Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein

Abstract

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

Figure 1

Alignment of the amino acid sequences (in single-letter code) predicted from a portion of the E1 and E2 genes of HCV, strain H77, spanning map positions 317–508 (13). H77 denotes the sample obtained from a plasmapheresis unit collected from patient H during the acute phase of posttransfusion NANB hepatitis (31), which represents the standard viral stock used for all the inocula, which had been previously titrated in chimpanzees (32). H77-1 and H77-2 denote two distinct sequences obtained independently in our laboratory by direct sequencing, following PCR amplification of cDNA from the same sample (H77). One of the two sequences was previously reported (23). The HVR1 of the E2 protein, spanning map position 384–414 (13), is shown in boldface type. The boxed sequences denote the sequence of the peptides (HVR1-A and HVR1-B) used for generating hyperimmune rabbit sera. Matches are indicated by vertical bars and changes are indicated by colons.

Figure 2

Course of HCV infection in chimpanzees after antibody-mediated in vitro neutralization. Details of the in vitro neutralization test, of the source of HCV, and of the antisera used in this study are provided in Materials and Methods. Neutralization of HCV was attempted with the preimmune rabbit serum and with an anti-HCV negative human plasma (A), with the hyperimmune rabbit anti-HVR1 serum (B), or with the H79 human plasma (C). The upper arrows indicate the time of challenge. The shaded areas indicate the values of serum ALT. Normal ALT values in chimpanzees range between 6 and 38 units/liter. Open bars indicate negative results for serum HCV RNA by PCR and solid bars indicate positive results. The horizontal bar indicates the time during which serum was positive for antibodies to HCV, as detected by second generation ELISA assay. Chimpanzee 1442 was described previously (11).

Figure 3

Predicted amino acid sequence alignment of 31 amino acids (in single-letter code) representing HVR1 of the E2 gene, spanning map position 384–414, of 104 molecular clones obtained from HCV, strain H77, which was used for inoculation. The sequence obtained by direct sequencing is indicated at the top for comparison. The standard viral stock used for all the inocula (H77) was obtained from patient H during the acute phase of posttransfusion NANB hepatitis (31). The remaining sequence of the 191 amino acids spanning map position 318–508, which is not shown, is available upon request.

Figure 4

Comparative sequence analysis of the sequence of the H77 virus used for inoculation and the viruses recovered from the chimpanzees 2 weeks after inoculation. The two sequences indicated for H77 were obtained independently by direct sequencing (Dir. seq.). For each chimpanzee the results of both direct sequencing and sequencing of 9 or 10 molecular clones are indicated.