Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo

Abstract

The hepatitis C virus (HCV) protease and helicase encompasses the nonstructural (NS) 3 protein and the cofactor NS4A, which targets the NS3/4A-complex to intracellular membranes. We here evaluate the importance of NS4A in NS3-based genetic immunogens. A full-length genotype 1 NS3/4A gene was cloned into a eucaryotic expression vector in the form of NS3/4A and NS3 alone. Transient transfections revealed that the inclusion of NS4A increased the expression levels of NS3. Subsequently, immunization with the NS3/4A gene primed 10- to 100-fold higher levels of NS3-specific antibodies as compared to immunization with the NS3 gene. Humoral responses primed by the NS3/4A gene had a higher IgG2a/IgG1 ratio (>20) as compared to the NS3 gene (3.0), suggesting a T helper 1-skewed response. Low dose i.m. (10 microg) immunization with the NS3/4A gene inhibited the growth of NS3/4A-expressing tumor cells in vivo, whereas the NS3 gene alone or NS3 protein did not. We then evaluated the efficiency of the NS3/4A gene administered by the gene gun, at the same doses used for humans, in priming cytotoxic T lymphocyte (CTL) responses. Three to four 4 microg doses of the NS3/4A gene primed CTL at a precursor frequency of 2-4%, which inhibited the growth of NS3/4A-expressing tumor cells in vivo. Thus, NS4A enhances the expression levels and immunogenicity of NS3, and an NS3/4A gene delivered transdermally could be a therapeutic vaccine candidate.

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

“…The enhanced expression of the wtNS3 gene seen when including NS4A in the SFV replicon system has been reported previously. 23 Staining of coNS3/4A plasmid-transfected, and SFVinfected, BHK cells revealed a similar perinuclear and cytoplasmic distribution of the NS3 as previously observed, confirming an unchanged subcellular localization ( Figure 1). 13,23 Effect of codon optimization and mRNA amplification on humoral responses…”

“…with the wtNS3-, wtNS3/4A-, or coNS3/4A-containing plasmids had Th1-skewed Th-cell responses evidenced by IgG2a/IgG1 ratios of 41 ( Figure 2). 23 Thus, codon optimization did not change the IgG subclass distribution. When genetically immunizing BALB/c mice with NS3/4A using the gene gun, the subclass ratio suggested a mixed Th1/Th2 response ( Figure 2).…”

“…13,23 To compare indirectly the T helper 1 (Th1) and Th2 skewing of the T-cell response primed by wtNS3/4A, coNS3/4A, and wtNS3/4A-SFV immunizations, the levels of NS3-specific IgG1 (Th2) and IgG2a (Th1) antibodies were analyzed ( Figure 2). The IgG2a/IgG1 ratio in mice immunized with rNS3 with or without adjuvant was always o1 regardless of the murine haplotype, 27 signaling a Th2-dominated response.…”

“…21,22 However, we could recently show that the presence of NS4A, by some mechanism, increases the expression levels of NS3, which explains the increased immunogenicity conferred by NS4A. 23 Interestingly, a recent report showed that the NS3/4A complex interferes with the interferon signaling pathways in transfected cells. 24 Apart from implying an immunomodulatory role in natural infection, this may further affect the NS3 expression in the presence of NS4A observed when using the Semliki forest virus (SFV) vector system.…”

“…24 Apart from implying an immunomodulatory role in natural infection, this may further affect the NS3 expression in the presence of NS4A observed when using the Semliki forest virus (SFV) vector system. 23 In addition to improving expression levels by the inclusion of NS4A or mRNA amplification by the SFV replicase, the immunogenicity of NS3 may possibly be improved by alternative modifications. One approach, which has been shown to sometimes improve expression levels, is codon optimization.…”