In accordance with NIH's decision to discontinue chimpanzee research we have not performed any studies with live chimpanzees. We have analyzed cryopreserved chimpanzee plasma, lymphocyte and liver biopsy biospecimens that were collected in previous years. This analysis has been completed for project 1 and the results are now published. Project 1: Innate and adaptive immune responses after low-dose exposure to HCV Successful hepatitis C virus (HCV) treatment is defined as absence of viremia six months after therapy cessation. We previously reported that trace amounts of HCV RNA, below the sensitivity of the standard clinical assay, re-appear sporadically in treatment responders (Veerapu et al., Gastroenterology 2011:140:676-685). To assess the infectivity of this RNA, we had in previous years infused three chimpanzees sequentially at 9-week intervals with plasma or PBMC from patients who tested positive for trace amounts of HCV RNA more than 6 months after the end of pegylated interferon-alpha/ribavirin therapy. A fourth chimpanzee had received HCV RNA-negative plasma and PBMC from healthy blood donors. We had shown that the three experimental chimpanzees but not the control chimpanzee generated HCV-specific T-cells specific against non-structural HCV sequences 6-10 weeks after the first infusion and during subsequent infusions. T-cell responses declined in one chimpanzee and high-level viremia developed at week 27. In the current year, cryopreserved plasma samples from the previous study were analyzed by deep sequencing of HCV. The results show that a minor HCV variant from the first infusion donor was transmitted and that it was able to persist for more than 6 months despite undetectable systemic viremia. Collectively, these results demonstrate (i) that trace amounts of HCV RNA, which appear sporadically in successfully treated patients, can be infectious and (ii) that transmission can be masked in the recipient by an extended eclipse phase prior to establishing high-level viremia. These results are now published (Veerapu et al., J Clin Invest 2014; 124:3469-78). The conclusions are supported by the detection of late relapse 8, 75 and 78 months after a sustained virological responses to interferon-based therapy in 3 of 103 patients followed in the Liver Diseases Branch. HCV sequence comparisons suggests that reappearance of HCV RNA years can be from relapse of the initial viral infection rather than reinfection from a different virus (Hara et al., J Infect Dis, 2014:209:38-45). We have also completed the virological and immunological analyses of cryopreserved blood and liver biopsy samples from chimpanzees that had been exposed to low amounts of HCV in previous studies. The results demonstrate that the induction of T cell responses by repeated low dose exposure does not protect upon subsequent HCV challenge. Rather, HCV&#61485;specific recall and de novo T cell responses as well as intrahepatic T cell recruitment and IFN-&#947; production were suppressed upon HCV challenge, concomitant to quantitative and qualitative changes in regulatory T (Treg) cells that began after subinfectious HCV exposure and increased after HCV challenge. In vitro Treg cell depletion restored HCV&#61485;specific T cell responses. Thus, T cells primed by trace amounts of HCV do not generate effective recall responses upon subsequent HCV infection. Rather, subinfectious HCV exposure predisposes to Treg cell expansion, which suppresses effector T cells during subsequent infection. Strategies to reverse this exposure&#61485;induced suppression should be examined to aid the development of T cell&#61485;based vaccines against HCV and other endemic pathogens. These results are now published (Park et al., Nat Medicine 2013; 19:1638-42). Project 2: Immunotherapy of chronic HBV and HCV infection We have continued the virological and immunological analyses of cryopreserved blood and liver biopsy samples from chimpanzee studies that have been conducted in previous years. These studies were designed to test whether effective antiviral T cell responses against HBV and HCV can be restored in chronic infection. In both HBV and HCV infections, virus-specific T cells are considered key factor in viral clearance, but their frequency is low and their function is impaired in those that have developed chronic infection. To test the hypothesis that successful antiviral T cell responses can be restored in chronic infection, we had in past years treated 2 chimpanzees with chronic HBV infection with autologous T cells that were transduce with a chimeric, antibody-based receptor (CAR) to HBsAg. We had also treated 2 chimpanzees with chronic HCV infection with autologous T cells that were transduced with T cell receptors from chimpanzees that had successfully cleared acute HCV infection. Several weeks after T cell transfer the HCV chimpanzees received a candidate vaccine to boost the T cell response. Whereas the studies with live chimpanzees had already been completed in previous years, we have now continued the analysis of cryopreserved biospecimens from those studies. Specifically, we have sequenced the virus to assess any sequence evolution/selection in T cell epitopes and we have studied the frequency and function of transferred HCV-specific T cells at multiple time points during the study. This analysis will continue in the next year.