[Show abstract][Hide abstract] ABSTRACT: Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1)
infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific
epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe
the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree
of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include
the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2-restricted
CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included
in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important
to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines.
Journal of Virology 03/2001; 75(3):1301-11. DOI:10.1128/JVI.75.3.1301-1311.2001 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.
[Show abstract][Hide abstract] ABSTRACT: Various peptide-based approaches to simultaneous induction of multiple cytotoxic T lymphocyte (CTL) responses were evaluated as part of ongoing efforts to develop immunotherapeutic vaccines for use in humans. To this end, HLA (human histocompatibility leukocyte antigen)-A2-restricted epitopes from several specific viral proteins were tested in an HLA-A2 transgenic mouse model system, which mimics human CTL responses to these viral proteins. Multiple CTL responses were elicited by immunization with either peptides emulsified in incomplete Freund's adjuvant (IFA), or lipidated peptides administered in phosphate buffered saline (PBS). In the case of lipidated peptides, induction of CTL responses was crucially dependent on the presence of helper T lymphocyte (HTL) epitopes, and most efficient in the case of lipidated covalently linked HTL-CTL epitope constructs. CTL could also be induced by immunization with lipidated HTL epitopes simply mixed with CTL epitopes and formulated in PBS. However, this approach was highly dependent on the particular lipidated HTL/CTL combination utilized, and was marginally effective for simultaneous priming of multiple CTL responses. By contrast, all HTL/CTL combinations were potent immunogens when delivered as lipidated, covalently linked molecules. This was the most effective of the approaches analysed in terms of multi-epitope priming, as demonstrated by the induction of simultaneous CTL responses to a pool of five different epitopes.
[Show abstract][Hide abstract] ABSTRACT: Transgenic mice expressing chimeric human (alpha1 and alpha2 HLA-A11 domains) and murine (alpha3, transmembrane, and cytoplasmic H-2Kb domains) class I molecules were derived. These mice were used as a model system to study the immunogenicity of human CTL epitopes and also to examine the aspects of Ag processing differences of mice vs man. Immunization of these mice with seven known HLA-A11-restricted CTL epitopes emulsified in IFA resulted in vigorous specific CTL responses. A larger panel of 45 A11-binding peptides was used to examine the relationship between immunogenicity in the HLA-A11/Kb transgenic mice and HLA-A11 binding capacity. Twenty-one of 28 (75%) peptides with high binding affinities (50% inhibitory concentration (IC50), 2-50 nM) and 7 of 13 (54%) intermediate binding peptides (IC50, 50-500 nM range) were immunogenic. In parallel, 19 of these peptides were used for in vitro primary immunizations of PBMC derived from HLA-A11 healthy human donors. It was found that 8 of 8 peptides that were able to elicit CTL in primary human in vitro cultures were also immunogenic in HLA-A11/Kb mice. Finally, HLA-A11/Kb transgenic mice were found to generate an A11/Kb restricted CTL response following immunization with influenza virus A/PR/8/34, suggesting that, at least to some extent, A11 epitopes are generated by transgenic mice as a result of natural in vivo processing and presentation.
The Journal of Immunology 12/1997; 159(10):4753-61. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent data demonstrate that HLA class I alleles can be grouped into superfamilies based on similarities of their peptide-binding motifs. In this study, we have tested the immunogenicity and antigenicity of peptides capable of degenerate binding to multiple HLA class I molecules of the A3-like superfamily. The assay systems utilized included both primary in vitro cultures of lymphocytes from healthy donors, as well as in vitro restimulation of lymphocytes from HIV-infected individuals. Several of the peptides capable of binding more than one HLA A3-like class I molecule were also found to be immunogenic in the context of this same group of A3-like molecules (degenerate CTL recognition). Furthermore, some of the CTL lines thus generated demonstrated promiscuous recognition of the cognate epitope in the context of MHC molecules from more than one member of the superfamily. The fine Ag specificity of this phenomenon was further analyzed using two promiscuous CTL clones derived from A3 and A11 individuals, respectively, and specific for an epitope in the HIV-1 reverse transcriptase. By the use of single-amino acid-substitution analogues, it was demonstrated that the fine specificity of the TCR is largely maintained between MHC-matched and MHC-mismatched presentation of peptide within the A3-like superfamily. These results indicate that the similar peptide-binding specificities among different members of the A3-like superfamily can be reflected in a remarkable similarity in the peptide-MHC complex structures engaged by the TCR and responsible for T cell activation.
