Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents
Neutralizing antibodies (NAbs) play a critical role in the prevention and treatment of COVID-19. However, most of the receptor-binding domain (RBD)-targeting antibodies already been escaped by the Omicron variants, indicating that previous NAb drug screening strategies are insufficient against the fast-evolving SARS-CoV-2.
In this article, we described a strategy to identify a broad sarbecovirus-neutralizing antibodies that would be hard to evade by future SARS-CoV-2 variants using high-throughput epitope determination. The resulting NAb pair named SA55+SA58 demonstrates high neutralizing potency and breadth against SARS-CoV-2 variants including the latest BQ.1.1 and XBB, and protects mice from BA.1 and BA.5 infection, making it a valuable bsNAb drug candidate for prophylactic or therapeutic usage.
A rational strategy finds broad SARS-CoV-2 NAbs that are hard to evade
Besides the previous antibody selecting criteria, “potent against the circulating variants at the time” and “2 antibodies forming a non-competing antibody cocktail”, we proposed the other two for identifying RBD-targeting broad NAb drugs against SARS-CoV-2: (1) broad NAbs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations; (2) their interacting antigen residues should be associated with sarbecovirus conserved sites and critical viral functions, making the antibody-escaping mutations less likely to appear.
We choose SARS-CoV-2-vaccinated SARS convalescents as the antibody source since their memory B cells are more likely to encode NAbs that target sarbecovirus conserved epitopes. Droplet-based single-cell V(D)J sequencing were used to get a larger collection of antibody sequence. The epitope distribution and escaping mutation analyses were performed by high-throughput epitope mapping based on deep mutational scanning (DMS) (Figure 1).
Broad NAbs from SARS-CoV-2-vaccinated SARS convalescents form non-competing cocktail
Among all the antibody groups, group E3, F1, and F2 antibodies demonstrate broad specificity to sarbecoviruses in all clades. E1+F2 and E1+F3 are the two practical strategies to build non-competing bNAb cocktails. In comparison, E1 and F3 antibodies only bind ACE2-utilizing sarbecoviruses, and are generally rare in SARS-CoV-2 convalescent individuals/vaccinees. Besides, the epitopes of E1 NAbs are centered around N343 glycan and F3 NAbs’ escaping mutations mainly focus on the binding interface of ACE2. SA58 and SA55 showed the highest authentic virus neutralization activity against both the SARS-CoV-2 ancestral strain and Omicron BA.1 among E1 and F3 antibodies, respectively.
We examined the escape profiles of the E1 and F3 NAb candidates (Figure 2). Most E1 NAbs are susceptible to mutations of T345 and R346. Mutations of T345 could lead to prominent escapes from SA58 since T345 is enclosed by Y105H, L94L, and W96L. F3 NAbs are mainly escaped by mutations of V503, G504, and D405. SA55 are susceptible to the changes on V503 and G504. Nevertheless, G504 is also critical for ACE2 binding as shown by DMS, and both V503 and G504 are conserved among sarbecoviruses, making it difficult for variants with mutations on these sites to prevail.
Structures of featured E1 and F3 NAbs in the complex of Spike or RBD are solved using Cryo-EM or crystallography, respectively (Figure 3). As expected, SA55 and SA58 can simultaneously bind the RBD. Antibodies in the same group bind to a similar epitope on RBD, with a slight variation in detailed interactions.
SA55+SA58 broadly and potently neutralizes SARS-CoV-2 variants and sarbecoviruses
In VSV-based pseudovirus neutralization assays, SA55+SA58 potently neutralizes circulating SARS-CoV-2 variants and multiple SARS-related coronaviruses (Figure 4). They could also neutralize SARS-CoV-2 ancestral strain and Omicron BA.1 authentic virus, and are cross-reactive to most ACE2-utilizing sarbecoviruses. As reported in another study, SA55 also exhibited high potency against various emerging SARS-CoV-2 variants with convergent mutations on RBD, including BQ.1.1 and XBB (Cao et al., 2022).
Escape mutants of SARS-CoV-2 against SA55/SA58 were further analyzed with replication-competent recombinant VSV (rVSV) and multiple VSV-based pseudoviruses harboring SARS-CoV-2 BA.2/ BA.2.12.1/BA.5 spike with additional substitutions on RBD. The results of DMS, structural analyses, rVSV-based mutation screening, and VSV-based pseudoviruses correspond well with each other. Specifically, the efficacy of SA55 was escaped by V503E and G504D, and SA58 was slightly affected by K356T, D339Y, and K444N; moderately affected by T345N, R346Q, and R346T; strongly affected by E340D; and escaped by E340K and K444E. The accumulation of mutations on R346 and K444 may affect the neutralization of SA58, but SA55 could efficiently neutralize such mutants, including recently emerging variants with convergent mutations on RBD such as BQ.1, BQ.1.1, and XBB (Figure 5).
SA55+SA58 protects mice from BA.1 and BA.5 infection
In SARS-CoV-2 BA.1 or BA.5 challenge experiments using hACE2-transgenic mice, SA55+SA58 displayed strong viral clearance and blocking efficacy in vivo against BA.1 and BA.5, especially in lungs and when delivered via intraperitoneal (Figure 6). Compared to mice in control groups, mice that received SA55+SA58 exhibited significantly lower viral load, for both prophylactic and therapeutic purposes.
Overall, SA55+SA58 exhibited strong resistance to RBD mutations and may be resistant to escape by future SARS-CoV-2 variants. In cooperation with Sinovac, SA55 and SA58 will be in clinical trials before the end of 2022 and serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.
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