Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients

The COVID-19 pandemic, caused by SARS-CoV-2, highlighted the threat of zoonotic coronaviruses. RaTG13, a bat coronavirus, shares 96% genome-wide sequence similarity with SARS-CoV-2, making it a crucial subject for understanding potential future outbreaks. While the receptor-binding domain (RBD) of RaTG13 shows amino acid substitutions compared to SARS-CoV-2, potentially affecting ACE2 receptor affinity, the antigenic differences and the ability of SARS-CoV-2-induced immunity to neutralize RaTG13 remain unclear. Understanding cross-reactivity is vital for predicting the potential for future outbreaks and informing vaccine development strategies. This research aimed to determine the efficacy of antibodies generated through natural SARS-CoV-2 infection or vaccination in neutralizing RaTG13 pseudotypes, compared to SARS-CoV-2 pseudotypes, and to explore the impact of specific amino acid substitutions in the RBD on neutralization. The study's importance stems from the potential for future zoonotic spillover events and the need to assess the breadth of protection offered by existing and future vaccines.

Previous studies have characterized the genomic similarity between RaTG13 and SARS-CoV-2, highlighting the close phylogenetic relationship. Research has also investigated the structural and functional aspects of the SARS-CoV-2 spike protein, focusing on its interaction with the ACE2 receptor and the role of specific amino acid residues in receptor binding and immune evasion. Existing literature also documents the escape mechanisms employed by variants of concern (VOCs) of SARS-CoV-2, such as the Beta variant, which demonstrates immune evasion capabilities. However, the extent of cross-neutralization between RaTG13 and SARS-CoV-2, and the role of specific mutations in influencing neutralization, remained largely unexplored prior to this study.

The study employed pseudotyped viruses bearing the spike protein of SARS-CoV-2, RaTG13, and their mutants. Neutralization assays were performed using convalescent sera from SARS-CoV-2-infected patients and vaccinated healthcare workers. The WHO International Reference Panel for anti-SARS-CoV-2 immunobiology was initially used, followed by a larger cohort of 25 convalescent sera. Sera from healthcare workers vaccinated with either ChAdOx1 or BNT162b2 were also tested. The assays involved measuring the reduction in luciferase activity, indicating viral neutralization. To further dissect the role of specific amino acid substitutions, mutant spike plasmids with single and combinatorial changes in the RBD were generated and tested. Statistical analyses, including Wilcoxon matched pairs signed rank tests and Student's t-tests, were used to compare neutralization titers between different virus types and mutants. HEK293T cells were used for pseudotype virus generation and titration, and the infectivity of pseudotyped viruses was assessed using target cells expressing human ACE2. Detailed procedures for cell culture, plasmid generation, pseudotype virus generation, virus titre determination, and statistical analyses were described in the Methods section.

The study revealed that RaTG13 was more efficiently neutralized than SARS-CoV-2 by convalescent sera from SARS-CoV-2 infected patients (2-fold change, p=0.0001). This trend was also observed in sera from vaccinated healthcare workers, with BNT162b2 vaccination showing a more substantial increase in neutralization against RaTG13 compared to ChAdOx1. Analysis of spike mutants showed that SARS-CoV-2 multi-RBD mutants were neutralized more efficiently than SARS-CoV-2 WT, while RaTG13 multi-RBD mutants showed slightly reduced neutralization compared to RaTG13 WT. Individual amino acid substitutions in RaTG13 had minimal effects on neutralization, except for K439N. In contrast, some individual substitutions in SARS-CoV-2 significantly increased neutralization. Importantly, the E484K mutation increased RaTG13 neutralization but decreased SARS-CoV-2 neutralization, highlighting the context-dependent impact of this mutation. The data suggest that a significant number of changes in the RBD are tolerated without loss of neutralization efficacy by SARS-CoV-2-specific antibodies.

The unexpected finding that RaTG13 was more effectively neutralized than SARS-CoV-2 by SARS-CoV-2-specific antibodies suggests the existence of shared antigenic epitopes between the two viruses. The differential effects of the E484K mutation in SARS-CoV-2 and RaTG13 emphasize the context-dependent nature of this mutation's impact on immune escape. The results highlight the possibility that pre-existing immunity to SARS-CoV-2 might offer cross-protection against RaTG13, potentially mitigating the risk of future spillover events. The observed tolerance of multiple RBD substitutions in SARS-CoV-2 without substantial loss of neutralization might be partially explained by a reduced receptor-binding affinity for human ACE2 in RaTG13, allowing for easier displacement by higher-affinity antibodies. Further research is needed to explore the broader implications of these findings for cross-neutralization among related sarbecoviruses and the design of future vaccines that offer more comprehensive protection against diverse coronaviruses.

This study demonstrated that RaTG13, despite its RBD differences from SARS-CoV-2, is effectively neutralized by antibodies elicited by SARS-CoV-2 infection or vaccination. This suggests that existing SARS-CoV-2 immunity might provide cross-protection against RaTG13 spillover. The findings underscore the complexity of coronavirus immunology and the need for continued surveillance of emerging variants to guide vaccine development and public health strategies. Future research should focus on the broader cross-neutralization potential of SARS-CoV-2-induced immunity against more distantly related sarbecoviruses.

The study focused on neutralization assays using pseudotyped viruses, which might not fully recapitulate the complexity of authentic virus infection. The sample size for some analyses could be considered relatively small, potentially limiting the statistical power of some comparisons. Further studies are needed to confirm these findings using authentic viruses and larger cohorts of patients and vaccinated individuals.