The Journal of Immunology 09/1997; 159(4):1648-57. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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-gamma and tumor necrosis factor alpha) 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
International Immunology 06/1996; 8(5). DOI:10.1093/intimm/8.5.651 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An HLA-A3-like supertype (minimally comprised of products from the HLA class I alleles A3, A11, A31, A*3301, and A*6801) has been defined on the basis of (a) structural similarities in the antigen-binding groove, (b) shared main anchor peptide-binding motifs, (c) the identification of peptides cross-reacting with most or all of these molecules, and (d) the definition of an A3-like supermotif that efficiently predicts highly cross-reactive peptides. Detailed secondary anchor maps for A3, A11, A31, A*3301, and A*6801 are also described. The biologic relevance of the A3-like supertype is indicated by the fact that high frequencies of the A3-like supertype alleles are conserved in all major ethnic groups. Because A3-like supertype alleles are found in most major HLA evolutionary lineages, possibly a reflection of common ancestry, the A3-like supermotif might in fact represent a primeval human HLA class I peptide-binding specificity. It is also possible that these phenomena might be related to optimal exploitation of the peptide specificity by human TAP molecules. The grouping of HLA alleles into supertypes on the basis of their overlapping peptide-binding repertoires represents an alternative to serologic or phylogenetic classification.
Human Immunology 03/1996; 45(2):79-93. DOI:10.1016/0198-8859(95)00173-5 · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HLA-A2.1-binding peptides (n = 38) were screened for immunogenicity with human peripheral blood mononuclear cells in cytotoxic T lymphocyte (CTL) induction experiments in vitro and with splenocytes from HLA-A2.1/Kb transgenic mice following immunization in vivo. These data were compiled and analyzed to determine the level of overlap between the A2.1-restricted CTL repertoire of A2.1/Kb-transgenic mice and A2.1+ humans. In both humans and mice, a major histocompatibility complex affinity threshold of approximately 500 nM appears to determine the capacity of a peptide to elicit a CTL response. Good concordance between the human data in vitro and mouse data in vivo was observed with 85% of the high-binding peptides, 58% of the intermediate binders, and 83% of the low/negative binders. Although some peptides immunogenic for mouse CTL but not for humans (and vice versa) could be identified, the data as a whole suggest an extensive overlap between T cell receptor repertoires of mouse and human CTL and support the use of HLA-transgenic mice for the identification of potential human CTL epitopes.
European Journal of Immunology 01/1996; 26(1):97 - 101. DOI:10.1002/eji.1830260115 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.
The Journal of Immunology 07/1995; 154(11):5934-43. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and β2-microglobulin (β2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18–27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4 + T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.
[Show abstract][Hide abstract] ABSTRACT: Antiviral cytotoxic T lymphocytes (CTL) may play a role in clearance of hepatitis C virus (HCV)-infected cells and thereby cause hepatocellular injury during acute and chronic HCV infection. The aim of this study was to identify HLA-A2.1-restricted HCV T-cell epitopes and to evaluate whether anti-HCV-specific CTL are present during chronic hepatitis C. Peripheral blood mononuclear cells from four HLA-A2-positive patients with chronic hepatitis C and from two individuals after recovery from HCV infection were tested against a panel of HCV-encoded peptides derived from different regions of the genome, including some peptides containing HLA-A2.1 binding motifs. HLA-A2-negative patients with chronic hepatitis C as well as healthy HLA-A2-positive (anti-HCV-negative) donors served as controls. Peripheral blood mononuclear cells stimulated repeatedly with several HCV-encoded peptides (three in core, one in NS4B, and one in NS5B) yielded cytolytic responses. All four HLA-A2-positive patients with active infection had CTL specific for at least one of the identified epitopes, whereas two patients who had recovered from HCV infection had almost no CTL responses. Monoclonal antibody blocking experiments performed for two epitopes demonstrated a class I- and HLA-A2-restricted CTL response. CTL epitopes could partially be predicted by HLA-A2 binding motifs and more reliably by quantitative HLA-A2.1 molecule binding assays. Most of the identified epitopes could also be produced via the endogenous pathway. Specific CTL against multiple, mostly highly conserved epitopes of HCV were detected during chronic HCV infection. This finding may be important for further investigations of the immunopathogenesis of HCV, the development of potential therapies against HCV on the basis of induction or enhancement of cellular immunity, and the design of vaccines.
