An ACE2-dependent Sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines

Abstract

Spillover of sarbecoviruses from animals to humans has resulted in outbreaks of severe acute respiratory syndrome SARS-CoVs and the ongoing COVID-19 pandemic. Efforts to identify the origins of SARS-CoV-1 and -2 has resulted in the discovery of numerous animal sarbecoviruses-the majority of which are only distantly related to known human pathogens and do not infect human cells. The receptor binding domain (RBD) on sarbecoviruses engages receptor molecules on the host cell and mediates cell invasion. Here, we tested the receptor tropism and serological cross reactivity for RBDs from two sarbecoviruses found in Russian horseshoe bats. While these two viruses are in a viral lineage distinct from SARS-CoV-1 and -2, the RBD from one virus, Khosta 2, was capable of using human ACE2 to facilitate cell entry. Viral pseudotypes with a recombinant, SARS-CoV-2 spike encoding for the Khosta 2 RBD were resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Our findings further demonstrate that sarbecoviruses circulating in wildlife outside of Asia also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2.

Fig 1. Khosta 2 and other RBD clade 3 sarbecoviruses use human ACE2 to infect cells.

(A) Sarbecovirus Receptor Binding Domain Cladogram based on amino acid sequences and rooted at the midpoint. Countries of origin and known host receptors are indicated to the right. Clade 1 viruses are shown in red and orange, clade 2 in grey, clade 3 in blue and clade 4 in purple. (B) Diagram of spike constructs used for this study. The SARS-CoV-1 RBD was replaced with RBDs from other sarbecoviruses. (C) Expression and incorporation of viral pseudotypes by westernblot. (D) Huh-7 cells were infected with pseudotypes in the presence of absence of trypsin. Cells were infected in triplicate. (E) BHK cells were transfected with receptors and infected in the absence (F) or presence of trypsin. (G) 293T cells stably over-expressing human ACE2 were infected with pseudotypes without trypsin. Cells were infected in triplicate or quadruplicate for experiments in D-G.

Fig 2. Modelled co-structure of Khosta 2 RBD and human ACE2.

(A) Crystal structure of SARS-CoV RBD bound to human ACE2 (PDB ID: 2AJF) with contact points indicated in light blue. (B) Crystal structure of SARS-CoV-2 RBD bound to human ACE2 (PDB ID: 6M0J) with contact points indicated in light blue. (C) Predicted structure of Khosta 2 RBD bound to human ACE2 with contact points identical to either SARS-CoV or SARS-CoV-2 spike indicated in light green and resides that are different indicated in red. (D) ACE2-contact point comparison between SARS-CoV, SARS-CoV-2 and Khosta 2 spike. Residues that are identical between Khosta 2 and SARS-CoV or SARS-CoV-2 are shaded in light green.

Fig 3. Full-length Khosta spikes infect human cells.

(A) Huh-7 or (B) 293T cells that stably express human ACE2 cells were infected with VSV pseudotypes bearing the indicated spike protein.

Fig 4. Chimeric SARS-CoV-2-Khosta 2 spike is resistant to current vaccines.

(A) The RBD from SARS-Cov-2 spike was replaced with Khosta 2 RBD. (B) Pseudotpyes with indicated chimeric spikes were used to infect 293T cells stably expressing human ACE2. Pseudotypes were combined with (C) bamlanivimab or (D) vaccinated patient serum at various concentrations and used to infect 293T-hACE2 cells.(E) Area under the curve analysis for data in panel D. p-value 0.0021 (**), 0.0002 (***), <0.0001 (****). (F) Pseudotypes with Khosta 2 or Omicron variant RBD were combined with serum from vaccinated patients with breakthrough Omicron infection and used to infect 293T-hACE2 cells. (G) Area under the curve analysis for data in panel F. p-value 0.0021 (**), 0.0002 (***), <0.0001 (****) (H) Sequence identity matrix for RBD amino acid sequences from Khosta 2 and known SARS-CoV-2 variants of concern.