Biomedical subjects

J Sidney

Publications and source records attributed to J Sidney.

At least 73 records · Page 4Linked to original sources

PubMed1995

The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis.

Cytotoxic T lymphocytes (CTL) are thought to contribute to viral clearance and liver cell injury during hepatitis B virus (HBV) infection. Using a strategy involving the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with HBV-derived synthetic peptides containing HLA-A2.1, -A31, and -Aw68 binding motifs, we have previously described CTL responses to several epitopes within the HBV nucleocapsid and envelope antigens in patients with acute hepatitis. In this study we define six HLA-A2-restricted CTL epitopes located in the highly conserved reverse transcriptase and RNase H domains of the viral polymerase protein, and we show that the CTL response to polymerase is polyclonal, multispecific, and mediated by CD8+ T cells in patients with acute viral hepatitis, but that it is not detectable in patients with chronic HBV infection or uninfected healthy blood donors. Importantly, the peptide-activated CTL recognize target cells that express endogenously synthesized polymerase protein, suggesting that these peptides represent naturally processed viral epitopes. DNA sequence analysis of the viruses in patients who did not respond to peptide stimulation indicated that CTL nonresponsiveness was not due to infection by viral variants that differed in sequences from the synthetic peptides. CTL specific for one of the epitopes were unable to recognize several naturally occurring viral variants, except at high peptide concentration, underlining the HBV subtype specificity of this response. Furthermore, CTL responses against polymerase, core, and envelope epitopes were detectable for more than a year after complete clinical recovery and seroconversion, reflecting either the persistence of trace amounts of virus or the presence of long lived memory CTL in the absence of viral antigen. Finally, we demonstrated that wild type viral DNA and RNA can persist indefinitely, in trace quantities, in the serum and PBMC after complete clinical and serological recovery, despite a concomitant, vigorous, and sustained polyclonal CTL response. Since viral persistence is not due to escape from CTL recognition under these conditions, the data suggest that HBV may retreat into immunologically privileged sites from which it can seed the circulation and reach CTL-inaccessible tissues, thereby maintaining the CTL response in apparently cured individuals and, perhaps, prolonging the liver disease in patients with chronic hepatitis.

Acute Disease↗

PubMed1995

Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype.

Direct MHC binding assays with radiolabeled peptides and HLA class I-expressing mammalian cells such as EBV-transformed B cell lines and PHA-activated blasts have been developed. Significant binding of the radiolabeled probe could be obtained if the target cells were preincubated overnight at 26 degrees C in the presence of beta 2-microglobulin. Under these conditions, up to a few percent of the HLA molecules expressed by either cell type could be bound by the labeled peptides. With these assays, the degree of cross-reactivity of the A*0201-restricted hepatitis B virus core 18-27 peptide with other A2 subtypes was examined. It was determined that this peptide epitope also binds the A*0202, A*0205, and A*0206 but not A*0207 subtypes. Inhibition experiments with panels of synthetic peptide analogues underlined the similar ligand specificities of the HLA-A*0201, A*0202, and A*0205 alleles. Analysis of the polymorphic residues that help form the B and F pockets of various HLA alleles allowed prediction of binding of the hepatitis B virus core 18-27 epitope to two other HLA alleles (HLA-A*6802 and A*6901). Thus, it appears that a family of at least six different HLA-A molecules may share overlapping ligand specificities (aliphatic residues in position 2 and at the C termini). These results suggest that broadly cross-reactive peptide epitopes can be identified and greatly enhance the prospective feasibility of peptide-based vaccination approaches.

Amino Acid Sequence↗

PubMed1995

Several HLA alleles share overlapping peptide specificities.

