Anti-envelope antibody responses in individuals at high risk of hepatitis C virus who resist infection

Abstract

Injection drug users uninfected by hepatitis C virus (HCV) despite likely repeated exposure through high-risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti-HCV-envelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty-two hepatitis C diagnostic antibody- and RNA-negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus-neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti-envelope antibodies. In addition to confirming viral exposure, the presence of specific anti-envelope antibodies may be a factor that helps these individuals resist HCV infection.

Keywords: E1E2; exposed uninfected (EU); injection drug user (IDU); neutralization; neutralizing antibodies.

Figure 1

EU individuals show elevated IgG reactivity to HCV envelope proteins which correlates with lifetime risk of exposure. (a+b) Purified IgGs were tested for their ability to bind HEK‐293T‐expressed E1E2 in a (a) GNA capture gt 1a and (b) GNA capture gt 3a ELISAs. For those individuals where serum from multiple time points was available, each time point was tested individually, but only one value for each individual (i.e. the first sample taken) is plotted. Statistical differences between the groups were calculated using the Wilcoxon rank sum test (*P<.05). (c+d) Correlation between IgG reactivity to either gt 1a (c) or gt 3a (d) and total number of lifetime injecting episodes (duration of reported injection drug use multiplied by the frequency of reported injection drug use) was plotted for 37 EU (injection data not available for the remaining 5)

Figure 2

IgG and IgM responses to soluble gt 1a E2 are evident in EU individuals. IgG (a) and IgM (b) absorbance to purified gt1a sE2 protein was determined for EU and HC individuals using a modified ELISA protocol described in the Supplementary Methods. Statistical differences between the groups were calculated using the Wilcoxon rank sum test. **P<.01, *** P<.001

Figure 3

Neutralization of HCVpp by purified IgGs from EU. Virus pseudoparticle neutralization assays were performed using HCVpp bearing E1E2 derived from (a and c) gt 1a strain H77 or (b) gt 3 HCV. (a and b) Percentage reduction in HCVpp entry after incubation with 400 μg mL ⁻¹ test IgG is plotted as evidenced by luciferase reading at 72 hours compared to control. (c) Percentage reduction in HCVpp entry after incubation with reducing concentrations of IgG as shown. The 50% cut‐off is shown as a dashed lined. All results represent the average of at least 3 separate technical replicates, with error bars representing the SEM

Figure 4

EU IgG fails to compete with conformational antibodies to known epitopes on gt 1a E2. IgGs from individuals with neutralizing ability in the HCV pp system were selected for testing by competition ELISA to determine competitive binding with monoclonal antibodies to known conformational epitopes on E2 (Table S1). Samples from chronically infected individuals (CHCV) with known neutralizing activity were also included as positive controls. Percentage reduction in absorbance of the monoclonal antibodies was calculated and plotted. Significant competition would be expected at a level of 50% inhibition