JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen

Ran is considered to be a promising target for tumor-specific immunotherapy because its protein is exclusively expressed in tumor tissues, though its mRNA can be expressed in most normal tissues. In our study, we obtained four candidate wild-type epitopes designated Ran1, Ran2, Ran3, and Ran4, derived from the Ran antigen with the highest predicted affinity with MHC-I, indicated by affinity prediction plots and molecular dynamics simulation. However, in vitro affinity assays of these epitopes showed only a moderate affinity with MHC-I. Thus, we designed altered peptide ligands (APLs) derived from Ran wild-type epitopes with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. Of the eight tested peptides, the 1Y analog had the strongest binding-affinity and lowest-dissociation rate to HLA-A*0201. Additionally, we investigated the CTLs activities induced by Ran wild-type peptides and the APLs in human PBMCs and in HLA-A*0201/K^b transgenic mice. Ran1 1Y was superior to other APLs and wild-type peptides in eliciting epitope-specific CTL immune responses both in vitro and in vivo. In summary, a wild-type epitope of the tumor-specific antigen Ran, expressed broadly in many tumors, was identified and designated Ran1. An APL of Ran1, Ran1 1Y, was further designed and verified in vitro and in vivo and found to elicit a stronger Ran-specific CTL response, indicating a potential anti-tumor application in the future.     

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.     

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope,M1.2

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (10⁹) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2⁺ MUC1⁺ MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were “consensus” agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-α, IFN-γ, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.     

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.     

Association of prolonged survival in HLA-A2+ progressive multifocal leukoencephalopathy patients with a CTL response specific for a commonly recognized JC virus epitope.

The role of JC virus (JCV)-specific CTL was explored in the immunopathogenesis of progressive multifocal leukoencephalopathy (PML). We identified a 9-aa epitope of the JCV capsid protein VP1, the VP1(p100) peptide ILMWEAVTL, which is recognized by CTL of HLA-A2+ HIV+/PML survivors. We then constructed an HLA-A*0201/VP1(p100) tetrameric complex that allowed us to assess by flow cytometry the PBMC of 13 PML patients and 11 control subjects for the presence of JCV-specific CTL. VP1(p100)-specific CTL were detected by tetramer binding in VP1(p100)-stimulated PBMC of five of seven (71%) PML survivors and zero of six PML progressors (p = 0.02). Two of three HIV+ patients with a leukoencephalopathy resembling PML, but with no virologic evidence of JCV infection, also had detectable VP1(p100)-specific CTL in their PBMC. PBMC of eight HIV+ patients with other neurologic diseases and healthy control subjects had no detectable JCV-specific CTL. These data suggest that the JCV-specific cellular immune response may be important in the containment of PML, and the tetramer-staining assay may provide a useful prognostic tool in the clinical management of these patients.     

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.     

Extension of HLA-A*0201-restricted minimal epitope by N epsilon-palmitoyl-lysine increases the life span of functional presentation to cytotoxic T cells

The delineation of the minimal requirements for efficient delivery of functional cytotoxic epitopes into APC could be a step toward the definition of "minimal length" lipopeptides for the modulation of CTL activity. Several analogues of the HLA-A*0201-restricted HIV-1 polymerase (pol476-484) minimal cytotoxic epitope were obtained by modifying P0, P1, or P10 positions by a single N epsilon-palmitoyl-lysine residue. The use of fluorescent derivatives confirmed the cell-permeating activities and suggested that a P0- and a P1-modified lipopeptide possessing ionizable extremities fulfills the structural requirements for MHC loading. The expressions of HLA-peptide complexes at the surface of TAP-deficient cells incubated with the parent epitope or lipopeptide derivatives were compared, in terms of intensity and stability. Both lipopeptides induced a considerably prolonged expression of conformationally correct complexes, which were dependent on the integrity of the exocytosis pathway, suggesting a dynamic mechanism of formation or reloading of the complexes from an intracellular pool. The agonistic activities of the different HLA-peptide complexes were evaluated using two independent T cell lines from HIV-infected donors. We report that a lipodecapeptide obtained by N-terminal addition of a N epsilon-palmitoyl-lysine to the pol476-484 epitope was able to increase the life span of functional presentation to cytotoxic T cells specific for the parent peptide.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

HLA-A*2402-restricted HIV-1-specific cytotoxic T lymphocytes and escape mutation after ART with structured treatment interruptions

Although a limited duration of immune activation of structured treatment interruptions (STIs) has been reported, the immune escape mechanism during STIs remains obscure. We therefore investigated the role of three immunodominant cytotoxic T lymphocyte (epitopes) in 12 HLA-A*2402-positive patients participating longitudinally during the clinical study of early antiretroviral treatment (ART) with five series of structured treatment interruptions (STIs). The frequency of HLA-A*2402-restricted CTLs varied widely and a sustained CTL response was rarely noted. However, a Y-to-F substitution at the second position in an immunodominant CTL epitope Nef138-10 (Nef138-2F), which was previously demonstrated as escape mutation, was frequently detected in seven patients primarily and emerged in the remaining five patients thereafter, and the existence of escape mutations was correlated with high pVL levels early in the clinical course. These findings suggest that escape mutation in the immunodominant CTL epitope may be one of the mechanisms to limit HIV-1-specific immune control in STIs.     

