Recognition of Core- and Polymerase-derived immunogenic peptides included in novel therapeutic vaccine by T cells from Chinese chronic hepatitis B patients.

Chronic hepatitis B (CHB) is one of the major public health challenges in the world. Due to a strong interplay between specific T-cell immunity and elimination of hepatitis B virus (HBV), efforts to develop novel immunotherapeutics are gaining attention. TG1050, a novel immunotherapy, has shown efficacy in an animal study. To support the clinical development of TG1050 in China, specific immunity to the fusion antigens of TG1050 was assessed in Chinese patients. One hundred and thirty subjects were divided into three groups as CHB patients, HBV spontaneous resolvers, and CHB patients with HBsAg loss after antiviral treatment. HBV-specific T-cell responses to pools of HBV Core or Polymerase genotype D peptides included in TG1050 were evaluated. HBV Core- or Polymerase-specific cells were detected in peripheral blood mononuclear cells (PBMCs) from the different cohorts. The frequencies and intensities of HBV Core-specific immune responses were significantly lower in CHB patients than in HBsAg loss subjects. In CHB patients, a dominant pool derived from Polymerase (Pol1) was the most immunogenic. CHB patients with low viral loads (<106 IU/mL) were more likely to have a positive response specific to the Core peptide pool. Overall, genotype D-derived peptides included in TG1050 could raise broad and functional T-cell responses in PBMCs from Chinese CHB patients infected with genotype B/C isolates. Core-specific immunogenic domains appeared as "hot spots" with the capacity to differentiate between CHB vs HBsAg loss subjects. These observations support the extended application and associated immune monitoring of TG1050 in China.

T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates.

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.

Vaccines and immunotherapies against hepatitis B and hepatitis C viruses.

Both hepatitis B and hepatitis C viruses (HBV and HCV) cause chronic infections worldwide that are associated with development of liver diseases ranging from mild liver inflammation to hepatocellular carcinomas. While efficient preventive vaccines are available for HBV, efforts are ongoing to develop one in case of HCV. Yet, both infections share the fact that therapeutic agents available to treat already established infections are yet poorly efficient, toxic or associated with development of resistance. Thus, novel immune-based therapies are actively being developed to complement or replace standard antiviral treatments. Among those, development of therapeutic vaccines represents a major effort. Peptide-, recombinant protein- or viral vector-based vaccines have been engineered and tested at preclinical and clinical levels. Means to adjuvant these vaccines are being pursued, including approaches based on combining vaccines of different nature. This review will outline major advances in the field of both HBV and HCV therapeutic vaccine development with a particular focus on candidates presented at the 12th International Symposium on Viral Hepatitis and Liver Disease (July 2006, Paris, France).

An accelerated vaccine schedule with a poly-antigenic hepatitis C virus MVA-based candidate vaccine induces potent, long lasting and in vivo cross-reactive T cell responses.

We designed and evaluated in HLA-class I transgenic mouse models a hepatitis C virus (HCV) T cell-based MVA vectored vaccine expressing three viral antigens known to be targets of potent CD8+- and CD4+-mediated responses. An accelerated (3 week-based) vaccination induced specific CD8+ T cells harboring two effector functions (cytolytic activity - both in vitro and in vivo- and production of IFNgamma) as well as specific CD4+ T cells recognizing all three vaccine antigens. Responses were long lasting (6 months), boostable by a fourth MVA vaccination and in vivo cross-reactive as demonstrated in a surrogate Listeria-based challenge assay. This candidate vaccine has now moved into clinical trials.

Primary and memory T cell responses induced by hepatitis C virus multiepitope long peptides.

To develop a vaccine against hepatitis C virus, we synthesized four long peptides from nonstructural proteins NS3, NS4 and NS5B containing HLA-class I and class II epitopes mainly inducing responses in natural infection. In HLA-A2.1 transgenic mice, the four peptides primed higher CTL responses to 6:7 minimal HLA-A2 epitopes than those induced by the minimal epitopes. HLA-A2.1/HLA-DR1 transgenic mice immunized with one peptide, containing a class II epitope implicated in viral resolution, developed IFNgamma-producing CD4+-T and CD8+-T cells. These peptides recalled HCV-specific IFNgamma-producing cells from HCV-infected patients' PBMC. This support the selection of these domains for inclusion in a vaccine formulation.

Genetic immunization and comprehensive screening approaches in HLA-A2 transgenic mice lead to the identification of three novel epitopes in hepatitis C virus NS3 antigen.

