Study isolated largest panel of β-CoV broadly neutralizing antibodies to date

In a recent study posted to the bioRxiv*preprint server, researchers isolated a large panel of broadly neutralizing antibodies (bnAbs) from coronavirus disease 2019 (COVID-19) recovered-vaccinated donors.

Study: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease. Image Credit: nobeastsofierce/Shutterstock
Study: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease. Image Credit: nobeastsofierce/Shutterstock

Background

The rapid emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concerns (VOCs) has triggered massive efforts on the development of vaccines and antibodies that specifically targets the most conserved regions of the SARS-CoV-2 spike (S) protein. The recently emerged SARS-CoV-2 Omicron variant showed escape from neutralizing antibodies (nAbs) targeting the more conserved regions of the receptor-binding domain (RBD).

The S2 region on the SARS-CoV-2 spike harbors neutralizing epitopes and is of interest as a target to generate vaccines effective against SARS-CoV-2 VOCs and specifically pan-betacoronavirus (b-CoV).

There is a need for a rational strategy in terms of the SARS-CoV-2 vaccine design that favors a bnAb panel rather than single a monoclonal antibody to precisely define broadly neutralizing epitopes and to determine nAb characteristics detrimental for broad neutralization.

Study design

In this study, the researchers collected sera samples from the convalescent COVID-19 donors, vaccinated donors, and COVID-19-recovered vaccinated donors. FreeStyle293-F, Expi293F, HEK293T, and HeLa-ACE2 cell lines were used, and flow cytometry of peripheral blood mononuclear cells (PBMCs) samples for B-cell profiling and monoclonal antibodies (mAbs) isolation were performed. Enzyme-linked immune sorbent assay (ELISA) was used to detect the reactivity of antibodies against single alanine substitution peptides, and the team also performed a pseudotyped viral neutralization assay.

Human epithelial type 2 (HEp2) polyreactive assay was used to estimate the monoclonal antibodies reactivity with HEp2. Biolayer interferometry (BLI) measured the binding of a monoclonal antibody with S-proteins. Antibody immunogenetic analysis of light and heavy chain sequences of mature antibodies was performed.

Findings

The findings of the study demonstrated that sera from COVID-19 recovered or vaccinated donors showed weak or no binding to the spike stem-helix peptides of human β-CoVs. However, 80% of sera from COVID-19 recovered and vaccinated donors demonstrated effective cross-reactive binding to the peptides.

The researchers sorted 36% B cells specific to SARS-CoV-2 S-protein of which a sizable portion was cross-reactive to the S-protein of MERS-CoV. From the ten convalescent donors, a total of 358 double-positive single B cells specific to SARS-CoV-2:MERS-CoV S-protein were recovered.

Stem-helix mAbs neutralization by clade 1a and clade 1b angiotensin-converting enzyme 2 (ACE2)-utilizing sarbecovirus and MERS-CoV was examined. It was noted that all the 32 stem-helix bnAbs lineages neutralized all five sarbecovirus with varying neutralizing potency. The bnAbs neutralized SHC014 clade 1a and SARS-CoV-2 clade 1b more adequately as compared to other sarbecovirus but few bnAbs neutralized all viruses with IC50 neutralization titer range of 0.1 to 3 µg/ml.

Among the 32 bnAbs lineages, 72% bnAbs neutralized MERS-CoV but with lower neutralization potency as compared to the Sarbecovirus. The selected bnAbs were effective against the following SARS-CoV-2 VOCs: Alpha, Beta, Gamma, Delta, and Omicron variants.

The immunogenetic analysis of stem-helix bnAbs sequences demonstrated 63% and 22% enrichment of immunoglobulin heavy chain (IGHV1-46) and IGHV3-23 gene, respectively, compared to the human germline frequencies at baseline. The germline gene IGHV1-46 was 78% enriched in stem-helix bnAbs that demonstrated neutralization of MERS-CoV in addition to sarbecovirus.

The team observed that the binding affinity of mAbs to stem-helix peptides of SARS-CoV-2 was higher than MERS-CoV. No association was noted with binding of light or heavy chain somatic hypermutations (SHMs) with MERS-CoV or SARS-CoV-2 stem-helix peptides or the corresponding virus neutralization. Overall, a significant contribution from residues of encoded germline to epitope binding was observed, which was consistent with the enrichment of a few gene features of antibody germline.

The findings revealed that although the level of SHM does not correlate with binding or neutralization, mutations in key antibodies were critical for the neutralization phenotype to achieve adequate affinity for neutralization.

The researchers performed binding of 32 stem-helix bnAbs with the alanine mutant of SARS-CoV-2 to measure its epitope specificities and the potential link with antibody immunogenetic features. They identified three hydrophobic core residues that form a core epitope and were important targets of spike stem-helix bnAbs.

The protective efficacy of two stem-helix bnAbs - CC68.109 and CC99.103 - was determined against SARS-CoV-2, SARS-CoV-1, and MERS-CoV in aged adults Balb/c mice. The stem-helix bnAb-treated animals showed considerable reduction in weight loss and hemorrhage and optimal pulmonary function, highlighting the protective role of the bnAbs in the betacoronaviruses challenge group with CC99.103 bnAbs eliciting a slightly higher protective effect as compared to the CC68.109 bnAbs. 

Conclusion

The findings of the study demonstrated the isolation of a huge panel of betacoronavirus bnAbs and revealed the molecular ground for the broad protection afforded by them. The bnAbs targeted a conserved S2 region on the fusion machinery of betacoronavirus spikes. The bnAbs showed strong in vivo protection against pan-betacoronaviruses - SARS-CoV-1, SARS-CoV-2, and MERS-CoV - that spilled over in the past 20 years into humans to cause severe disease.

The anti-S2 bnAbs furnished a detailed framework of new opportunities for antibody-based interventions and can be used to counter spillovers of betacoronaviruses by developing pan-betacoronavirus vaccines.

*Important notice

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

Journal reference:
Sangeeta Paul

Written by

Sangeeta Paul

Sangeeta Paul is a researcher and medical writer based in Gurugram, India. Her academic background is in Pharmacy; she has a Bachelor’s in Pharmacy, a Master’s in Pharmacy (Pharmacology), and Ph.D. in Pharmacology from Banasthali Vidyapith, Rajasthan, India. She also holds a post-graduate diploma in Drug regulatory affairs from Jamia Hamdard, New Delhi, and a post-graduate diploma in Intellectual Property Rights, IGNOU, India.

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