Evidence for protection against chronic hepatitis C virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus.

Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved HCV infection. The difference in the rate of chronicity between the immunized and the control animals was close to statistical significance (P = 0.067). Immunized animals developed vigorous gamma interferon enzyme-linked immunospot responses and moderate proliferative responses. To investigate cross-genotype protection, the immunized recovered chimpanzees were challenged with a pool of six major HCV genotypes. During the acute phase after the multigenotype challenge, all animals had high-titer viremia in which genotype 4 dominated (87%), followed by genotype 5 (13%). However, after fluctuating low-level viremia, the viremia finally turned negative or persisted at very low levels. This study suggests the potential efficacy of replicating recombinant vaccinia virus-based immunization against chronic HCV infection.

Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees.

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-naïve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.

Attempted therapeutic immunization in a chimpanzee chronic HBV carrier with a high viral load.

BACKGROUND: We previously reported successful therapeutic immunization in a chimpanzee having a relatively low viral load, which was immunized with recombinant plasmid hepatitis B surface antigen (HBsAg) DNA and boosted with recombinant HBsAg encoding canarypox virus. In the present study, we attempted to confirm these findings in an animal with a high virus load. METHODS AND RESULTS: We tested three immunization strategies successively over a 3-year period. In the first of these, we administered four monthly injections of DNA encoding HBsAg + PreS2 + hepatitis B core antigen (HBcAg) + DNA encoding interleukin (IL)-12, (given 3 days later), and boosted with canarypox expressing all of the above HBV genes 6 months after initial immunization. No reduction in viral load was observed. In the second trial, we administered lamivudine for 8 weeks, and then began monthly DNA-based immunization with plasmids expressing the above viral genes; however, viral loads rebounded 1 week after termination of lamivudine therapy. In a third trial, we continued lamivudine therapy for 30 weeks and immunized with vaccinia virus expressing the above viral genes 18 and 23 weeks after the start of lamivudine therapy. Again viral loads rebounded shortly after cessation of lamivudine treatment. Analysis of cell-mediated immune responses, and their avidity, revealed that DNA-based immunization produced the strongest enhancement of high avidity T-cell responses, while recombinant vaccinia immunization during lamivudine therapy enhanced low avidity responses only. The strongest low and high avidity responses were directed to the middle surface antigen. CONCLUSIONS: Three strategies for therapeutic immunization failed to control HBV viremia in a chronically infected chimpanzee with a high viral load.

gC1qR expression in chimpanzees with resolved and chronic infection: potential role of HCV core/gC1qR-mediated T cell suppression in the outcome of HCV infection.

Chimpanzee is a unique animal model for HCV infection, in which about 50% of infections resolve spontaneously. It has been reported that the magnitude of T cell responses to HCV core in recovered chimpanzees is greater than that in chronically infected ones. However, the mechanism(s) by which the chimpanzees with resolved infection overcome core-mediated immunosuppression remains unknown. In this study, we examined the effect of HCV core on T cell responsiveness in chimpanzees with resolved and chronic HCV infection. We found that core protein strongly inhibited T cell activation and proliferation in chimpanzees with chronic infection, while this inhibition was limited in chimpanzees with resolved infection. Notably, the level of gC1qR, as well as the binding of core protein, on the surface of T cells was lower in recovered chimpanzees when compared to chimpanzees with chronic HCV infection. Intriguingly, the observed differences in gC1qR expression levels and susceptibility to core-induced suppression amongst HCV-chronically infected and recovered chimpanzees were observed prior to HCV challenge, suggesting a possible genetic determination of the outcome of infection. These findings suggest that gC1qR expression on the surface of T cells is crucial for HCV core-mediated T cell suppression and viral clearance, and that represents a novel mechanism by which a virus usurps host machinery for persistence.

Individual donor nucleic acid amplification testing for detection of West Nile virus.

