Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery

A recent study posted to bioRxiv* identified neutralizing antibodies (nAbs) against conserved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epitopes.

Study: Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2. Image Credit: Yurchanka Siarhei/Shutterstock
Study: Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2. Image Credit: Yurchanka Siarhei/Shutterstock

Background

Most nAbs against the SARS-CoV-2 target the spike protein’s N-terminal (NTD) and receptor-binding (RBD) domains. Nonetheless, changes in the amino acid (AA) sequences in the NTD and RBD due to mutations diminish the efficacy of monoclonal antibodies (mAbs) and vaccines. Therefore, there is a need for innovative strategies and interventions that remain effective despite viral evolution.

The study and findings

In the present study, researchers leveraged a coldspot-guided antibody discovery approach to identify nAbs against conserved viral epitopes. They speculated that some spike regions might be under selective pressure to conserve AA sequences for structural and functional integrity. More than 10.4 million SARS-CoV-2 sequences obtained from the Global Initiative for Sharing Avian Influenza Data (GISAID) repository were analyzed to test this.

The team identified 15 coldspots, AA sequences longer than 17 residues with a substitution frequency of less than 0.1%. One coldspot encompassed the S2’ cleavage site and a part of fusion peptide (FP), the substrate for cathepsin and transmembrane protease, serine 2 (TMPRSS2). Another coldspot was at the stem helix preceding the heptad repeat 2 (HR2). Three coldspots spanned the spike subdomain 1 (SD1). The HR2 and FP coldspots were unchanged in SARS-CoV-2 variants of concern (VOCs).

Enzyme-linked immunosorbent assay (ELISA) of plasma specimens from CoV disease 2019 (COVID-19) convalescent individuals identified high immunoglobulin G (IgG) antibodies against FP and HR2. In contrast, low IgG levels were observed in the plasma of COVID-19-naïve controls, most COVID-19 vaccinated individuals, and pre-COVID-19 samples after infection with common cold CoVs. FP- and HR2-specific B cells were isolated using flow cytometry from subjects with high IgG levels.

Fifty-five and 100 paired IgG heavy and light chain sequences were obtained, and 29 mAbs (11 against FP and 18 against HR2) were recombinantly produced. Ten anti-FP antibodies bound to the FP with half-maximal effective concentration (EC50) values ranging from 25 ng/ml to 119 ng/ml. Their EC50 values were more than four-fold higher against spike. All anti-HR2 antibodies bound to the HR2 with similar EC50 (up to 117 ng/ml), with 1.3-fold higher EC50 values against spike.

Most FP antibodies could recognize CoVs from alpha, beta, gamma, and delta genera, including all human CoVs, while some HR2 antibodies were cross-reactive with alpha-, beta-, and gamma-CoVs. In a SARS-CoV-2 pseudovirus neutralization assay, the half-maximal inhibitory concentration (IC50) values of fp.006 and hr2.016, the most potent antibodies, were 737 ng/ml and 10 ng/ml, respectively.

Moreover, some HR2 and FP antibodies effectively neutralized SARS-CoV-2 VOC pseudoviruses, including Omicron, and even protected mice against ancestral SARS-CoV-2/Omicron challenge. The binding of fp.006 to cell surface-expressed spike protein increased upon angiotensin-converting enzyme 2 (ACE2) attachment. The addition of soluble ACE2 had a synergistic effect on the neutralizing potency of fp.006. Thus, ACE2 can cause conformation changes in the spike, exposing the FP epitope and promoting neutralization.

Similarly, ACE2 attachment improved the binding of hr2.016 and hr2.023 antibodies to ancestral and Omicron spike proteins but did not affect fp.007 binding. This implied that fp.007-like antibodies do not require ACE2-induced conformational changes for optimal FP recognition. Further, 25 anti-SD1 mAbs were cloned and synthesized. ELISA confirmed the binding of these mAbs to SD1, with 16 being specific for SD1. Six SD1 antibodies were cross-reactive with SD1-RBD from all SARS-CoV-2 VOCs.

The pseudovirus assay identified sd1.040 as the most potent and broadly cross-reactive SD1 antibody. sd1.040 failed to prevent spike-ACE2 binding in ELISA, suggesting that neutralization does not involve receptor-binding inhibition. The researchers measured fp.006 binding to spike by flow cytometry, wherein ACE2 attachment augments fp.006 binding to spike, which was inhibited by the addition of sd1.040. This suggested that sd1.040 might interfere with conformational changes in the spike downstream of ACE2 attachment.

Finally, based on the synergistic effects of sd1.040 and rbd.042 antibodies against SARS-CoV-2 VOCs, the researchers developed a bispecific antibody (bsAb), namely CoV-X4042, which integrated sd1.040 and rbd.042 moieties. The two arms of this bsAb could concurrently engage the same SD1-RBD molecule. The CoV-X4042 bsAb exhibited significant synergy in neutralizing VOC pseudoviruses. Besides, mice treated with CoV-X4042 were protected against ancestral SARS-CoV-2 and Omicron challenge.

Conclusions

The researchers discovered antibodies against SARS-CoV-2 spike HR2, FP, and SD1 regions. Notably, FP and HR2 antibodies were detected in convalescents and recipients of inactivated virus vaccines but not in those vaccinated with the mRNA or adenovirus vector vaccine. This implied that not all vaccines might elicit antibodies against these highly conserved neutralizing epitopes. Together, the coldspot-guided discovery of antibodies in COVID-19 convalescent individuals revealed nAbs that were broadly cross-reactive with SARS-CoV-2 VOCs and other CoVs.

*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:
Tarun Sai Lomte

Written by

Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.

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