The ultra-long complementarity determining region H3 of cattle was found to cross-react with sarbecoviruses

The ultra-long complementarity determining region H3 of cattle was found to cross-react with sarbecoviruses

In a recently published study in the Journal of Biological Chemistry Researchers conducted an in vitro analysis to isolate ultra-long bovine heavy chains that showed binding with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses (CoVs).

Studie: Ein boviner Antikörper, der über eine ultralange komplementaritätsbestimmende Region CDRH3 verfügt, zielt auf ein hochkonserviertes Epitop in Sarbecovirus-Spike-Proteinen ab. Bildnachweis: Andrii Yarovsky/Shutterstock

background

Studies have shown that broadly neutralizing antibodies (Abs) have enormous potential as antiviral therapeutics due to their ability to identify highly conserved epitopes that are rarely mutated in virus variants. A bovine Ab subset possesses an ultralong complementarity determining region (CDR)H3, which is ideal for identifying conserved viral epitopes; However, their activity against sarbecovirus spike (S) proteins is not well characterized and requires further investigation.

About the study

In the present proof-of-principle study, researchers aimed to isolate ultra-long bovine heavy chains that could bind to sarbecovirus S proteins in vitro.

Mammalian cell surface screening was used to screen ultralong CDRH3 Ab libraries. The variable exons of cow leukocyte gDNA (genomic deoxyribonucleic acid) were amplified to generate a library of ultralong bovine paratopes. Enrichment of the ultralong CDRH3 regions was then performed by polymerase chain reaction (PCR) and size selection to create a library of ultralong CDRH3s.

Next, the team inserted the amplicons into the pBovShow cassette. It examined the ultra-long single chain variable fragment (scFv) protein library to identify the S-linkage of SARS-CoV-2 by transiently transfecting the scFv library into the 293T cells and performing FC analysis. To increase the efficiency of S-binding scFv(s) isolation, the library was cloned into LV vectors (lentivirus), and LV particles pseudotyped with vesicular stomatitis virus (VSV) were generated and transduced in the 293T cells to obtain combined scFv sequences for each cell.

A total of 15 SCCs (single cell clones) showed S-interaction, three of which contained three scFvs with an identical nucleotide sequence, designated B9-scFv. To locate the epitope of B9-scFv, the S subunits, S1, S2 and S1 receptor binding domain (RBD) of SARS-CoV-2 were purified using IMAC (immobilized metal affinity chromatography) analysis. To investigate the mechanism of antibody binding, differential hydrogen-deuterium exchange mass spectrometry (MS) was performed.

Results

A broadly reactive and an ultra-long scFv (B9-scFv) CDRH3 epitope was isolated from a SARS-CoV-2 naive heavy chain library that showed binding to SARS-CoV-2 RBD, all SARS-CoV-2 variants of concern (VOCs) and SARS-CoV RBD. The epitope neutralized SARS-CoV-S pseudotyped viruses, but not through competition with ACE2 (angiotensin converting enzyme 2) receptor binding.

Rather, the epitope neutralized pseudotyped SARS-CoV LVs that were transiently available via S protein movements between domains and destabilized the prefusion complex. The epitope localized to a cryptic cleft on the inner surface of the RBD, a location that overlapped with footprints of a few broad anti-SARS-CoV-2 Abs such as S2H97, 7D6/6D6 and FD20.

The broadly active CDRH3 was isolated from a modest sequence diversity library, highlighting the enormous potential of the bovine system as a source for obtaining broadly active Abs that can provide protection against novel pathogenic organisms and their mutant variants. B9-scFv comprised 53% of the scFvs obtained from LV-transduced 293T cells after a single S protein binding selection, which increased to 83% upon further FC enrichment. The results showed that B9-scFv was largely responsible for the anti-S activity in the library.

Cells transiently expressing B9-scFv showed binding to S, RBD, and S1, but not to S2, indicating that the B9-scFv binding site was located at RBD amino acid residue 319 to 591. The binding of B9-scFv to S protein-transfected cells was concentration dependent and enriched compared to ultralong scFv controls.

Notably, B9-scFv showed no reactivity with untransfected cells, even at concentrations of five mM over one hour, strongly indicating a specific S-Ab interaction. B9-scFv-S binding was maintained via mutations such as N501Y, D614G, Y453F, E484K, K417N and L452R in SARS-CoV-2 VOCs such as Beta, Alpha, Delta, Gamma, Omicron and Gamma. The results indicated broad cross-reactivity between B9 and scFv.

Binding affinity to SARS-CoV-2 variant RBDs was comparable compared to wild-type (wt) S, supporting the observation of B9 scFv binding to a highly targeted and conserved epitope. SARS-targeted human CR3022 scFv and B9 scFv were relatively unreactive with the Middle East Respiratory Syndrome CoV (MERS-CoV) RBD, suggesting that B9 scFv was SARS-CoV specific.

Only a slight difference was observed in B9-scFv binding to 200 nM and 2.0 μM SARS-CoV RBD, indicating nanomolar binding affinity. B9-scFv almost completely (98%) neutralized LV particles pseudotyped with SARS-CoV S (Urbani strain) at a concentration of 70 μg/ml, but showed no such effects at equivalent SARS-CoV-2 titers. The half-maximal inhibitory concentration value (IC50) for pseudotyped SARS-CoV LV neutralization by B9-scFv was 468 nM.

Overall, the study results highlighted the potential of in vitro expressed bovine Abs with ultralong CDRH3s to isolate novel and broadly effective therapeutics to combat emerging pathogenic organisms and their variants, as well as to identify prime epitopes for vaccine development. The team supported previously published findings that ultralong CDRH3 regions pair with relatively invariant Vλ light chains to create a scFv template on which ultralong heavy chain libraries can be cloned and expressed.

Reference:

• Burke MJ, Scott JNF, Minshull TC, Gao Z, Manfield I, Savic S, Stockley PG, Calabrese AN, Boyes J, Ein boviner Antikörper mit einer ultralangen komplementaritätsbestimmenden Region CDRH3 zielt auf ein hochkonserviertes Epitop in Sarbecovirus-Spike-Proteinen ab. (2022). Journal of Biological Chemistr. doi: https://doi.org/10.1016/j.jbc.2022.102624 https://www.sciencedirect.com/science/article/pii/S0021925822010675