A heterologous prime/boost vaccination strategy enhances the immunogenicity of therapeutic vaccines for hepatitis C virus.

BACKGROUND: We explored the concept of heterologous prime/boost vaccination using 2 therapeutic vaccines currently in clinical development aimed at treating chronically infected hepatitis C virus (HCV) patients: prime with a DNA-based vaccine expressing HCV genotype 1a NS3/4A proteins (ChronVac-C) and boost with a modified vaccinia virus Ankara vaccine expressing genotype 1b NS3/4/5B proteins (MVATG16643). METHODS: Two ChronVac-C immunizations 4 weeks apart were delivered intramuscularly in combination with in vivo electroporation and subsequently 5 or 12 weeks later boosted by 3 weekly subcutaneous injections of MVATG16643. Two mouse strains were used, and we evaluated quality, magnitude, and functionality of the T cells induced. RESULTS: DNA prime/MVA boost regimen induced significantly higher levels of interferon γ (IFN-γ) or interleukin 2 (IL-2) ELISpot responses compared with each vaccine alone, independent of the time of analysis and the time interval between vaccinations. Both CD8⁺ and CD4⁺ T-cell responses as well as the spectrum of epitopes recognized was improved. A significant increase in polyfunctional IFN-γ/tumor necrosis factor α (TNF-α)/CD107a⁺ CD8⁺ T cells was detected following ChronVac-C/MVATG16643 vaccination (from 3% to 25%), and prime/boost was the only regimen that activated quadrifunctional T cells (IFN-γ/TNF-α/CD107a/IL-2). In vivo functional protective capacity of DNA prime/MVA boost was demonstrated in a Listeria-NS3-1a challenge model. CONCLUSIONS: We provide a proof-of-concept that immunogenicity of 2 HCV therapeutic vaccines can be improved using their combination, which merits further clinical development.

MIT CAACB Risk Assessment Case Study: Assessing Virus Cross-Contamination Risk between Two Simultaneous Processes in an Open Biomanufacturing Facility.

Some members of MIT's Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) previously published content on the "Quality Risk Management in the Context of Viral Contamination", which described tools, procedures, and methodologies for assessing and managing the risk of a potential virus contamination in cell culture processes. To address the growing industry interest in moving manufacturing toward open ballrooms with functionally closed systems and to demonstrate how the ideas of risk management can be leveraged to perform a risk assessment, CAACB conducted a case study exercise of these new manufacturing modalities. In the case study exercise, a cross-functional team composed of personnel from many of CAACB's industry membership collaboratively assessed the risks of viral cross-contamination between a human and non-human host cell system in an open manufacturing facility. This open manufacturing facility had no walls to provide architectural separation of two processes occurring simultaneously, specifically a recombinant protein perfusion cell culture process using the human cell line, HEK-293 (Process 1) and a downstream postviral filtration unit operation (Process 2) of a recombinant protein produced in CHO cells. This viral risk assessment focused on cross-contamination of the Process 2 filtration unit operation after the Process 1 perfusion bioreactor was contaminated with a virus that went undetected. The workflow for quality risk management that is recommended by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) was followed, which included identifying and mapping the manufacturing process, defining the risk question, risk evaluation, and risk control. The case study includes a completed Failure Mode and Effects Analysis (FMEA) to provide descriptions of the specific risks and corresponding recommended risk reduction actions.

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein.

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.

Single lysophosphatidylcholine components exhibit adjuvant activities in vitro and in vivo.