Herein we describe the establishment of assays to measure peptide binding to purified HLA-B*0701, -B*0801, -B*2705, -B*3501-03, -B*5401, -Cw*0401, -Cw*0602, and -Cw*0702 molecules. The binding of known peptide epitopes or naturally processed peptides correlates well with HLA restriction or origin, underscoring the immunologic relevance of these assays. Analysis of the sequences of various HLA class I alleles suggested that alleles with peptide motifs characterized by proline in position 2 and aromatic or hydrophobic residues at their C-terminus shared key consensus residues at positions 9, 63, 66, 67, and 70 (B pocket) and residue 116 (F pocket). Prediction of the peptide-binding specificity of HLA-B*5401, on the basis of this consensus B and F pocket structure, verified this hypothesis and suggested that a relatively large family of HLA-B alleles (which we have defined as the HLA-B7-like supertype) may significantly overlap in peptide binding specificity. Availability of quantitative binding assays allowed verification that, indeed, many (25%) of the peptide ligands carrying proline in position 2 and hydrophobic/aromatic residues at the C-terminus (the B7-like supermotif) were capable of binding at least three of five HLA-B7-like supertype alleles. Identification of epitopes carrying the B7-like supermotif and binding to a family of alleles represented in over 40% of individuals from all major ethnic groups may be of considerable use in the design of peptide vaccines.

Alleles↗

PubMed1995

Class I MHC-peptide interactions: structural requirements and functional implications.

In this chapter, we have defined the structural motifs that dictate the capacity of peptides to bind to five different HLA-A alleles that represent some of the most common alleles found in different ethnic populations. In general, these peptide motifs were very specific for the individual HLA-A alleles, with the exception of HLA-A degree 0301 and HLA-A degree 1101, for which the motifs were very similar. When these motifs were tested against an unbiased and complete set of nonamer peptides derived from human papillomavirus E6 and E7 proteins, it was found that the vast majority of high and intermediate binding peptides contained the appropriate motif. Furthermore, using the dominant anchor residues, together with the amino acid positions that interact with secondary anchor residues, it was possible to predict high and intermediate binders. Finally, the finding that there is a direct correlation between binding affinity for MHC and immunogenicity suggests a practical application of being able to predict those peptides that have a high affinity binding for a particular MHC allele--that is, in the design of peptide based vaccines for prophylactic or therapeutic use.

Alleles↗

PubMed1994

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

Amino Acid Sequence↗

PubMed1994

Definition of a DQ3.1-specific binding motif.

A quantitative peptide binding assay using purified DQA1*0301/DQB1*0301 (DQ3.1) molecules was developed and validated by examining the correlation between the data obtained in the binding assay with those obtained in inhibition of Ag presentation assays. By the combined use of large libraries of synthetic peptides and of substitution and truncation analogues, a putative DQ3.1 motif was defined. Its most prominent feature is the requirement for two small and/or hydrophobic residues spaced at positions i + 2 and i + 4. This motif is quite different from the motif recognized by DR molecules, but similar to the motif previously defined for certain IA alleles (the putative mouse homologue of DQ). These data suggest that various class II isotypes have evolved to present different peptide structures to each other, thus maximizing the repertoire of different epitopes available to T cell scrutiny.

Amino Acid Sequence↗

PubMed1994

Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins.

We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.

Alleles↗

PubMed1994

Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations.

An equilibrium binding assay was used to directly measure the relative affinities of naturally processed 9-mer, 10-mer, and 12-mer peptides for the human class I MHC molecule HLA-A2.1. The peptides exhibited a range of affinities with IC50 values of 11 to 214 nM. The mode of interaction between these peptides and HLA-A2.1 was examined using peptides in which Asp had been substituted for suspected anchor residues. Regardless of length, the previously identified Leu at position 2 relative to the amino terminus was critical for peptide binding. While the carboxyl terminal residue was also critical for the binding of a 9-mer peptide, it was much less important in the binding of longer peptides. Additional residues close to the carboxyl terminus that contained aliphatic hydrocarbon side chains were of similar or greater importance in peptide binding. In addition, residue at position 3 also appeared to be important for the binding of longer peptides. The data suggest that different naturally occurring longer peptides can bind in different conformations to class I MHC molecules. While one of these is similar to the kinked conformation described by others, another conformation would involve an extension of the carboxyl terminus out of the class I binding site. The ability of MHC molecules to accommodate the same peptide in different conformations would appear to have distinct advantages to the immune system.

Amino Acid Sequence↗

PubMed1994

Peptide binding to the most frequent HLA-A class I alleles measured by quantitative molecular binding assays.

