This meta-analysis provides important data for future development, expectations and assessment of HCV prophylactic vaccines. We show statistically significant differences in viral kinetics between primary and secondary infections in recovered chimpanzees. This statistical analysis is consistent with previous observations demonstrating that although infection can occur upon re-exposure following clearance, viral kinetics are very different with rapid clearance in the majority of cases18;48;55;56
. Similar significant decreases in maximum HCV RNA titer and duration of infection for secondary infections in intravenous drug users were recently reported57
. Interestingly, in both chimpanzees () and humans57
the HCV clearance rate was 83% of reinfected subjects, this compares to ~25% in patients57
and ~38% in chimpanzees () with primary infections. These data together with studies on immune correlates of clearance support the argument that memory immune responses are induced during primary infections, that these responses are recalled following secondary infections and that the responses are primarily T-cell based (reviewed in ref 58
All of the chimpanzee vaccine approaches listed in induced HCV-specific immune responses and HCV kinetics were significantly impacted in vaccinees compared to naïve animals. The peak viral titers were significantly decreased (P<0.001) and viral replication was controlled earlier (P<0.0001) regardless of the final outcome of infection. This shows that vaccination against HCV induces memory immune responses that are recalled upon exposure to virus and are effective at controlling viral replication. However, the duration of viremia in vaccinated animals was not significantly improved over naïve animals; suggesting vaccination was not able to consistently produce immune responses qualitatively similar to those induced during spontaneous clearance. Thus, not only does previous infection with HCV induce immune responses that protect from secondary infections but this natural immunity is superior to vaccine-induced immunity.
Our meta-analysis indicates that the rate of viral persistence is not increased following vaccination. However, the T-cell vaccine approach that includes NS proteins does not significantly (P=0.227) decrease the rate of persistence (45.8%) compared to naïve animals (61.9%) and results in a significantly higher rate compared to vaccines that target the structural region alone (P=0.015). Vaccines with the greatest success at leading to resolved infections included all or part of the HCV envelope region inducing either neutralizing antibody33;52-54
, E1E2 T-cell responses34
, suggesting that neutralizing antibodies can play a role in protection but also that this region may contain T-cell epitopes that are important for clearance. However, the induction of neutralizing antibodies or T-cell responses to the envelope region does not necessarily guarantee success of a vaccine and absence of these antigens from a vaccine was not always associated with persistence.
There are a number of mechanisms that may account for our observed difference in outcome based upon the vaccine antigen. The choice or use of adjuvants may have influenced the quality of the immune response and memory cells induced. Most of the vaccines targeting structural proteins used adjuvants () but a number of vaccines targeting the NS proteins also included adjuvants e.g. CpGs or recombinant viruses expressing co-stimulatory molecules. An analysis of animals vaccinated with adjuvants compared to those vaccinated without adjuvants showed no significant difference (p=0.51) in the rate of persistence. The type of recombinant virus used for the T-cell based vaccines also did not impact the outcome of infection after challenge. The rate of persistence in animals immunized with Vaccinia virus vectors compared to those immunized with Adenovirus vectors was not significantly different (p=0.64). It is more difficult to analyze the use of viral vectors on the outcome of infection as the vaccines designed to induce T-cells mainly used recombinant viruses (). This approach is unlikely to change unless significant drawbacks can be associated with viral vectors as these are one of the most efficient means of inducing T-cell responses. It is also possible that antibody plays a role in successful clearance through antibody-dependent cell-mediated cytotoxicity (ADCC). This has been shown to be important in a number of viral infections59;60
and also to be induced by a recent HIV vaccine that showed a reduced risk of HIV infection in vaccinees61;62
It is not possible to predict from this meta-analysis which vaccines should be developed for future clinical trials, but vaccines with a T-cell component targeting NS proteins should not be dismissed. It should be remembered that all the vaccines trials induced immune responses that modified viral replication and in order to protect against heterologous viruses responses to the more conserved NS proteins will almost certainly be required. However, more extensive studies need to be performed at the level of T-cell function in order to obtain a better understanding of the types of T-cells induced and provide reliable biomarkers to predict vaccine success.
Vaccine failure may also be caused by immune escape from the induced response. The persistence of HCV in some vaccine studies has been associated with immune escape from CD4+63
T-cells and with higher viral mutation rates64
although immune escape from neutralizing antibody has not yet been demonstrated in vaccine studies. These data suggest that in cases where the immune response cannot rapidly clear the virus there remains an environment for immune pressure.
Ideally, the goal of prophylactic vaccines is to prevent infection upon exposure. However, from our data analysis sterilizing immunity seems to be unrealistic for hepatitis C. Most efficacy trials use reduction of clinical disease as the endpoint and not elimination of all evidence of infection. With the highly sensitive assays such as PCR, it is likely that low level replication of the infectious agent may be detected in successfully vaccinated individuals. HCV differs from many viruses in that it causes persistent infection which leads to chronic liver disease. A vaccine that leads to low-level HCV titers long-term would probably not be considered successful. Although low-level HCV titers increases the chances of virus elimination under the current standard of care (SOC)65
the risk to benefit ratio in vaccinees would be considered too high and a clinical trial demonstrating a significant beneficial effect in vaccinees when combined with treatment would also be difficult. However, eliminating persistent infections while reducing acute phase viral titers would eventually prevent the major disease burden and most transmissions66;67
such that any prophylactic hepatitis C vaccine could target prevention of chronic infections as the primary endpoint.
Clinical development of HCV vaccine candidates is challenging. It is now known that treatment of acute HCV infections has a high probability of success. At present, the SOC recommends monitoring patients for ~12 weeks after diagnosis of acute HCV infection prior to initiating treatment with pegylated interferon-α, usually in combination with ribavirin, if the individual has not cleared the virus68
. In clinical trials there is active monitoring; if treatment is initiated at the time of diagnosis it would be difficult to determine if the vaccine can prevent chronic infections. Our meta-analysis suggests that this difficult problem is not unsolvable. In clinical vaccine trials, the subjects would be monitored periodically after the last dose of vaccine, typically at 6 month intervals. Given our data analysis, the median duration of viremia in vaccinees that clear HCV is 77 days (IQR=75); for rechallenged animals it is 28 days (IQR=39) and naive animals it is 102 days (IQR=51). These data indicate that if vaccine-induced immune responses are as efficient at clearing HCV as the immunity induced by natural infection there is a high probability that vaccinees will have cleared HCV within 3 months of exposure and significant differences would be seen between vaccine and placebo groups. If vaccines are developed that do not decrease the duration of viremia but decrease the rate of virus persistence differences could still be seen between vaccine and placebo groups if sufficient numbers were included in the studies.
This meta-analysis provides values for viral kinetic parameters that can be used to judge the success of future vaccine studies. However, it also demonstrates that there are scientific questions that still need to be resolved in HCV vaccine development, specifically correlates of protection. However, substantial progress has been made: vaccines can induce immune responses that impact viral replication; clinical studies are feasible and we have the means to judge efficacy. The development of effective HCV vaccines is a very real possibility.