We have developed an economical, high-throughput nucleic acid amplification test (NAT) for blood-borne viruses, suitable for use in the screening of plasma samples from individual blood donors. This assay system includes a semiautomated procedure, using 96-well glass fiber plates for the extraction of viral nucleic acids from plasma and "universal beacon" technology which permits the detection of all genotypes of highly variable viruses (e.g., human immunodeficiency virus and hepatitis C virus). In this detection system, two fluorescent- detection technologies were employed successfully in a single tube: molecular beacon for West Nile virus (WNV) detection using a 6-carboxyfluorescein fluorophore and TaqMan for internal control detection using a VIC fluorophore. To establish proof of concept, we focused on the development of a robust individual donor NAT for WNV. The assay showed no reactivity to 15 other viruses tested or to 420 blood donor samples from the WNV pre-epidemic season. No cross-contamination was observed on an alternating positive-/negative-well test. The sensitivity (limit of detection, 95%) of the assay for WNV is between 3.79 and 16.3 RNA copies/ml, depending on which material was used as a standard. The assay detected all positive blood donation samples identified by the Roche WNV NAT. The assay can be performed qualitatively for screening and quantitatively for confirmation.

Protection against chronic hepatitis C virus infection after rechallenge with homologous, but not heterologous, genotypes in a chimpanzee model.

An open question for hepatitis C virus (HCV) vaccine development is whether the various genotypes of this virus protect against the development of chronic infection after heterologous infection with different genotypes. We approached this question by challenging chimpanzees that had recovered from HCV genotype 1a or 1b infection with 6 heterologous genotypes as well as with a homologous genotype (for chimpanzees originally infected with genotype 1a). All 9 chimpanzees rechallenged with a homologous genotype developed self-limited infections. Of 11 chimpanzees challenged with 100 chimpanzee infectious doses of heterologous genotypes, 6 developed self-limited infections, with peak viral loads in acute-phase serum that were ~5-fold lower than those seen during primary infections. One chimpanzee (which had recovered from genotype 1b infection and was rechallenged with genotype 6a) did not develop viremia but did show an anamnestic cell-mediated immune response after rechallenge. Four of the 11 chimpanzees rechallenged with heterologous genotypes developed chronic infections with the genotypes used for rechallenge. These findings suggest that a universally protective HCV vaccine may need to incorporate epitopes from multiple genotypes.

Sustained E2 antibody response correlates with reduced peak viremia after hepatitis C virus infection in the chimpanzee.

Immune correlates of protection against hepatitis C virus (HCV) infection are not well understood. Here we investigated 2 naive and 6 immunized chimpanzees before and after intravenous challenge, 12 weeks after the last immunization, with 100 50% chimpanzee infectious doses (CID(50)) of heterologous genotype 1b HCV. Vaccination with recombinant DNA and adenovirus vaccines expressing HCV core, E1E2, and NS3-5 genes induced long-term HCV-specific antibody and T-cell responses and reduced peak viral load about 100 times compared with controls (5.91 +/- 0.38 vs. 3.81 +/- 0.71 logs, respectively). There was a statistically significant inverse correlation between peak viral loads and envelope glycoprotein 2 (E2)-specific antibody responses at the time of challenge. Interestingly, one vaccinee that had sterilizing immunity against slightly heterologous virus generated the highest level of E2-specific total and neutralizing antibody responses as well as strong NS3/NS5-specific T-cell proliferative responses. The other four vaccinees with low levels of E2-specific antibody had about 44-fold reduced peak viral loads but eventually developed persistent infections. In conclusion, vaccine-induced E2-specific antibody plays an important role in prevention from nonhomologous virus infection and may provide new insight into the development of an effective HCV vaccine.

Exposure to low infective doses of HCV induces cellular immune responses without consistently detectable viremia or seroconversion in chimpanzees.

In hepatitis C virus (HCV) infection, there is accumulating data suggesting the presence of cellular immune responses to HCV in exposed but seemingly uninfected populations. Some studies have suggested cross-reactive antigens rather than prior HCV exposure as the main reason for the immune responses. In this study we address this question by analyzing the immune response of chimpanzees that have been sequentially exposed to increasing doses of HCV virions. The level of viremia, as well as the immune responses to HCV at different times after virus inoculation, were examined. Our data indicate that HCV infective doses as low as 1-10 RNA (+) virions induce detectable cellular immune responses in chimpanzees without consistently detectable viremia or persistent seroconversion. However, increasing the infective doses of HCV to 100 RNA (+) virions overcame the low-inoculum-induced immune response and produced high-level viremia followed by seroconversion.