Quantitative assays to measure the binding of defined synthetic antigenic peptides and purified MHC class I molecules are described for several common human HLA-A alleles (A1, A2.1, A3, A11 and A24). Under appropriate conditions, the binding of radiolabeled peptides to purified MHC class I molecules is very effective, highly specific, and appears to be dependent on the specific sequence motif of the peptide as defined by critical anchor residue positions. Establishment and optimization of the assay reveals that a relatively high fraction of the MHC class I molecules isolated from EBV transformed B cell line sources is capable of binding exogenously added peptide. Scatchard analysis for all alleles yields 5-10% occupancy values. There is a stringent peptide size requirement that is reflected by the direct influence of peptide length on the binding affinity. The peptide-MHC class I interactions demonstrate remarkable similarity to peptide-MHC class II interactions, both in overall affinity and kinetic behavior. The immunological relevance of the peptide-MHC class I binding assay is also demonstrated by measuring the affinity of a panel of previously described HLA restricted peptides for their HLA restriction element. In 91% (10/11) of the cases, the peptides bound with affinities of 50 nM or less, and in the remaining 9% (1/11) of the cases, in the 50 to 500 nM range. Thus, these data provide the first quantitative estimate of what level of HLA-A binding affinity is associated with a diverse panel of immunodominant CTL epitopes in man.

Amino Acid Sequence↗

PubMed1994

Identification of potential CTL epitopes of tumor-associated antigen MAGE-1 for five common HLA-A alleles.

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 (Kd < or = 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.

Amino Acid Sequence↗

PubMed1994

Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides.

Pan DR-binding peptides engineered by introducing anchor residues for different DR motifs within a polyalanine backbone bound 10 of 10 DR molecules tested, with affinities, in most cases, in the nanomolar range. Because of the small methyl group exposed for T cell recognition, these peptides were poor immunogens but effective blockers of DR-restricted antigen presentation. Introduction of bulky and charged residues at positions accessible for T cell recognition yielded extremely powerful Pan DR epitope peptides (PADRE). These peptides elicited powerful responses in vitro from human peripheral blood mononuclear cells (PBMC). Because these cells also cross-react on certain mouse class II alleles, we could also demonstrate that PADRE peptides are active in vivo. In one example of their capacity to elicit T help, they were approximately 1000 times more powerful than natural T cell epitopes. We propose that PADRE peptides may be useful in the development of subunit vaccines.

Alleles↗

PubMed1994

Class I MHC-peptide interaction: structural and functional aspects.

The structural requirements for the interaction between antigens and class I molecules was investigated through the use of a quantitative assay to measure peptide binding to different MHC class I alleles. We determined the permissiveness of the main anchors reported by Rammensee and his group for peptide binding and defined an extended motif for peptides binding to the HLA-A2.1 allele, including the role of non-anchor positions. It was found that the main anchors were necessary, but not sufficient, for good binding. Certain non-anchor positions contributed significantly to overall binding and were referred to a secondary anchors. This finding allowed a better prediction of high affinity binding peptides selected from libraries of different viral and tumor proteins. Furthermore, our data allowed correlation of the structural requirements for binding of peptides with crystallographic data of the MHC molecule. In order to characterize allele-specific motifs for a larger number of alleles, the HLA-A alleles A1, A3, A11, and A24, which represent some of the most common alleles found in different ethnic populations, were chosen. Here, most motifs were found to be highly exclusive; however, HLA-A3 and A11 shared a common motif. The defined motifs were validated further by using naturally processed peptides. Those peptides were also synthesized and tested for binding to the appropriate HLA alleles, giving a binding affinity from 0.3 to 200 nM for sequences of naturally processed peptides. Finally, a set of all possible 9-mer peptides from HPV 16 proteins were synthesized and tested for binding to the five class I alleles. For each allele, high affinity binders were identified, thus allowing for selection of possible peptide candidates for a CTL based vaccine.

Alleles↗

PubMed1993

HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions.

