FIG. 4.

Human serum or HDL protects HCVpp from neutralization. (A) Effects of sera from a cohort of acutely infected HCV patients (24) on infectivity of the indicated HCVpp. Data were obtained over a time interval of 16 weeks and are shown for four patients who are representative of the cohort. The HCV RNA kinetics for each patient were measured weekly after inclusion in the cohort (HCV RNAs [given in IU/ml], analyzed by means of a third-generation, branched-DNA-based assay [Versant HCV RNA 3.0 assay; Bayer Diagnostics, Tarrytown, N.J.]). The 13-patient cohort consisted of two groups. The first group (7/13 patients), represented by patients 3 and 4 (Pt-3 and Pt-4), showed a >3- to 4-log reduction in HCV RNA titers in the second half of the study period (24). Patients 8 and 9 (Pt-8 and Pt-9) represent the second group (6/13 patients), in whom replication levels remained high (<1-log decrease in HCV RNA titers) throughout the entire study period (24). Serum samples chosen from the beginning, middle, and end of the study period from these four patients were investigated in neutralization assays at 1/50 dilutions with HCVpp (10⁴ IU) of genotype 1b, strain CG1b (HCVpp-1b); HCVpp of genotype 1a, strain H77 (HCVpp-1a); or their HVR1-deleted counterparts (HCVpp-del1b and HCVpp-del1a, respectively) by incubation for 30 min at room temperature before infection of Huh-7 target cells. The results are expressed as mean percentages (±standard deviations [SD]; n = 3) of inhibition of the infectious titers relative to inhibition from incubation with medium devoid of patient sera. Note that no or poor neutralization was detected in Pt-3 and Pt-4 (group 1) with HCVpp-1a, and no neutralization at all was detected in Pt-8 and Pt-9 (group 2) with both HCVpp-1a and HCVpp-1b (Table 2). The specificity of neutralization was controlled with RD114pp, against which no antibodies were detected in HS (3). Nonspecific inhibition of RD114pp (data not shown) over a value of ±20% was never detected. (B) Titration of neutralizing antibodies in total IgG purified from chronically infected HCV patients. Neutralization assays were performed in the absence of HS (no HS) or in the presence of 2.5% HS, 39 μg/ml LDL, or 6 μg/ml HDL. The results are expressed as mean percentages (±SD; n = 4) of inhibition of the infectious titers relative to inhibition from incubation with medium devoid of antibodies. (C) Neutralization curves of the AP33 monoclonal HCV E2 antibody in the absence (no HS) or presence of 2.5% HS using HCVpp harboring the indicated point mutations in HVR1. The results are expressed as the mean percentages (±SD; n = 3) of inhibition of the infectious titers relative to incubation with medium devoid of antibodies. ID₅₀ values are indicated by dotted lines.