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
Introduction
Approximately 3% of the world's population is infected with hepatitis C virus (HCV) [1] and about 80% of infected people develop a chronic infection leading to liver failure in 4% cases [2]. Standard treatment combining interferon α (IFNα) and ribavirine is effective in about half of the treated patients, however associated with significant toxicity and cost, and remains counter-indicated in a non-neglectable number of cases [3]. Novel therapies are in development, mainly targeting the viral protease or polymerase [4]. However, preliminary clinical data indicate that these new antivirals display low efficiency when used as stand-alone therapy, and it is becoming clear that the HCV therapeutic field is moving towards a complex association of multiple, costly drugs. The need for alternative therapeutic strategies, relying on complementary mechanisms than those currently exploited by antiviral molecules candidate, is well recognized.
Studies in humans and chimpanzees have indicated that failure to generate broad and long-lasting HCV-specific CD4+ and CD8+ T lymphocyte mediated immune responses during the acute phase of infection correlates with development of chronicity [5]. Conversely, patients displaying a functional and maintained Th1 CD4+ T lymphocyte-mediated response, associated with the mounting of matured and multifunctional effector CD8+ T lymphocytes, exert a more efficient control of viremia and are prone to evolve towards recovery [6], [7], [8], [9], [10], [11]. Multiple studies have established that non-structural antigens, and in particular NS3, are the preferential targets of responses associated with natural or therapeutic viral clearance [12], [13], [14]. In contrast, although the field is moving quickly due to novel assays recently developed, the contribution of anti-HCV antibodies in infection outcome remains controversial as these antibodies are typically present in face of ongoing chronicity [15], [16], [17].
Over the last 10 years, a wide variety of HCV vaccine efforts have been pursued. Various formulations, ranging from classical adjuvanted-recombinant proteins to dendritic cell-based vaccines, have been tested in mice, macaques and for a few, in chimpanzees [18]. It is striking to observe that only a few vector-based vaccines have been evaluated so far such as recombinant DNA [19], [20], recombinant bacteria [21] or adenoviruses [22], [23]. Most surprisingly, one of the safest known vaccine vector used to-date in the clinic, namely the modified non-replicative vaccinia virus Ankara strain (MVA), has seldom been evaluated towards the development of HCV vaccines. This highly attenuated strain of vaccinia virus that has been used in the campaign for eradication of smallpox has demonstrated a safety profile in more than 100,000 people [24], [25]. In the case of HCV, only two pre-clinical studies based on MVA vaccines have so far been reported: one describing MVA candidates expressing HCV envelop glycoproteins E1 and E2, either as wild-type or membrane-targeted immunogens [26], the other reporting on a vaccine combining two MVA expressing the three structural proteins (core, E1 and E2) as well as the non-structural protein 3 [20]. However, encouraging results with MVA-based vaccines have been observed for example, in the field of HIV or malaria vaccine development, where numerous studies involve MVA vaccine candidates either used alone or in prime-boost combinations [27], [28], [29]. These studies have run from evaluations performed in HLA-A2 transgenic murin models to small non-human primates and to clinical trials [30], [31]. Another poxvirus that has been tested in the HCV vaccine field is a HCV recombinant canarypox virus reported to induce potent T cell immune responses although this candidate has been only tested in a DNA prime-canarypox virus boost regimen [32]. The superior safety profile of MVA combined with its powerful immunogenic potential; argue unambiguously in favour of developing a potent HCV MVA-based vaccine, both for prophylactic and therapeutic application.
With the aim to develop a safe, poly-antigenic, T cell-based HCV vaccine, we have engineered and preclinically evaluated a recombinant MVA vaccine candidate encoding for HCV non-structural (NS) proteins NS3, NS4 and NS5B. We report here that an accelerated schedule of vaccination using this vaccine is able to induce CD4+ and CD8+ T lymphocyte-mediated responses targeted at all three vaccine immunogens and recognizing class I T cell epitopes recognized during the natural infection. Potent and specific CD8+ T cell-mediated responses are long lasting (detectable up to 6 months) and can be efficiently recalled when boosted at a later time with the original MVA NS34–NS5B. Using a challenge model based on HCV recombinant Listeria monocytogenes that can infect liver, we show that the accelerated schedule of vaccination with the MVA NS34–NS5B results in in vivo cross-reactive responses.