In the present study, we describe the definition of a DRB1*0401 (DR4w4)-specific motif. The strategy used entailed a three-step process: 1) screening a large set of unrelated peptide ligands to identify good MHC binders; 2) truncation analysis of several DR4w4 binding peptides of high affinity to identify the crucial core-binding regions; 3) the use of single amino acid substitutions of the DR4w4-binding peptide hemagglutinin (HA) 307-319 to elucidate the specific residues crucial for binding. The DR4w4 motif is characterized by the presence of a hydrophobic or aromatic (F, W, Y, L, I, V, M) anchor residue in position 1, and a second hydroxyl (S, T) or aliphatic (L, I, V, or M) anchor residue in position 6. Furthermore, positive charges (R, K) are not allowed in positions 4, 7, and 9, and negative charges (D, E) are not allowed in position 9. This motif was present in 92% of good (IC50 < or = 100 nM) DR4w4-binding peptides, but less than 25% of the negative (IC50 > 45 microM) binders, indicating that the presence of the motif is necessary, but not sufficient for good DR4w4 binding capacity. The results of the present study are discussed in relation to previous work defining binding motifs and rules for other DR alleles, illustrating how different DR alleles bind variations on a similar structural theme. Finally, using two different peptide ligands [tetanus toxoid 830-843 and HA 307-319] as model systems, it is demonstrated how the fine allelic specificity of the DR binders can be predictably modulated by introducing subtle changes in the primary amino acid sequence.

Amino Acid Sequence↗

PubMed1993

Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules.

The functional determinants of histocompatibility leukocyte antigen (HLA)-A2.1-peptide interactions have been detailed by the use of quantitative molecular binding assays and a chemically synthesized library of naturally occurring epitopes. The importance of hydrophobic anchor residues in position 2 and the C-terminus was confirmed. These anchors are necessary, but not sufficient, for high affinity binding, as the predictions based solely on these anchors are only about 30% accurate. Prominent roles for several other positions (1, 3, and 7) were also demonstrated. The location of these residues within the peptides matches secondary A2.1 pockets previously demonstrated by X-ray crystallography. From a functional standpoint, similar dominant negative effects on binding were observed for charged residues in both nonamers and decamers, while positive effects differed between nonamers and decamers. An extended motif taking into account secondary anchors increased the predictability of A2.1-binding epitopes to a level of 70%, underscoring the practical usefulness of extended motifs.

Acid Phosphatase↗

PubMed1993

T cell receptor antagonism mediated by interaction between T cell receptor junctional residues and peptide antigen analogues.

A major objective of new rational immunosuppressive therapies is to be able to inhibit deleterious T cell responses in an Ag-specific manner. Recently, a novel approach to inducing Ag-specific nonresponsiveness in T cells, termed receptor or TCR antagonism, has been described. Several analogues of the influenza hemagglutinin (H3) peptide HA307-319 were shown to block recognition of the native HA307-319 peptide by a DR1-restricted T cell clone at concentrations 1,000- to 10,000-fold less than required to competitively inhibit HA307-319 binding to DR1. These Ag analogues that inhibited T cell recognition differed from the stimulatory antigenic peptide only at single positions. How such limited changes affect the ternary complex interactions of TCR, peptide, and DR remains unclear. In the present study, we have utilized two different DR5-restricted T cell clones that exhibit mutually exclusive specificities for HA peptides with or without substitutions at position 313 to investigate the molecular requirements for receptor antagonism. In this reciprocal model system, we show that a single peptide/DR complex can antagonize one T cell clone while stimulating another. A comparison of nucleotide sequences derived from the TCR of the T cell clones that were either antagonized or stimulated by the peptide analogue/DR5 complex indicated that the different responses of these T cells result from minor differences in the amino acid residues in junctional regions that most likely interact directly with position 313 of the peptide analogues. Our results suggest that TCR antagonism is an Ag-specific phenomenon in which T cells are inhibited by interactions involving TCR residues required for the recognition of conventional Ag and the altered residues in peptide analogues.

Amino Acid Sequence↗

PubMed1993

Functional consequences of engagement of the T cell receptor by low affinity ligands.

