Nick K. Jones1,2*, Lucy Rivett1,2*, Chris Workman3, Mark Ferris3, Ashley Shaw1, Cambridge COVID-19 Collaboration1,4, Paul J. Lehner1,4, Rob Howes5, Giles Wright3, Nicholas J. Matheson1,4,6¶, Michael P. Weekes1,7¶1 Cambridge University NHS Hospitals Foundation Trust, Cambridge, UK2 Clinical Microbiology & Public Health Laboratory, Public Health England, Cambridge, UK3 Occupational Health and Wellbeing, Cambridge Biomedical Campus, Cambridge, UK4 Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK5 Cambridge COVID-19 Testing Centre and AstraZeneca, Anne Mclaren Building, Cambridge, UK6 NHS Blood and Transplant, Cambridge, UK7 Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK*Joint first authorship¶Joint last authorshipCorrespondence: [email protected] UK has initiated mass COVID-19 immunisation, with healthcare workers (HCWs) given early priority because of the potential for workplace exposure and risk of onward transmission to patients. The UK’s Joint Committee on Vaccination and Immunisation has recommended maximising the number of people vaccinated with first doses at the expense of early booster vaccinations, based on single dose efficacy against symptomatic COVID-19 disease.1-3At the time of writing, three COVID-19 vaccines have been granted emergency use authorisation in the UK, including the BNT162b2 mRNA COVID-19 vaccine (Pfizer-BioNTech). A vital outstanding question is whether this vaccine prevents or promotes asymptomatic SARS-CoV-2 infection, rather than symptomatic COVID-19 disease, because sub-clinical infection following vaccination could continue to drive transmission. This is especially important because many UK HCWs have received this vaccine, and nosocomial COVID-19 infection has been a persistent problem.Through the implementation of a 24 h-turnaround PCR-based comprehensive HCW screening programme at Cambridge University Hospitals NHS Foundation Trust (CUHNFT), we previously demonstrated the frequent presence of pauci- and asymptomatic infection amongst HCWs during the UK’s first wave of the COVID-19 pandemic.4 Here, we evaluate the effect of first-dose BNT162b2 vaccination on test positivity rates and cycle threshold (Ct) values in the asymptomatic arm of our programme, which now offers weekly screening to all staff.Vaccination of HCWs at CUHNFT began on 8th December 2020, with mass vaccination from 8th January 2021. Here, we analyse data from the two weeks spanning 18thto 31st January 2021, during which: (a) the prevalence of COVID-19 amongst HCWs remained approximately constant; and (b) we screened comparable numbers of vaccinated and unvaccinated HCWs. Over this period, 4,408 (week 1) and 4,411 (week 2) PCR tests were performed from individuals reporting well to work. We stratified HCWs <12 days or > 12 days post-vaccination because this was the point at which protection against symptomatic infection began to appear in phase III clinical trial.226/3,252 (0·80%) tests from unvaccinated HCWs were positive (Ct<36), compared to 13/3,535 (0·37%) from HCWs <12 days post-vaccination and 4/1,989 (0·20%) tests from HCWs ≥12 days post-vaccination (p=0·023 and p=0·004, respectively; Fisher’s exact test, Figure). This suggests a four-fold decrease in the risk of asymptomatic SARS-CoV-2 infection amongst HCWs ≥12 days post-vaccination, compared to unvaccinated HCWs, with an intermediate effect amongst HCWs <12 days post-vaccination.A marked reduction in infections was also seen when analyses were repeated with: (a) inclusion of HCWs testing positive through both the symptomatic and asymptomatic arms of the programme (56/3,282 (1·71%) unvaccinated vs 8/1,997 (0·40%) ≥12 days post-vaccination, 4·3-fold reduction, p=0·00001); (b) inclusion of PCR tests which were positive at the limit of detection (Ct>36, 42/3,268 (1·29%) vs 15/2,000 (0·75%), 1·7-fold reduction, p=0·075); and (c) extension of the period of analysis to include six weeks from December 28th to February 7th 2021 (113/14,083 (0·80%) vs 5/4,872 (0·10%), 7·8-fold reduction, p=1x10-9). In addition, the median Ct value of positive tests showed a non-significant trend towards increase between unvaccinated HCWs and HCWs > 12 days post-vaccination (23·3 to 30·3, Figure), suggesting that samples from vaccinated individuals had lower viral loads.We therefore provide real-world evidence for a high level of protection against asymptomatic SARS-CoV-2 infection after a single dose of BNT162b2 vaccine, at a time of predominant transmission of the UK COVID-19 variant of concern 202012/01 (lineage B.1.1.7), and amongst a population with a relatively low frequency of prior infection (7.2% antibody positive).5This work was funded by a Wellcome Senior Clinical Research Fellowship to MPW (108070/Z/15/Z), a Wellcome Principal Research Fellowship to PJL (210688/Z/18/Z), and an MRC Clinician Scientist Fellowship (MR/P008801/1) and NHSBT workpackage (WPA15-02) to NJM. Funding was also received from Addenbrooke’s Charitable Trust and the Cambridge Biomedical Research Centre. We also acknowledge contributions from all staff at CUHNFT Occupational Health and Wellbeing and the Cambridge COVID-19 Testing Centre.