Journal of Virology 05/1995; 69(4):2462-70. · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many human leukemias are characterized by chromosomal translocations yielding hybrid RNAs capable of encoding fusion chimeric proteins. The unique amino acid sequences found in these oncogenic fusion proteins represent true tumor-specific antigens that are potentially immunogenic. Although these leukemia-specific fusion proteins have an intracellular location, they might be recognized immunologically by T lymphocytes if peptides derived from the unique sequences are capable of presentation by the major histocompatibility complex (MHC) molecules on leukemic cells. The ability of a series of synthetic peptides corresponding to the junctional sequences of chronic myelogenous leukemia (CML)-derived bcr-abl and acute promyelocytic leukemia (APL)-derived PML-RAR alpha fusion proteins to bind to purified class I molecules was studied. A series of 152 peptides 8, 9, 10, and 11 amino acids in length, spanning the b3a2 and b2a2 breakpoints for CML and PML-RAR alpha A and B breakpoints for APL were analyzed for HLA A1, A2.1, A3.2, A11, A24, B7, B8, and B27 binding motifs. Twenty-one CML peptides and 4 APL peptides were predicted to be potential HLA class I binders. The peptides were tested for binding to appropriate purified HLA molecules in a competition radioimmunoassay. Four peptides derived from b3a2 CML breakpoint bound with high (< 50 nmol/L) or intermediate (< or = 500 nmol/L) affinity to HLA A3, A11, and B8. None of the CML b2a2 or PML-RAR alpha A or B junctional peptides showed affinity of this magnitude for the HLA class I molecules tested. This is the first evidence that tumor-specific breakpoint peptides can bind human MHC class I molecules and provides a rationale for developing a therapeutic vaccine strategy.
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic T lymphocytes (CTLs) have been implicated in the control and elimination of various viral and bacterial infections, tumors, and some parasitic diseases. CTLs that are characterized by the presence of the CD8 antigen generally recognize peptide fragments derived from intracellular processing of various antigens in the form of a complex with MHC class I molecules expressed on the cell surface (Germain and Margulies, 1993). This recognition may result in the lysis of the cell bearing the MHC-peptide antigen complex in an antigen-specific, MHC-restricted manner (Germain and Margulies, 1993). In addition, CTLs also generate a number of different lymphokines including gamma interferon (IFN-γ) and tumor necrosis factor (TNF), which are either directly cytolytic, regulate viral replication, or function to amplify the ongoing immune response (van der Bruggen and Van den Eynde, 1992; Jassoy et al., 1993; Rosenberg et al., 1988).
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We have described previously the generation of seven HIV-SF2 Nef-specific, CD4+ T-cell clones, identification of epitopes within which are recognized by these clones, and the MHC alleles that restrict their responses. In this study, we have extended this characterization to include evaluation of antigen-processing and presentation requirements and cytotoxic activity. Clones were generated from five HIV-1 uninfected donors by in vitro stimulation of peripheral blood mononuclear cells with purified recombinant Nef1. In experiments with fixed cells, with the exception of two clones, recognition of Nef, but not Nef peptides, required processing. Also, at higher concentrations of antigen, the clones themselves were capable of presenting Nef peptides, but not soluble Nef. All clones had the ability to specifically lyse autologous, Epstein-Barr virus-transformed lines sensitized with Nef synthetic peptides, or, in some cases, soluble Nef. The cytotoxic activity mapped to the same epitopes identified for the proliferative response (a.a. 14-22, 47-53, 68-77, 70-77, 195-203 and 185-192) and was restricted by the same HLA class II molecules (DRw6, DQw7, DRw15(2), DR1 and DP5). Sensitization of the cytolytic clones with specific Nef peptides, but not soluble Nef, resulted in autolysis.
