Anne Fournillier

Comparative Vaccine Studies in HLA-A2.1-Transgenic Mice Reveal a Clustered Organization of Epitopes Presented in Hepatitis C Virus Natural Infection

ABSTRACT 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.

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.

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.

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

ABSTRACT 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.