Repertoire and immunofocusing of CD8 T cell responses generated by HIV-1 gag-pol and expression library immunization vaccines

Several gene-based vaccine approaches are being tested to drive multivalent cellular immune responses to control HIV-1 viral variants. To compare the utility of these approaches, HLA-A*0201 transgenic mice were genetically immunized with plasmids encoding wild-type (wt) gag-pol, codon-optimized (CO) gag-pol, and an expression library immunization (ELI) vaccine genetically re-engineered to express non-CO fragments of gag and pol fused to ubiquitin for proteasome targeting. Equimolar delivery of each vaccine into HLA-A*0201 transgenic mice generated CD8 T cell responses, with the ELI vaccine producing up to 10-fold higher responses than the wt or CO gag-pol plasmids against cognate and mutant epitopes. All three vaccines generated multivalent CD8 responses against varying numbers of epitopes after priming. However, when the animals were immunized again, the wt and CO gag-pol vaccines boosted only the responses against a subset of epitopes and attenuated the responses against all other Ags including epitopes from clade and drug-resistant viral variants. In contrast, the ELI vaccine boosted CD8 responses against all of the gag-pol Ags and against mutant epitopes from clade and drug-resistant variants. These data suggest that HIV-1 vaccines expressing structurally intact gag and pol proteins drive immunofocused CD8 responses that reduce the repertoire of T cell responses. In contrast, the genetically re-engineered ELI vaccine appears to better maintain the multivalent CD8 responses that may be required to control HIV-1 viral variants.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8(+) T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.     

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.     

HIV-1-specific CD8+ T cell responses and viral evolution in women and infants

CD8+ T lymphocyte responses play an important role in controlling HIV-1 replication but escape from CD8+ T cell surveillance may limit the effectiveness of these responses. Mother-to-child transmission of CD8+ T cell escape variants may particularly affect CD8+ T cell recognition of infant HIV-1 epitopes. In this study, amino acid sequence variation in HIV-1 gag and nef was examined in five untreated mother-infant pairs to evaluate the potential role of CD8+ T cell responses in the evolution of the viral quasispecies. Several CD8+ T cell escape variants were detected in maternal plasma. Evaluation of infant plasma viruses at 1-3 mo documented heterogeneity of gag and nef gene sequences and mother-to-child transmission of CD8+ T cell escape variants. Infant HLA haplotype and viral fitness appeared to determine the stability of the escape mutants in the infant over time. Changes in CD8+ T cell epitope sequences were detected in infants' sequential plasma specimens, suggesting that infants are capable of generating virus-specific CD8+ T cell responses that exert selective pressures in vivo. Altogether, these studies document that HIV-1-specific CD8+ T cell responses contribute to the evolution of the viral quasispecies in HIV-1-infected women and their infants and may have important implications for vaccine design.     

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.     

Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins.

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.     

Induction of HIV-1-specific T-helper responses and type 1 cytokine secretion following therapeutic vaccination of macaques with a recombinant fowlpoxvirus co-expressing interferon-gamma

Preventive and/or therapeutic vaccines against Human Immunodeficiency Virus (HIV-1) are urgently required. Induction of cellular immunity is favoured since these responses correlate with control of HIV-1. Recombinant fowlpoxvirus (FPV) vaccines encoding both HIV-1 gag/pol and interferon-gamma (FPV gag/pol-IFNgamma) were hypothesised to enhance HIV-specific cellular immunity and were further evaluated in macaques previously infected with HIV-1. A novel assay to detect IFNgamma secretion following HIV antigen stimulation of whole blood was developed to further assess the safety and immunogenicity of the FPV gag/pol-IFNgamma vaccine. Immunisation with FPV gag/pol-IFNgamma safely enhanced HIV-specific IFNgamma secretion following ex vivo stimulation of whole blood, greater than that observed following FPV gag/pol vaccination not co-expressing IFNgamma. Both HIV-specific IFNgamma-spot-forming cells by ELISPOT and CD69 expression by CD4+ lymphocytes were also enhanced following FPV gag/pol-IFNgamma vaccination. Hence, the FPV-HIV vaccine co-expressing IFNgamma stimulated HIV-specific T cell responses in macaques, and should be further evaluated as a therapeutic or preventive HIV vaccine.     

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.     

HLA-A0201 positive pancreatic cell lines: new findings and discrepancies

Pancreatic cancer is being pursued as an immunotherapy target using antigen-specific vaccine approaches activating CD8(+) CTL and CD4(+) T-helper cells. CD8(+) CTL exert their anti-tumor effects in an HLA-restricted manner and only tumor cells carrying a matched HLA class I sub-type are targets for antigen-specific CTL. In the process of characterizing CD8(+) T cell responses against pancreatic cancer, we screened a number of human pancreatic tumor cell lines for HLA-A0201 positive (HLA-A2(+)) cell lines to be used in the evaluation of CTL function. This analysis revealed some new findings and discrepancies in the literature on the HLA sub-type of some commonly used pancreatic cell lines. We found that Capan-1 cells, originally reported to be HLA-A0201(+), actually only express HLA-A010101 and HLA-A300101 and were targets for HLA-A0201-restricted CTL only after transduction with an HLA-A0201-expressing lentivirus. Panc-1 cells were found to be HLA-A0201 positive, in agreement with published reports, while CF-Pac-1 cells were found to express both HLA-A020101 and HLA-A030101. We also found a normal human pancreatic ductal epithelial cell line, HPDE, to be HLA-A0201 positive. Our findings were verified with two different sequence-based typing methods, antibody staining followed by flow cytometry analysis, and functional analysis using an HLA-A0201-restricted peptide-specific T cell response.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

