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.
The SIV-infected rhesus macaque (
Macaca mulatta)
is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The ...understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-
B*039:01
, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-
B*039:01
. A similar motif was previously described for the D
d
mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-
B*052:01
, shares this same motif. These “G2” alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the “G2” alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.
Characterization of the T-cell epitope that causes anti-GBM glomerulonephritis.
We have demonstrated that a single T-cell epitope pCol(28-40) (SQTTANPSCPEGT) alone, which is derived from NC1 domain ...of α3 chain of type IV collagen (Col4α3 NC1), can induce severe glomerulonephritis in Wistar Kyoto rats. This study further characterized this T-cell epitope.
A series of synthetic peptides derived from pCol (28-40) were tested in vivo and in vitro for their T-cell epitope activity and nephritogenicity. Major histocompatability complex (MHC) class II molecules in Wistar Kyoto rats were cloned, and MHC restriction of pCol(28-40) was determined.
The T-cell epitope pCol(28-40) was restricted by rat MHC class II RT.1Bl. Ten amino acid residues (29 to 38) were mapped to be the minimum core of the T-cell epitope, which was capable of inducing the T-cell response and severe glomerulonephritis. Only three residues were identified as absolutely critical for the T-cell epitope: position 31 (T) was an anchor residue to the class II molecule, and positions 33 (N) and 34 (P) contributed to the specificity of the T-cell epitope. Thus, only substitution at those positions completely abrogated nephritogenicity of the T-cell epitope. Interestingly, pCol (28-40) also bound to human MHC class II human MHC class II molecule HLA-DRB*1501, which has been linked to human anti-glomerular basement membrane (GBM) disease, suggesting that human homologue of pCol(28-40) could be a potential human T-cell epitope.
Our study demonstrated that only few residues in the nephritogenic T-cell epitope pCol(28-40) were critical. Our finding also revealed that pCol(28-40) is a potential nephritogenic T-cell epitope in Goodpasture's syndrome.
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.
MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the ...present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.
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.
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.
The cytotoxic T cell response against lymphocytic choriomeningitis virus (LCMV) in BALB/c (H-2d) mice is predominantly directed against a single immunodominant Ld-restricted epitope in the viral ...nucleoprotein (NP118-126). Here we report that the immunodominance of this peptide can be in part attributed to its very high affinity for Ld class I molecules. By employing motif searches and sensitive MHC class I binding assays, we also identified 5 Kd-binding peptides in the viral nucleoprotein and glycoprotein among 16 Kd motif-fitting peptides. The nucleoprotein and glycoprotein sequences also contained 18 Dd motif-fitting peptides, three of which bound Dd with weak affinity. Two of the Kd-binding peptides, residues 99-108 and residues 283-291 from the viral glycoprotein, are subdominant epitopes. Although these peptides did not sensitize target cells for direct ex vivo killing by primary antiviral CTL, secondary responses against these peptides were readily detected in BALB/c mice after acute LCMV infection. BALB/c mice that had cleared a long-term LCMV infection showed more sustained CTL responses against these subdominant epitopes, suggesting that subdominant responses might play a role in clearance of chronic infections. One of the subdominant epitopes, GP283-291, conferred partial protection against persistent viral infection after peptide vaccination.
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.
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 TriGridTM 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 super(+) T cell responses to protection against influenza infection.
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