Section snippets
Synthetic peptides and recombinant proteins
All synthetic peptides and recombinant proteins used were derived from a genotype 1b sequence (HCV-JA) [33]. Peptides (Eurogentec) were derived from NS3: CVNGVCWTV (referred as CVN, corresponding to aa 1073–1081 on the HCV polyprotein), GLLGCIITSL (GLL, aa 1038–1047), KLTGLGLNAV (KLT, aa 1406–1415), WPAPPGARSM (WPA10, aa 1111–1121), LSPRPVSYLK (LSP10, aa 1152–1162) or NS5B: ALYDVVSTL (ALY, aa 2594–2602) antigens. A peptide derived from the HCV core: DLMGYIPLV (DLM, aa 132–140) was used as
Design and in vitro expression of a single recombinant MVA encoding for HCV NS3, NS4 and NS5B proteins
Two recombinant MVA vectors, encoding each for HCV NS3NS4 proteins under the ph5r promoter and NS5B protein under the p7.5 promoter, were designed. Both expression cassettes were cloned in the deletion III of the MVA backbone, either in same or in opposite orientation. Western blot analysis revealed an enhanced expression of all three cloned antigens when the two expression cassettes were inserted in the same orientation (data not shown). Thus, the MVA vector containing both expression
Discussion
In the present study, we designed a HCV vaccine candidate based on the vaccine strain MVA expressing three HCV antigens, NS3, NS4 and NS5B, from a genotype 1b viral strain (MVA NS34–NS5B). Injected according to an “accelerated” immunization schedule based on three injections performed 1 week apart in various HLA-transgenic or commercial mouse models, we show that this candidate vaccine induces simultaneously specific CD8+ T cells able to produce IFNγ and to lyse cells as well as specific CD4+ T
Acknowledgments
We would like to thank J.D. Abraham for helpful advice, F. Penin and D. Moradpour for stimulating discussions as well as J.Y. Bonnefoy for his constant support during the course of this work. This work was supported, in part, by a grant from the Lyonbiopôle (“Biotherapic”). It was performed, in part, with the technical platforms provided by the IFR128, Lyon, France.
References (61)
- et al.
Global epidemiology of hepatitis C virus infection
Lancet Infect Dis
(2005) - et al.
High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection
Gastroenterology
(2004) - et al.
Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens
Hepatology
(2001) - et al.
Hepatitis C vaccines
Drug Discov Today
(2006) - et al.
DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface
Vaccine
(1999) - et al.
Oral immunization with HCV-NS3-transformed Salmonella: induction of HCV- specific CTL in a transgenic mouse model
Gastroenterology
(2001) - et al.
Vaccination with an adenoviral vector-encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus
Vaccine
(2002) - et al.
Comparative immunogenicity analysis of modified vaccinia Ankara vectors expressing native or modified forms of hepatitis C virus E1 and E2 glycoproteins
Vaccine
(2004) - et al.
Enhanced CD8 T cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes
Vaccine
(2002) - et al.
Different hepatitis C virus non-structural protein 3 (NS3)-DNA-expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope
Hepatology
(2001)
Modulation of cellular immune responses against hepatitis C virus non-structural protein 3 by cationic liposome encapsulated DNA immunization
Hepatology
Recombinant ovine atadenovirus induces a strong ans sustained T cell response against the hepatitis C virus NS3 antigen in mice
Vaccine
Natural history of chronic hepatitis C
Hepatology
Past, present, and future hepatitis C treatments
Semin Liver Dis
New therapies for chronic hepatitis C virus infection
Curr Gastroenterol Rep
Cell-mediated immunity and the outcome of hepatitis C virus infection
Annu Rev Microbiol
Analysis of successful immune responses in persons infected with hepatitis C virus
J Exp Med
Determinants of viral clearance and persistence during acute hepatitis C virus infection
J Exp Med
Adaptative immune responses in acute and chronic hepatitis C virus infection
Nature
Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C
Hepatology
Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals
Eur J Immunol
Immunodominant CD4+ T-cell epitope within non-structural protein 3 in acute hepatitis C virus infection
J Virol
Differential antigenic hierarchy associated with spontaneous recovery from hepatitis C virus infection: implications for vaccine design
J Infect Dis
In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes
Proc Natl Acad Sci USA
Neutralizing antibody response during acute and chronic hepatitis C virus infection
Proc Natl Acad Sci USA
Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1
Proc Natl Acad Sci USA
Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response
J Virol
A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees
Nat Med
The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism
Zentralbl Bakteriol
Experiences with immunization against orthopox viruses of humans and animals using vaccine strain MVA
Bert Muench Tieraerztl Wochenschr
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