Hepatitis B and C viral infections account for a heavy burden of chronic liver disease in much of the developing world, particularly in Africa and large parts of Asia. To investigate a promising approach to low-cost immunization against these viruses, we attempted to use polyvalent recombinant vaccinia viruses, as was first suggested by Perkus et al. (48
). Upon HBV challenge at 4 weeks after a single PolyVax immunization, two of the immunized animals developed no detectable HBV infection, one developed transient low-level viremia, and one reached a high peak viral load for reasons which remain unexplained (data not shown). It is likely that stronger protection would have been observed if prime boosting had been used, as was done for HCV.
The initial HCV challenge contained 2.5 CID50
and did not induce viremia. The animals were therefore rechallenged with 24 CID50
, which resulted in acute-phase viremia in all animals. It is unlikely that the low-dose challenge was responsible for the protection against chronic infection in the immunized animals, as both controls developed chronic infection after this mild infection. Although low-dose challenge alone was not enough to provide protection, we could not rule out the possibility that combination of our vaccination regimen with low-dose challenge somehow influenced the outcome of HCV infection, possibly by enhancing HCV-specific T-cell responses. This possibility was investigated by IFN-γ ELISPOT and proliferation assays at 6 weeks after low-dose challenge, and a boosting effect was not observed (data not shown). In control chimpanzee 407, we found HCV-specific T-cell responses 2 weeks after parental VV immunization. This chimpanzee may have had a previous subclinical exposure to HCV or may have had cross-reactive T-cell responses to other viral antigens (65
After challenge with 24 CID50
all animals rapidly developed viremia. However, peak viral loads in the immunized animals were about 1.3 logs lower than in the controls, indicating that a degree of immunity had been induced. This was further indicated by the fact that none of the four immunized animals, but both controls, developed chronic infection. This difference was statistically on the borderline of significance (P
= 0.067) and has yet to be confirmed due to the relatively small number of animals used in this study. All of the immunized animals developed relatively strong cell-mediated immune responses after the booster immunization. These were predominantly directed to NS3. Neutralizing antibody responses after the vaccine booster were relatively weak and inconsistent. Thus, our findings are consistent with previous reports indicating that cell-mediated immune responses are critical for prevention of chronic HCV infections (52
We previously reported that convalescent chimpanzees resist chronic infection when rechallenged with homologous but not heterologous genotypes (49
). This suggested that an HCV vaccine derived from only one genotype might not provide cross-protection against heterologous genotypes. However, a degree of cross-genotype protection has also been reported by others (32
). When the immunized animals were rechallenged with a pool containing six major genotypes, PVL (5.79 logs) reached titers similar to those seen in secondary infections rather than in primary infection. Upon homologous genotype 1b (bk) challenge, we observed PVLs of 7.32 and 6.05 logs in the control and immunized animals, respectively. This suggested partial controlling immunity to acute infection by heterologous genotypes. This speculation was supported by the multiple fluctuations of viremia at low levels, which implicate a dynamic interaction between host immune surveillance and viral escape. Alternatively, it is also possible that simultaneous replication of multiple genotypes may result in lower levels of acute-phase viremia than challenge with a single genotype, due to competition among different genotypes.
During the late phase, a very low titer or undetectable viremia was observed in three of the immunized animals, although one had clearly detectable viremia of 3.82 logs up to 102 weeks after multigenotype challenge. This may correspond to the “occult HCV” infections recently reported by Carreno et al. (11
). It is not known whether these infections are really chronic and long-lasting or whether these will eventually resolve. It is also not clear whether such low-level infections are clinically significant. Unfortunately, the animals in our study were released to retirement islands; thus, we were not able to follow them longer.
To gain an insight regarding the correlation of T-cell immunity with protection, we were interested in the immunogenicity compared with previous chimpanzee studies that had achieved different rates of protection. Notably, the current recombinant vaccinia virus-induced immunogenicity was no higher than that of other studies (17
). Due to many variables that affect the results of cell-mediated immunological assays, our previous study was considered ideal for the comparison, because only two of six vaccinated chimpanzees were protected and results were obtained under similar experimental conditions (69
). Unexpectedly, there was no significant difference in immunogenicity as determined by IFN-γ ELISPOT and proliferation assays, and the previous study induced even higher immunogenicity than the current one, which suggests that results under the current T-cell assay conditions may not completely represent protective immunity. There are at least three possibilities to explain the discrepancy, and these are not mutually exclusive. First, current immunological assays may not directly represent in vivo T-cell immunity. Stimulation with exogenous antigen to detect IFN-γ-secreting cells may far exceed the concentrations which occur in vivo, and thus T-cell responses may be overestimated (7
). Second, other types of HCV-specific T cells that are multifunctional or do not exhibit readouts in conventional T-cell assays, such as cytokine production or proliferation, may affect the protection. Lastly, there may be other types of host responses than antibody and T-cell responses that better correlate with the outcome of HCV infection, as suggested from the discordance between acute-phase control of HCV replication and chronic protection (54
The genotype shifting from 4 to eventually 1a may also result from a significant difference in the biological behavior of the different genotypes, a combined consequence of immune escape and viral fitness. As well-documented in previous reports for T-cell dysfunction during persistent lymphocytic choriomeningitis virus (66
) and HCV infection (2
), continued HCV replication might have resulted in dysfunction of preexisting HCV-specific T cells. As the degree of T-cell dysfunction increases, which results in a decrease of immune selection pressure, so does the contribution of virological factors on genotype distribution, such as “fitness cost.” Genotype 1 has been reported to replicate more efficiently (9
), be more pathogenic (16
), and be IFN resistant (15
) and thus is considered to have greater “fitness” than other genotypes in HCV-superinfected patients (33
). This may explain why genotype 1a was eventually fixed in multigenotype-challenged chimpanzee 414. This phenomenon is likely to correspond to viral evolution of diverse quasispecies in humans, but with much broader diversity, in which both escape from host immune responses and fitness to viral replication determine dominant quasispecies (14
It has yet to be determined why genotype 4 was predominant during the very early phase. It is possible that sequence differences in critical T-cell epitopes (12
) or in flanking regions (55
) may prevent successful recognition of genotypes by 1b-specific T cells.
We conclude that the use of replicating recombinant VV vectors provides protection against highly variable viruses, such as HCV. The WR strain of vaccinia virus is clearly too virulent for use in human immunization, particularly in immunosuppressed individuals. Thus, we are now investigating the use of highly attenuated VV strains, which we hope will retain immunogenicity and protectivity, as vectors for human use.