Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis c virus infections uncover molecular determinants for E2 targeting and vaccine design

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1–6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1–6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.</div

Substitution L665W in E2 of HCV increased resistance against antibody AR5A.

<p>(A) Huh7.5 cells were transfected with in vitro transcribed RNA of the indicated recombinants. Supernatants were collected and HCV infectivity titers were determined as indicated in Materials and Methods. (B) 1st passages of the indicated viruses were subjected to a ten-fold dilution series of AR5A starting at 50 μg/ml. The virus/antibody mixes along with virus only were added to Huh7.5 cells and after 48 hour infection the cells were immunostained and the number of FFUs per well were counted. Neutralization data are shown as the mean of four replicates normalized to 8 replicates of virus only. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Error bars represents the standard errors of the mean. *Virus harbored the substitution T534A (E2).</p

The substitutions L665S and S680T in E2 are both required to confer complete AR5A resistance to J6/JFH1_ΔHVR1 without affecting virus fitness.

<p>Huh7.5 cells were transfected with in-vitro transcribed RNA of the recombinant viruses with (A) single or (B) combined substitutions I345V, L665S, and S680T. Supernatants were collected at the indicated time-points and HCV infectivity titers were determined. 1st passages of the viruses with (C) single or (D) combined substitutions I345V, L665S and S680T were subjected to a ten-fold dilution series of AR5A starting at 50 μg/ml. The virus/antibody mixes along with virus only were added to Huh7.5 cells and after 48 hour infection the cells were immunostained and the number of FFUs per well were counted as described in Materials and Methods. Neutralization values are the mean of four replicates and normalized to eight replicates of virus only. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Error bars represent the standard error of the mean.</p

Culturing J6/JFH1_ΔHVR1 with AR5A resulted in the selection of a resistant virus.

<p>(A) Huh7.5 cells were infected with virus J6/JFH1_ΔHVR1 and treated with 20, 10, or 5 μg/ml of AR5A antibody during the indicated period of time. (B) 1st passage of virus supernatant from the 20 μg/ml treatment and the untreated control were subjected to a ten-fold dilution series of AR5A starting at 50 μg/ml. The virus/antibody mixes along with virus only were added to Huh7.5 cells and after 48 hour infection the cells were immunostained and the number of FFUs per well were counted. Neutralization data are shown as the mean of 4 replicates. Neutralization was related to infection in the absence of antibody. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Error bars represent the standard error of the mean.</p

Substitution L665W increased AR5A resistance of HCV genotypes 1–6.

<p>1st passage virus stocks of the indicated viruses were subjected to a dilution series of AR5A and the IC₅₀ was determined for virus with (A) and without HVR1 (B). Numbers represent the number of times that the IC₅₀ of a HCV virus with L665W is higher than its parental virus. J6/JFH1_L665W harbored the substitution I262L, S52/JFH1_L665W harbored the substitution I355F and S52/JFH1_ΔHVR1/L665W harbored the substitutions A372V, Q454H/q and F580V. *IC₅₀ could not be determined as less than 50% neutralization was observed at the highest tested AR5A concentration.</p

L665W decreased AR5A binding to H77/JFH1 particles and to the E1/E2 complex in H77/JFH1 infected cells, but did not alter receptor dependency.

<p>(A) Immunoprecipitation was carried out using anti-E1/E2 antibodies AR5A, AR4A, or the anti-E2 antibody, AR3A, or an irrelevant IgG as described in Material and Methods. RNA was measured in duplicates by RT-PCR (values given in international units, IU). The results represent the mean of the total amount of RNA in each sample. The error bar represents standard deviation. *HCV RNA titer below assay cut-off. (B) Huh7.5 cells were infected with virus H77/JFH1 or H77/JFH1_L665W. After 48 hours cells were fixed and incubated with primary antibodies against NS5A (9E10) and E1/E2 (AR5A). Nuclei were counter-stained using Hoechst. Antibody binding was visualized using specific secondary antibodies coupled to fluorophores Alexa488 or Alexa594. Images were acquired using a Zeiss Axio Observer Z1. (C-E) Huh7.5 cells were incubated for 1 hour with dilution series of blocking antibodies against either (C) CD81, (D) SR-BI, or (E) LDLr or control antibody (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006214#sec014" target="_blank">Material and Methods</a> for specific antibodies). The indicated virus supernatants were added to Huh7.5 cells and incubated for 4 hour prior to wash and addition of fresh medium. Following a total of 48 hour infection the cells were immunostained and the number of FFUs per well were counted. Values are means of four replicates and normalized to 8 replicates of virus only. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Errors bars represent the standard errors of the mean.</p