The mechanisms involved in TCR antagonism by Ag analog/MHC have been analyzed. A detailed structure-activity relationship study indicated that modification of any of the major T cell contact residues of the peptide molecule can yield a powerful antagonist. It was also shown that as the analog structure increased in similarity to the Ag, the capacity to antagonize Ag-TCR interaction increased up to the point that the analogs themselves became antigenic. These data strongly suggest an affinity-related mechanism whereby a certain affinity is required for signaling through the TCR, and that below this level there can be sufficient affinity to engage the receptor such that triggering does not occur and antagonism can be detected. Taking advantage of this information, antagonist peptides active down to the 10 nM range were engineered. Thus, this approach demonstrates for the first time a rational approach to designing effective, selective low m.w. compounds with high potential in treatment of allergies and autoimmune diseases.

Amino Acids↗

PubMed1993

MHC class II molecules bind indiscriminately self and non-self peptide homologs: effect on the immunogenicity of non-self peptides.

Synthetic peptides spanning the entire sequence of both human and mouse beta 2-microglobulin (beta 2M) have been tested for their capacity to bind to three different mouse (I-Ad, I-Ed, and I-Ak) or human (DR1, DR2, and DR5) class II molecules. The results demonstrate that class II molecules do not discriminate between self and non-self peptides. When the immunogenicity of the human beta 2M peptides was measured by their ability to prime H-2d mice for in vitro T cell proliferation, it was found that peptides incapable of binding class II molecules in vitro were also non-immunogenic in vivo. Interestingly, however, several binders, including the human beta 2M peptide 1-16, the best binder in this series to Iad molecules, were found to be non-immunogenic. Since the corresponding mouse beta 2M peptide 1-16 was also capable of binding to Iad molecules, this suggested that lack of responsiveness to the non-self peptide could arise either from central or peripheral tolerance induced by the self homolog. Alternatively, lack of responsiveness could arise from other mechanisms, such as negative selection by other non-homolog sequences or lack of suitable T cell receptor genes. To discriminate between these possibilities, H-2d mice with disrupted beta 2M genes were immunized with the human beta 2M peptide 1-16. This peptide also failed to prime for T cell responsiveness in beta 2M-negative mice, suggesting that a hole in the T cell repertoire for this antigen was not mediated by negative selection or peripheral tolerance induced by self beta 2M peptides.

Amino Acid Sequence↗

PubMed1993

Binding of myelin basic protein peptides to human histocompatibility leukocyte antigen class II molecules and their recognition by T cells from multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease in which myelin proteins have been implicated as autoantigens recognized by pathogenic autoreactive T cells. To study the relationship between human myelin basic protein (hMBP) and HLA alleles associated to MS susceptibility, such as DRB1*1501, the binding of synthetic peptides spanning the entire hMBP sequence to 10 purified HLA-DR molecules was determined. All the hMBP peptides tested showed binding affinity for at least one of the DR molecules analyzed, but three hMBP peptides, included in sequences 13-32, 84-103, and 144-163 were found capable of binding to three or more DR molecules. The hMBP peptide 84-103 was the most degenerate in binding, in that it bound to 9 out of 10 DR molecules tested. Interestingly, it bound with highest affinity to DRB1*1501 molecules. To correlate the binding pattern of hMBP peptides to HLA class II molecules with their recognition by T cells, 61 hMBP-specific T cell lines (TCL) were established from the peripheral blood of 20 MS patients, who were homozygous, heterozygous, or negative for DRB1*1501. Analysis of hMBP epitopes recognized by these TCL and their HLA restriction demonstrated a very good correlation between binding data and T cell proliferation to hMBP peptides. Although virtually all hMBP peptides tested could be recognized by at least one TCL from MS patients, three immunodominant T cell epitopes were apparent among the TCL examined, corresponding exactly to the hMBP peptides capable of binding to several DR molecules. No major difference could be detected in the recognition of immunodominant hMBP peptides by TCL from DRB1*1501 positive or negative MS patients. These results have implications for the role of hMBP as relevant autoantigen, and of DRB1*1501 as susceptibility allele in MS.

Adult↗