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic T lymphocytes (CTLs) recognize peptide antigens associated with cell surface major histocompatibility complex (MHC) molecules. The identification of tumor cell-derived peptides capable of eliciting anti-tumor CTL responses would enable the design of antigen-specific immunotherapies. Our strategy to identify such potentially therapeutic peptides relies on selecting high-affinity MHC binders from known tumor-associated antigens. These peptides are subsequently tested for their ability to induce CTLs capable of killing tumor cells. With this strategy, we have identified a nine-residue epitope, derived from the product of the tumor-associated gene MAGE-3, which has the capacity to induce in vitro CTLs that kill melanoma and other tumor cell lines. These results show the primary in vitro induction of tumor-specific human CTLs and illustrate the feasibility of ex vivo antigen-specific approaches to the immunological therapy of cancer.
Proceedings of the National Academy of Sciences 04/1994; 91(6):2105-9. DOI:10.1073/pnas.91.6.2105 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human T-cell clones with specificity to the HIV-1 nef protein were generated by the in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-seronegative donors with purified nef from the HIV-SF2 isolate produced in genetically engineered yeast. Here the characterization is described of a total of seven discrete clones derived from five different donors. Each clone was CD3+ CD4+ CD8- as determined by FACS analysis. The epitopes recognized by these clones were identified using synthetic overlapping peptides spanning the entire length of nef. Six discrete helper T-cell epitopes located in five distinct regions of nef were identified by this approach. Three of these epitopes are more than 80% conserved among all HIV-1 nef proteins for which sequence data are available. The remaining epitopes are in regions of nef that vary among isolates. Many of the epitopes recognized by our clones overlap T-cell epitopes identified by others examining T-cell responses to nef in HIV-1-infected patients and immunized animals. Using partially class II-matched EBV-transformed B-cell lines, we were able to identify five different HLA class II alleles which encode restricting elements for the in vitro nef-specific proliferative response of these clones (DR1, DRw15(2), DRw6, DQw7, DP5).
[Show abstract][Hide abstract] ABSTRACT: To further understand and evaluate the phenomenon of TCR antagonism, we wished to determine whether analogs of an antigenic determinant could antagonize a specific polyclonal response. To this end, the ability of TCR antagonist peptides to inhibit a panel of five different DR4w4-restricted, influenza hemagglutinin 307-319-specific T cell lines was examined. An analysis of their V beta and J beta usage indicated that each of these five T cell lines expressed different TCR. A series of HA 307-319 single amino acid substituted analogs were used to determine the fine Ag specificities of the different lines. Ag analogs that demonstrated little or no stimulatory capacity were then examined for their ability to act as TCR antagonists by inhibiting the proliferative response of these five lines. Overall, 17 different peptide analogs capable of antagonizing at least one T cell line were identified. Although no single analog was capable of inhibiting all five T cell lines, two different analogs were identified that were capable of inhibiting four of five of the T cell specificities examined.
The Journal of Immunology 01/1994; 151(12):6815-21. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peptides that bind with high affinity to major histocompatibility complex molecules could represent useful tools in treating class II-associated autoimmune diseases such as rheumatoid arthritis, type 1 diabetes and multiple sclerosis. Although the concept has been validated in experiments with both purified receptor systems in vitro and cellular systems in vivo, many challenging problems need to be resolved before efficacious therapeutic agents are obtained.
Current Opinion in Biotechnology 03/1992; 2(6):877-81. DOI:10.1016/S0958-1669(05)80125-9 · 7.12 Impact Factor