Safety Profile of a Multi-Antigenic DNA Vaccine Against Hepatitis C Virus

Abstract

Despite direct acting antivirals (DAAs) curing >95% of individuals infected with hepatitis C (HCV), in order to achieve the World Health Organization HCV Global Elimination Goals by 2030 there are still major challenges that need to be overcome. DAAs alone are unlikely to eliminate HCV in the absence of a vaccine that can limit viral transmission. Consequently, a prophylactic HCV vaccine is necessary to relieve the worldwide burden of HCV disease. DNA vaccines are a promising vaccine platform due to their commercial viability and ability to elicit robust T-cell-mediated immunity (CMI). We have developed a novel cytolytic DNA vaccine that encodes non-structural HCV proteins and a truncated mouse perforin (PRF), which is more immunogenic than the respective canonical DNA vaccine lacking PRF. Initially we assessed the ability of the HCV pNS3-PRF and pNS4/5-PRF DNA vaccines to elicit robust long-term CMI without any adverse side-effects in mice. Interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay was used to evaluate CMI against NS3, NS4 and NS5B in a dose-dependent manner. This analysis showed a dose-dependent bell-curve of HCV-specific responses in vaccinated animals. We then thoroughly examined the effects associated with reactogenicity of cytolytic DNA vaccination with the multi-antigenic HCV DNA vaccine (pNS3/4/5B). Hematological, biochemical and histological studies were performed in male Sprague Dawley rats with a relative vaccine dose 10-20-fold higher than the proposed dose in Phase I clinical studies. The vaccine was well tolerated, and no toxicity was observed. Thus, the cytolytic multi-antigenic DNA vaccine is safe and elicits broad memory CMI.

Keywords: DNA vaccine; Hepatitis C Virus; Pre-clinical; cell death; immune breadth; pathology; perforin; toxicology.

Figure 1

Schematic map of the vaccine constructs. (A) pVAX empty construct, (B) pVAX encoding NS3, 4A, 4B and 5B downstream of the CMV promoter (pNS3/4A/4B/5B) and (C) pNS3/4A/4B/5B encoding Perforin (PRF) downstream of the SV40 promoter (pNS3/4A/4B/5B-PRF).

Figure 2

Titration of pNS3 in C57BL/6 mice. Mice were vaccinated three times at two week intervals with pNS3 and splenocytes were harvested 14 days post final vaccination. Splenocytes were re-stimulated in duplicate with overlapping peptides representing the complete HCV NS3 protein (gt3a) and IFN-γ secretion detected by ELISpot analysis. Splenocytes from vaccinated animals were re-stimulated with overlapping peptides representing (A) NS3 pool 1 (B) NS3 pool 2 (C) NS3 pool 3 (D) NS3 combined analysis. Each symbol represents an individual mouse and data are shown as mean ± SEM of SFU per 10⁶ splenocytes. Significance shown between control and dose groups. No significant differences were observed between dose groups. * p ≤ 0.05 (Mann–Whitney test non-parametric t-test).

Figure 3

Titration of pNS4B/5B in C57BL/6. Mice were vaccinated three times at two week intervals with pNS4B/5B and splenocytes were harvested 14 days post final vaccination. Splenocytes were re-stimulated with overlapping peptides representing the HCV (A) NS4B (B) NS5B Pool 1 (C) NS5B Pool 2 (D) NS5B Pool 3. (E) NS4B and NS5B combined analysis. Each symbol represents an individual mouse and data are shown as mean ± SEM of SFU per 10⁶ splenocytes. Significance shown between control and dose groups. No significant differences were observed between dose groups. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (Mann–Whitney test non-parametric t-test).

Figure 4

Weight of Sprague Dawley rats after vaccination. Rats were injected with 150 μg of DNA on Day 0 followed by 450 μg DNA on Days 5 and 15, then killed and necropsies performed on Day 23. Weight of (A) rat was measured daily and increase graphed as a percentage and (B) spleen as a percentage of total rat weight. Data shown as median (n = 10) and interquartile range. ** p ≤ 0.01 (Mann–Whitney test).

Figure 5

Histological representation of site of injection in Sprague Dawley rats. Histological representation of lymphocyte infiltration in the (A) dermis and (B) subcutaneous layer at the site of injection in rats vaccinated with Saline, pNS3/4A/4B/5B, and pNS3/4A/4B/5B-PRF. Images on left are 10× objective, insets on the right are 40× magnification.