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. 2013 Sep;21(9):1787-95.
doi: 10.1038/mt.2013.122. Epub 2013 Jun 18.

The novel replication-defective vaccinia virus (Tiantan strain)-based hepatitis C virus vaccine induces robust immunity in macaques

Bo Wen  1 , Yao Deng, Hong Chen, Jie Guan, Xia Chuai, Li Ruan, Wei Kong, Wenjie Tan
Affiliations

The novel replication-defective vaccinia virus (Tiantan strain)-based hepatitis C virus vaccine induces robust immunity in macaques

Bo Wen et al. Mol Ther. 2013 Sep.

Abstract

The induction of a robust neutralizing antibody (nAb) response is likely to be as essential as specific cell-mediated immunity (CMI) against multiple antigens for the development of effective preventive and therapeutic vaccines against hepatitis C virus (HCV) infection in humans. To date, no data on the immunogenicity of the replication-defective vaccinia virus (derived from the Tiantan strain) (rNTV)-based HCV vaccine in primates have been reported. This study describes in detail the immunogenicity of various vaccine candidates in rhesus macaques, including rNTV-based and replication-defective recombinant adenoviral (rAd)-based HCV vaccines, as well as HCV pseudotyped virus-like particles (HCVpp). Our data showed that rAd-HCV vaccine boosting induced robust CMI, while priming or boosting with HCVpp enhanced the antigen-specific nAb response after rAd-HCV vaccination; however, CMI was not enhanced. Vaccination includes rNTV-HCV priming induced robust antigen-specific antibody, particularly nAbs, and CMI responses. Furthermore, more robust and longer-lasting CMI and higher cytokine levels (both Th1 and Th2 types, especially IFN-γ) resulted from boosting with rAd-HCV. We conclude that the rNTV-based HCV vaccine induces robust nAbs and CMI when combined with a heterogeneous primer-booster strategy, which shows promise for development of a human HCV vaccine.

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Figures

Figure 1
Figure 1
Schematic genomic diagrams of hepatitis C virus (HCV) vaccine vectors. (a) Insert region sketch maps of recombinant rNTV-HCV. HCV CE1E2 proteins (1–746 aa) and the full-length NS3 (aa1024-1658) gene are indicated by the dark box. (b) Diagram of the genomes of recombinant adenovirus vectors. HCV CE1E2 and C44P proteins are enclosed by a box that indicates the amino acid sites, C44p is a recombinant protein comprising HCV NS3' (1201-1465aa) with Core' (1–34 aa) in its C-terminal. (c) Schematic diagram of pseudotyped virus-like particles (HCVpp). HCV E1E2 (161–746 aa) are packed as envelope proteins, and NS3 protein genes in ssRNA form are wrapped by p24 and pol of HIV inside the particle. Further details can be found in the Materials and Methods section; the names of the recombinant vectors are indicated above the genome diagrams.
Figure 2
Figure 2
Schedule of recombinant hepatitis C virus (HCV) vaccine immunization and analysis. Twelve rhesus macaques were immunized I.M. with the vaccines indicated in Table 1. Blood samples were assessed for HCV-antigen-specific cell-mediated immunity (CMI) (ELISPOT, Luminex) at the (filled triangle) indicated time points (Luminex detection of multiple cytokines only at week 38). Antibody (IgG) and neutralizing antibody (nAb) titres were assessed every 2 weeks after immunization (inverted triangle) (nAb analysis at weeks 26 and 38). Only four monkeys were immunized with rAd-HCV and were assessed in the weeks indicated by italicized numbers.
Figure 3
Figure 3
Humoral immune responses measured by ELISA. Twelve rhesus macaques divided into three groups were immunized with rAd-HCV, rNTV-HCV, and HCVpp, as described in Table 1. Results are expressed as (a,d,g,j) anti-E1 IgG, (b,e,h,k) anti-E2 IgG, and (c,f,i,l) anti-NS3 IgG titres determined using serial twofold dilutions of sera. All sera were negative pre-immunization when tested at a 1:100 dilution and are plotted below this detection limit. The first three rows (a–i) indicate the titre changes in an individual, and the median response with standard error (represented by bars) of each group is shown at the end of the line (j–l). In all experiments (a–i), the mean of triplicate determinations of each sample is shown. An asterisk denotes a significant difference in comparison with the control response at that time point. Associated P values are provided in the text: *P < 0.05, **P < 0.01 (by Wilcoxon signed-rank test).
Figure 4
Figure 4
E1 and E2 antibodies and cross-neutralizing antibodies titres at weeks 26 and 38. Serum samples were collected at weeks (a,c) +26 and (b,d) +38 from macaques. Differences among the three groups in hepatitis C virus (HCV) E1E2-specific antibody titres are shown in a and b. *P < 0.05, **P < 0.01 (one-way analysis of variance). (c,d) cross-neutralization of samples was tested at a dilution of 1:50 for their capacity to neutralize infection with HCVpp of various strains and genotypes. Huh-7 cells were infected with 100 TCID50 (median tissue culture infective dose). Neutralization of VSVGpp was performed in parallel as a control for specificity. Data are expressed as the means ± SD of the percentage of neutralization of HCVpp by sera tested in triplicate. P values from Student's t-tests comparing neutralization of HCVpp with that of control VSVGpp: *P < 0.05, **P < 0.01.
Figure 5
Figure 5
Cellular immune responses in rhesus macaques quantified by IFN-γ ELISPOT. (a–c) mean IFN-γ ELISPOT responses to the hepatitis C virus (HCV) insert antigen (summed responses of all individual peptide pools) in PBMCs of each group over time are shown. (d) Breakdown of the total T-cell IFN-γ response into separate peptide pools; bars represent the median group response with each animal displayed as a single point. Data were analyzed using a two-way analysis of variance with a post hoc Bonferroni test; asterisks denote statistical significance: *P < 0.05, **P < 0.01.
Figure 6
Figure 6
Multiple cytokine detection in the culture supernatants of PBMCs stimulated with peptides at week 38. PBMCs (5 × 105) from vaccinated rhesus macaques were cocultured with 4 μg/ml peptides, with culture medium as the negative control. After 2 days in culture, cytokines in supernatants were assayed by Luminex. (a,b) Th1 (TNF-α, IFN-γ, IL2, and IL12) and Th2 (IL4, IL5, IL10, and IL6) cytokines were quantitated. (c) The cytokine levels of activated samples were divided by those of the negative control to calculate the enhancement rate. The enhancement rates among the three groups were compared by two-way analysis of variance. Cytokines whose expression was significantly different between the groups are indicated by asterisks (*P < 0.05, **P < 0.01).
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