Characterization of linear epitope specificity of antibodies potentially contributing to spontaneous clearance of hepatitis C virus

Abstract

Background: Around 30% of the HCV infected patients can spontaneously clear the virus. Cumulative evidence suggests the role of neutralizing antibodies in such spontaneous resolution. Understanding the epitope specificity of such antibodies will inform the rational vaccine design as such information is limited to date. In addition to conformational epitope targeted antibodies, linear epitope specific antibodies have been identified that are broadly cross reactive against diverse HCV strains. In this study, we have characterized the potential role of three conserved linear epitopes in the spontaneous clearance of HCV.

Methods: We tested the reactivity of sera from chronic patients (CP) and spontaneous resolvers (SR) with linear peptides corresponding to three conserved regions of HCV envelope protein E2 spanning amino acids 412-423, 523-532 and 432-443 using ELISA. Subsequently, we characterized the dependency of HCV neutralization by the reactive serum samples on the antibodies specific for these epitopes using pseudoparticle-based neutralization assay. In ELISA most of the CP sera showed reactivity to multiple peptides while most of the SR samples were reactive to a single peptide suggesting presence of more specific antibodies in the SR sera. In most of the HCVpp neutralizing sera of particular peptide reactivity the neutralization was significantly affected by the presence of respective peptide. HCV neutralization by CP sera was affected by multiple peptides while 75% of the HCVpp neutralizing SR sera were competed by the 432 epitope.

Conclusions: These findings suggest that individuals who spontaneously resolve HCV infection at the acute phase, can produce antibodies specific for conserved linear epitopes, and those antibodies can potentially play a role in the spontaneous viral clearance. The epitope present in the 432-443 region of E2 was identified as the primary neutralizing epitope with potential role in spontaneous viral clearance and this epitope potentiates for the design of immunogen for prophylactic vaccine.

Fig 1. ELISA-based reactivity of three peptides corresponding to conserved linear epitopes, with CP and SR serum samples.

ELISA was performed using peptides corresponding to three conserved linear epitopes, the 412-epitope, the 432-epitope and the 523-epitope, against three different serum dilutions (10-fold, 100-fold, 1000-fold) of (a) 30 serum samples from chronic patients and (b) 49 serum samples from spontaneous resolvers. Dotted line represents the cutoff value using the mean value from five healthy serum controls plus three times the standard deviation.

Fig 2. Comparison of the CP and the SR sera for their reactivity to a single epitope or multiple epitopes.

Fig 3. Dose response curves of selected CP serum samples to measure their ED₅₀ values in HCVpp neutralization assay.

Dose response curves showing neutralization of HCVpp by serum samples of chronic patients. Serum samples at varying dilutions were mixed with HCVpp at 37°C and subsequently added to the wells containing Huh7.5 cells. After 72 h, cells were lysed and luciferase activity was measured. Subsequently, percent neutralization of each dilution was calculated by comparing with the control containing no serum.

Fig 4. Dose response curves of selected SR serum samples to measure their ED₅₀ values in HCVpp neutralization assay.

Dose response curves showing neutralization of HCVpp by serum samples of spontaneous resolvers. Serum samples at varying dilutions were mixed with HCVpp at 37°C and subsequently added to the wells containing Huh7.5 cells. After 72 h, cells were lysed and luciferase activity was measured. Subsequently, percent neutralization of each dilution was calculated by comparing with the control containing no serum.

Fig 5. Effect of peptides corresponding to different conserved linear epitopes on HCVpp neutralization by the SR serum samples.

HCVpp neutralizing serum samples of spontaneous resolvers were incubated at 1:50 dilution with different peptides at 125 μg/ml concentration at 37°C for 2 hr before performing HCVpp neutralization assay. Column bars labeled with HCVpp represent 100% infection; column bars labeled with “Serum” represent HCV neutralization by the respective serum sample in the absence of peptide; and column bars labeled with “Serum+412”, “Serum+432” or “Serum+523” represent neutralization by the serum samples after incubation of that serum with respective peptides. Plus and minus signs at the tops of each graph indicates the presence or absence of ELISA reactivity of that serum sample with individual peptides indicated by colors. HCVpp neutralization by the AP33 monoclonal antibody at 1 μg/ml concentration and the effect of the 412 peptide on this neutralization was also determined and shown in the bottom left panel. significance was defined as *p≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.