IntroductionHuman cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that persistently infects the majority of the human population worldwide (Cannon et al., 2010). Following primary infection under the control of a healthy immune system, a latent infection is established that persists lifelong (Reeves et al., 2005). Although primary infection is mostly asymptomatic in healthy individuals, HCMV may lead to significant morbidity or mortality in immunocompromised patients, particularly transplant recipients and AIDS patients (Griffiths et al., 2015). Vertical transmission of HCMV is a leading cause of congenital infection, resulting in deafness and intellectual disability in newborns (Manicklal et al., 2013). Existing therapies that either target the viral polymerase or terminase are associated with significant toxicity and/or sporadic resistance (El Helou and Razonable, 2019). The identification and characterisation of critical facets of host innate immunity that are targeted for degradation by HCMV proteins thus has important implications for antiviral therapy, since such interactions may be inhibitable by small-molecules, facilitating endogenous inhibition of viral replication (Nathans et al., 2008).HCMV has been reported to disrupt interferon (IFN) production, neutralise the IFN response (Le-Trilling and Trilling, 2015;Goodwin et al., 2018), inhibit natural killer (NK) cell activation (Patel et al., 2018), and avoid T cell surveillance via downregulation of MHC molecules (Jackson et al., 2011). A common final pathway for many host protein targets is proteasomal or lysosomal degradation (Halenius et al., 2015). For example, HCMV facilitates viral replication by degrading components of cellular promyelocytic leukemia nuclear bodies (PML-NB) Sp100, MORC3 and DAXX that act as restriction factors (Kim et al., 2011;Tavalai et al., 2011;Schreiner and Wodrich, 2013;Sloan et al., 2016).We previously developed three orthogonal proteomic/transcriptomic screens to quantify protein degradation early during HCMV infection, identifying 133 degraded proteins that were enriched in antiviral restriction factors. The power of this approach was demonstrated by our identification of helicase-like transcription factor (HLTF) as a novel restriction factor that potently inhibited early viral gene expression and was targeted by the HCMV protein UL145 (Nightingale et al., 2018). However, a global approach to identify the mechanism of HCMV-induced protein degradation is lacking. Our previous study employed the broad, non-selective inhibitor MG132, which is known to affect lysosomal cathepsins in addition to the proteasome (Wiertz et al., 1996), and leupeptin which is a naturally occurring protease inhibitor that can inhibit some proteasomal proteases in addition to the lysosome (Nightingale et al., 2018).In this study, we used the selective proteasome inhibitor bortezomib (Chen et al., 2011) to identify proteins specifically targeted for proteasomal degradation during HCMV infection. This identified that the majority of proteins rescued from degradation by MG132 were also rescued by bortezomib, highlighting the role of viral subversion of the proteasome in immune evasion. Our data additionally provide a shortlist of proteins degraded by the proteasome early during infection that are enriched in known antiviral factors for further investigation.