Monoclonal antibodies from COVID-19 convalescent donors target conserved sites

In a recent study published in bioRxiv* Researchers, a preprint server, have discovered six monoclonal antibodies (mAbs) that bind to the spike (S) protein of all human coronavirus (CoV).

study: Widely neutralizing antibodies target coronavirus fusion peptides.. Image Credit: Slow Area / Shutterstock.com

Background

Coronaviruses belong to the genera Alpha, Beta, Gamma, and Delta and can infect a variety of birds and mammals. There are seven human CoVs identified to date, of which HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East Respiratory Syndrome (MERS)- CoV, and SARS-CoV-2. HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 are endemic viruses that cause mild disease, while SARS-CoV, MERS-CoV, and SARS-CoV-2 have been used for the past 20 years. Caused a serious outbreak.

SARS-CoV-2 causes coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. SARS-CoV-2 mutants (VOCs) show at least partial resistance to many therapeutic antibodies and vaccines.

Vaccine-induced protection is primarily provided by Neutralizing antibody It is directed to the receptor-binding domain (RBD) of the (nAbs) SARS-CoV-2 spike (S) protein, which is also a target for therapeutic mAbs. The latest Omicron BA.1 and BA.2 variants contain highly mutated RBDs, so most mAbs have no effect on them. Therefore, other sites of the S protein need to be investigated for treatment breakthroughs.

Investigation result

Researchers have identified widely responsive mAbs from COVID-19 convalescent donors in current studies. We examined plasma samples from 142 convalescent donors and examined B cells that produce a wide range of mAbs.

The reactivity of immunoglobulin G (IgG) with the S protein of human CoV was evaluated. Approximately 19 donors were identified for mAb isolation and characterization.

IgG⁺ Memory B cells (MBCs) were screened in a two-step manner that prioritized broadly reactive mAbs and identified 60 mAbs that were reactive to a minimum of 3 CoVs. Further investigation reveals that only 6 mAbs react with all 7 S proteins, including COV44-62, COV91-27, COV44-79, COV77-39, COV77-04, and COV78-36. became.

The ability to neutralize the six mAbs has been tested against the SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-NL63, and genuine HCoV-OC43 pseudoviruses. The two mAbs, COV44-62 and COV44-79, show the broadest functional reactivity of beta (HCoV-OC43, SARS-CoV, SARS-CoV-2) and alpha CoV (HCoV-229E, HCoV-NL63). Neutralized both. In addition, two mAbs neutralized the BA.1 and BA.2 variants of SARS-CoV-2.

Only COV44-62 showed neutralization of MERS-CoV, but other mAbs failed. Interestingly, COV77-39 did not have neutralizing activity, even though it had the same broad binding reactivity as the remaining mAbs. All six mAbs were observed to bind to the S2 subunit rather than the RBD or N-terminal domain (NTD) of the SARS-CoV-2 S protein.

A surface plasmon resonance (SPR) -based high-throughput analysis was performed to map 15 mer of overlapping peptides over the full length of the SARS-CoV-2S2 subunit. SPR analysis revealed that mAbs bound to peptides 42-44 ₈₁₅RSFIEDLLF₈₂₃ A motif in the fusion peptide region of SARS-CoV-2.

Studying 35 virus isolates representing each CoV genus, ₈₁₅RSFIEDLLF₈₂₃ motif. The motif was preserved in more than 90% of the selected isolates.

Widely neutralizing antibodies target the coronavirus associated with human disease. (A) Analysis of the frequency of MBCs expressing widely reactive antibodies from n = 19 donors. The values ​​in parentheses represent the percentage of SARS-CoV-2 reactive supernatant that also binds to a specified subset of coronavirus spikes other than SARS. A total of 10,356 MBC culture supernatants (50-100 B cells / well) were screened. (B) The phylogenetic relationship of the entire coronavirus spike protein targeted by the broadly responsive mAb was speculated by the neighbor-joining method of MEGA11 using the full-length amino acid sequence of the CoV spike protein. The bootstrap values ​​from 500 samplings are displayed in the branch. (C) A heat map representing the binding of widely responsive mAbs to peplomers from coronaviruses throughout the genus Alpha, Beta, and Deltacoronavirus. H1 hemagglutinin is included as a negative control in mAb binding experiments, and the subcurve area (AUC) value for each antigen is shown after subtracting the value for the negative control antigen CD4. (D) The value is Antibody titer At 50% neutralization (NT₅₀) Against SARS-CoV-2 Wuhan Hu-1, SARS-CoV-1, MERS-CoV, HCoV-NL63, HCoV-229E Envelope Pseudo-Lentivirus, and Genuine HCoV-OC43. NT50 values ​​were calculated using a dose-response inhibition model using the GraphPad Prism 9 5-parameter Hill Slope equation. (E) Neutralization of the SARS-CoV-2 mutant (pseudovirus) of concern by COV44-62 and COV44-79.

An alanine scan was performed focusing on the amino acids (AA) targeted by COV44-62 and COV44-79. To this end, the team discovered that the E819, D820, L822, and F823 residues are essential for COV44-62 binding. Similar to COV44-79, E819, D820, F823, and R815 residues were important for binding. These five residues were one of the conserved sites of the S protein throughout CoV.

Polymeric sera (IgG) from convalescent donors, messenger ribonucleic acid (mRNA) -1273 (Moderna) vaccinated, and SARS-CoV-2 inexperienced individuals are obtained to the SARS-CoV-2 fusion peptide. I tested the join. As a cohort, convalescent donors responded less than vaccinated subjects. However, some convalescent subjects showed the highest response of the three cohorts. This indicates that natural infections can elicit a stronger antibody response to the fusion peptide of a particular individual.

Evaluated using a Syrian hamster model Effectiveness of COV4-79 and COV44-62 In vivo.. Hamsters treated with COV44-79 recovered faster and showed reduced weight loss than control hamsters. Four of the six hamsters receiving COV44-62 showed clinical symptoms by day 6 after infection. In contrast, hamsters treated with COV44-79 showed respiratory rate on day 6.

Conclusion

The authors found 6 mAbs that bind to all 7 human CoVs and 2 mAbs that neutralize at least 6 CoVs from convalescent individuals. All mAbs are identical in the current SARS-CoV-2 VOC and are directed against fusion peptides conserved across the four CoV genera, with their vital role and potential as candidate sites for vaccine design. Emphasizes sex. Despite its potential benefits, this peptide has not received much attention, probably due to the relatively weak binding of mAbs to intact S proteins. This will only improve if the S1 cap is removed.

Overall, this study provides valuable insights into widely acting mAbs targeting fusion peptides that are highly conserved sites throughout CoV and requires further research in this area as potential vaccine candidates. It was made.

*Important Notices

bioRxiv publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information.