Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus

Abstract

Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 x 10(-3) and 1.48 x 10(-3) nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence.

FIG. 1

Clinical, virologic, and serologic responses in Ch1535 (A and B) and Ch1536 (C and D) following inoculation with RNA transcripts representing the HCV infectious clone. (A and C) Serum RNA, ALT, and inflammatory responses (numbers across the top of the graph), graded from 0 to 4 (representing none, minimal, mild, moderate, and marked). (B and D) Antibody responses to recombinant E1E2 antigen (open squares) and positivity in commercial EIAs [EIA (+)]. Anti-E1E2 antibody responses are represented as P/N ratios, with the cutoff of 2 (twice the value of the preserum sample) indicated by the dotted line.

FIG. 2

(A) HVR-1-specific antibody responses in Ch1535 and Ch1536 as determined by peptide ELISA, with a 20-mer peptide representing the predicted amino acid sequence encoded by the HCV infectious clone p90 (HVR1-p90). The values are expressed as P/N, with the cutoff of 2 indicated by the dotted line. The data are representative of three separate experiments. (B) ELISA blocking assay. HVR1-specific antibodies were blocked in serum from Ch1536 obtained at week 60 p.i. HVR1-p90 represents the sequence expressed by the infectious clone. H8 represents a negative peptide that does not cross-react with antibody to HVR1-p90 and is not recognized by serum from Ch1536. The data are representative of three separate experiments. pre, preimmune serum.

FIG. 3

Predicted amino acid sequence of HVR1 in Ch1535 (A) and Ch1536 (B) serum, liver, and PBMCs at selected time points following inoculation with the infectious clone. The sequence p90 corresponds to the predicted amino acid sequence encoded by the RNA transcripts used for inoculations. HVR1 is underlined. The sequence displayed is the majority sequence as determined by analysis of purified PCR product. Dashes represent identical residues. aa, amino acid.

FIG. 4

Reactivity of serum from Ch1535 at week 17 p.i. following serial dilution with peptides p90, 1536-51, and H3 (negative or nonreactive peptide sequence).