Broadly neutralizing antibodies abrogate established hepatitis C virus infection

Abstract

In most exposed individuals, hepatitis C virus (HCV) establishes a chronic infection; this long-term infection in turn contributes to the development of liver diseases such as cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. We demonstrate that three broadly nAbs-AR3A, AR3B, and AR4A-delivered with adeno-associated viral vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a therapeutic approach to interfere with HCV infection by exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes to sustain a chronic infection.

Figure 1. Prophylactic efficacy of broadly neutralizing anti-HCV antibodies

(a) A pool of AAV vectors expressing the three nAbs AR3A, 3B and 4A or control nAb B12 were injected intramuscularly in immunodeficient NRG mice and human IgG in mouse serum was measured by ELISA (b) FVB mice were injected with AAV vectors expressing the nAbs AR3A, 3B, 4A or control nAb B12 or a luciferase expressing AAV (luc2) and serum human IgG levels were measured by ELISA. (c) Sera from FVB mice that were injected with the AAV-nAb was used for in vitro neutralization assays of intergenotypic HCVcc on Huh-7.5 hepatoma cells. Indicated are the genotypes and origin of the structural proteins of the challenge strains. IC50 values are depicted at mg/ml of human IgG in mouse serum. (d) R26-Fluc mice were administered AAV-nAbs. Once nAb reached peak titers, HCV entry factors were adenovirally delivered to the liver and challenged with HCVcc expressing Cre recombinase, after which bioluminescence was measured. P values comparing B12 to AR antibodies, one way ANOVA. (e) Highly engrafted human liver chimeric FNRG mice were either injected with the pool of three nAb expressing AAV vectors (n=3) or control B12 AAV (n=3), or received three injections of a pool of purified nAbs (n=2) or b6 control nAb (n=3) and challenged with low dose H77. HCV RNA copies in mouse serum were measured by qRT-PCR. P=0.012, two way ANOVA for pooled experiments.

Figure 2. Combination treatment of primary human hepatocyte cultures with broadly neutralizing anti-HCV antibodies abrogate established HCV infection

(a) The three nAbs AR3A, 3B and 4A alone or combined (ARx3) were used for neutralization assays on human fetal liver cultures (HFLCs) and compared to the previously described nAb 3/11 or control mAb b6. Antibodies at 1μg/ml were added to cultures 1 hour prior to infection with HCVcc-GLuc and 6 hours later cultures were washed and maintained in media without nAbs for serial luminescence sampling. P values for ARx3 vs b6 on day 2 and day 9, unpaired t-test. (b) HFLC were infected with HCVcc-Gluc and after three days nAbs were added at 1μg/ml, which were maintained in the media for the duration of the experiment. (c) Pooled end point luminescence data from 3 out of 4 HFLCs that supported viral replication and received nAbs starting three days after infection with HCVcc-Gluc. P values by unpaired t-test.

Figure 3. Combination treatment of human liver chimeric mice with broadly neutralizing anti-HCV antibodies clears established HCV infection

(a) Highly engrafted huFNRG mice were infected with HCVcc (J6/JFH) and 25 days after infection, when all mice were viremic, were injected with either the pool of nAb AR3A, 3B and 4A or control mAb b6 at time points indicated by the grey arrows. Human IgG levels in mouse serum were determined by ELISA longitudinally until they became undetectable. (b) In the same group of mice from figure (a) hAlb levels in mouse serum were measured by ELISA for the duration of the experiment. (c) Starting one day after nAb were injected, HCV serum infectivity as measured in a limiting dilution assay on Huh-7.5 hepatoma cells. (d) HCV RNA was determined in mouse serum by qRT-PCR for the duration of the experiment. On day 163 post infection and 56 days after nAb levels had fallen below the LOD, the three surviving mice were challenged with HCV (H77) (red arrow) and their viremia since rechallenge is depicted by red circles. Each dot represents an individual liver chimeric animal. Lowest P value for individual time point is 0.01 on treatment day 13 by unpaired t-test, P=0.067 for experiment by two-way ANOVA. (e) Repeat experiment in which J6/JFH viremic huFNRG mice were treated with injections of nAb (grey arrows) and HCV RNA serum levels were determined by qRT-PCR. One surviving mouse was rechallenged with H77 (red arrow) and serum RNA is depicted in red circle. Lowest P value for individual time point is 0.016 on day 39 by unpaired t-test, (f) Combined graph of two experiments with H77 infected huFNRG mice. Viremic mice received 5 (squares) or 7 (circles) injections of nAb (arrows) and HCV RNA in mouse serum was determined by qRT-PCR.