Screening and engineering of potent and broad neutralizing antibodies against SARS-CoV-2 infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the Betacoronavirus genus, has caused the COVID-19 pandemic with over 450 million infections and 6 million deaths. To fight the pandemic, it was necessary to discover effective measures for SARS-CoV-2 prevention and treatment. Besides vaccines, worldwide research efforts have been focused on potent neutralizing antibodies (NAbs). In this study, we hypothesize that potent and broad NAbs are essential for SARS-CoV-2 prevention and immunotherapy. We used single memory B cell antibody gene cloning to generate SARS-CoV-2 receptor binding domain (RBD)-specific monoclonal NAbs from the peripheral blood mononuclear cells (PBMCs) of infected individuals and vaccinees in Hong Kong. In the first panel of NAbs, the NAb B8 showed the strongest affinity (EC50 0.018 μg/ml) and neutralization (IC50 0.0095μg/ml). The in vivo efficacy of B8 was determined in golden Syrian hamsters against authentic wildtype SARS-CoV-2. Single injected B8 pre-treatment or post-exposure treatment protected lungs with reduced viral load and infected foci. There was, however, high viral load and infected foci in nasal turbinates (NT). These findings suggested insufficient protection in NT after systemic NAb treatments, which explained the high transmissibility of SARS-CoV-2 among vaccinated people.

As NT belong to airway mucosa where enough protective IgG antibody is difficult to reach, we hypothesize that neutralizing IgA is likely needed for mucosal protection. We then engineered B8 into monomeric IgA1 (B8-mIgA1), monomeric IgA2 (B8-mIgA2), dimeric (B8-dIgA1) and dimeric IgA2 (B8-dIgA2) with comparable in vitro neutralization potency. After intranasal SARS-CoV-2 challenge, we found that intranasal B8-dIgA pre-treatment reduced lung infections, decreased NT viral loads at 1 dpi but enhanced NT viral loads at 4 dpi compared with control hamsters. In contrast, systemic administration of B8-dIgA protected NT partially with reduced viral loads but had less protective efficacy in lungs. These findings demonstrated that different forms and delivery routes of IgA antibodies exbibit distinct characteristics in inhibiting SARS-CoV-2 infection, providing critical insights for mucosal immunity and protection.

Next, we hypothesize that engineering of bi-specific antibodies targeting different epitopes in RBD and non-RBD domains may improve antibody neutralization potency. Indeed, we engineered and demonstrated that a bispecific β-E5-B8 in form of dual-variable domain immunoglobin (DVD-Ig) had remarkable neutralization potency against SARS-CoV-2, providing a novel strategy for future research and development.

Lastly, after emergence of SARS-CoV-2 variants of concern (VOCs), we hypothesize that vaccinees with broad neutralizing antibody activities may contain broad monoclonal NAb. We successfully cloned another panel of NAbs from a BNT162b2 vaccinee. An RBD-specific NAb ZCB11 neutralized all live SARS-CoV-2 VOCs, including Omicron and OmicronR346K with low IC50 concentrations of 36.8 and 11.7 ng/mL, respectively. Pseudovirus-based mapping of 57 naturally occurred single mutations or deletions revealed that only S371L resulted in 11-fold neutralization resistance, but this phenotype was not observed in Omicron variants. Furthermore, prophylactic administration of ZCB11 protected lungs against the circulating Delta and Omicron variants in hamsters, respectively. These results demonstrated that BNT162b2-induced ZCB11 is a potent and broad NAb against pandemic VOCs.