Characterization of Anti-HCV Antibodies in IL-10-Treated Patients

Abstract

There is limited information on the direct role of the neutralizing antibody responses against hepatitis C virus (HCV) infection or methodologies to study them. Previously we have demonstrated that interleukin-10 (IL-10) administered to chronic hepatitis patients led to a decrease in disease activity, but an increase in HCV viral burden. The mechanism behind this is unknown. The objective of this study was to examine the antibody response in IL-10-treated patients. To establish a neutralization antibody assay, HCV-positive and HCV-negative sera were collected and incubated with HCV strain JFH-1 particles before culture with Huh 7.5 cells. Viral replication was measured a week later by either indirect immunofluorescence assay (iIFA) or real-time reverse transcriptase polymerase chain reaction (RT-PCR). After validation of the methodology, the sera from 30 previously-described subjects of a group previously treated with IL-10 were tested for the neutralization capacity of their antibodies. The amount of total anti-HCV antibody in the sera was also measured by direct staining of HCV full-length replicon cells. With this validated neutralization assay for anti-HCV antibodies we found that HCV-neutralizing antibodies are universally present, but with significantly different titers. In patients who were treated with IL-10, the total anti-HCV antibody titers appear to be constant, but with significantly decreased antibody neutralization activity. Our study validates an assay to quantitatively determine the presence and strength of HCV-specific neutralizing antibodies. We have found that IL-10-treated patients have significantly lower HCV antibodies, but maintain the total anti-HCV antibody titer, suggesting a novel mechanism by which IL-10 treatment increases viral load in patients.

FIG. 1.

Representative iIFA images of HCV neutralization with patient serum. Cells were seeded at 5000 cells per well in a 24-well plate and infected with either 100 μL of virus/100 μL diluents (positive control), 200 μL diluents (negative control), or 100 μL virus with 100 μL of sera at different dilutions. Cellular nuclei are stained with DAPI (blue), and HCV NS5A protein is stained with FITC (green). Representative figure out of 150. Color images available online at www.liebertonline.com/vim.

FIG. 2.

Neutralization of HCV-JFH1 by patient sera. HCV-positive or HCV-negative patient sera neutralization assay cell counts from immunofluorescence were averaged for each dilution. (A) Neutralization with HCV-positive sera. (B) Neutralization with HCV-negative sera. (C) Average of the HDL and LDL levels that were reported for the patient sera tested (normal values are: male HDL >40 mg/dL; female HDL >50 mg/dL; LDL <100 mg/dL for both genders). In A and B error bars represent the SEM of 150 and 5 patient sera experiments, respectively. In C error bars represent the standard deviation of the reported values.

FIG. 2.

Neutralization of HCV-JFH1 by patient sera. HCV-positive or HCV-negative patient sera neutralization assay cell counts from immunofluorescence were averaged for each dilution. (A) Neutralization with HCV-positive sera. (B) Neutralization with HCV-negative sera. (C) Average of the HDL and LDL levels that were reported for the patient sera tested (normal values are: male HDL >40 mg/dL; female HDL >50 mg/dL; LDL <100 mg/dL for both genders). In A and B error bars represent the SEM of 150 and 5 patient sera experiments, respectively. In C error bars represent the standard deviation of the reported values.

FIG. 3.

Genotype independence of the neutralizing antibodies. The EC50 dilution distribution was tabulated for each separate genotype available out of 150 patients. (A) Distribution of genotype 1. (B) Distribution of genotype 2. (C) Distribution of genotype 3. (D) Distribution of genotype 4. (E) Comparison of the average log EC50 of each genotype (and the separated combination for genotypes 1a and 1b). Error bars indicate the SEM of all of the experiments done.

FIG. 4.

Patient serum neutralizing activity in vitro is decreased after 12 mo of treatment with rIL-10, as observed by increased viral replication: A neutralization assay was performed in which patient sera were incubated at a 1:1 ratio with HCV-JFH1 viral particles (100 μL of sera per 100 μL of 1 × 10⁵ ffu/mL of HCV-JFH1 for 30 min at room temperature). Afterwards, each mixture was added to previously-seeded plates (5000 Huh 7.5 cells per well) and cultured for 24 h. At this point the wells were washed, and fresh media (virus-free) was added to the cultures and incubated for 6 more days. RNA was collected by Trizol isolation and real-time RT-PCR was run for HCV, with the housekeeping gene GAPDH as an internal control. Each point was calculated by the ΔΔCt method and compared against the positive control (virus alone in Huh 7.5 cells). Neutralization is represented as the log₁₀ of the inverse of those results. (A) HCV antibody neutralization at 0 and 12 mo for the three dosing groups. (B, C, and D) Sera was added directly to Huh 7.5 cells under the same conditions as described for the neutralization assay. HCV replication from patient sera compared to negative Huh 7.5-uninfected cells, error bars represent the SEM of triplicate determinations for each patient sample (*p < 0.05 as calculated by Student's t-test compared to 0-mo control).

FIG. 5.

HCV antibody titer in patient sera increases after 12 mo of IL-10 treatment. To test for HCV antibody titer in the patient sera we performed three dilutions of sera and used a secondary antibody (anti-human IgG conjugated with Alexa 488 [green]). Nuclei were counterstained with DAPI (blue). (A) Negative control (no sera). (B) A 1:1 dilution of patient sera. (C) Positive control, Huh 7.5 cells stained for NS5A antibody. Secondary antibody is FITC conjugated. (D) The calculated log EC50 of anti-HCV antibodies based on the iIFAs. Error bars indicate the SEM of triplicate determinations for each patient out of 10 patients per group. A statistical comparison by Student's t-test was done for each dose (*p < 0.05).

FIG. 5.

HCV antibody titer in patient sera increases after 12 mo of IL-10 treatment. To test for HCV antibody titer in the patient sera we performed three dilutions of sera and used a secondary antibody (anti-human IgG conjugated with Alexa 488 [green]). Nuclei were counterstained with DAPI (blue). (A) Negative control (no sera). (B) A 1:1 dilution of patient sera. (C) Positive control, Huh 7.5 cells stained for NS5A antibody. Secondary antibody is FITC conjugated. (D) The calculated log EC50 of anti-HCV antibodies based on the iIFAs. Error bars indicate the SEM of triplicate determinations for each patient out of 10 patients per group. A statistical comparison by Student's t-test was done for each dose (*p < 0.05).