Immunopathogenesis of HCV: Both CD4+ and CD8+ memory T cells are important in providing protective immunity from HCV reinfection. CD4+ T cells with a distinct cytokine profile contribute to HCV resolution in acute infections of chimpanzees. A subset of these cells that were CD4+ and CD8+ were specific for HCV,expressed IFN-gamma and IL-4. These highly activated but not cytopathic cells were important in the clearance of HCV in acute infections. HCV Vaccine studies: Vaccines based on the envelope glycoproteins of HCV induced only transient antibody responses in a chimpanzee. This animal became infected on rechallenge when the antibody titer dropped and the animal developed a persistent infection. A similar vaccine was used in a recovered chimp and it induced a sustained high level antibody response which protected the animal. The chimp had only a very brief very low level viremia detected on a single week without significant liver disease. A vaccine designed to induce T cell immunity to non-structural region antigens was shown also to provide protection from serious infection of HCV with a short lived viremia and minimal disease observed after challenge. This same vaccine was used in a chronic HCV carrier chimpanzee and a T cell response was observed without a significant change observed in the animal's clinical or virologic status. Dendritic cell based vaccines intended for immunotherapy are being tested in the chimpanzee model.~~~~~ Project Number: Z01 BK 04002-10 LHV Title: Biology and Molecular Biology of Hepatitis C Virus. Principal Investigator: Stephen Feinstone Summary: Two chimpanzees were infected in 1996 with RNA transcripts from cDNA clones of HCV, both animals developed persistent infections. The RNA, encoding a single polyprotein sequence, eliminated the quasispecies nature of standard virus-containing inocula and has allowed us to study in detail the molecular evolution of HCV in vivo beginning with a single sequence. We have previously reported the molecular evolution of the circulating HCV genomes after one year of infection and observed mutation rates of approximately 1.5E-3.0 nucleotide (nt) substitutions/site/year. These mutations localized to the envelope region, but not HVR1, and NS3 or NS5. Sequence analysis of the HCV genomes from both animals has been carried out periodically and we have observed an accumulation of amino acid mutations over time (4 and 6 at wk. 22, increasing to 14 and 16 at wk. 130). Early in infection most of the amino acid changes were located in E1E2 and NS3 but were included in other gene products by later time points. However, viral genomes from both animals at week 130 contained proportionately more mutations in E1E2 (50% and 62.5% for 1535 and 1536, respectively). These appeared to coincide with increased anti-E1E2 antibody titers, suggesting a response to immune pressure. While substitutions did occur in HVR1 these did not seem to represent immune escape mutants based upon cross reactivity data from an HVR1 peptide ELISA. These studies confirm that the persistence of infection is not solely due to changes in HVR1 and that extensive mutations are not incorporated into the HVR1 even following long-term infection in the presence of antibody, this seems to occur more in other regions of E1E2. These data contrast with many previous publications that have demonstrated rapid divergence of the HVR1 in humans and chimpanzees. After 2.5 years of infection the calculated nucleotide substitution rate remains close to that observed after 1 year, 1.6E-3.0 (1535) and 1.1E-3.0 (1536) nt substitutions/site/year. However, the amino acid (aa) substitution rate appears to decrease with time. The number of aa substitutions/site/year decreased from 4E-3.0 (1535) and 3.1E-3.0 (1536) at wk 26 to 2.6E-3.0 (1535) and 2.9E-3.0 (1536) at wk 60 to 1.9E-3.0 (1535) and 2.1E-3.0 (1536) at wk 130. These animals have now been persistently infected for 4 years and provide a unique means to study HCV mutation in vivo. Data will be presented on SSCP analysis to study the diversity of the circulating genomes with particular emphasis on the HVR1. Micro array analysis of HCV influence on gene regulation in the liver is being pursued in the chimpanzee model. Prospecitvely collected liver biopsies beginning prior to inoculation are being studied by a high density micro array system with colleagues at Stanford University. The kinetics of HCV infection in the chimpanzee is being studied with Avidan Neumann and others. Using real time quantiative RT-PCR (TaqMan) we have quantitated the HCV RNA in serum at weekly from prior to infection through the actute infection to recovery or into the chronic phase. Mathematical modeling is genreating extremely interesting insights into how HCV replicates and is controlled by host factors including the immune response and interferon. The molecular mechanisms of HCV interferon resistance are being investigated by several techniques. We are using a replicon to analyze the role of various HCV proteins such as NS5A and E2 to inhibit the interferon pathway at various points. Persistent HCV infection may be due, in part, to a high rate of resistance to interferon (IFN). The HCV envelope protein, E2, is an endoplasmic reticulum (ER)-bound protein that contains a region of sequence homology with the double-stranded (ds)RNA-activated protein kinase, PKR and its substrate, the eukaryotic translation initiation factor 2 (eIF2). E2 modulates global translation through inhibition of the IFN-induced antiviral protein, PKR. The PKR-eIF2 phosphorylation-site homology domain (PePHD) within E2 is required for inhibition of PKR. Both PKR and the PKR-like ER-resident kinase (PERK) induce the cessation of translation through phosphorylation of serine 51 on the alpha subunit of eIF2. ER stress is characterized by cellular responses that attempt to reduce the accumulation of aggregated and misfolded proteins. Signal transduction that results in the upregulation of chaperone gene expression and an arrest in protein synthesis are two features that help the cell cope with ER stress. PERK facilitates the arrest of protein synthesis during ER stress, which occurs during viral infection. The ER chaperone, BiP, maintains PERK in an inactive state at the ER membrane and HCV E2 activates BiP during the unfolded protein response (UPR). We found that E2 binds to PERK and inhibits autophosphorylation and phosphorylation of its substrates. In a chloramphenicol acetyl transferase (CAT) reporter assay overexpressed PERK resulted in a decrease in CAT expression. E2, however, stimulated translation of CAT in the presence of overexpressed PERK. The PePHD of E2 was required for the rescue of general translation inhibited by activated PERK. We report the inhibition of a second eIF2 alpha kinase by E2, which is consistent with a pseudosubstrate mechanism of inhibition. These findings may explain the relative resistance of HCV to IFN therapy and its mode of promoting persistent infection by overcoming the cellular IFN and ER stress responses.
