Targeted transcriptional downregulation of MYC using epigenomic controllers demonstrates antitumor activity in hepatocellular carcinoma models.

Dysregulation of master regulator c-MYC (MYC) plays a central role in hepatocellular carcinoma (HCC) and other cancers but remains an elusive target for therapeutic intervention. MYC expression is epigenetically modulated within naturally occurring DNA loop structures, Insulated Genomic Domains (IGDs). We present a therapeutic approach using an epigenomic controller (EC), a programmable epigenomic mRNA medicine, to precisely modify MYC IGD sub-elements, leading to methylation of MYC regulatory elements and durable downregulation of MYC mRNA transcription. Significant antitumor activity is observed in preclinical models of HCC treated with the MYC-targeted EC, as monotherapy or in combination with tyrosine kinase or immune checkpoint inhibitors. These findings pave the way for clinical development of MYC-targeting epigenomic controllers in HCC patients and provide a framework for programmable epigenomic mRNA therapeutics for cancer and other diseases.

Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing.

The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro , implying a key role of the interaction in 'unhooking' during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB's stimulation of SNM1A's activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB's stimulation of SNM1A's nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.

Digital endpoints in clinical trials: emerging themes from a multi-stakeholder Knowledge Exchange event.

BACKGROUND: Digital technologies, such as wearable devices and smartphone applications (apps), can enable the decentralisation of clinical trials by measuring endpoints in people's chosen locations rather than in traditional clinical settings. Digital endpoints can allow high-frequency and sensitive measurements of health outcomes compared to visit-based endpoints which provide an episodic snapshot of a person's health. However, there are underexplored challenges in this emerging space that require interdisciplinary and cross-sector collaboration. A multi-stakeholder Knowledge Exchange event was organised to facilitate conversations across silos within this research ecosystem. METHODS: A survey was sent to an initial list of stakeholders to identify potential discussion topics. Additional stakeholders were identified through iterative discussions on perspectives that needed representation. Co-design meetings with attendees were held to discuss the scope, format and ethos of the event. The event itself featured a cross-disciplinary selection of talks, a panel discussion, small-group discussions facilitated via a rolling seating plan and audience participation via Slido. A transcript was generated from the day, which, together with the output from Slido, provided a record of the day's discussions. Finally, meetings were held following the event to identify the key challenges for digital endpoints which emerged and reflections and recommendations for dissemination. RESULTS: Several challenges for digital endpoints were identified in the following areas: patient adherence and acceptability; algorithms and software for devices; design, analysis and conduct of clinical trials with digital endpoints; the environmental impact of digital endpoints; and the need for ongoing ethical support. Learnings taken for next generation events include the need to include additional stakeholder perspectives, such as those of funders and regulators, and the need for additional resources and facilitation to allow patient and public contributors to engage meaningfully during the event. CONCLUSIONS: The event emphasised the importance of consortium building and highlighted the critical role that collaborative, multi-disciplinary, and cross-sector efforts play in driving innovation in research design and strategic partnership building moving forward. This necessitates enhanced recognition by funders to support multi-stakeholder projects with patient involvement, standardised terminology, and the utilisation of open-source software.

Chronic thromboembolic pulmonary hypertension is an uncommon complication of COVID-19: UK national surveillance and observational screening cohort studies.

BACKGROUND: Pulmonary embolism (PE) is a well-recognised complication of coronavirus disease 2019 (COVID-19) infection, and chronic thromboembolic pulmonary disease with and without pulmonary hypertension (CTEPD/CTEPH) are potential life-limiting consequences. At present the burden of CTEPD/CTEPH is unclear and optimal and cost-effective screening strategies yet to be established. METHODS: We evaluated the CTEPD/CTEPH referral rate to the UK national multidisciplinary team (MDT) during the 2017-2022 period to establish the national incidence of CTEPD/CTEPH potentially attributable to COVID-19-associated PE with historical comparator years. All individual cases of suspected CTEPH were reviewed by the MDT for evidence of associated COVID-19. In a separate multicentre cohort, the risk of developing CTEPH following hospitalisation with COVID-19 was calculated using simple clinical parameters at a median of 5 months post-hospital discharge according to existing risk scores using symptoms, ECG and N-terminal pro-brain natriuretic peptide. RESULTS: By the second year of the pandemic, CTEPH diagnoses had returned to the pre-pandemic baseline (23.1 versus 27.8 cases per month; p=0.252). Of 334 confirmed CTEPD/CTEPH cases, four (1.2%) patients were identified to have CTEPH potentially associated with COVID-19 PE, and a further three (0.9%) CTEPD without PH. Of 1094 patients (mean age 58 years, 60.4% male) hospitalised with COVID-19 screened across the UK, 11 (1.0%) were at high risk of CTEPH at follow-up, none of whom had a diagnosis of CTEPH made at the national MDT. CONCLUSION: A priori risk of developing CTEPH following COVID-19-related hospitalisation is low. Simple risk scoring is a potentially effective way of screening patients for further investigation.

