The Omicron BA.1 and BA.2 variants, responsible for widespread COVID-19 infections globally, evolved independently from earlier variants. BA.2 has surpassed BA.1 in prevalence. The SARS-CoV-2 spike protein, a trimeric glycoprotein, mediates cell entry via binding to the host receptor ACE2. The spike protein consists of S1 (containing the receptor-binding domain, RBD, and an N-terminal domain) and S2 (containing a fusion peptide for membrane fusion) subunits. The RBD exists in open (up) and closed (down) conformations; the open conformation is required for ACE2 binding. The Omicron variants exhibit numerous mutations in the spike protein (37 in BA.1 and 31 in BA.2), many located on surfaces known to interact with neutralizing antibodies, contributing to immune evasion. BA.2 shows even higher transmissibility than BA.1, necessitating investigation into its enhanced infectivity and immune evasion mechanisms. The potential involvement of intermediate hosts, particularly mice, based on mutational analysis, remains to be explored.

Previous studies on Omicron BA.1 demonstrated its increased binding affinity to human ACE2 and reduced stability, favoring the open RBD conformation. The higher ACE2 binding affinity was linked to mutations in the RBD. Moreover, previous research on JMB2002, a therapeutic antibody, highlighted its broad-spectrum neutralizing activity against several SARS-CoV-2 variants, including BA.1. However, the efficacy of JMB2002 against BA.2 and the structural basis for the increased infectivity and immune evasion of BA.2 remained unexplored. This paper aimed to address these knowledge gaps by investigating BA.2’s structural characteristics, binding to ACE2, and sensitivity to JMB2002, alongside investigating the role of intermediate animal hosts in Omicron's evolution.

The study employed cryo-electron microscopy (cryo-EM) to determine the three-dimensional structures of the Omicron BA.2 spike trimer in complex with human ACE2 (hACE2) and the therapeutic antibody JMB2002. The binding affinities of BA.1, BA.2, and wildtype (WT) spike trimers to hACE2 were evaluated using biolayer interferometry (BLI). Similar BLI experiments were conducted to investigate the interaction of the spike trimers with mouse ACE2 (mACE2), cat ACE2, and other species' ACE2 proteins to explore possible intermediate hosts. Additionally, the neutralizing activity of JMB2002 against BA.2 pseudovirus was assessed using a pseudovirus neutralization assay. The thermal stability of the spike trimers was analyzed using a thermal shift assay (TSA). Cryo-EM data processing involved motion correction, CTF estimation, particle picking, 2D and 3D classification, and refinement using cryoSPARC. Model building involved fitting initial models into the cryo-EM density maps using Chimera, followed by iterative manual adjustments and automated rebuilding using Coot and Phenix. The final models were validated using Phenix.

The study found that Omicron BA.2 spike trimer binds to hACE2 with an approximately 11-fold higher affinity than the WT and nearly a 2-fold higher affinity than BA.1. Cryo-EM structures revealed two states for BA.2-hACE2 complex: one with all three RBDs in the open conformation bound to hACE2, and the other with two RBDs up. The higher hACE2 binding affinity was attributed to mutations such as Q493R, Q498R, and K417N, which introduce new salt bridges or lose existing ones. The BA.2 RBD showed higher thermal stability than BA.1, possibly contributing to its enhanced binding affinity. Importantly, JMB2002 was highly effective against BA.2, displaying similar potency to its action against BA.1. Cryo-EM structures of JMB2002 Fab bound to BA.2 demonstrated that JMB2002 binding to all three RBDs blocks hACE2 access. Surprisingly, BA.1 and BA.2 spike trimers bound to mACE2 with high affinity, whereas WT did not. This high binding to mACE2, along with the binding of both BA.1 and BA.2 to cat ACE2, but not to rat or dog ACE2, supports the hypothesis of an evolutionary pathway involving mice and cats as intermediate hosts. Cryo-EM structures of BA.1 and BA.2 spike trimers complexed with mACE2 revealed that mutations Q493R, Q498R, and N501Y were critical for mACE2 binding. These mutations have been observed in mouse-adapted SARS-CoV-2, further suggesting mice as a potential intermediate host.

The findings highlight the increased transmissibility of BA.2, primarily attributable to its elevated binding affinity to hACE2, a structural consequence of several key mutations in the RBD. The efficacy of JMB2002 against BA.2 offers a promising therapeutic avenue, although further research is needed to optimize antibody therapies. The unexpected high affinity of BA.1 and BA.2 spike trimers to mACE2 significantly strengthens the hypothesis of an evolutionary pathway involving mice and cats. The results support a possible scenario of initial human infection, followed by spillover to cats and mice, further evolution in mice, and re-emergence into the human population. This emphasizes the need to consider animal reservoirs in the prevention and control of future outbreaks.

This study provides crucial structural and biochemical insights into the enhanced transmissibility and immune evasion of the Omicron BA.2 variant. The high affinity binding to hACE2, the effectiveness of JMB2002, and the notable binding to mACE2 are key findings. The proposed evolutionary pathway involving mice and cats underscores the importance of considering animal reservoirs in the fight against SARS-CoV-2. Future research should focus on developing more effective antiviral strategies targeting the specific interactions revealed in this study.

The study primarily focused on in vitro experiments, and further in vivo studies are needed to validate the findings. While the mouse model provides insights into a possible evolutionary pathway, the complexity of viral evolution could involve other intermediate hosts and selective pressures not fully captured in this study. The pseudovirus neutralization assay might not perfectly reflect the neutralization efficacy in real-world infections. Additional studies using different animal models and more diverse antibody panels would provide further validation.