Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.     

In vivo hierarchy of immunodominant and subdominant HLA-A*0201-restricted T-cell epitopes of HBx antigen of hepatitis B virus

A polyepitopic CD8+ T-cell response is critical for the control of hepatitis B virus (HBV) infection. The HBV X protein (HBx) is a multifunctional protein that is important for the viral life cycle and for host-virus interactions. The aim of this study was to analyze the immunogenicity and dominance of various HLA-A*0201-restricted HBx-derived epitopes. For this purpose, we immunized HLA-A*0201-transgenic mice with HBx-derived peptides and DNA. This is a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, as these mice possess a cytotoxic T lymphocyte (CTL) repertoire representative of HLA-A2.1 individuals. We used cytotoxic tests and enzyme-linked immunosorbent spot (ELISPOT) assays to study the induction of specific cytotoxic and interferon (IFN)-gamma-secreting T cells. This allowed us to classify the HBx epitopes according to their T-cell activation capacity. After endogenous processing of the antigen synthesized in vivo after DNA-based immunization, we found that the HBx-specific T-cell response is targeted against one immunodominant epitope. Furthermore, following peptide immunization, we identified six additional novel subdominant T-cell epitopes. Inclusion of well-characterized epitopic sequences of HBx in a new vaccine for chronic HBV infections could help to broaden the T-cell response.     

Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy

The cytotoxic T-cell response in chronic hepatitis B virus (HBV) infection has been described as weak and mono- or oligospecific in comparison to the more robust virus-specific T-cell response present in resolved infection. However, chronic hepatitis B is a heterogeneous disease with markedly variable levels of virus replication and liver disease activity. Here we analyzed (both directly ex vivo and after in vitro stimulation) the HBV-specific CD8 T-cell responses against structural and nonstructural HBV proteins longitudinally in patients with different patterns of chronic infections. We found that the profiles of virus-specific CD8(+)-T-cell responses during chronic infections are highly heterogeneous and influenced more by the level of HBV replication than by the activity of liver disease. An HBV DNA load of <10(7) copies/ml appears to be the threshold below which circulating multispecific HBV-specific CD8(+) T cells are consistently detected. Furthermore, CD8(+) T cells with different specificities are differentially regulated during chronic infections. HBV core-specific CD8(+) T cells are associated with viral control, while CD8(+) T cells specific for envelope and polymerase epitopes can occasionally be found in the setting of high levels (>10(7) copies) of HBV replication. These findings have implications for the design of immunotherapy for chronic HBV infections.     

Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T-cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection.

Overcoming hepatitis B virus infection essentially depends on the appropriate immune response of the infected host. Among the hepatitis B virus antigens, the core (HBcAg) and e (HBeAg) proteins appear highly immunogenic and induce important lymphocyte effector functions. In order to investigate the importance of HBcAg/HBeAg-specific T lymphocytes in patients with acute and chronic hepatitis B and to identify immunodominant epitopes within the HBcAg/HBeAg, CD4+ T-cell responses to hepatitis B virus-encoded HBcAg and HBcAg/HBeAg-derived peptides were studied in 49 patients with acute and 39 patients with chronic hepatitis B. The results show a frequent antigen-specific CD4+ T-cell activation during acute hepatitis B infection, a rare HBcAg/HBeAg-specific CD4+ T-cell response among HBeAg+ chronic carriers, and no response in patients with anti-HBe+ chronic hepatitis. An increasing CD4+ T-cell response to HBcAg/HBeAg coincides with loss of HBeAg and hepatitis B virus surface antigen (HBsAg). Functional analysis of peptide-specific CD4+ T-cell clones revealed a heterogeneous population with respect to lymphokine production. Epitope mapping within the HBcAg/HBeAg peptide defined amino acids (aa) 1 to 25 and aa 61 to 85, irrespective of the HLA haplotype, as the predominant CD4+ T-cell recognition sites. Other important sequences could be identified in the amino-terminal part of the protein, aa 21 to 45, aa 41 to 65, and aa 81 to 105. The immunodominant epitopes are expressed in both proteins, HBcAg and HBeAg. Our findings lead to the conclusion that activation of CD4+ T lymphocytes by HBcAg/HBeAg is a prerequisite for viral elimination, and further studies have to focus on the question of how to enhance or induce this type of T-cell response in chronic carriers. The immunodominant viral sequences identified may have relevance to synthetic vaccine design and to the use of peptide T-cell sites as immunotherapeutic agents in chronic infection.     