SNM1A is crucial for efficient repair of complex DNA breaks in human cells.

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.

Cell-active small molecule inhibitors validate the SNM1A DNA repair nuclease as a cancer target.

The three human SNM1 metallo-ß-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.

Associate Principal Investigators and the HEAL-COVID trial: good for trainees, good for trials.

BACKGROUND: The NIHR's Associate Principal Investigator (API) Scheme in the United Kingdom was expanded nationally in 2020 with the aim of training clinicians to become Principal Investigators for clinical research in the future. The HEAL-COVID adaptive platform trial is an urgent public health study registered with the API Scheme. Within eighteen months of opening, the trial had recruited almost 1200 patients with over 100 active sites. Here we describe our experiences of APIs working on the trial with two broad objectives. Firstly, we aim to explore through qualitative methods the impact that the scheme has had on the APIs' professional development. Secondly, we aim to quantify the impact that the APIs have had on the recruitment of patients into the trial. METHODS: The professional backgrounds of the APIs are described from data from their application forms to the scheme. The HEAL-COVID API Network is described from records of the monthly meetings. The APIs' experiences are reviewed from data from the NIHR exit surveys at 6 months and from a reflective practice exercise at the final network meeting. Data of patient recruitment to HEAL-COVID was analysed for centres with and without APIs via a multivariate analysis. RESULTS: Forty-two APIs were registered with the HEAL-COVID trial with a diversity of backgrounds in terms of gender, country, profession, grade and specialty. Eleven monthly network meetings took place with the dual objectives of facilitating trial activity and providing educational content. Fourteen APIs completed the NIHR survey with all reporting Good Clinical Practice completion, local promotional activity of the trial, patient recruitment and support from their respective PI. Sites with at least one API recruited over 3.5 times more patients than sites without an API (medians 4 vs 14.5, p < 0.05), independent of factors including type of hospital or number of inpatient beds. DISCUSSION: This study adds to the growing literature that the NIHR's API Scheme is effective in meeting its objectives in providing research training to clinicians, thus building a workforce of future clinical researchers. Moreover, data from the HEAL-COVID trial shows that sites with an API are associated with higher recruitment. Overall, registering a trial with the API Scheme not only trains future clinical researchers, but it is also likely to increase the number of patients recruited (amongst other benefits), increasing the efficiency of trials and improving access for patients.

In vivo dose response and efficacy of the ß-lactamase inhibitor, durlobactam, in combination with sulbactam against the Acinetobacter baumannii-calcoaceticus complex.

Multi-drug resistant (MDR) Acinetobacter baumannii is emerging as a pathogen of increasing prevalence and concern. Infections associated with this Gram-negative pathogen are often associated with increased morbidity and mortality and few therapeutic options. The ß-lactamase inhibitor sulbactam used commonly in combination with ampicillin demonstrates intrinsic antibacterial activity against A. baumannii acting as an inhibitor of PBP1 and PBP3, which participate in cell wall biosynthesis. The production of ß-lactamases, particularly class D oxacillinases, however, has limited the utility of sulbactam resorting to increased doses and the need for alternate therapies. Durlobactam is a non-ß-lactam ß-lactamase inhibitor that demonstrates broad ß-lactamase inhibition including class D enzymes produced by A. baumannii and has shown potent in vitro activity against MDR A. baumannii, particularly carbapenem-resistant isolates in susceptibility and pharmacodynamic model systems. The objective of this study is to evaluate the exposure-response relationship of sulbactam and durlobactam in combination using in vivo neutropenic thigh and lung models to establish PK/PD exposure magnitudes to project clinically effective doses. Utilizing established PK/PD determinants of %T>MIC and AUC/MIC for sulbactam and durlobactam, respectively, non-linear regressional analysis of drug exposure was evaluated relative to the 24-hour change in bacterial burden (log10 CFU/g). Co-modeling of the data across multiple strains exhibiting a broad range of MIC susceptibility suggested net 1-log10 CFU/g0 reduction can be achieved when sulbactam T>MIC exceeds 50% of the dosing interval and durlobactam AUC/MIC is 10. These data were ultimately used to support sulbactam-durlobactam dose selection for Phase 3 clinical trials.

