₃₃₂ stimulation, thus showing a Th1‐type cytokine profile. Furthermore, HLA‐DRB1*1501 or ‐DRB1*1502‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines responded to WT1‐expressing transformed cells in an HLA‐DRB1‐restricted manner, which is consistent with our previous finding that WT1₃₃₂ is a naturally processed peptide. These results indicate that the natural peptide, WT1₃₃₂, is a promiscuous WT1‐specific helper epitope. WT1₃₃₂ is expected to apply to cancer patients with various types of HLA class II as a WT1‐specific helper peptide in combination with HLA class I‐restricted WT1 peptides.     

Antigen-specific inhibition of CD4+ T-cell responses to β-lactoglobulin by its single amino acid-substituted mutant form through T-cell receptor antagonism

T cell responses can be antagonized by some single amino acid-substituted analogs of a peptide ligand for T-cell receptors (TCR), and these are called TCR antagonists. In this study, we addressed the question of whether TCR antagonism can be elicited by a whole protein antigen carrying a mutated T-cell determinant region corresponding to a TCR antagonist peptide. To clarify this, we examined the ability of a single amino acid-substituted mutant form of bovine β-lactoglobulin (β-Lg) to inhibit three CD4⁺ T-cell clones recognizing a peptide corresponding to an immunodominant determinant region 119-133 of β-Lg (p119-133). First, we identified pD129A, an analog of p119-133 with a substitution of Ala for ¹²⁹Asp, as an antagonist which can inhibit the response of two of the three T-cell clones. Then, using a yeast expression system, we prepared a mutant β-Lg (mutD129A) with the same substitution of Ala for ¹²⁹Asp as that in pD129A. This mutant protein could inhibit the proliferation of the two T-cell clones in a manner similar to the effect of pD129A. From these results we can demonstrate that TCR antagonism can be elicited by peptides naturally processed from a single-substituted mutant protein as well as by the corresponding peptides added exogenously. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tumour-specific MHC-class-II-restricted responses after in vitro sensitization to synthetic peptides corresponding to gp100 and Annexin II eluted from melanoma cells

In a search for potentially tumour-specific MHC-class-II-restricted antigens, the immunogenicity of endogenous peptides that had been eluted from HLA-DR molecules of the human melanoma cell line FM3 (HLA-DRB1*02x, DRB1*0401) was tested in vitro. Two 16-mers representing gp100 positions 44–59, and annexin II positions 208–223 bound well to isolated DRB1*0401 molecules and are discussed here. HLA-DR-matched normal donors' T cells were cultured with peptide-pulsed artificial antigen-presenting cells (CHO cells cotransfected with genes for HLA-DRB1*0401 and CD80 and coexpressing high levels of both human molecules). Specific sensitization was achieved against both peptides, as measured in assays of autocrine proliferation and interleukin-2 secretion. Moreover, responses to native autologous melanoma cells but not to autologous B cells were also observed. In view of the expression of fas by the activated T cells and of fas ligand by the melanoma cells, blockade of potential fas/fas-ligand interactions was undertaken using monoclonal antibodies (mAb). The antagonistic fas-specific mAb M3, but not the fas agonist M33, caused a markedly enhanced T cell response to FM3 cells. These results demonstrate that synthetic peptide antigens are able to sensitize T cells in vitro for effective MHC-class-II-restricted recognition of melanoma cells. Received: 12 April 1998 / Accepted: 23 April 1998     

Magnitude of structural changes of the T-cell receptor binding regions determine the strength of T-cell antagonism: molecular dynamics simulations of HLA-DR4 (DRB1*0405) complexed with analogue peptide

In our model system, we generated T cell clones specific for the HLA-DR4 (DRB1*0405)-index peptide (YWALEAAAD) complex. Based on response patterns of the T cell clones, analogue peptides containing single amino acid substitutions of the index peptide were classified into three types, agonists, antagonists or null peptides (non-agonistic and non-antagonistic peptides). Subtle structural changes induced by the antagonists in the T-cell receptor (TCR) binding regions have already been explained using the root mean square (r.m.s.) deviations from the DR4-index peptide complex in the molecular dynamics (MD) trajectory. In this work, we performed additional MD simulations at 300 K with explicit solvent molecules to reveal the structural character of the HLA-DR4 complexed with the analogue peptides. We examined the r.m.s. deviations of the TCR-binding sites and the exposed areas of the bound peptides. Remarkable differences of the r.m.s. deviations among the DR4-antagonist complexes, together with our previous data, suggest that the magnitude of structural changes of TCR-binding regions would determine the strength of TCR antagonism. The simulations also indicate that TCR could discriminate null peptides from other ligands mainly through the changes of exposed side chains of the bound peptide, rather than the conformational changes of TCR-binding surfaces on HLA molecule.     

T cell epitopes of human myelin oligodendrocyte glycoprotein identified in HLA-DR4 (DRB1*0401) transgenic mice are encephalitogenic and are presented by human B cells.

Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97-108 (core 99-107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.     

Dendritic Cell Mediated Delivery of Plasmid DNA Encoding LAMP/HIV-1 Gag Fusion Immunogen Enhances T Cell Epitope Responses in HLA DR4 Transgenic Mice

This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1×10⁵ immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-γ ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets.     

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

 HLA-B^*3501 and -B^*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B^*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B^*3501 and HLA-B^*5101 molecules, we generated HLA-B^*3501 mutant molecules carrying Tyr at residue 116 (B^*3501–116Y) and tested the binding of a panel of nonamer peptides to the B^*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B^*3501–116Y molecules was much higher than that to HLA-B^*3501 and HLA-B^*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B^*3501 and HLA-B^*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B^*3501 and HLA-B^*5101 may be responsible for the low peptide binding property of the latter. Received: 18 April 1997 / Revised: 18 September 1997     

Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501

 A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8⁺ T-cell response. Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide that stimulates one individual’s CD8⁺ cytotoxic T lymphocytes will be presented by a second individual’s different class I molecules. Therefore, to facilitate precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry to assign precise peptide binding signatures to individual class I HLA molecules. In applying this strategy to HLA-B^*1501, we isolated milligram quantities of B^*1501-bound peptides and mapped them using mass spectrometry. Repeated analyses consistently assign the same peptide binding signature to B^*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of their peptide signatures. Received: 3 October 1996 / Revised: 20 November 1996