Interferon-gamma (IFN gamma)-producing CD8+ T cells have been shown to play a key role in the control or eradication of hepatitis C virus (HCV) infections. In particular, T cells specific of the non-structural protein 3 (NS3) are often associated with control of viremia. The aim of the study was to identify novel HLA-A2 restricted CD8+ T cell epitopes specific of NS3 using a combination of comprehensive approaches. HLA-A2.1 transgenic mice were immunized with a DNA vaccine optimized for NS3 specific epitope presentation and induced CD8+ T cell reactivity was screened using 42 algorithm-predicted peptides as well as a library of 78 overlapping 15-mer peptides spanning the whole protein. Three epitopes mapping within the NS3 protease (GLL: aa 1038-1047) or helicase (ATL: aa 1260-1268 and TLH: aa 1617-1625) were identified. These epitopes, which display similar and high in vitro binding capacities to soluble HLA-A2 molecules, are able to induce either cytotoxic T lymphocytes (CTL) and/or IFN gamma-producing T cells. Comparative in vitro target cell sensitization studies revealed a higher immunogenicity of the GLL peptide as compared with both ATL and TLH peptides. This peptide was capable to recall in vitro HCV-specific IFN gamma and IL-10-producing T cells from peripheral blood mononuclear cells (PBMC) of chronically infected patients. These data increase the pool of NS3-specific CD8+ T cell epitopes available to analyze HCV associated immunity and could contribute to the design and evaluation of candidate vaccines.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles.

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2.6x10(5) to 1:1x10(6)). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.

Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA-expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope.

The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.10(5) cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope.

Natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) is not impaired in patients suffering from chronic hepatitis C.

BACKGROUND/AIMS: Recent observations suggest that natural killer (NK) cell activity might be impaired in chronic hepatitis C. However, to date antibody-dependent cellular cytotoxicity (ADCC) has not been studied in chronic hepatitis C in detail. METHODS: Therefore, we investigated spontaneous and cytokine-induced (interleukin-2 and interferon-gamma) natural cytotoxicity and ADCC in 29 patients suffering from chronic hepatitis C and 19 healthy controls. Cytotoxicity was determined with a flow-cytometric assay, which can also assess monocyte cytotoxicity. As target cells we used the colorectal tumor cell line HT29 and the lymphoma cell line Raji. RESULTS: We found no significant differences with respect to spontaneous cytotoxicity (HCV versus healthy controls (32 vs. 46%) and 17-1A specific ADCC (59 vs. 48%), even if isolated monocytes or NK cells were studied. Preincubation and stimulation of effector cells with cytokines increased both natural cytotoxicity and ADCC by 20-30%. However, natural cytotoxicity and ADCC after stimulation did not differ between the two groups. CONCLUSIONS: Our data obtained with a long-term cytotoxicity assay do not reveal impaired cytolytic capacity of the innate immune system in chronic hepatitis C, even when isolated monocytes and NK cells were studied as effector cells.

Induction of hepatitis C virus E1 envelope protein-specific immune response can be enhanced by mutation of N-glycosylation sites.

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.

[Dendritic cells and hepatitis C virus]. / Cellules dendritiques et virus de l'hépatite C.

Hepatitis C virus (HCV) induces chronic persistent infection that can lead to the development of hepatocellular carcinoma. We have searched for the presence of HCV genomic RNA in cells from hematopoietic origin and have, among others, documented such sequences in B cells as well as dendritic cells (DC) derived from monocytes. The allostimulatory capacity of these latter cells was found altered in chronic patients while it appeared restored in long term responders to therapy.

DNA vaccines for hepatitis C virus.

Hepatitis C virus is an RNA encoded virus of the Flaviviridae family. In most cases, infections develop into a chronic carrier stage that can result in the onset of cirrhosis and hepatocellular carcinoma over a 20- to 30-year period. Because existing therapies are still of limited benefit and expensive, the development of a vaccine represents a priority to prevent further spreading of the infection. Immune correlates of protection remain poorly defined although increasing evidence suggests that both humoral and cellular immune responses are likely to contribute to protection and/or neutralization of the virus. Current DNA-based vaccines, while capable of generating the latter, appear limited in their capacity to induce a strong and long-lasting antibody response.

Lipidation of T helper sequences from hepatitis C virus core significantly enhances T-cell activity in vitro.

Successful elimination of the hepatitis C virus (HCV) during acute infection has been linked to strong HCV-specific in vitro T-cell proliferation, whereas T cells from patients with chronic hepatitis C respond only weakly to HCV antigens. Lipid-coupled peptides are immunostimulants, which might provide a basis for novel therapeutic strategies against HCV. Therefore, in 20 patients with chronic hepatitis C, we studied whether tri-palmitoyl-S-cysteine-coupled peptides could modify in vitro T-cell proliferation (by [3H]thymidine uptake) in response to virus core and NS4. The lipopeptides corresponded to five immunodominant T helper epitopes of HCV core. Contrary to unmodified peptides, the lipopeptides specifically enhanced [3H]thymidine uptake in response to HCV antigens but not to a non-HCV related control antigen. They increased the frequency of responders (stimulation index, SI > or = 4) to core (13/20 versus 2/20; p = 0.0008) and NS4 (20/20 versus 7/20; p < 0.0001) among our patients with chronic hepatitis C. This immunostimulatory effect was dose-dependent, and was observed specifically with lipopeptides corresponding to the HCV epitopes. Our data demonstrate that the poor in vitro T-cell proliferation of patients with chronic hepatitis C can be improved when T cells are co-stimulated with HCV core-derived T helper lipopeptides, while the same peptides in unlipidated form had no effects. Thus, lipopeptides corresponding to HCV T-cell epitopes may offer novel immunomodulatory strategies against HCV.

Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.

BACKGROUND & AIMS: Dendritic cells (DC), which play an essential role in the triggering of primary antiviral immune reactions, may also contribute, in some viral models, to the propagation of viral infection and the pathogenesis of viral disease. During natural infection with hepatitis C virus (HCV), the interactions between the virus and DC may contribute to viral persistence, a general feature of HCV infection. METHODS: We compared the phenotypical and biological functions of monocyte-derived DC from patients with chronic hepatitis C (HCV-DC; n = 6), seronegative individuals (naive-DC; n = 8), long-term responders to antiviral therapy (LTR-DC; n = 8), and a group of patients with non-HCV-hepatic disorders (n = 11). The presence and the nature of HCV sequences during the DC cultures was assessed by reverse transcription-polymerase chain reaction and the analysis of the viral quasispecies distribution. RESULTS: Although HCV-DC displayed a normal morphology, phenotype, and capacity to capture antigen, their ability to stimulate the proliferation of allogeneic T cells was dramatically impaired in comparison with naive-DC (P = 0.0013). Mixing experiments revealed that HCV-DC did not affect the proliferation of T cells induced by naive-DC. Remarkably, the allostimulatory function of LTR-DC or DC from patients with non-HCV-hepatic disorders did not show any impairment. The presence of HCV genomic sequences could be documented for 5 of 6 HCV carriers either in the cells and/or the supernatants of the DC cultures. The presence of HCV sequences was found in the DC cultures from one patient showing a dramatic allostimulation defect. For that patient, extensive analysis of the viral quasispecies distribution revealed the presence, in the DC cultures, of genomic sequences of a unique nature, distinct from those identified in the patient's mononuclear cells, serum, or liver. CONCLUSIONS: Overall, these results indicate that chronic infection by HCV is associated with an allostimulatory defect of monocyte-derived DC, possibly because these cells constitute an extrahepatic reservoir for the virus. Although the exact mechanism responsible for such an alteration remains to be unraveled, our observations argue against an active immunosuppression-based mechanism.

[Role of neutralizing antibodies and cellular immunity in hepatitis C virus infection]. / Rôle des anticorps neutralisants et de l'immunité cellulaire dans l'infection à VHC.

Hepatitis C virus (HCV) is responsible for the development of a chronic carrier state that can lead to the induction of cirrhosis and liver cancer. These clinical manifestations are believed to be the direct consequence of the viral persistence and the incapacity of the host to develop vigorous and sustained immune responses. Still contradictory data suggest the existence of neutralizing antibodies specifically directed at the second glycoprotein (E2). These antibodies may nonetheless play only a minor role in the control of infection. In contrast, it is now generally recognized that cellular-mediated immune responses, CD4+ and CD8+ mediated, if in place early enough, of a vigorous and polyclonal nature as well as long-lasting, appear by themselves competent enough to control an infection. One of the mechanisms possibly responsible for the establishment and persistence of a chronic infection could be the alteration of the ability of antigen-presenting cells (dendritic cells) to stimulate a T cell response. Such alteration could be the result of a direct infection of these cells by HCV.

Flow cytometric analysis of peptide binding to major histocampatibility complex class I for hepatitis C virus core T-cell epitopes.

BACKGROUND/METHODS: To characterize the repertoire of T-cell epitopes on the hepatitis C virus (HCV) core protein, we studied major histocompatibility complex (MHC) class I binding of 75 decapeptides on 20 human B-cell lines and murine spleen cells using a flow cytometric assay. The results were compared with MHC class I stabilization on T2 cells, the SYFPEITHI algorithm, and known T-cell epitopes from the literature. RESULTS: Binding of peptides proved to be specific for MHC class I molecules. We observed peak fluorescence signals at positions amino acids (aa) 35-44, aa 87-96, aa 131-140, and aa 167-176 in virtually all HLA-A2-positive cell lines. These sites corresponded to T-cell epitopes predicted by SYFPEITHI and the positions of known T-cell epitopes, whereas T2 stabilization was at variance for two peptides. The assay was applied to HLA-A2-negative cells and murine spleen cells without further modification, and identified additional peptides, corresponding to known T-cell epitopes. CONCLUSIONS: Peptide binding to different MHC class I alleles can be mapped rapidly by a flow cytometric assay and enables a first orientation on the sites of possible T-cell epitopes. Application of this assay to HCV core suggests a rather limited repertoire of epitopes in the Caucasoid population.

Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens.

Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1-100 microgram) did not show an increased efficacy of the core-SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.

A genomic approach of the hepatitis C virus generates a protein interaction map.

The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.