Multiepitopic HLA-A*0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization

CTL together with anti-envelope Abs represent major effectors for viral clearance during hepatitis B virus (HBV) infection. The induction of strong cytotoxic and Ab responses against the envelope proteins after DNA-based immunization has been proposed as a promising therapeutic approach to mediate viral clearance in chronically infected patients. Here, we studied the CTL responses against previously described hepatitis B surface Ag (HBsAg)-HLA-A*0201-restricted epitopes after DNA-based immunization in HLA-A*0201 transgenic mice. The animal model used was Human Human D(b) (HHD) mice, which are deficient for mouse MHC class I molecules (beta(2)-microglobulin(-/-) D(b-/-)) and transgenic for a chimeric HLA-A*0201/D(b) molecule covalently bound to the human beta(2)-microglobulin (HHD(+/+)). Immunization of these mice with a DNA vector encoding the small and the middle HBV envelope proteins carrying HBsAg induced CTL responses against several epitopes in each animal. This study performed on a large number of animals described dominant epitopes with specific CTL induced in all animals and others with a weaker frequency of recognition. These results confirmed the relevance of the HHD transgenic mouse model in the assessment of vaccine constructs for human use. Moreover, genetic immunization of HLA-A2 transgenic mice generates IFN-gamma-secreting CD8(+) T lymphocytes specific for endogenously processed peptides and with recognition specificities similar to those described during self-limited infection in humans. This suggests that responses induced by DNA immunization could have the same immune potential as those developing during natural HBV infection in human patients.     

Immune and viral profile from tolerance to hepatitis B surface antigen clearance: a longitudinal study of vertically hepatitis B virus-infected children on combined therapy

The aim of the study was to investigate longitudinally hepatitis B virus (HBV)-specific T-cell reactivity and viral behavior versus treatment response in tolerant children during combined antiviral therapy. Twenty-three children with infancy-acquired hepatitis B (HBeAg(+)) belonging to a published pilot study of 1-year treatment with lamivudine/alpha interferon (IFN-α) were investigated. Five seroconverted to anti-HBs (responders). Nine were HLA-A2(+) (4 responders and 5 nonresponders). Mutations within the HBV core gene were determined at baseline in liver and in serial serum samples by direct sequencing at baseline; during treatment week 2 (TW2), TW9, TW28, and TW52; and after follow-up week 24 (FUW24) and FUW52. HBV-specific reactivity was evaluated by T-cell proliferation with 16 HBV core 20-mer overlapping peptides and by HLA-A2-restricted core(18-27) pentamer staining and CD8(+) IFN-γ enzyme-linked immunospot (ELISPOT) assay. HBV core-specific T-cell proliferative and CD8 responses were more vigorous and broader among responders than among nonresponders at TW28 and TW52, while the number of mutations within HBV core gene immunodominant epitopes was lower at TW28 and was negatively associated with HBV-specific T-cell proliferative responses at both time points. The HBV DNA viral load was negatively associated with HBV-specific T-cell proliferative and CD8 responses during treatment, especially at TW28. Treatment-induced transition from immunotolerance to HBV immune control is characterized by the emergence of efficient virus-specific immune responses capable of restraining mutations and preventing viral evasion.     

Patterns of CD8+ immunodominance may influence the ability of Mamu-B*08-positive macaques to naturally control simian immunodeficiency virus SIVmac239 replication

Certain major histocompatibility complex (MHC) class I alleles are strongly associated with control of human immunodeficiency virus and simian immunodeficiency virus (SIV). CD8(+) T cells specific for epitopes restricted by these molecules may be particularly effective. Understanding how CD8(+) T cells contribute to control of viral replication should yield important insights for vaccine design. We have recently identified an Indian rhesus macaque MHC class I allele, Mamu-B*08, associated with elite control and low plasma viremia after infection with the pathogenic isolate SIVmac239. Here, we infected four Mamu-B*08-positive macaques with SIVmac239 to investigate why some of these macaques control viral replication. Three of the four macaques controlled SIVmac239 replication with plasma virus concentrations below 20,000 viral RNA copies/ml at 20 weeks postinfection; two of four macaques were elite controllers (ECs). Interestingly, two of the four macaques preserved their CD4(+) memory T lymphocytes during peak viremia, and all four recovered their CD4(+) memory T lymphocytes in the chronic phase of infection. Mamu-B*08-restricted CD8(+) T-cell responses dominated the acute phase and accounted for 23.3% to 59.6% of the total SIV-specific immune responses. Additionally, the ECs mounted strong and broad CD8(+) T-cell responses against several epitopes in Vif and Nef. Mamu-B*08-specific CD8(+) T cells accounted for the majority of mutations in the virus at 18 weeks postinfection. Interestingly, patterns of viral variation in Nef differed between the ECs and the other two macaques. Natural containment of AIDS virus replication in Mamu-B*08-positive macaques may, therefore, be related to a combination of immunodominance and viral escape from CD8(+) T-cell responses.     

