Both innate and adaptive immunity mediate protective immunity against hepatitis C virus infection in chimpanzees

Abstract

Understanding the immunological correlates associated with protective immunity following hepatitis C virus (HCV) reexposure is a prerequisite for the design of effective HCV vaccines and immunotherapeutics. In this study we performed a comprehensive analysis of innate and adaptive immunity following HCV reexposure of two chimpanzees that had previously recovered from HCV-JFH1 infection. One of the chimpanzees, CH10274, became protected from active viremia by repeated challenges with homologous HCV-JFH1 and developed neutralizing antibodies, but was later infected with high-level viremia by a heterologous challenge with the HCV H77 virus that persisted for more than 1 year. The other chimpanzee, CH10273, was protected from a similar, heterologous H77 challenge without any evidence of neutralizing antibodies. Peripheral HCV-specific T-cell responses were present in both chimpanzees after challenges and, interestingly, the overall magnitude of response was lower in uninfected CH10273, which, however, exhibited a more robust CD8+ T-cell response. CH10273 showed higher hepatic expression of CD8 and CD56 (natural killer) markers than CH10274 did shortly after inoculation with H77. The heightened T-cell response was associated with an enhanced hepatic production of interferons (both type I and II) and interferon-stimulated genes (ISGs) in CH10273. Therefore, protection or clearance of HCV reinfection upon heterologous rechallenge depends on the activation of both intrahepatic innate and cellular immune responses. Furthermore, our results suggest that serum neutralizing antibodies may contribute to early control of viral replication and spread after homologous HCV rechallenges but may not be sufficient for a long-term protective immunity.

Conclusion: Our study shows that protective immunity against HCV reinfection is orchestrated by a complex network of innate and adaptive immune responses.

Figure 1. Clinical and virologic course of homologous and heterologous HCV re-challenges

CH10273 was re-challenged with heterologous H77 (genotype 1a) at week 0. CH10274 was re-challenged three times with homologous JFH1-HCVcc (genotype 2a) at week 0, 6, and 12 and heterologous H77 (genotype 1a) at week 22. The course of infection was monitored by testing for HCV RNA (qualitative RT-PCR: top horizontal bar, blue as positive; real-time quantitative RT-PCR: black bars), HCV antibodies by ELISA, and ALT levels. Green horizontal bar indicates seroconversion. Arrows and circles indicate the time points of the re-challenges. ALT normal values (determined by using ten ALT determinations prior to the study): CH10273 < 76 U/L; CH10274 < 73 U/L.

Figure 2. Induction of cross-reactive neutralizing antibodies following homologous HCV re-challenge

CH10273 was challenged with heterologous H77 at week 0. CH10274 was re-challenged three times with homologous JFH1-HCVcc (genotype 2a) at week 0, 6, and 12 and heterologous challenge with H77 (genotype 1a) at week 22. Serum samples of both chimpanzees were tested at the indicated weeks for the presence of neutralizing antibodies. For the determination of neutralizing antibodies, the percent of pseudoparticle infection bearing the JFH1 (genotype 2a) or the H77 (genotype 1a) envelope proteins was measured and compared to pseudoparticle infection in the presence of human control sera (=100%). Experiments were done in triplicates, and the standard deviation is indicated. The neutralizing activity was defined as ≥ 50% reduction in HCVpp entry indicated by a dashed line. The arrows and circle indicate the time points of the first homologous and heterologous HCV re-challenges.

Figure 3. Peripheral HCV-specific T cell response following HCV re-challenge

Frequencies of IFN-γ and IL-2 producing T cells in response to HCV genotype 2a, genotype 1a overlapping peptide pools (OLPs) and HCV proteins (genotype 1) are shown as spot-forming units (SFU) per 2.5 × 10⁵ PBMCs. T cell responses to OLPs of HCV genotype 2a comprise core, NS3 and NS5B, and of HCV genotype 1a comprise core, NS3, NS5A and NS5B. T cell responses to HCV proteins comprise core, helicase, NS5A and NS5B. Antigen-specific SFU was calculated by subtracting the average of background values (typically fewer than 10 spots) from that of the antigen-stimulated sample. Arrows and circles indicate the time points of the re-challenges. The weeks analyzed are indicated at the bottom of each graph. Weeks that are circled in black represent repeated JFH inoculations and week circled in red represents H77 challenge.

Figure 3. Peripheral HCV-specific T cell response following HCV re-challenge

Frequencies of IFN-γ and IL-2 producing T cells in response to HCV genotype 2a, genotype 1a overlapping peptide pools (OLPs) and HCV proteins (genotype 1) are shown as spot-forming units (SFU) per 2.5 × 10⁵ PBMCs. T cell responses to OLPs of HCV genotype 2a comprise core, NS3 and NS5B, and of HCV genotype 1a comprise core, NS3, NS5A and NS5B. T cell responses to HCV proteins comprise core, helicase, NS5A and NS5B. Antigen-specific SFU was calculated by subtracting the average of background values (typically fewer than 10 spots) from that of the antigen-stimulated sample. Arrows and circles indicate the time points of the re-challenges. The weeks analyzed are indicated at the bottom of each graph. Weeks that are circled in black represent repeated JFH inoculations and week circled in red represents H77 challenge.

Figure 4. Intracellular cytokine staining of IFN-γ+ CD4 and CD8 T cells after HCV re-challenge

The upper histogram shows the percentage of IFN-γ secreting CD4 (blue) and the bottom CD8 (green) cells in the CD3+ lymphocyte population. The percentage in red indicates value that is significantly above the DMSO background sample (>0.25%) and at least twice of the baseline value (week 0 for CH10273 and week 22 for CH10274). FSC - forward scatter.

Figure 4. Intracellular cytokine staining of IFN-γ+ CD4 and CD8 T cells after HCV re-challenge

The upper histogram shows the percentage of IFN-γ secreting CD4 (blue) and the bottom CD8 (green) cells in the CD3+ lymphocyte population. The percentage in red indicates value that is significantly above the DMSO background sample (>0.25%) and at least twice of the baseline value (week 0 for CH10273 and week 22 for CH10274). FSC - forward scatter.

Figure 5. Analyses of intrahepatic immune response during HCV re-challenge by quantitative real-time PCR

Serial samples of liver biopsies from both chimpanzees were obtained and used to isolated total RNA. cDNA synthesis and TaqMan real-time PCR was performed as described in Methods. Relative levels of 17 different gene expression, markers for T cells (CD3, CD4, CD8b), NK cells (CD56) and dendritic cells (CD11c, CD304), interferons (IFN-α, IFN-β, and IFN-γ), and ISGs (OAS, Mx1, ISG15, IFIT1-3, IFI44, RSAD2), were analyzed. The y-axis illustrates the relative gene expression levels where each gene expression was normalized to GAPDH and determined relatively to week 0 value that is set as 1.