Vigorous but short-term gamma interferon T-cell responses against a dominant HLA-A*02-restricted measles virus epitope in patients with measles

The absolute and relative abundance of major histocompatibility complex class I-presented viral epitopes is important in the induction and maintenance of antiviral cytotoxic-T-lymphocyte (CTL) responses. We demonstrate that the supra-abundant HLA-A*0201-restricted peptide KLWESPQEI of the measles virus nonstructural C protein induces strong gamma interferon CD8(+)-T-cell responses in children with acute measles. However, longitudinal analysis indicates that these responses are only short-lived. Thus, some viral epitopes that can be immunodominant during primary infection may fail to establish memory CTL responses.     

Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a StepRank scheme

Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well.     

Modulation of HLA-A*0201-restricted T cell responses by natural polymorphism in the IE1(315-324) epitope of human cytomegalovirus

Cytotoxic T lymphocytes play a central role in the control of persistent human CMV (HCMV) infection and reactivation. In healthy virus carriers, the specific CD8(+) CTL response is almost entirely directed against the virion tegument protein pp65 and/or the 72-kDa major immediate early protein, IE1. Studies that included a large panel of HCMV(+) donors suggested that immunorelevance of pp65 and IE1 was directly related with individual HLA haplotype difference. Nevertheless, there are no data on the incidence of HCMV natural polymorphism on virus-specific CTL responses. To assess the impact of IE1 polymorphism on CTL response, we have sequenced in 103 clinical isolates the DNA region corresponding to IE1(315-324), an immunodominant epitope presented by HLA-A*0201 molecules. Seven peptidic variants were found with extensive difference in their frequencies. The response of four HLA-A*0201-restricted anti-IE1 T lymphocyte clones, which were previously generated from one donor against autologous B lymphoblastoid cells expressing a recombinant clinical variant of IE1, was then evaluated using target cells loaded with mutant synthetic peptides or expressing rIE1 variants. One of four clones, which have been sorted 19 times among 22 clones targeted against IE1(315-324), recognized six of the seven tested variant epitopes. All three other clones showed distinct reactivity patterns to target cells loaded with the different mutant peptides or expressing IE1 variants. Therefore, in the HLA-A2 context, clonal expansions of anti-IE1 memory CTLs may confer a protection against HCMV successive infections and reactivations by killing cells presenting most of the naturally occurring IE1(315-324) epitope variants.     

Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.

The Ag specificity of the CTL response against CMV is directed almost entirely to a single CMV tegument protein, the phosphoprotein pp65. We report the identification of three peptides derived from the protein pp65 that displayed a high or intermediate binding to HLA-A*0201 molecules, which were also able to induce an in vitro CTL response in peripheral blood lymphocytes from CMV seropositive individuals. The peptide-specific CTLs generated were capable of recognizing the naturally processed pp65 either presented by CMV-infected cells or by cells infected with an adenovirus construct expressing pp65 in an HLA-A*0201-restricted manner. Thus, we were able to demonstrate responses to subdominant CTL epitopes in CMV-pp65 that were not detected in polyclonal cultures obtained by conventional stimulations. We also found that the amino acid sequences of the three peptides identified as HLA-A*0201-restricted CTL epitopes were conserved among different wild-type strains of CMV obtained from renal transplant patients, an AIDS patient, and a congenitally infected infant, as well as three laboratory strains of the virus (AD169, Towne and Davis). These observations suggest that these pp65 CTL peptide epitopes could potentially be used as synthetic peptide vaccines or for other therapeutic strategies aimed at HLA-A*0201-positive individuals, who represent approximately 40% of the European Caucasoid population. However, strain variation must be taken in consideration when the search for CTL epitopes is extended to other HLA class I alleles, because these mutations may span potential CTL epitopes for other HLA molecules, as it is described in this study.     

In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.     

Adjuvant activities of novel cytokines, interleukin-23 (IL-23) and IL-27, for induction of hepatitis C virus-specific cytotoxic T lymphocytes in HLA-A*0201 transgenic mice

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-gamma)-producing, HCV-specific CD8+ cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-gamma-producing, HCV-specific CD8+ cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.     

New epitope peptides derived from hepatitis C virus (HCV) 2a which have the capacity to induce cytotoxic T lymphocytes in HLA-A2+ HCV-infected patients

Because cytotoxic T lymphocytes (CTLs) play an important role in the specific immunotherapy of hepatitis C virus (HCV) infection, a series of CTL epitopes has been defined from HCV genotype 1a or 1b protein. Here, we attempted to identify HCV2a-derived epitopes that are capable of inducing HLA-A2-restricted and peptide-specific CTLs. Peripheral blood mononuclear cells (PBMCs) of HLA-A2+ HCV2ainfected patients or healthy donors were stimulated in vitro with each of the HCV2a-derived peptides, which were prepared based on the HLA-A2-binding motif, and their peptide-specific and HLA-A2-restricted cytotoxicities were examined. The HCV2a 432-441, HCV2a 716-724, and HCV2a 2251-2260 peptides were found to efficiently induce peptide-specific CTLs from the PBMCs of HLA-A2+ HCV2ainfected patients. Cytotoxicity was mainly mediated by CD8+ T cells in a HLA class I-restricted manner. These results indicate that the HCV2a 432-441, HCV2a 716-724, and HCV2a 2251-2260 peptides might be applicable for peptide-based immunotherapy of HLA-A2+ HCV2a-infected patients.     

The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr59-67) that is targeted by acute-phase HIV-1-specific CD8+ T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I60L variant all developed these responses. The I60L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.