A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics

Abstract

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54^CHO, ZV54^WT, and ZV54^ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54^CHO and ZV54^ΔXF showed significant ADE activity while ZV54^WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54^WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Keywords: Fc effector function; Zika virus; antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent enhancement of infection (ADE); glycosylation; monoclonal antibody (mAb); plant-made antibody; plant-made pharmaceutical.

Figure 1

ZIKV neutralization by the ZV54 mAb glycovariants. The ZV54 mAb glycovariants were serially diluted and co-incubated with ZIKV (100 PFU) and Vero cells. Plaques were counted after 3 days of incubation, and the percent neutralization and IC50 were calculated with GraphPad. The experiments were repeated at least twice, with technical triplicates. The error bars represent the SDs of the means.

Figure 2

ADE activity of the ZV54 mAb glycovariants for DENV and ZIKV infection. DENV-2 (A) or ZIKV (B) samples were mixed with serial dilutions of the ZV54 mAb glycovariants or a generic IgG negative control and then incubated with FcγRIIa⁺ K562 cells. The cells were then washed, fixed, permeabilized, and stained with an anti-flavivirus E antibody. The DENV-2 or ZIKV infected cells were identified by flow cytometry. The experiments were performed at least twice, with technical triplicates for each sample of the presented results (means ± SDs).

Figure 3

NK cell-mediated ADCC activity of the ZV54 mAb glycovariants. The ZIKV-infected Vero cells (A) or uninfected Vero cells (B) were incubated with NK cells (E:T ratio = 5:1) in the presence of the ZV54 glycovariants or an IgG isotype negative control (hIgG). The percent of Vero cell lysis (mAb–NK cell-mediated ADCC target-cell killing) and the percent increase in ZV54 ADCC compared to the negative control hIgG were calculated. * and ns indicate p values of <0.05 and >0.05, respectively.

Figure 4

In vivo activity of the plant-made ZV54 glycovariants in mice. Five-weeks-old Ifnar1^−/− mice were inoculated with ZIKV and administered ZV54WT or PBS (negative control) intraperitoneally 24 h after ZIKV infection. Blood was collected on day 3 post-infection, and viremia was quantitated by RT-qPCR (A). Mice were further monitored daily for survival to 20 days post-infection (B). The results are representative of two independent experiments. ** indicates a p value of 0.0065.