Neutralizing antibody response during acute and chronic hepatitis C virus infection

Abstract

Little is known about the role of Abs in determining the outcome of hepatitis C virus (HCV) infection. By using infectious retroviral pseudotypes bearing HCV glycoproteins, we measured neutralizing Ab (nAb) responses during acute and chronic HCV infection. In seven acutely infected health care workers, only two developed a nAb response that failed to associate with viral clearance. In contrast, the majority of chronically infected patients had nAbs. To determine the kinetics of strain-specific and crossreactive nAb emergence, we studied patient H, the source of the prototype genotype 1a H77 HCV strain. An early weak nAb response, specific for the autologous virus, was detected at seroconversion. However, neutralization of heterologous viruses was detected only between 33 and 111 weeks of infection. We also examined the development of nAbs in 10 chimpanzees infected with H77 clonal virus. No nAb responses were detected in three animals that cleared virus, whereas strain-specific nAbs were detected in six of the seven chronically infected animals after approximately 50 weeks of infection. The delayed appearance of high titer crossreactive nAbs in chronically infected patients suggests that selective mechanism(s) may operate to prevent the appearance of these Abs during acute infection. The long-term persistence of these nAbs in chronically infected patients may regulate viral replication.

Fig. 1.

HCV-specific nAb response. (A) Plasma samples from uninfected individuals (cont 1 and cont 2) and those chronically infected with HCV genotypes 1, 2, 3, and 4, with low (+, <10³ copies per ml) and high (++, >10³ copies per ml) viral RNA levels, were tested for their ability to neutralize pseudotype viruses bearing H (HIV-HCV H) and MLV (HIV-MLV) gps at plasma dilutions of 1/200 and 1/1,000. The graph depicts infectivity, expressed as luciferase RLU, of HIV-HCV H and HIV-MLV (RLU ×10) in the presence of various plasma levels. Values are the mean of quadruplicate wells with the standard deviation shown. (B) Plasma from two chronically HCV-infected (79 and 552) individuals were tested for neutralization of HIV-HCV H at final dilutions of 1/200 and 1/1,000 before and after depletion of IgG. As a specificity control, virus infection in the presence of plasma from an uninfected individual (cont 1) is shown. Virus infectivity is shown as RLU, and values are the mean of quadruplicate wells with the standard deviation shown. (C) Neutralization of HIV-HCV H by plasma IgG purified from chronically infected patient 552. Data are shown as percentage neutralization, derived from quadruplicate wells with the standard deviation shown.

Fig. 2.

Strain-specific and crossreactive nAb responses in patient H. (A) Sequential plasma samples from chronically infected patient H were monitored for their ability to neutralize pseudotype viruses bearing H77, H, and HCJ4 gps at final dilution of 1/200. Data are shown as percentage neutralization. (B) Neutralization titer of plasma for HIV-HCV H77, defined as the dilution of plasma able to reduce virus infectivity by 90%. (C) Plasma samples (tested at a dilution of 1/100) were tested for anti-NS3 (circle) and anti-E1E2 (square) IgM (open symbols) and IgG (filled symbols) responses. Data are represented as a P/N ratio, calculated by dividing the OD value of a test serum by that obtained with an irrelevant HCV-negative human serum. P/N values >2 were considered positive. All infections were performed in quadruplicate, and the data are representative of two independent experiments.

Fig. 3.

The nAb response in experimentally infected chimpanzees. Sequential plasma samples from six chronically H77-virus-infected chimpanzees were monitored for viral RNA levels, nAb for pseudotype virus bearing autologous H77 gp (HIV-HCV H77), and anti-E1E2 and anti-HVR reactivity. All plasma samples were tested at a dilution of 1/100. Data are shown as percentage neutralization. The anti-E1E2 and anti-HVR Ab data are represented as a P/N ratio, calculated by dividing the OD value of the test sera by that obtained with a preimmune serum. P/N values >2 were considered positive. All assays were performed in quadruplicate, and the data are representative of two independent experiments.

Fig. 4.

Comparative neutralization titer of plasma from chronically infected chimpanzees and patients. Plasma from chronically infected chimpanzees 1536, 6412, and 6475 collected at various weeks after infection (shown in parentheses) and two patients, 79 and 513, were tested for their ability to neutralize pseudotype virus bearing H77 gps (HIV-HCV H77). Data are shown as percentage neutralization. All assays were performed in quadruplicate, and the data are representative of two independent experiments.