The development of an HCV vaccine is challenged by the fact that HCV can infect patients that previously recovered from HCV infection 19, 20
suggesting that complete protection appears difficult to achieve. Likewise, studies in chimpanzees demonstrated that animals re-challenged with homologous or heterologous strains of HCV are not consistently protected against re-infection following acute resolving infection 15
. Aiming to better understand the immunological determinants of protective immune responses to HCV infection, we performed an extensive analysis of the innate and adaptive immune response in two chimpanzees that had previously cleared HCV and were re-challenged with homologous and/or heterologous strains of HCV.
Chimpanzee 10274 was re-challenged three times with the JFH1 homologous virus derived from cell culture. The first challenge produced detectable HCV RNA lasting only 2 weeks. The chimpanzee was not infected following the subsequent challenge. The chimpanzee became virus-positive at a low level 10 weeks after the third re-challenge. Unfortunately the chimpanzee was re-challenged with the H77 virus on the same day per protocol and we were not able to follow this course of viremia. The homologous JFH1cc re-challenges were associated with the development of neutralizing antibodies and the induction of HCV-specific T cells, probably contributing to the rapid control of viral infection. The viral clearance was not associated with a significant increase of serum ALT level suggesting that cytolytic mechanisms were not involved in viral clearance or that the number of virus-infected cells in the liver was very low. We also did not detect any intrahepatic innate immune response in this animal. Our results are in line with several previous studies in chimpanzees demonstrating the importance of T cells in viral clearance and protection after re-challenge 11–13, 15, 21
. It is interesting to note that the H77 virus overcame the protective immune responses against JFH1, dominated over the concurrent low-level JFH1 viremia, and developed into a high-viremic infection, suggesting that the protective immunity noted above was rather strain-specific.
The role of the humoral immune response in protection against HCV infection is less well defined. Prospective studies demonstrated that viral clearance in acute HCV infection did not correlate with the development of neutralizing antibodies in chimpanzees 15, 22
. In our study, CH10274 sero-converted and developed neutralizing antibodies against the homologous re-challenge strain (JFH-1, genotype 2a) and a heterologous strain (genotype 1a), indicating the production of genotype cross-reactive antibodies. However such antibodies were not able to prevent re-infection with the H77 strain. Thus, neutralizing antibodies may be capable of preventing low-level subclinical infection, such as the JFH-1cc infection 16
, but they are not sufficient to control robust high-viremic infection like the H77 infection 18
Following challenge with H77 virus in CH10274 and CH10273, we observed two distinct clinical courses. CH10273 had what appeared to be protective immunity because no viremia was detected. By contrast, CH10274 was infected with a fluctuating course of viremia and viral clearance almost a year later. In humans, chronic HCV infection is characterized by 1–2 log decrease in viral load followed by a viral load stabilization in most cases of persistent infection within several months. However, fluctuating viremia in both patients with resolution of infection and those with chronic infection including intermittent negative HCV RNA test results after initial viremia have been observed. Furthermore, although most patients with an acute and self-limited course of HCV infection clear infection within 6 months, viral clearance has been also reported 1 and 2 years after diagnosis of acute infection 23
As discussed above, although CH10274 possessed antibodies with neutralizing activity against the re-challenging viral strain, the antibodies appeared insufficient to prevent re-infection. We did not observe any significant level of neutralizing antibodies in CH10273 following heterologous challenge suggesting that the observed sterilizing immunity was not associated with the development of neutralizing antibodies. Although both animals demonstrated HCV-specific T cell responses in the blood, the magnitude of the HCV-specific T cell response was higher in CH10274 who became re-infected. Since there is an ongoing redistribution and migration of T cells between blood, lymph nodes and liver, we examined the intrahepatic immune response in both animals. Compared to other organs, the liver is particularly enriched with cells of the innate immune system, including natural killer (NK), natural killer T (NKT) cells, Kupffer cells (KC) and dendritic cells (DCs), and T cells, which participate in adaptive immune responses 24
. The protective immunity in CH10273 was associated with a rapid and durable increase of specific T, NK and NKT cell markers and increased level of IFN-γ mRNA in the liver suggesting an intense infiltration of activated T, NK and NKT cells into the liver and/or the activation of resident liver T, NK and NKT cells. CD8+ T cells, NK and NKT cells exert their effector functions in viral infection either by direct cytotoxicity or the release of IFN-γ which inhibits viral replication 24
. Since we observed only a mild evaluation of ALT level following heterologous HCV re-challenge, control of HCV replication was probably mediated by non-cytolytic mechanisms. In contrast, CH10274 who became re-infected displayed a weak enhancement of T, NK and NKT cell markers with marginally induced IFN-γ mRNA in the liver. This relative inability of virus-specific T and innate immune cells to enter the liver and be activated may account initially for the inefficient control of HCV replication in this animal. However this animal did develop a strong secondary infiltration of a different T cell response much later, leading to eventual viral clearance. The underlying mechanism that contributes to the weak or delayed movement of HCV-specific T cells from the blood into the liver of CH10274 remains unknown. It would be of interest to examine and correlate the intrahepatic HCV-specific T cell responses in theses chimpanzees. However the currently available technique in studying intrahepatic T cells involves artificial T cell expansion and cloning, which is inadequate in providing a global analysis of the T cell response.
