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

The study investigates antigenic relationships between SARS-CoV-2 and its close bat-derived relative RaTG13, focusing on whether antibodies elicited by SARS-CoV-2 infection or vaccination can neutralise RaTG13. Given substantial sequence similarity but notable RBD amino acid differences that affect ACE2 binding, the research question asks if these differences also translate into altered neutralisation susceptibility and potential immune escape. Understanding cross-neutralisation informs spillover risk assessment and the breadth of protection conferred by current SARS-CoV-2 vaccines.

Prior work has established that certain SARS-CoV-2 variants of concern (e.g., B.1.351/Beta) escape neutralising antibodies, with residues such as E484, N501, Q493, and others in the RBD implicated in modulating antibody binding and ACE2 affinity. E484K, in particular, is associated with immune evasion. Studies also suggest sarbecoviruses can adapt to human ACE2, and that cross-neutralising antibodies can exist across related coronaviruses. However, detailed antigenic comparison between SARS-CoV-2 and RaTG13, including the impact of RaTG13-specific RBD substitutions, had been less explored.

• Study design: Comparative pseudovirus neutralisation (pMN) assays using SARS-CoV-2 and RaTG13 spike-pseudotyped viruses, including chimeric 'Multi RBD' constructs and individual point mutants reflecting reciprocal RBD substitutions.
• Sera panels: WHO International Reference Panel (NIBSC 20/268); convalescent sera from patients and healthcare workers (HCWs) infected during the first UK wave (n=25); sera from HCWs after first vaccine dose (ChAdOx1 n=12; BNT162b2 n=9); additional doubly vaccinated HCWs (n=20) for specific mutant analyses.
• Cell culture: HEK293T/17 cells maintained in DMEM with 10% FBS and antibiotics; HEK293T/17 transiently made permissive via expression of ACE2 and TMPRSS2 for titration/infection.
• Plasmids: Expression constructs for SARS-CoV-2 and RaTG13 spikes, chimeras swapping RBD regions, and single amino acid mutants; Beta (B.1.351) spike variant included. Constructs synthesised commercially and subcloned into pcDNA3.1+/pcGAGS.
• Pseudotype generation: Co-transfection of HEK293T/17 with plasmids encoding HIV gag, luciferase reporter, and spike variants using FuGENE HD; harvest 48–72 h post-transfection, filtration (0.45 µm), storage at −70°C.
• Neutralisation assay: Serial dilution of sera incubated with pseudoviruses, infection of permissive cells, luciferase readout after 48 h; IC50/EC50 derived via non-linear regression (GraphPad Prism 8/9). Lower limit of detection denoted; biological replicates independent.
• Mutant panels: Multi RBD constructs with multiple RBD substitutions reciprocal between SARS-CoV-2 and RaTG13; single-residue mutants tested individually (some not assessed due to low titres, e.g., RaTG13 Y498Q).
• Statistics: Wilcoxon matched-pairs signed-rank tests for paired neutralisation comparisons; Student’s t-tests for fold-change analyses in mutant panels; significance thresholds reported with p-values.

• RaTG13 pseudovirus was neutralised more efficiently than SARS-CoV-2 by convalescent sera: approximately 2-fold higher neutralisation (p=0.0001).
• The Beta (B.1.351) variant showed significantly reduced neutralisation compared to SARS-CoV-2 (∼4.0-fold decrease, p=0.0001).
• Vaccine-derived sera after a single dose showed enhanced neutralisation of RaTG13 relative to SARS-CoV-2: BNT162b2 ∼2.1-fold (p=0.001); ChAdOx1 ∼1.2-fold (p=0.0061). Stratification by prior infection suggested differences in magnitude; no significant differences were observed in some no-prior-infection subsets (per supplementary data).
• Multi RBD constructs implicated RBD substitutions in modulating neutralisation: SARS-CoV-2 Multi RBD was neutralised more efficiently than SARS-CoV-2 WT (reported as significant; p=0.0005); RaTG13 Multi RBD was neutralised slightly less than RaTG13 WT (∼1.2-fold reduction; p=0.0043).
• Single-residue effects:
  • In the RaTG13 background, most single substitutions had minimal impact; K439N increased neutralisation ∼1.2-fold (p=0.0194). RaTG13 Y498Q could not be evaluated due to insufficient titres.
  • In the SARS-CoV-2 background, Q493Y increased neutralisation ∼1.2-fold (p=0.0088) and N501D ∼2.9-fold (p=0.0069).
  • E484K in SARS-CoV-2 significantly reduced neutralisation (p=0.00001), consistent with immune escape.
  • The analogous 484K substitution in RaTG13 led to a modest, non-significant increase in neutralisation (p=0.1054).
  • T403R in RaTG13 reduced neutralisation significantly (p=0.00001).
• Data support an 'affinity model' whereby lower ACE2 binding affinity (e.g., RaTG13 WT relative to SARS-CoV-2) may render spikes more susceptible to antibody-mediated neutralisation; conversely, substitutions that enhance ACE2 affinity (e.g., T403R) can decrease neutralisation.

The findings address whether SARS-CoV-2-induced immunity neutralises the related bat coronavirus RaTG13. Contrary to expectations based on extensive RBD differences, RaTG13 was neutralised as well as or better than SARS-CoV-2 by both convalescent and vaccinee sera, indicating conserved antigenic epitopes are targeted by human antibodies. Mutational analyses suggest that several RBD residues modulate neutralisation, and that SARS-CoV-2-specific antibodies can tolerate substantial sequence variation within the RBD without a dramatic loss of neutralisation. The contrasting effects of position 484 in the two backbones (immune escape for SARS-CoV-2 E484K versus neutralisation increase for RaTG13 484K) underscore the context dependence of antigenic effects. Results align with an affinity-based mechanism: spikes with reduced human ACE2 affinity (e.g., RaTG13) are more easily inhibited by antibodies, whereas substitutions increasing ACE2 usage (e.g., T403R) reduce neutralisation. These data suggest pre-existing SARS-CoV-2 immunity could reduce the risk posed by spillover of closely related sarbecoviruses like RaTG13 and inform vaccine strategies focused on breadth.

RaTG13 is efficiently neutralised by antibodies elicited through SARS-CoV-2 infection and vaccination, implying that pre-existing immunity in humans may mitigate spillover risk from RaTG13-like sarbecoviruses. Despite numerous RBD differences, cross-neutralisation remains robust, and current SARS-CoV-2-based vaccines likely confer meaningful protection against closely related viruses. The study highlights that a limited set of substitutions can substantially influence neutralisation, reinforcing surveillance priorities for SARS-CoV-2 variants carrying potent escape mutations. Future work should define the breadth of this cross-neutralisation across a wider panel of sarbecoviruses (e.g., including SARS-CoV-1 and other bat coronaviruses), dissect structural bases of epitope conservation, and assess live-virus correlates of pseudovirus findings.

• Use of pseudotyped viruses rather than live viruses may not fully capture all aspects of neutralisation and entry.
• Neutralisation assays focus on humoral responses; cellular immunity was not assessed.
• Some analyses used modest sample sizes (e.g., n=4 for certain single-mutant assays), which may limit statistical power.
• Certain mutants could not be evaluated due to low pseudovirus titres (e.g., RaTG13 Y498Q).
• Cross-neutralisation breadth beyond RaTG13 (to more distantly related sarbecoviruses) was not directly tested in this study.
• Affinity-based interpretations are inferential, based on infectivity proxies and literature, rather than direct biophysical ACE2 binding measurements within this work.