Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C

Abstract

In contrast to a detailed understanding of antiviral cellular immune responses, the impact of neutralizing antibodies for the resolution of acute hepatitis C is poorly defined. The analysis of neutralizing responses has been hampered by the fact that patient cohorts as well as hepatitis C virus (HCV) strains are usually heterogeneous, and that clinical data from acute-phase and long-term follow-up after infection are not readily available. Using an infectious retroviral HCV pseudoparticle model system, we studied a cohort of women accidentally exposed to the same HCV strain of known sequence. In this single-source outbreak of hepatitis C, viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection. Neutralizing antibodies decreased or disappeared after recovery from HCV infection. In contrast, chronic HCV infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection despite the induction of cross-neutralizing antibodies in the late phase of infection. These data suggest that rapid induction of neutralizing antibodies during the early phase of infection may contribute to control of HCV infection. This finding may have important implications for understanding the pathogenesis of HCV infection and for the development of novel preventive and therapeutic antiviral strategies.

Fig. 1.

Synthesis and characterization of HCVpp derived from HCV isolate AD78. (A) HCV protein expression in 293T-transfected cells. 293T cells were transfected as described in Materials and Methods, including an expression vector coding for HCV envelope glycoproteins of isolate AD78 from the contaminated anti-D Ig batches. 293T-transfected cells, expressing HCVpp H77C, HCVpp AD78, HCVpp J, or control pp, were lysed, and proteins were incubated in the presence or absence of endoglycosidase H (EndoH) and separated by SDS/PAGE, followed by immunoblotting with anti-E1 mAb (11B7) and anti-E2 mAb (3E5). The positions of the molecular markers in kilodaltons are shown. (B) Infectivity of HCVpp. Huh7 cells were incubated with HCVpp for 3 h at 37°C. HCVpp H77C (■), HCVpp AD78 (◇), HCVpp J (▲), and control pp (□) entry into Huh7 cells was determined by measuring expression of GFP reporter gene by flow cytometry after 72 h. (C) Inhibition of HCVpp AD78 by anti-E2 antibodies. Antibodies (50 μg/ml) were preincubated with HCVpp AD78 for 1 h at room temperature. This mixture was then added to Huh7 cells and incubated for 3 h at 37°C. HCVpp entry into Huh7 cells was determined as described above. (D) Dose-dependent inhibition of HCVpp AD78 entry by mAb AP33. HCVpp AD78 were preincubated with different concentrations of mAb AP33 (▲), ALP 98 (◇) or control IgG (■) for 1 h. This mixture was then used to infect Huh7 cells as described above. (E) Dose-dependent inhibition of HCVpp AD78 entry by an anti-HCV positive serum from a patient with chronic hepatitis C. HCVpp AD78 were preincubated for 1 h with different dilutions of sera from a patient with chronic HCV (■) or from an anti-HCV negative individual (◇). This mixture was then used to infect Huh7 cells as described above. Data are expressed as means of triplicate determinations ± SD from one of at least three independent experiments. HCVpp, HCVpp containing HCV envelope glycoproteins; control pp, control pp without envelope glycoproteins; E1+ and E2+, deglycosylated forms of E1 and E2.

Fig. 2.

Neutralizing antibodies in patients with resolved or chronic hepatitis C. Anti-HCVpp neutralizing titers were determined by endpoint dilution of sera. HCVpp AD78 or control pp were preincubated for 1 h with serial serum dilutions before infection of Huh7 target cells. The endpoint titers of the early phase (1–6 months after infection) and late-phase (10–17 years after infection) serum samples are shown as scatter plots. The median titer is marked by a line. Data are expressed as means of two independent experiments performed in duplicate. Samples showing a titer of <1/20 were considered negative. The cutoff titer 1/20 is indicated by a dashed line.

Fig. 3.

Neutralizing antibodies in serial serum samples of individual patients. (A) Kinetic of neutralizing antibodies in serial serum samples of individual patients. Titers of anti-HCVpp neutralizing antibodies were determined by endpoint dilution of serial serum samples from 16 patients with resolved and 23 patients with chronic hepatitis C. Patients' identification numbers are indicated in boxes. Titers of neutralizing antibodies present in the early and late phase of infection in individual patients are connected through lines. (B) Plotting of sampling time (in days after infection) vs. neutralizing antibody titer during the acute phase of infection. Samples showing a titer of <1/20 were considered negative. The cutoff titer 1/20 is indicated by a dashed line.