Distinct subsets of DCs, including myeloid and plasmacytoid DCs are present in the liver and there is a continuous influx of DCs from the blood into the liver 25
. Analysis of DC markers revealed a decrease in plasmacytoid and myeloid DCs in both animals following re-challenges suggesting that liver resident DCs migrated to the draining lymph node. Recruitment of DCs to lymph nodes is pivotal for the initiation of adaptive immune responses 25
Interferons (IFNs) are key mediators of the host innate antiviral immune response. Interferon stimulated gene (ISG) products can prevent the translation of viral RNAs and thereby limiting the initial viral spread in the liver until viral clearance occurs by HCV-specific T cells 26
. In CH10273, prevention of re-infection was associated with an early and extensive induction of the ISGs in the liver, coinciding with the enhanced NK, NKT and T markers and IFN-γ. Infected hepatocytes are probably the primary cell types in the liver involved in type I IFN and ISG expression. However, since we did not dissect the cellular origin of the type I IFN production and ISG expression in the liver, DCs may also be involved in IFN-α/β production. Although, DCs appear not to be directly infected or stimulated by HCV to produce type I IFNs in vitro, recent studies demonstrated that HCV-infected hepatocyte cell lines have the capability to stimulate pDCs to produce large amounts of type 1 IFN via Toll-like receptor 7 (TLR7) signaling that is induced by direct cell-to-cell contact with HCV-infected cells 27
. Gene expression analysis of liver biopsy samples from CH10273 revealed strong induction of interferon antiviral pathways, e.g. ISG15, Mx, RSAD2, IFI44, IFIT1 and OAS. Since these pathways are involved in blocking viral transcription, degrading viral RNA, inhibiting translation and modifying protein functions 26
, the induced vigorous IFN response in CH10273 appeared to control virus replication and spread in the liver. The data are in line with previous reports that demonstrate the induction of the IFN response pathways in chimpanzees during acute resolving HCV infection 28–30
CH10274 also exhibited induction of ISGs in the liver shortly after re-infection by H77 virus. However the magnitude and breadth was weaker than that of CH10273. This induction of ISGs occurred in the absence of a robust increase in intrahepatic T and NK cell markers, suggesting that this response is probably secondary to a high level of viral replication in the liver of this chimpanzee but insufficient to clear the viral infection. However this chimpanzee was able to mount a more vigorous T cell response with induction of ISGs in the liver later prior to viral clearance. These observations suggest that the timing and the breadth of the innate and adaptive intrahepatic immune responses is a critical factor in determining the outcome of HCV infection. It can be assumed that the earlier and robust ISG response observed in CH10273 inhibited HCV replication and spread in the liver. Furthermore, the ISG response in this animal was supported by a robust intrahepatic NK and T cell response which probably cleared infected cells. As observed in CH10274, the weak ISG response and intrahepatic immunity led to a continued HCV replication and a poor or inefficient activation of the intrahepatic T cell response. It was probably the second wave of the intrahepatic innate and cellular responses in CH10274 that finally controlled the heterologous HCV re-challenge. The reason for the variation in the immune response of the two animals is unknown. However, it could be due to the different re-challenges protocol but may also reflect inter-individual variability. As discussed above, CH10274 had a low-level subclinical infection with HCV JFH1cc at the time of the heterologous H77 re-challenge.
In conclusion, although the number of animals studied was limited and we used different re-challenge protocols, our study, which included multiple sequential samples of the liver and blood, demonstrates that protective immunity against HCV infection likely depends primarily on the activation of both intrahepatic innate and cellular immune responses. Our data indicate that regardless of the infection outcome following heterologous HCV re-challenge, peripheral T cell responses are present. However, a rapid onset of the complex and coordinated interplay between innate immune cells and T cells in the liver appears to be critical for protection against HCV infection after re-challenge with heterologous genotypes. Miscueing of this coordinated immune response in the liver leads to failure of viral control and favours persistent viral infection. In addition, our results suggest that neutralizing antibodies contribute to the initial protection after re-exposure with homologous HCV probably by interfering with the early steps of the HCV life cycle such as viral binding and entry. However, despite the evidence for cross-reactivity of these antibodies, they appear to not to provide protection against the heterologous HCV strain. Development of an effective preventive vaccine and immunotherapeutics would have to target multiple pathways of immune response for optimal effect.