A broad cocktail of sarbecovirus-neutralizing antibodies

In a recent study published on the bioRxiv* preprint server, researchers in China present a rational approach to identify broad neutralizing antibody (NAb) cocktails targeted by the protein receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ).

Study: Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails in SARS convalescents. Image Credit: Kateryna Kon/Shutterstock.com

Background

The continued emergence of immunocompromised SARS-CoV-2 variants, such as the concerning Omicron variant (VOC) and its sublines, justifies the need for more potent and broader neutralization of the SARS-CoV-2 sarbecovirus NAb (bsNAb) with epitopes at conserved sarbecovirus sites.

Several bsNAbs targeting SARS-CoV-2 S subunit 2 (S2) demonstrated broad neutralization of SARS-CoV-2; however, their potency has been weak. In contrast, bsNAbs targeting the SARS-CoV-2 RBD showed high potency; however, most of these antibodies escaped neutralization by Omicron BA.2 and/or Omicron BA.4/BA.5.

About the study

In the present study, researchers propose a rational strategy to discover broad Nabs that are highly resistant to SARS-CoV-2 RBD mutations.

Fluorescence-activated cell sorting (FACS), high-throughput deep mutation scanning (DMS), and droplet-based single-cell scV(D)J sequencing (scVDJ-seq) analyzes were performed to isolate B cells cross-reactive memory from SARS-CoV-2 vaccinated convalescents and screen for escaped mutation profiles of 314 nAbs.

Epitope cluster analysis was performed to identify antibody epitopes. In vitro screening for escape mutations using SARS-CoV-2BA.1-S pseudotyped recombinant vesicular stomatitis virus (rVSV) and Vero cells was also performed.

Next-generation sequencing (NGS) analysis was used to identify the mutations, while the international ImMunoGeneTics (IMGT) and Global Initiative on Sharing Full Details of Influenza (GISAID) databases were were referred for analysis. Three-dimensional reconstructed structures of complex BD55-3546/Delta S6P, BD55-4637/BA.1 S6P and BD55-5514+ BD55-5840 (SA55+SA58)/BA.1 S6P were generated through the use of cryo-electron microscopy (EM).

Structural models of bsNAbs were prepared to visualize the multiple sequence alignment (MSA) aligned structures of S309, S2H97, S304, BD55-1239, ADG-2 in epitope groups E1, E3, F1, F2 and F3. Human angiotensin-converting enzyme 2 (hACE2) transgenic mice were challenged with SARS-CoV-2 BA.1 before or after intraperitoneal (IP) and intramuscular (IM) SA55+SA58 treatment to assess the anti-SARS-CoV-2 efficacy of SA55+SA58 live.

The mice’s body weight changes were assessed, in addition to their lung and trachea samples which were subjected to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis to determine viral loads.

Study results

Approximately 13,000 clusters of differentiation (DC) 19 + CD27 + immunoglobulin M (IgM) – memory B cells with cross-linking to SARS-CoV-1 and SARS-CoV-2 RBDs were obtained from 28 convalescents who received two doses of CoronaVac vaccine and a booster dose of ZF2001 in 2021. By scVDJ-seq analysis, 2,838 matched Ab sequences were extracted from memory B cells, of which 1,413 sequences comprising the IgG1 heavy chain constant region were chosen for the in vitro experiences.

In epitope clustering analysis, 286 bsNAbs of F1, F3, E1, E3, and F2 epitopes and with sarbecovirus neutralization titers were observed. However, only 12 bsNAbs with non-competing E1, E3 and F3 anti-epitope Abs were analyzed in more detail, among which bsAbs F1 and E3 showed weak neutralization and were not considered for the development of NAb drugs. NAbs from groups E1 and F2 showed high and moderate neutralization potency, respectively.

Group E1 antibodies were related to clade 1a/1b sarbecoviruses. F3 NAbs (ADG-2 Ab) have shown binding to sarbecovirus clades that utilize ACE2.

BD55-5840 showed the most potent neutralization of ancestral strain SARS-CoV-2 and Omicron BA.1 with 90% inhibition concentrations (CI90) values ​​of 5.6 ng/mL and 24 ng/mL, respectively. Conversely, BD55-5514 showed potent neutralization of BA.1 with IC90 of 23 ng/mL. Neutralization of SARS-CoV-2 by F2 group NAbs was lower than that of F3 and E1 Abs.

SA55 was bound only in the RBD up configuration, while SA58 was bound in the RBD up and down configurations. The E1 group Abs formed non-overlapping NAb cocktails with the F2/F3 groups; thus, the E1 + F3 and E1 + F2 Ab cocktails were analyzed in more detail.

NAbs E1 and F3 centered on the binding site of glycan N343 and SARS-CoV-2 S-ACE2, respectively. BD55-3546 was escaped by the T345 and N440 mutations, while BD55-5585 and BD55-5549 were escaped by the R346, T345, and L441 mutations.

BD55-3546 showed five to 10-fold lower activity against Omicron variants containing S371F. BD55-5549 but not BD55-5840 could be escaped by L441 mutations, whereas BD55-5840 was slightly affected by S371F.

Furthermore, BD55-5840 exhibited high potency and lower susceptibility to R346 mutations and glycan displacement. Thus, BD55-5840 was the selected E1 group bsNAb.

BD55-3372, BD55-5483 and BD55-5514 were susceptible to V503 and G504 mutations, while BD55-4637 was susceptible to N439T/S and Y508 mutations. BD55-5514 was selected as the final bsNAb F3 due to its highest neutralization potency.

BD55-5840 and BD55-5514 showed synergy such that BD55-5840 could engage both up and down RBDs, while BD55-5514 blocked ACE2. The SA55+SA58 cocktail showed robust anti-SARS-CoV-2 efficacy live and was chosen as the final non-competitive bsNAb cocktail.

conclusion

The results of the study demonstrate that the non-competing antibody cocktail SA55 + SA58 exhibits broad sarbecovirus neutralization and high potency against all Omicron sublines of BA.1, BA.2, BA.2.12.1 and BA.4/BA.5, making it a valuable prophylactic and therapeutic drug candidate against sarbecoviruses. Future drug candidates should be targeted to rare and conserved RBD epitopes associated with essential SARS-CoV-2 functions such as ACE2 binding and/or glycosylation.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

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