Successful anti-scavenger receptor class B type I (SR-BI) monoclonal antibody therapy in humanized mice after challenge with HCV variants with in vitro resistance to SR-BI-targeting agents

Abstract

Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect.

Conclusion: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting.

Figure 1. In vitro neutralization assay

Huh7.5 cells were pre-treated with 20 μg/ml mAb1671 (A) and 2 μM ITX-5061 (small molecule SR-BI antagonist) (B) before infection with Jc1wt, Jc1ΔHVR1, Jc1G451R, Jc1mtCD81 and J6/JFH1 Clone2. After two days the number of HCV-positive clusters was counted and normalized to control. The effect of mAb1671 on the infectivity of Jc1wt, ΔHVR1 and mtCD81 was evaluated in ten separate wells over four different experiments, while the effect on Jc1G451R and J6/JFH1 Clone2 was assessed over eight separate wells in three different experiments. The data of these experiments was merged and the means are shown. The asterisks (*: P<0.05; and ***: P<0.001) indicate that the effect of mAb1671 on Jc1ΔHVR1, Jc1G451R, Jc1mtCD81 and J6/JFH1 Clone2 differs significantly from its effect on Jc1wt infectivity. The effect of ITX-5061 was assessed in one experiment and the means of duplicates are shown (this limited sample size did not allow statistical analysis). (C) HCVcc infectivity under increasing concentrations of mAb1671. All conditions were tested in quadruplicate and the mean values are shown. (D) Box-and-whisker presentation of cell-to-cell spread. While mAb1671 (20 μg/ml) and ITX-5061 (2 μM) efficiently inhibit direct cell-to-cell transmission of Jc1wt, only a minor effect can be observed against Jc1ΔHVR1 (***: P<0.001). For each condition, the amount of infected target cells per cluster was determined in at least 100 clusters and normalized to the median of the control. The box extends from the 25^th and 75^th percentile, while the whiskers indicate the 10^th and 90^th percentile. The red horizontal line indicates the median. Error bars in panel A, B and C represent the standard error of the mean.

Figure 2. Efficacy of the SR-BI-specific antibody mAb1671 in blocking HCV dissemination in humanized mice

Within a 2-week period (indicated by the gray area) the animals received 6 intraperitoneal injections, each containing 400 μg of the antibody. The antibody was tested in two different settings: (A) a prevention experiment where the first antibody dose was administered one day before viral challenge; and (B–D) a post-exposure setup where the anti-SR-BI therapy was initiated three days post-viral challenge. Antibody-treated mice are indicated with a dotted line, whereas non-treated control animals are represented by solid lines. Chimeric mice were challenged at day 0 with Jc1wt (B), Jc1ΔHVR1 (A and C), Jc1G451R (D) or Jc1mtCD81 (E). Each data point represents the plasma HCV RNA level (IU/ml) of an individual chimeric mouse at a given time point. The limit of detection (LOD) equals 750 IU/ml.

Figure 3. Buoyant density gradient analysis of HCV produced in cell culture and in humanized mice

Serum was collected over a two month infection period from humanized mice inoculated with cell culture produced (HCVcc) Jc1wt and Jc1ΔHVR1. Pooled serum containing mouse-passaged HCV, designated mHCV, and culture supernatant containing HCVcc was ultracentrifuged over an iodixanol gradient. Twelve fractions were collected from the top of the gradient and analysed for cell culture infectivity (in triplicates) expressed as FFU/ml.

Figure 4. In vitro anti-SR-BI mAb1671 sensitivity determination

(A) Huh7.5 cells were pre-treated with 2 μg/ml mAb1671 before infection with cell culture and mouse-passaged Jc1wt (green) and Jc1ΔHVR1 (red). Two days after infection, HCV-positive clusters were enumerated. (B–D) The different density fractions with in vitro detectable infectivity were incubated with mAb1671-pretreated Huh7.5 cells. Two days later the number of HCV-positive cell clusters was determined. All conditions were tested in quadruplicate and the means are shown. Error bars represent standard error of the mean.

Figure 5. Effect of HDL and VLDL on in vitro HCVcc neutralization

Huh7.5 cells were pre-treated with 230 μg human HDL cholesterol/ml or 180 μg human VLDL cholesterol/ml alone or in combination with 20 μg/ml mAb1671 (HDL and VLDL concentrations correspond with levels detected in serum from humanized uPA-SCID mice) before infection with Jc1wt, Jc1ΔHVR1, Jc1G451R, Jc1mtCD81 and J6/JFH1 Clone2. After two days, the number of HCV-positive clusters was enumerated. The data shown for Jc1wt, Jc1ΔHVR1 and Jc1mtCD81 originates from three (HDL and combination with mAb1671) and two (VLDL and combination with mAb1671) individual experiments, whereas the effect on Jc1G451R and J6/JFH1 Clone2 was assessed over two experiments (HDL and combination with mAb1671) and in one individual experiment (VLDL and combination with mAb1671). In each experiment all conditions were tested in duplicate and different batches of VLDL or HDL were used. Error bars represent standard error of the mean. The asterisks (*: P<0.05, **: P<0.01 and ***: P<0.001) indicate statistically significant differences, whereas “ns” stands for not significantly different.

Figure 6. In vitro effect of HDL in combination with different anti-receptor therapies

Huh7.5 were pre-treated with 4 μM ITX-5061, 0.2 μg/ml JS81 and 20 μg/ml mAb1671 alone or in combination with 230 μg human HDL cholesterol/ml before infection with Jc1wt. After two days, the number of HCV-positive clusters was enumerated. All conditions were tested in duplicate and error bars represent standard error of the mean.