T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates

Abstract

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.

Figure 1

Immunization schedule. Three groups of four macaques per group were immunized at the four time points indicated by arrows (at weeks 0, 6, 14 and 20), with the vaccine immunogens as indicated, further referred to as DNA+HuAd5 (DDAA), DNA+MVA (DDMM) and SFV+MVA (SSMM). The composition of vectors and their transgenes used for each immunization (DNA, HuAd5, MVA or SFV) is indicated.

Figure 2

Lymphoproliferation responses induced against all four HCV proteins (graph) and the proportional contribution of each antigen (pie charts) after priming and boosting. The cumulated SI to all four HCV antigens of each animal is shown, each diamond representing one animal, 2 weeks after the two DNA or SFV injections, and 2 weeks after HuAd5 or MVA injections. Individual values mentioned in the text are indicated. The geometric mean+s.d. of each group at both time points is also represented as horizontal bars. The contribution of each antigen-specific lymphoproliferation is represented on top of each vaccine regimen, each pie chart representing the geometric mean lymphoproliferation of each group to each antigen as indicated in the legend. Lymphoproliferation was performed using recombinant proteins.

Figure 3

Enumeration of IFN-γ (a), IL-2 (b) and IL-4 (c) producing cells by ELISPOT against all four HCV proteins (graphs) and the proportional contribution of each antigen (pie charts) after HuAd5 or MVA-booster injections. (a–c) The cumulated HCV-specific Spot Forming Units/million cells per animal are shown after two HuAd5 or MVA injections as indicated, each diamond representing one animal. The mean+s.d. of each group is also represented as horizontal bars. Some individual values (black) and average group values (red) are indicated for ease of comparison. * indicates statistical significant difference by ANOVA. The contribution of each antigen-specific cytokine production is represented on top of each regimen, each pie chart representing the mean of IFN-γ (a), IL-2 (b) and IL-4 (c) producing cell number of the group to the corresponding antigen as indicated in the colored legend. (d) Pie charts of the geometric mean cytokine-producing cells showing the contribution of each cytokine response to all four antigens for each regimen. The ELISPOT assays were performed using recombinant proteins.

Figure 4

Individual E1 (a) and NS3 (b–d) peptide pool-specific IFN-γ production by PBMCs. IFN-γ production to E1 or NS3 peptide pools as tested by ELISPOT is represented for each animal from each group post HuAd5 or MVA immunization (a and b, respectively). Results are expressed as mean number of spots of triplicate assay per one million cells minus the mean number of spots obtained with the medium cultured cells (also in triplicate assays)+2s.d. (c) The time course of NS3-specific response is shown for the three groups as indicated in the legend, as the geometric mean numbers of cytokine-producing cells per group. Gray arrows indicate the timing of DNA or SFV injections, and black arrows indicate the timing of HuAd5 or MVA injections. (d) Individual responses to NS3pp1 covering aa 1028–1346 and NS3pp2 covering aa 1340–1659 after HuAd5 or MVA immunizations are shown, along with the geometric mean of the group.

Figure 5

Serum antibody responses to E1 and E2 recombinant proteins. The geometric mean titers+s.d. of the E1- (a) and E2 (b) -specific antibody responses in each of the three groups is represented over time. Black arrows indicate the time points of immunizations (DNA or SFV at weeks 0 and 6, HuAd5 or MVA at weeks 14 and 20). (c) Individual responses to E2 at week 22, each dot representing one animal and the horizontal lines representing the geometric mean of the group. Differences of statistical significance between groups are indicated by *. ELISA was performed with recombinant proteins.