A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

Abstract

The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >104 postimmunization. With respect to CD8+ T cell responses, the coNS5A gene primed more potent IFN-γ–producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8+ T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8+ T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

Alphavirus-based Vaccines Encoding Nonstructural Proteins of Hepatitis C Virus Induce Robust and Protective T-cell Responses

“…Increasing the size of foreign transgenes allows broadening of the spectrum of T-cell responses which is considered essential for the induction of protective HCV immunity. [18][19][20] T-cell responses against HCV 2252-2259 ILDSFDPL, an epitope recently identified by Holmstrom, F and colleagues, 16 was not detectable this study. It should however be noted that the ILDSFDPL-specific T-cell responses were identified in mice immunized with DNA plasmid expressing NS5A only.…”

Molecular Characterization of HCV NS5A in Patients Receiving Interferon Therapy

“…The intrinsic immunogenicity of NS5A has been highlighted by post-immunization production of high titres of NS5A-specific IgG antibodies and proposes its utilization in vaccine composition. NS5Abased DNA vaccine has been shown to induce specific T cell production and it might be a suitable candidate for therapeutic vaccine in chronic HCV infection [20]. This is supported by the study on HIV-1 that determined that a single mutation of alanine to threonine in the V3 loop at position 21 vanishes neutralizing epitope that constitutes target of one of the monoclonal antibodies.…”

Long-term functional duration of immune responses to HCV NS3/4A induced by DNA vaccination

“…The RMA-S stabilization assay was performed exactly as described previously. 40 Antibodies against mouse H-2K d , H-2D d and H-2L d was used to detect stabilized MHC class I molecules. The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate-conjugated H-2K d (clone SF1-1.1) and H-2D d (clone 34-2-12), phycoerythrin-conjugated anti-mouse IFN-γ (clone XMG1.2) and fluorescein isothiocyanate-conjugated anti-mouse CD8 (clone 53–6.7), CyChrome-conjugated CD4 (clone RM4-5) and CyChrome-conjugated B220 (clone RA3-6B2), and anti-CD16/CD32 (Fc block, 2.4G2).…”