In a recent study published in the Journal of Biological Chemistry, researchers performed an in vitro assay to isolate ultra-long bovine heavy chains that showed binding to SARS-CoV-2 and related acute respiratory syndrome coronaviruses (CoVs). severe
Study: A bovine antibody possessing an ultra-long CDRH3 complementarity-determining region targets a highly conserved epitope in sarbecovirus spike proteins. Image credit: Andrii Yarovsky/Shutterstock
background
Studies have reported that broadly neutralizing antibodies (Abs) have immense potential as antiviral therapeutic agents due to their ability to identify highly conserved epitopes rarely mutated in viral variants. A subset of bovine Ab possesses an ultralong complementarity-determining region (CDR) H3 highly capable of identifying conserved viral epitopes; however, its activity against Sarbecovirus (S) spike proteins has not been well characterized and requires further investigation.
About the study
In the present proof-of-principle study, the researchers aimed to isolate ultra-long bovine heavy chains that could bind to Sarbecovirus S proteins in vitro.
A mammalian cell surface screen was used to screen ultra-long CDRH3 Ab libraries. Variable exons from bovine leukocyte gDNA (genomic deoxyribonucleic acid) were amplified to generate an ultra-long bovine paratope library. Subsequently, enrichment of the ultra-long CDRH3 regions was performed by polymerase chain reaction (PCR) and size selection, to produce an ultra-long CDRH3 library.
The team then inserted the amplicons into the pBovShow cassette. Screened the ultralong single-chain variable fragment (scFv) protein library to identify SARS-CoV-2 S binding by transiently transfecting the scFv library into 293T cells and performing FC analysis. To increase the isolation efficiency of S-binding scFv(s), the library was cloned into LV vectors (lentivirus) and vesicular stomatitis virus (VSV) pseudotyped LV particles were generated and transduced into 293T cells to obtain pooled scFv sequences for each cell.
A total of 15 SCCs (single cell clones) showed S-interaction, of which three contained three scFvs with an identical nucleotide sequence, which was named B9-scFv. To locate the epitope of B9-scFv, SARS-CoV-2 S subunits, the S1, S2, and S1 receptor binding domain (RBD) were purified by IMAC (metal affinity chromatography immobilized). Differential hydrogen-deuterium exchange mass spectrometry (MS) was performed to assess the mechanism of antibody binding.
results
An ultra-long, broadly reactive scFv CDRH3 epitope (B9-scFv) was isolated from a naive SARS-CoV-2 heavy chain library that showed binding to SARS-CoV-2 RBD, all variants of SARS- CoV-2 of concern (VOC) and SARS-CoV RBD. The epitope neutralized pseudotyped SARS-CoV S viruses, but not by competing with binding to the ACE2 (angiotensin-converting enzyme 2) receptor.
Rather, the epitope neutralized SARS-CoV pseudotyped LVs, transiently available through interdomain S protein movements and destabilizing the prefusion complex. The epitope was located in a cryptic cleft on the inner surface of the RBD, a site that overlapped with the footprints of a few broad anti-SARS-CoV-2 Abs such as S2H97, 7D6/6D6, and FD20.
Broadly active CDRH3 was isolated from a modest library of sequence diversity, underscoring the enormous potential of the bovine system as a source for broadly active Abs that can confer protection against novel pathogens and their mutant variants. B9-scFv comprised 53% of the scFvs obtained from LV-transduced 293T cells after a single protein S binding selection, which rose to 83% after further FC enrichment. The findings indicated that B9-scFv largely accounted for the anti-S activity in the library.
Cells that transiently expressed B9-scFv showed binding to S, RBD, and S1, but not to S2, indicating that the B9-scFv binding site was localized to the amino acid residue RBD 319 at residue 591. Binding of B9-scFv to protein S -transfected cells was concentration-dependent and enriched compared to ultralong scFv controls.
Notably, B9-scFv showed no reactivity with non-transfected cells, even at concentrations of five mM for one hour, strongly indicating specific S-Ab interaction. Binding of B9-scFv-S was maintained across mutations such as N501Y, D614G, Y453F, E484K, K417N and L452R in SARS-CoV-2 COVs such as Beta, Alpha, Delta, Gamma, Omicron and Gamma. The findings indicated extensive cross-reactivity of B9-scFv.
The binding affinity to the RBDs of the SARS-CoV-2 variant compared to wild-type (wt) S was comparable, reinforcing the observation of B9-scFv binding to a highly targeted and conserved epitope. Human SARS-targeted CR3022-scFv and B9-scFv were relatively unreactive with Middle East respiratory syndrome CoV (MERS-CoV) RBD, indicating that B9-scFv was specific to SARS-CoV.
Only a minor difference was observed for B9-scFv binding to 200 nM and 2.0 μM SARS-CoV RBD, indicative of nanomolar binding affinity. B9-scFv almost completely (98%) neutralized SARS-CoV S (strain Urbani) pseudotyped LV particles at a concentration of 70 μg/ml, but did not show these effects for equivalent titers of SARS-CoV-2. The half maximal inhibitory concentration (IC50) value for neutralization of SARS-CoV pseudotyped LV by B9-scFv was 468 nM.
Overall, the findings of the study highlighted the potential of bovine Abs expressed in vitro with ultralong CDRH3 to isolate novel and broadly active therapeutic agents to combat emerging pathogenic organisms and their variants and to identify key epitopes for developing vaccines . The team supported previously reported findings that ultra-long CDRH3 regions combine with relatively invariant Vλ light chains to generate a scFv template onto which libraries of ultra-long heavy chains can be cloned and expressed.