Allogeneic HLA-A*02-restricted WT1-specific T cells from mismatched donors are highly reactive but show off-target promiscuity

T cells recognizing tumor-associated Ags such as Wilms tumor protein (WT1) are thought to exert potent antitumor reactivity. However, no consistent high-avidity T cell responses have been demonstrated in vaccination studies with WT1 as target in cancer immunotherapy. The aim of this study was to investigate the possible role of negative thymic selection on the avidity and specificity of T cells directed against self-antigens. T cell clones directed against the HLA-A*0201-binding WT1(126-134) peptide were generated from both HLA-A*02-positive (self-HLA-restricted) and HLA-A*02-negative [nonself (allogeneic) HLA [allo-HLA]-restricted] individuals by direct ex vivo isolation using tetramers or after in vitro priming and selection. The functional avidity and specificity of these T cell clones was analyzed in-depth. Self-HLA-restricted WT1-specific clones only recognized WT1(126-134) with low avidities. In contrast, allo-HLA-restricted WT1 clones exhibited profound functional reactivity against a multitude of HLA-A*02-positive targets, even in the absence of exogenously loaded WT1 peptide, indicative of Ag-binding promiscuity. To characterize this potential promiscuity, reactivity of the T cell clones against 400 randomly selected HLA-A*0201-binding peptides was investigated. The self-HLA-restricted WT1-specific T cell clones only recognized the WT1 peptide. In contrast, the allo-HLA-restricted WT1-reactive clones recognized besides WT1 various other HLA-A*0201-binding peptides. In conclusion, allogeneic HLA-A*02-restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts but can show specific off-target promiscuity of potential clinical importance. As a result of this, administration of WT1-specific T cells generated from HLA-mismatched donors should be performed with appropriate precautions against potential off-target effects.     

HLA-A*0201-restricted CD8+ cytotoxic T lymphocyte epitopes identified from herpes simplex virus glycoprotein D

Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53-61, gD70-78, and gD278-286, significantly stabilized HLA-A*0201 molecules on T2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-gamma ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD53-61, gD70-78, and gD278-286 epitopes. In addition, CD8+ T cell lines generated by gD53-61, gD70-78, and gD278-286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD53-61, gD70-78, and gD278-286 epitopes were induced in HLA-A*0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.     

Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.     

Induction of HIV-1-specific T-helper responses and type 1 cytokine secretion following therapeutic vaccination of macaques with a recombinant fowlpoxvirus co-expressing interferon-gamma

Preventive and/or therapeutic vaccines against Human Immunodeficiency Virus (HIV-1) are urgently required. Induction of cellular immunity is favoured since these responses correlate with control of HIV-1. Recombinant fowlpoxvirus (FPV) vaccines encoding both HIV-1 gag/pol and interferon-gamma (FPV gag/pol-IFNΓ) were hypothesised to enhance HIV-specific cellular immunity and were further evaluated in macaques previously infected with HIV-1. A novel assay to detect IFNΓ secretion following HIV antigen stimulation of whole blood was developed to further assess the safety and immunogenicity of the FPV gag/pol-IFNΓ vaccine. Immunisation with FPV gag/pol-IFNΓ safely enhanced HIV-specific IFNΓ secretion following ex vivo stimulation of whole blood, greater than that observed following FPV gag/pol vaccination not co-expressing IFNΓ. Both HIV-specific IFNΓ-spot-forming cells by ELISPOT and CD69 expression by CD4⁺ lymphocytes were also enhanced following FPV gag/pol-IFNΓ vaccination. Hence, the FPV-HIV vaccine co-expressing IFNΓ stimulated HIV-specific T cell responses in macaques, and should be further evaluated as a therapeutic or preventive HIV vaccine.     

The immunodominant CD8 T cell response to the human cytomegalovirus tegument phosphoprotein pp65(495-503) epitope critically depends on CD4 T cell help in vaccinated HLA-A*0201 transgenic mice

Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-A(b+)/A2-HHD-II and HLA-DR1(+)/A2-DR1 mice the HLA-A*0201-restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ(+) CD8 T cell frequencies to the pp65(495-503)/(e6) epitope and low responses to the pp65(320-328)/(e3) and pp65(522-530)/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65(Δ501-503) DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65(487-503) domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.     

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.     

Identification and characterization of HLA-B*5401-restricted HIV-1-Nef and Pol-specific CTL epitopes

The identification of HIV-1 cytotoxic T lymphocyte (CTL) epitopes presented by each HLA allele and the characterization of their CTL responses are important for the study of pathogenesis of AIDS and the development of a vaccine against it. In the present study, we focused on identification and characterization of HIV-1 epitopes presented by HLA-B*5401, which is frequently found in the Asian population, because these epitopes have not yet been reported. We identified these epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef. Seven of these 17-mer peptides induced HLA-B*5401-restricted CD8+ T cell responses. Only five HLA-B*5401-restricted Pol- or Nef-specific CD8+ T cell responses were detected in the analysis using 11-mer overlapping peptides. Three Pol and two Nef optimal peptides were identified by further analysis using truncated peptides. These epitope-specific CTLs effectively killed HLA-B*5401-expressing target cells infected with HIV-1 recombinant vaccinia virus, indicating that these peptides were naturally processed by HLA-B*5401 in HIV-1-infected cells. These epitope-specific CD8+ T cells were elicited in more than 25% of chronically HIV-1-infected individuals carrying HLA-B*5401. Therefore, these epitopes should prove useful for studying the pathogenesis of AIDS in Asia and developing a vaccine against HIV-1.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.