In a recent article posted to the bioRxiv* preprint server, researchers identified an ultralong bovine antibody that neutralizes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV).
Background
The coronavirus disease 2019 (COVID-19) pandemic has brought to light the enormous threat that zoonotic and highly transmissible pathogens pose to the global populations, specifically when prior immunity is limited.
Although COVID-19 vaccinations have shown to be quite effective in decreasing SARS-CoV-2 transmission and averting severe illness, this might not be the case for all pathogens. Furthermore, not everyone develops sufficient immunity following SARS-CoV-2 vaccinations, necessitating the development of additional therapies that can be implemented quickly in the event of a new pathogen.
Broadly neutralizing antibodies target conserved epitopes and have a lot of potential as antibody-based treatments, especially given the continuous evolution of SARS-COV-2 antigens. Some bovine antibodies are highly proficient at binding conserved, glycosylated epitopes because of their ultralong complementarity determining region heavy chain 3 (CDRH3). Moreover, the longest known CDRH3 domains are possessed by bovine ultralong antibodies.
About the study
In the present study, the scientists isolated ultralong bovine H-chains with neutralizing capacity against SARS-CoV-2 and similar CoVs. Based on prior reports that the ultralong bovine H-chains couple with a comparatively invariable Vλ light chain, the team built a single-chain variable fragment (scFv) framework to which ultralong H-chain-only libraries could be cloned and produced.
The researchers employed polyhistidine-tagged (His-tagged) SARS-CoV-2 spike (S) glycoprotein and mammalian cell surface display to isolate an ultralong scFv from a SARS-CoV-2-naïve H-chain library that engages with the receptor-binding domains (RBDs) of SARS-CoV and all the existing SARS-CoV-2 variants. Further, human embryonic kidney 293T (HEK293T) cells were used for mammalian cell culture. The researchers utilized site-directed mutagenesis and differential hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the mechanism of neutralization of lentiviruses pseudotyped with SARS-CoV-2 S and SARS-CoV S protein by the identified bovine paratope.
Findings and discussions
The study results demonstrated the discovery of a bovine extensively reactive CDRH3, named B9-scFv, from a library of <1 x 104 SARS-CoV-2-naïve H-chain sequences. The isolated ultralong B9-scFv interacted with RBDs of SARS-CoV and all prevailing SARS-CoV-2 variants. Additionally, the ultralong CDRH3 bound to SARS-CoV RBD approximately 50-times more strongly than SARS-CoV-2.
The bovine antibody-targeted epitope was found in a cryptic cleft on the inner region of the SARS-CoV and SARS-CoV-2 RBD, which was only accessible transiently due to inter-domain motions. This epitope was only discovered once previously as the target for two different pan-sarbecovirus antibodies (7D6 and 6D6) when several repeated vaccinations were necessary.
In addition, five vaccinations of mice with either a combination of SARS-CoV and SARS-CoV-2 S protein and the Middle East Respiratory Syndrome CoV (MERS-CoV) RBD protein or SARS-CoV-2 S-2 subunit protein (S-2P) were required for the isolation of the 7D6 and 6D6 antibodies. On the contrary, from a limited library of bovine CDRH3s, B9-scFv was isolated in the present study. Furthermore, the 7D6 and 6D6 antibodies demonstrated destabilization of the perfusion S complex.
B9-scFv did not demonstrate competition with SARS-CoV pseudotyped lentiviruses for angiotensin-converting enzyme 2 (ACE2) interaction. Yet, B9-scFv neutralized SARS-CoV pseudotyped viruses with a half-maximal inhibitory concentration (IC50) of 468 nM, most likely by disrupting the stability of the prefusion complex via a crucial glycan contact interference. Mutagenesis and HDX data depicted numerous critical contacts which were shared between the epitope of B9-scFv and 7D6 and 6D6 antibodies, including 457-467 residues on the β7-β8 loop of the RBD.
B9-scFv demonstrated a truncated descending and ascending β-stranded stalk, with few residues at the 101RD102 variable diversity (VD) junction and no alternate tyrosine motif at the 3’ terminus of D8-2. These characteristics would most likely affect the stalk’s angle, length, and flexibility and the manner in which the disulfide-bonded loops of the B9 knob domain interact with the CoVs RBD proteins.
Conclusions
The study findings demonstrated the identification of a bovine ultralong CDRH3, named B9-scFc, that neutralized viruses pseudotyped with SARS-CoV and all current SARS-CoV-2 variants’ S protein but not via competing with viral RBD for ACE2 receptor binding.
The researchers found that the presently discovered ultralong CDRH3 neutralized the SARS-CoV S protein via the identification of a conserved, seldom seen, and cryptic epitope that overlaps the target of 7D6 and 6D6 pan-sarbecovirus antibodies. The B9-scFc-targeted epitope in SARS-CoV-2 and SARS-CoV was glycan-shielded and only became temporarily accessible through inter-domain movements.
Overall, the present investigation identified the first bovine anti-sarbecovirus paratope and illustrated the potential of the bovine system for rapidly discovering widely reactive new ultralong CDRH3s targeting conserved vulnerable sites on emerging viral pathogens and their variants.
*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.