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.     

The differential ability of HLA B*5701+ long-term nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences

Although the HLA B(*)5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57(+) progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restricted gag epitopes by CD8(+) T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B(*)57(+) patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions of gag. Furthermore, CD8(+) T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4(+) T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8(+) T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restricted gag epitopes and are not the primary mechanism of virus evasion from immune control in B(*)5701(+) HIV-infected patients. Qualitative features of the virus-specific CD8(+) T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B(*)5701(+) LTNPs and progressors to restrict virus replication.     

Identification of novel avian influenza virus derived CD8+ T-cell epitopes

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

HLA-A2-restricted T-cell epitopes specific for prostatic acid phosphatase

Prostatic acid phosphatase (PAP) has been investigated as the target of several antigen-specific anti-prostate tumor vaccines. The goal of antigen-specific active immunotherapies targeting PAP would ideally be to elicit PAP-specific CD8+ effector T cells. The identification of PAP-specific CD8+ T-cell epitopes should provide a means of monitoring the immunological efficacy of vaccines targeting PAP, and these epitopes might themselves be developed as vaccine antigens. In the current report, we hypothesized that PAP-specific epitopes might be identified by direct identification of pre-existing CD8+ T cells specific for HLA-A2-restricted peptides derived from PAP in the blood of HLA-A2-expressing individuals. 11 nonamer peptides derived from the amino acid sequence of PAP were used as stimulator antigens in functional ELISPOT assays with peripheral blood mononuclear cells from 20 HLA-A2+ patients with prostate cancer or ten healthy blood donors. Peptide-specific T cells were frequently identified in both groups for three of the peptides, p18–26, p112–120, and p135–143. CD8+ T-cell clones specific for three peptides, p18–26, p112–120, and p299–307, confirmed that these are HLA-A2-restricted T-cell epitopes. Moreover, HLA-A2 transgenic mice immunized with a DNA vaccine encoding PAP developed epitope-specific responses for one or more of these three peptide epitopes. We propose that this method to first identify epitopes for which there are pre-existing epitope-specific T cells could be used to prioritize MHC class I-specific epitopes for other antigens. In addition, we propose that the epitopes identified here could be used to monitor immune responses in HLA-A2+ patients receiving vaccines targeting PAP to identify potentially therapeutic immune responses.     

Cellular immune responses associated with occult hepatitis C virus infection of the liver

Occult hepatitis C virus (HCV) infection is a type of recently identified chronic infection that is evidenced only by detection of HCV RNA in liver; patients consistently test negative for antibodies to HCV and HCV RNA in serum. Using ex vivo and in vitro measures of T-cell responses, we have identified functional virus-specific memory CD4(+) and CD8(+) T cells in the peripheral blood of patients with occult HCV infection. The features of the virus-specific T cells were consistent with immune surveillance functions, supporting previous exposure to HCV. In addition, the magnitudes of CD4(+) and CD8(+) T-cell responses were in parallel and correlated inversely with the extent of liver HCV infection. The detection of HCV-specific T cells in individuals in whom HCV RNA can persist in the liver despite the absence of viremia and antibodies indicates that HCV replication is prolonged in the face of virus-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that HCV-specific cellular immune responses are markers not only of previous exposure to and recovery from HCV but also of ongoing occult HCV infection.     

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.     

HBcAg-specific CD4+CD25+regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are accompanied by increased T cell responses to hepatitis B core and e antigens (HBcAg/HBeAg). Why patients are immunotolerant (IT) to the virus and why AEs occur spontaneously on the immunoactive phase remain unclear. The role of HBcAg-specific CD4(+)CD25(+) regulatory T (T(reg)) cells in AE and IT phases was investigated in this study. The SYFPEITHI scoring system was employed to predict MHC class II-restricted epitope peptides on HBcAg overlapping with HBeAg that were used for T(reg)-cell cloning and for the construction of MHC class II tetramers to measure T(reg) cell frequencies (T(reg) f). The results showed that HBcAg-specific T(reg) f declined during AE accompanied by increased HBcAg peptide-specific cytotoxic T lymphocyte frequencies. Predominant Foxp3-expressing T(reg) cell clones were generated from patients on the immune tolerance phase, while the majority of Th1 clones were obtained from patients on the immunoactive phase. T(reg) cells from liver and peripheral blood of CH-B patients express CD152 and PD1 antigens that exhibit suppression on PBMCs proliferation to HBcAg. These data suggest that HBcAg peptide-specific T(reg) cells modulate the IT phase, and that their decline may account for the spontaneous AEs on the natural history of chronic hepatitis B virus infection.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Selection of T-cell epitopes from foot-and-mouth disease virus reflects the binding affinity to different cattle MHC class II molecules

