Resistance of Russian Rhinolophus bats containing ACE2-dependent sarbecoviruses to COVID-19 vaccines

By Pooja Toshniwal PahariaSep 26 2022Reviewed by Aimee Molineux

In a recent study published in PLoS Pathogens, researchers investigated the receptor tropism and serological cross-reactivity for spike (S) protein receptor-binding domains (RBDs) from two clade 3 sarbecoviruses found in Russian Rhinolophus (horseshoe) bats: Khosta-1 in Rhinolophus ferrumequinum and Khosta 2 in R. hipposideros, that are divergent from receptor-binding domains (RBDs) of severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Zoonotic spillover of the SARS-CoV-2 sarbecovirus has led to the coronavirus disease 2019 (COVID-19) pandemic. Several sarbecoviruses have been discovered among Asian bats; however, most of them do not infect humans. Researchers have accelerated viral detection efforts globally, expanding genomic databases with novel zoonotically transmitted and circulating sarbecoviruses.

The authors and other researchers have categorized sarbecovirus RBDs into three clades: clade 1 viruses among Asian bats do not contain deletions and bind to the hACE2 (human angiotensin-converting enzyme 2) receptor; clade 2 viruses among Asian bats contain two deletions and don’t bind to hACE2; clade 3 viruses among European and African bats contain one deletion and bind to hACE2. In 2021, viruses were detected among Chinese bats, comprising clade 4, that also bind with the hACE2.

About the study

In the present study, researchers investigated host cell infection by Russian Rhinolophus bat sarbecoviruses’ S and their neutralization by monoclonal antibodies (mAb) and vaccinated donor sera.

The full-length Khosta S gene was synthesized, SARS-CoV-1 S RBD was substituted with that of Khosta viruses and chimeric S expression plasmids were generated. In addition, chimeric S RBDs from other previously tested clade 3 viruses (BM48-31, Uganda), SARS-CoV-2 and related RaTG13 viruses were included for comparative analysis. The chimeric S constructs generated VSV (vesicular stomatitis virus)-pseudotyped VLPs (virus-like particles) carrying chimeric SARS-CoV-2 S with Khosta RBDs to mimic the potential recombinant threat from Khosta viruses.

Further, the Huh-7 (human liver cell line) cells were infected with the VLPs bearing the chimeric Khosta S RBDs to test viral compatibility with human cells. For characterizing potential Khosta virus receptors, a receptor tropism test was performed, wherein baby hamster kidney (BHK) cells were transfected with human orthologues of known CoV receptors and subsequently infected with the viral pseudotypes. Human entry efficiency between the viral S proteins and human ACE2 (hACE2) was further assessed by infecting hACE2-expressing 293T- cells.

The 293T ‘producer cells’ were lysed, and the lysates were subjected to Western blot analysis. To explore protein interactions between Khosta 2 and hACE2 in comparison with other clade 1 S RBDs, Khosta 2 RBD structure predictions were made based on previously published data, which was then aligned by multiple sequence alignment (MSA) to ACE2-bound SARS-CoV-1 and SARS-CoV-2 co-structures for phylogenetic assessments.

Pseudotype experiments were repeated with sera obtained from vaccinated individuals. To compare Khosta 2 and SARS-CoV-2 variants of concern (VOCs) neutralization, the SARS-CoV-2 Omicron VOC S RBD was generated and tested against serum samples obtained from six vaccinated (doubly-vaccinated by the Pfizer vaccines or Moderna vaccines), four non-infected donor indiviudals and three vaccinees with breakthrough Omicron infections.

Human orthologs of ACE2, APN (Aminopeptidase N) and DPP4 (dipeptidyl peptidase IV) and plasmid S sequences from human CoV (HCoV)-229E, the Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-1 were obtained. Neutralization assays were performed using vaccinated donors’ sera and bamlanivimab, a SARS-CoV-2 RBD-specific mAb.

Results and discussion

The exogenous protease mediated Khosta-1 RBD entry into Huh-7 cells; however, protease addition did not facilitate Khosta-1 RBD entry in BHK cells transfected with several CoV receptors, indicating that trypsin-dependent Khosta-1 entry in Huh-7 cells did not depend on hACE2. Contrastingly, trypsin addition enhanced receptor-dependent entry signals for SARS-CoV-2 RBDs and Khosta 2 RBD, indicating that Khosta 2 RBDs utilized hACE2 receptors for cell infection. However, the full-length Khosta 2 S was less infectious than the chimeric SARS-CoV-based S.

Khosta 2 RBD infected hACE2-expressing cells with cell entry levels comparable to that of RaTG13, an hACE2-binding bat sarbecovirus highly similar to SARS-CoV-2 RBD. Both African clade 3 RBDs also showed hACE2 binding, although with considerably lower efficiency than SARS-CoV-2. Contrary to hACE2 receptor findings, only HCoV-229E and MERS-CoV infected APN-expressing and MERS-CoV DPP4-expressing cells, respectively.

Of surprise, Omicron S was neutralized effectively by bamlanivimab, whereas SARS-CoV-2 S with the Khosta 2 RBD showed slight resistance (and complete resistance to the Wuhan-Hu-1 strain), even at high serum dilutions, indicative of slight cross-reactivity between the two RBDs. Similar findings were observed when vaccinated donors’ sera were used.

The higher resistance to neutralization by Khosta 2 than SARS-CoV-2 could be since Khosta 2 RBD and SARS-CoV-2 RBD are only 60% similar, and the vaccination-induced neutralization responses were primarily RBD-directed. Bamlanivimab contacts 17 SARS-CoV-2 Wuhan-Hu-1 strain residues, 10 of which are shared by Khosta 2.  Further, bamlanivimab escape could be due to Omicron mutations Q493R and E484A; Khosta 2 encodes G435 at a site analogous to E484 on SARS-CoV-2.

Overall, the study findings highlighted hACE2 receptor preference in Khosta 2 clade 3 viruses using pseudotyped VLPs with chimeric and full-length clade 3 S proteins. Pseudotyped VLPs containing recombinant SARS-CoV-2 S-encoding Khosta 2 RBD resisted neutralization, indicating that new recombinant sarbecoviruses circulating beyond Asia could threaten current COVID-19 vaccine efficacy.