ABSTRACT
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 10(7)-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.
Subject(s)
Carcinoembryonic Antigen/immunology , Epitopes, T-Lymphocyte/immunology , Gene Products, pol/immunology , Hepatitis B Core Antigens/immunology , Histocompatibility Antigens Class I/immunology , Peptides/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Gene Products, pol/chemical synthesis , HIV-1/immunology , HLA-A2 Antigen/immunology , HT29 Cells , Hepatitis B virus/immunology , Humans , Interferon-gamma/biosynthesis , Interleukin-10/metabolism , Interleukin-5/metabolism , Jurkat Cells , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Peptide Biosynthesis , Peptides/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.
Subject(s)
Antigens, Viral/immunology , Gene Products, env/immunology , Gene Products, gag/immunology , Gene Products, pol/immunology , Gene Products, tax/immunology , HLA-B Antigens/immunology , Human T-lymphotropic virus 1/immunology , Retroviridae Proteins, Oncogenic/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Female , Gene Products, env/chemical synthesis , Gene Products, gag/chemical synthesis , Gene Products, pol/chemical synthesis , Gene Products, tax/chemical synthesis , HLA-B Antigens/genetics , Humans , Mice , Mice, Inbred C3H , Mice, Inbred ICR , Mice, Transgenic , Peptides/chemical synthesis , Peptides/immunology , Retroviridae Proteins, Oncogenic/chemical synthesis , gag Gene Products, Human Immunodeficiency VirusABSTRACT
A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.