Improving vaccine immunogenicity by developing new adjuvant formulations has long been a goal of vaccinologists. It has previously been shown that a natural mix of lysophosphatidylcholine (LPC) from chicken eggs promotes mature dendritic cell (DC) generation in vitro and primes antigen-specific immune responses in mice. In the present study, we dissected the adjuvant potentials of five individual LPC components found in the chicken egg mixture. In vitro analyses of the impact of the individual components on the maturation of human DCs were performed by means of phenotypic analysis, chemokine secretion analysis, and analysis of the ability of mature DC to stimulate T lymphocytes. Two components, C16:0-LPC and C18:0-LPC, were identified to be capable of the upregulation of expression of CD86, HLA-DR, and CD40 on in vitro-cultured monocyte-derived DCs from healthy donors. Both induced the release of chemokines to high concentrations (macrophage inflammatory protein 1, monocyte chemoattractant protein 1) or moderate concentrations (interleukin-8 [IL-8], gamma interferon-inducible protein 10). In addition, C16:0-LPC engaged naïve T cells to produce gamma interferon. This suggests that C16:0-LPC and C18:0-LPC have the capacity to promote, at least in vitro, a Th1-oriented response. The intravenous injection of C16:0-LPC or C18:0-LPC into mice resulted in the detectable secretion of IL-6 and IL-5 in sera. Both LPC components were tested for their capacities to act as adjuvants for two selected immunogens: the hepatitis B virus surface antigen and the hepatitis C virus NS3 helicase. The secretion of specific IgG1 was observed with either or both C16:0-LPC and C18:0-LPC, depending on the immunogen tested, and was observed at an efficiency comparable to that of alum. These data identify C16:0-LPC and C18:0-LPC as the active components of the LPC natural mixture. Although discrepancies between the results of the in vitro and in vivo analyses existed, studies with animals suggest that these components can trigger significant and specific humoral-mediated immunity.

A vector-based minigene vaccine approach results in strong induction of T-cell responses specific of hepatitis C virus.

Multiepitope-based vaccines against hepatitis C virus (HCV) were designed in the form of three minigenes encompassing four domains of the NS3, NS4 and NS5B proteins that contain multiple class I/II restricted epitopes. The polyEp-WT minigene encodes all four domains in fusion, the polyEp-C minigene encodes the same fusion but optimised for mammalian translation and the polyEp-E3 minigene has an additional endoplasmic reticulum targeting sequence. Whereas the minigenes vectorised by DNA were poorly immunogenic, adenovirus vectorisation induced strong and broader IFNgamma-ELISpot and CTL responses in HLA-A2 transgenic mice. In addition, polyEp-WT and polyEp-E3 responses were found cross-reactive in a recombinant Listeria-NS3-based surrogate challenge. This study illustrates the potency of vectorised minigenes in the field of HCV vaccine development.

Expression of the alternative reading frame protein of Hepatitis C virus induces cytokines involved in hepatic injuries.

Hepatitis C virus (HCV) Core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Discovery of different alternative reading frame proteins (ARFPs) expressed from the HCV Core coding sequence challenges properties assigned to Core. This study was designed to evaluate the immunomodulatory functions of Core and ARFPs in monocytes, dendritic cells (DCs), macrophages (Mphi) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived Mphi and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (CDeltaARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1beta, whereas production of these chemokines with AdCDeltaARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1beta in Mphi. Wild-type Core sequence, but not CDeltaARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42-44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.

Analysis of B-lymphocyte differentiation in patients infected with hepatitis C virus.

To clarify whether some of the functions of B lymphocytes could be affected during hepatitis C virus (HCV) infection, phenotypic characteristics of B lymphocytes from HCV-infected patients and their capacity to differentiate into immunoglobulins (Ig)-secreting cells were studied. B lymphocytes differentiation was investigated for patients untreated and non-responders (n=9), treated and non-responders (n=6), responders (n=6), long-term responders (n=9) to therapy and seronegative controls (n=14) following in vitro stimulation with S. aureus strain Cowan I mitogen. HCV sequences in purified B lymphocytes were detected by RT-PCR. It was found that HCV-patients harbor a similar mean percentage of B cells and a normal level of naïve B cells (% IgM+/IgD+ cells=79.7 +/- 15.4 for untreated non-responders, 57.1 +/- 22.9 for treated non-responders, 44.3 +/- 29.1 for responders, 75.7 +/- 16 for long-term responders) as compared with controls. It was also found that peripheral blood mononuclear cells (PBMCs) of patients or controls produced similar amounts of IgG, A, and M in vitro. A total of 57% of untreated non-responders versus 17% of treated non-responders were able to produce HCV-specific antibodies. Interestingly, B lymphocytes from PBMCs able to secrete anti-HCV antibodies contained HCV positive strand RNA, although no systematic detection of the negative strand was found. These data suggest that signaling through the B cell receptor (BCR) in B lymphocytes of HCV-infected patients appears normal whatever their response to therapy. The capacity to secrete HCV-specific IgG seemed to be linked to the presence of positive strand RNA rather than virus replication.

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.

Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection.

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.