A team of international scientists has recently isolated and characterized pan-coronavirus neutralizing antibodies from individuals with pre-existing immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These antibodies target the spike fusion peptide of all human alpha and beta coronaviruses and exhibit broad neutralizing activity. The study is currently available on the bioRxiv* preprint server.
Human coronaviruses are of two types including alpha- and beta-coronaviruses. All lethal members of the human coronavirus family including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) are beta-coronaviruses. Among non-lethal seasonal human coronaviruses, NL63 and 229E belong to the alpha-coronavirus family and OC43 and HKU1 belong to the beta-coronavirus family.
The spike glycoprotein expressed on the coronavirus envelop shares about 30% sequence similarity between alpha- and beta-coronaviruses. It mediates viral entry by binding to a designated host cell receptor. In SARS-CoV-2, the spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the host cell surface to initiate viral entry.
In the current study, the scientists have isolated and characterized monoclonal antibodies from individuals with pre-existing anti-SARS-CoV-2 immunity induced either by infection or vaccination. The basic aim was to identify cross-reactive antibodies that bind to both alpha- and beta-coronaviruses.
Isolation of antibodies
The scientists collected peripheral blood mononuclear cells from a total of 43 SARS-CoV-2 immune individuals and experimentally stimulated the cells to produce antibodies. To select anti-spike binding antibodies, they conducted enzyme-linked immunosorbent assay (ELISA) using recombinant spike proteins of alpha- and beta-coronaviruses.
Of all SARS-CoV-2-positive cultures, six were identified to have cross-reactivity against both alpha- and beta-coronaviruses, indicating that the memory B cells producing pan-coronavirus antibodies are present at very low frequencies. Using a high-throughput screening method, the scientists finally selected seven monoclonal antibodies that showed broad cross-reactivity against all human coronaviruses.
Epitope mapping of pan-coronavirus antibodies
The epitope mapping of all seven monoclonal antibodies identified a specific sequence in the spike S2 subunit. The sequence spans the S2 cleavage site and the N-terminal region of the fusion peptide, which is highly conserved across all coronaviruses and is required for membrane fusion.
By comparing the binding affinity of monoclonal antibodies to their unmutated common ancestors, the scientists observed that fusion peptide-specific pan-coronavirus antibodies acquire high epitope-binding affinity and cross-reactivity through somatic mutations.
Neutralizing efficacy of pan-coronavirus antibodies
The scientists tested neutralizing efficacy of fusion peptide-specific monoclonal antibodies by infecting cells with pseudotyped alpha- and beta-coronaviruses. Based on the findings, they identified two antibodies that neutralized all tested coronaviruses, indicating broad neutralizing cross-reactivity. In addition, they identified one antibody that efficiently neutralized all beta-coronaviruses, including the original SARS-CoV-2 strain and the omicron variant.
Furthermore, the scientists assessed the protective efficacy of two antibodies: the one with high neutralizing efficacy and the other one with broad neutralizing efficacy. They administered high doses of each antibody to hamsters infected with the gamma variant of SARS-CoV-2.
The findings revealed that both antibodies are capable of significantly reducing viral RNA and lung viral load and ameliorating lung pathologies.
Structural analysis of antigen-antibody complex
The scientists determined the crystal structures of five monoclonal antibodies bound to the fusion peptide. They observed that all antibodies have overlapping epitopes in the fusion peptide and the epitope interaction occurs via heavy and light chains.
With further structural analysis, they observed that the epitope targeted by tested antibodies is buried at the core of the spike trimer. This makes the epitope inaccessible to the antibodies.
The scientists further investigated how the fusion peptide epitope is exposed to facilitate antibody binding. Using spike-expressing cells, they observed that all antibodies bind marginally to the native spike protein. By adding soluble ACE2 to the cells, they observed a remarkable induction in the epitope binding ability of tested antibodies. This observation indicates that the binding of ACE2 induces a conformational change in the spike protein, which in turn exposes the fusion peptide for antibody binding.
The study identifies a panel of seven pan-coronavirus monoclonal antibodies that are able to bind highly conserved fusion peptides of all alpha- and beta-coronaviruses. The unmasking of fusion peptide through ACE2-induced conformational changes in the spike protein is required for antibody binding.
Despite targeting the same epitope, only some identified antibodies exhibit cross-reactive neutralizing ability against alpha- and beta-coronaviruses.
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.