The major histocompatibility complex (MHC)-restricted selection of T-cell epitopes of foot-and-mouth disease virus (FMDV) by individual cattle MHC class II DR (BoLA-DR) molecules was studied in a direct MHC-peptide binding assay. By in vitro priming of T lymphocytes derived from animals homozygous for both MHC class I and II, five T-cell epitopes were analyzed in the context of three MHC class II haplotypes. We found that the presentation of these T-cell epitopes was mediated by DR molecules, since blocking this pathway of antigen presentation using monoclonal antibody TH14B completely abolished the proliferative responses against the peptides. To study the DR-restricted presentation of these T-cell epitopes, a direct MHC-peptide binding assay on isolated cattle DR molecules was developed. Purified cattle MHC class II DR molecules of the BoLA-DRB3*0201, BoLA-DRB3*1101, and BoLA-DRB3*1201 alleles were isolated from peripheral blood mononuclear cells. For each allele, one of the identified T-cell epitopes was biotinylated, and used as a marker peptide for the development of a competitive MHC-peptide binding assay. Subsequently, the T-cell epitopes of FMDV with functionally defined MHC class II specificity were analyzed in this binding assay. The affinity of the epitopes to bind to certain DR molecules was significantly correlated to the capacity to induce T-cell proliferation. This demonstrated at the molecular level that the selection of individual T-cell epitopes found at the functional level was indeed the result of MHC restriction.     

Cellular immune responses to the hepatitis B virus polymerase

CD4 T cells play an important role in hepatitis B virus (HBV) infection by secretion of Th1 cytokines that down-regulate HBV replication, and by promoting CD8 T cell and B cell responses. We have identified and characterized 10 CD4 T cell epitopes within polymerase and used them to analyze the immunological effects of long-term antiviral therapy as compared with spontaneous recovery from HBV infection. Candidate epitopes were tested for binding to 14 HLA-DR molecules and in IFN-gamma ELISPOT and cytotoxicity assays using peripheral blood lymphocytes from 66 HBV-infected patients and 16 uninfected controls. All 10 epitopes bound with high affinity to the most prevalent HLA-DR Ags, were conserved among HBV genomes, and induced IFN-gamma responses from HBV-specific CD4+ T cells. Several epitopes contained nested MHC class I motifs and stimulated HBV-specific IFN-gamma production and cytotoxicity of CD8+ T cells. HBV polymerase-specific responses were more frequent during acute, self-limited hepatitis and after recovery (12 of 18; 67%) than during chronic hepatitis (16 of 48 (33%); p=0.02). Antiviral therapy of chronic patients restored HBV polymerase and core-specific T cell responses during the first year of treatment, but thereafter, responses decreased and, after 3 years, were no more frequent than in untreated patients. Decreased T cell responsiveness during prolonged therapy was associated with increased prevalence of lamivudine-resistant HBV mutants and increased HBV titers. The data provide a rationale for the combination of antiviral and immunostimulatory therapy. These newly described HBV polymerase epitopes could be a valuable component of a therapeutic vaccine for a large and ethnically diverse patient population.     

Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity.

CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8(+) T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8(+) CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.     

The CD8 T-cell response against murine gammaherpesvirus 68 is directed toward a broad repertoire of epitopes from both early and late antigens

Infection of mice with murine gammaherpesvirus 68 (MHV-68) robustly activates CD8 T cells, but only six class I major histocompatibility complex (MHC)-restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the cytotoxic T-lymphocyte response in MHV-68-infected C57BL/6 mice, we screened for H-2K(b)- and H-2D(b)-restricted epitopes using a set of 384 candidate epitopes in an MHC tetramer-based approach and identified 19 new epitopes in 16 different open reading frames. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T-cell-specific responses for the remaining three. The peak of the CD8 T-cell response to most peptides occurs between 6 and 10 days postinfection. The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62L(lo) and CD44(hi)) and produce gamma interferon upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T-cell repertoire and more-rapid activation than those previously recorded.