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.

Respiratory sequelae of COVID-19: pulmonary and extrapulmonary origins, and approaches to clinical care and rehabilitation.

Although the exact prevalence of post-COVID-19 condition (also known as long COVID) is unknown, more than a third of patients with COVID-19 develop symptoms that persist for more than 3 months after SARS-CoV-2 infection. These sequelae are highly heterogeneous in nature and adversely affect multiple biological systems, although breathlessness is a frequently cited symptom. Specific pulmonary sequelae, including pulmonary fibrosis and thromboembolic disease, need careful assessment and might require particular investigations and treatments. COVID-19 outcomes in people with pre-existing respiratory conditions vary according to the nature and severity of the respiratory disease and how well it is controlled. Extrapulmonary complications such as reduced exercise tolerance and frailty might contribute to breathlessness in post-COVID-19 condition. Non-pharmacological therapeutic options, including adapted pulmonary rehabilitation programmes and physiotherapy techniques for breathing management, might help to attenuate breathlessness in people with post-COVID-19 condition. Further research is needed to understand the origins and course of respiratory symptoms and to develop effective therapeutic and rehabilitative strategies.

Androgen Receptor Status in Triple Negative Breast Cancer: Does It Correlate with Clinicopathological Characteristics?

Purpose: Triple negative breast cancer (TNBC) is a breast carcinoma subtype that neither expresses estrogen (ER) and progesterone receptors (PR) nor the human epidermal growth factor receptor 2 (HER2). Patients with TNBC have been shown to have poorer outcomes mainly owing to the limited treatment options available. However, some studies have shown TNBC tumors expressing androgen receptors (AR), raising hopes of its prognostic role. Patients and Methods: This retrospective study investigated the expression of AR in TNBC and its relationship with known patient demographics, tumor and survival characteristics. From the records of 205 TNBC patients, 36 had available archived tissue samples eligible for AR staining. For statistical purposes, tumors were classified as either "positive" or "negative" for AR expression. The nuclear expression of AR was scored by measuring the percentage of stained tumor cells and its staining intensity. Results: AR was expressed by 50% of the tissue samples in our TNBC cohort. The relationship between AR status with age at the time of TNBC diagnosis was statistically significant, with all AR positive TNBC patients being greater than 50 years old (vs 72.2% in AR negative TNBC). Also, the relationship between AR status and type of surgery received was statistically significant. There were no statistically significant associations between AR status with other tumor characteristics including "TNM status", tumor grade or treatments received. There was no statistically significant difference in median survival between AR negative and AR positive TNBC patients (3.5 vs 3.1 years; p = 0.581). The relationship between OS time and AR status (p = 0.581), type of surgery (p = 0.061) and treatments (p = 0.917) were not statistically significant. Conclusion: The androgen receptor may be an important prognostic marker in TNBC, with further research warranted. This research may benefit future studies investigating receptor-targeted therapies in TNBC.

ChAdOx1 nCoV-19 vaccination generates spike-specific CD8+ T cells in aged mice.

Effective vaccines have reduced the morbidity and mortality caused by severe acute respiratory syndrome coronavirus-2 infection; however, the elderly remain the most at risk. Understanding how vaccines generate protective immunity and how these mechanisms change with age is key for informing future vaccine design. Cytotoxic CD8+ T cells are important for killing virally infected cells, and vaccines that induce antigen-specific CD8+ T cells in addition to humoral immunity provide an extra layer of immune protection. This is particularly important in cases where antibody titers are suboptimal, as can occur in older individuals. Here, we show that in aged mice, spike epitope-specific CD8+ T cells are generated in comparable numbers to younger animals after ChAdOx1 nCoV-19 vaccination, although phenotypic differences exist. This demonstrates that ChAdOx1 nCoV-19 elicits a good CD8+ T-cell response in older bodies, but that typical age-associated features are evident on these vaccine reactive T cells.

Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development.

NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.

Tfh cells and the germinal center are required for memory B cell formation & humoral immunity after ChAdOx1 nCoV-19 vaccination.

Emergence from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been facilitated by the rollout of effective vaccines. Successful vaccines generate high-affinity plasma blasts and long-lived protective memory B cells. Here, we show a requirement for T follicular helper (Tfh) cells and the germinal center reaction for optimal serum antibody and memory B cell formation after ChAdOx1 nCoV-19 vaccination. We found that Tfh cells play an important role in expanding antigen-specific B cells while identifying Tfh-cell-dependent and -independent memory B cell subsets. Upon secondary vaccination, germinal center B cells generated during primary immunizations can be recalled as germinal center B cells again. Likewise, primary immunization GC-Tfh cells can be recalled as either Tfh or Th1 cells, highlighting the pluripotent nature of Tfh cell memory. This study demonstrates that ChAdOx1 nCoV-19-induced germinal centers are a critical source of humoral immunity.

ChAdOx1 nCoV-19 (AZD1222) or nCoV-19-Beta (AZD2816) protect Syrian hamsters against Beta Delta and Omicron variants.

ChAdOx1 nCoV-19 (AZD1222) is a replication-deficient simian adenovirus-vectored vaccine encoding the spike (S) protein of SARS-CoV-2, based on the first published full-length sequence (Wuhan-1). AZD1222 has been shown to have 74% vaccine efficacy against symptomatic disease in clinical trials. However, variants of concern (VoCs) have been detected, with substitutions that are associated with a reduction in virus neutralizing antibody titer. Updating vaccines to include S proteins of VoCs may be beneficial, even though current real-world data is suggesting good efficacy following boosting with vaccines encoding the ancestral S protein. Using the Syrian hamster model, we evaluate the effect of a single dose of AZD2816, encoding the S protein of the Beta VoC, and efficacy of AZD1222/AZD2816 as a heterologous primary series against challenge with the Beta or Delta variant. Minimal to no viral sgRNA could be detected in lungs of vaccinated animals obtained at 3- or 5- days post inoculation, in contrast to lungs of control animals. In Omicron-challenged hamsters, a single dose of AZD2816 or AZD1222 reduced virus shedding. Thus, these vaccination regimens are protective against the Beta, Delta, and Omicron VoCs in the hamster model.

Neutralizing antibody activity against 21 SARS-CoV-2 variants in older adults vaccinated with BNT162b2.

SARS-CoV-2 variants may threaten the effectiveness of vaccines and antivirals to mitigate serious COVID-19 disease. This is of most concern in clinically vulnerable groups such as older adults. We analysed 72 sera samples from 37 individuals, aged 70-89 years, vaccinated with two doses of BNT162b2 (Pfizer-BioNTech) 3 weeks apart, for neutralizing antibody responses to wildtype SARS-CoV-2. Between 3 and 20 weeks after the second vaccine dose, neutralizing antibody titres fell 4.9-fold to a median titre of 21.3 (neutralization dose 80%), with 21.6% of individuals having no detectable neutralizing antibodies at the later time point. Next, we examined neutralization of 21 distinct SARS-CoV-2 variant spike proteins with these sera, and confirmed substantial antigenic escape, especially for the Omicron (B.1.1.529, BA.1/BA.2), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 spike variants. By combining pseudotype neutralization with specific receptor-binding domain (RBD) enzyme-linked immunosorbent assays, we showed that changes to position 484 in the spike RBD were mainly responsible for SARS-CoV-2 neutralizing antibody escape. Nineteen sera from the same individuals boosted with a third dose of BNT162b2 contained higher neutralizing antibody titres, providing cross-protection against Omicron BA.1 and BA.2. Despite SARS-CoV-2 immunity waning over time in older adults, booster vaccines can elicit broad neutralizing antibodies against a large number of SARS-CoV-2 variants in this clinically vulnerable cohort.

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

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.