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. 2019 Jun 25;9(1):9251.
doi: 10.1038/s41598-019-45461-z.

Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery

D Christiansen  1 , L Earnest-Silveira  1 , B Grubor-Bauk  2 , D K Wijesundara  2 , I Boo  3 , P A Ramsland  3   4   5   6 , E Vincan  7 , H E Drummer  3   8 , E J Gowans  2 , J Torresi  9
Affiliations

Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery

D Christiansen et al. Sci Rep. .

Abstract

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Overview of the HCV VLP quadrivalent vaccine immunisation, bleeding and testing schedule. Two groups of 3 pigs were immunized with 3 doses 21 days apart of quadrivalent vaccine by intradermal (Group 1) or intramuscular (Group 2) vaccination. Each dose consisted of a total of 300 µg of HCV VLP, containing 75 µg of each genotype. The assays performed at each sampling point are shown.
Figure 2
Figure 2
Antibody responses elicited by immunization with quadrivalent vaccine. White Landrace pigs (n = 3/group) were vaccinated with quadrivalent vaccine by intradermal microneedle injection or intramuscular injection. Antibody levels were determined by ELISA using heat-inactivated sera prepared from blood taken from all pigs at each of the indicated time points. Serum was diluted 1 in 500 in BSA5PBST and 50 µl of the diluted serum was added to VLP coated wells. The mean value and standard deviation is shown, this was calculated from the 3 pigs in each treatment group and is shown for each time point. P values in this, and other subsequent experiments were calculated using one-way ANOVA.
Figure 3
Figure 3
Time course of VLP specific antibody responses following quadrivalent vaccination via intradermal microneedle injection. Anti-VLP antibody levels at days 21 (A), 42 (B), 56 (C), 70 (D) and 84 (E) were determined by ELISA using serially diluted heat-inactivated sera from each of the individual pigs. The mean value and standard deviation is shown for triplicate samples for each dilution. Geometric mean antibody titres peaked at day 42 and were maintained through to day 84 (F).
Figure 4
Figure 4
IgG1 and IgG2 responses generated after immunization with quadrivalent vaccine via intradermal microneedle injection. IgG1 and IgG2 antibody levels were determined by ELISA using heat-inactivated sera prepared from blood from all pigs at each time point. Serum was diluted 1 in 500 in BSA5PBST and 50 µl of the diluted serum was added to VLP coated wells. The mean value and standard deviation is shown, this was calculated from the 3 pigs in the group and is shown for each bleed.
Figure 5
Figure 5
Genotype-specific antibody responses elicited by immunization with quadrivalent vaccine. Serum was diluted 1 in 500 in BSA5PBST and 50 µl of the diluted serum was added to genotype specific HCV VLP (Gt1a, Gt1b, Gt2a or Gt3a) coated wells. The mean value and standard deviation is shown, this was calculated from the 3 pigs at each time point.
Figure 6
Figure 6
(A) Neutralizing antibody responses to quadrivalent VLP. Percentage HCV inhibition was determined by preincubating HCV infectious cell culture virus (HCVcc) genotype 2a or 3a with an equal amount of diluted immune serum from pigs inoculated by intradermal injection with quadrivalent VLP. Sera from the day 56 time point were used for all HCVcc neutralisation assays. Several serum dilutions (1:40, 1:140, 1:400 and 1:1200) were tested. The concentrations (μg/ml) of the MAb24 used for each dilution series are as indicated. The negative control was pig IgG purified from pooled serum collected from pigs 10–12 at day 0. Individual animals are presented for each group, with the mean value being represented by the horizontal bar. (B) Cross-neutralising antibody responses to quadrivalent VLP. Percent neutralization was determined by pre-incubating FITC labelled VLP (Gt1b, Gt1a, Gt2a or Gt3a) with increasing dilutions of day 56 serum (1:80 to 1:2560) from pigs 10–12. Anti-CD81 and non- immunized pooled pig IgG were used as positive and negative controls respectively. The mean value and standard deviation is shown, this was calculated from pigs 10–12 and is shown for each dilution. Immune serum compared to negative pooled pig serum; *p < 0.001; **p < 0.01.
Figure 7
Figure 7
Memory B cell responses following immunization with quadrivalent vaccine. (A) PBMCs were harvested on each of the days as indicated from the 3 individual pigs (10–12) and frequencies of HCV specific memory B cells were determined by B cell ELISpot assay. The y-axis shows memory B cells number/million PBMCs. The mean value and standard deviation is shown. This was calculated from the 3 pigs and is shown for each time point. (B) The total number of IgG memory B cells on days 42 and 84 following R848 stimulation (a TLR9 agonist).
Figure 8
Figure 8
T cell response following immunization with quadrivalent vaccine (A) IFNγ T cell responses. Frequencies of IFNγ secreting cells were determined by ELISpot assay. The y-axis shows spots/million PBMCs. The mean value and standard deviation is shown, this was calculated from the 3 pigs at each bleed. (B) Granzyme B responses. The concentration of granzyme B (pg/ml) present in pig sera at the indicated time points was determined with reference to a standard curve. The mean value and standard deviation is shown from the 3 pigs at each of the time points. (C) Cytokine profiles. Serum cytokine profiles were analysed at each time point using a Porcine Cytokine 13-Plex Discovery Assay. Yellow to red colour gradation represents increasing concentration of each cytokine (pg/ml).
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