TRIM7 restricts Coxsackievirus and norovirus infection by detecting the C-terminalglutamine generated by 3C protease processing (preprint)

TRIM7 catalyses the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7s extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The helix-FQ degron motif recognised by TRIM7 is reminiscent of the N-end degron system and is found in ~ 1% of cellular proteins. These features, together with TRIM7s restricted tissue expression and lack of immune regulation suggest that viral restriction may not be its physiological function.

Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis (preprint)

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

SARS-COV-2 Vaccine Responses in Renal Patient Populations (preprint)

BackgroundDialysis patients and immunosuppressed renal patients are at increased risk of COVID-19 and were excluded from vaccine trials. We conducted a prospective multicentre study to assess SARS-CoV-2 vaccine antibody responses in dialysis patients and renal transplant recipients, and patients receiving immunosuppression for autoimmune disease. Methods Patients were recruited from three UK centres (ethics:20/EM/0180) and compared to healthy controls (ethics:17/EE/0025). SARS-CoV-2 IgG antibodies to spike protein were measured using a multiplex Luminex assay, after first and second doses of Pfizer BioNTech BNT162b2(Pfizer) or Oxford-AstraZeneca ChAdOx1nCoV-19(AZ) vaccine.Results692 patients were included (260 dialysis, 209 transplant, 223 autoimmune disease (prior rituximab 128(57%)) and 144 healthy controls. 299(43%) patients received Pfizer vaccine and 379(55%) received AZ.  Following two vaccine doses, positive responses occurred in 96% dialysis, 52% transplant, 70% autoimmune patients and 100% of healthy controls. In dialysis patients, higher antibody responses were observed with the Pfizer vaccination. Predictors of poor antibody response were triple immunosuppression (adjusted odds ratio [aOR]0.016;95%CI0.002-0.13;p<0.001) and mycophenolate mofetil (MMF) (aOR0.2;95%CI 0.1-0.42;p<0.001) in transplant patients; rituximab within 12 months in autoimmune patients (aOR0.29;95%CI 0.008–0.096;p<0.001) and patients receiving immunosuppression with eGFR 15-29ml/min (aOR0.031;95%CI 0.11–0.84;p=0.021). ConclusionsAmongst dialysis, kidney transplant and autoimmune populations SARS-CoV-2 vaccine antibody responses are reduced compared to healthy controls. A reduced response to vaccination was associated with rituximab, MMF, triple immunosuppression CKD stage 4. Vaccine responses increased after the second dose, suggesting low-responder groups should be prioritised for repeated vaccination. Greater antibody responses were observed with the mRNA Pfizer vaccine compared to adenovirus AZ vaccine in dialysis patients suggesting that Pfizer SARS-CoV-2 vaccine should be the preferred vaccine choice in this sub-group.

Tracking SARS-COV-2 Variants Using Nanopore Sequencing in Ukraine in Summer 2021 (preprint)

Since spring 2020, Ukraine has experienced at least two COVID-19 waves and has just entered a third wave in autumn 2021. The use of real-time genomic epidemiology has enabled the tracking of SARS-CoV-2 circulation patterns worldwide, thus informing evidence-based public health decision making, including implementation of travel restrictions and vaccine rollout strategies. However, insufficient capacity for local genetic sequencing in Ukraine and other Lower and Middle-Income countries limit opportunities for similar analyses. Herein, we report local sequencing of 24 SARS-CoV-2 genomes from patient samples collected in Kyiv in July 2021 using Oxford Nanopore MinION technology. Together with other published Ukrainian SARS-COV-2 genomes sequenced mostly abroad, our data suggest that the second wave of the epidemic in Ukraine (February-April 2021) was dominated by the Alpha variant of concern (VOC), while the beginning of the third wave has been dominated by the Delta VOC. Furthermore, our phylogeographic analysis revealed that the Delta variant was introduced into Ukraine in summer 2021 from multiple locations worldwide, with most introductions coming from Central and Eastern European countries. This study highlights the need to urgently integrate affordable and easily-scaled pathogen sequencing technologies in locations with less developed genomic infrastructure, in order to support local public health decision making.

Heat Inactivation of Clinical COVID-19 Samples on an Industrial Scale for Low Risk and Efficient High-throughput qRT-PCR Diagnostic Testing (preprint)

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 120 million confirmed cases, over 3.8 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as ‘direct to PCR’ assays that do not involve RNA extraction or chemical neutralisation methods.

The impact of hypoxia on B cells in COVID-19 (preprint)

Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This brought to mind the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. We demonstrated the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes we also observed in B cell VHL-deficient mice. This was corroborated by hypoxia-related transcriptional changes in COVID-19 patients, and by similar B cell abnormalities in mice kept in hypoxic conditions, including reduced marginal zone and germinal center B cells. Thus hypoxia might contribute to B cell pathology in COVID-19, and in other hypoxic states. Through this mechanism it may impact on COVID-19 outcome, and be remediable through early oxygen therapy.

Early Immune Pathology and Persistent Dysregulation Characterise Severe COVID-19 (preprint)

In a study of 207 SARS-CoV2-infected individuals with a range of severities followed over 12 weeks from symptom onset, we demonstrate that an early robust immune response, without systemic inflammation, is characteristic of asymptomatic or mild disease. Those presenting to hospital had delayed adaptive responses and systemic inflammation already evident at around symptom onset. Such early evidence of inflammation suggests immunopathology may be inevitable in some individuals, or that preventative intervention might be needed before symptom onset. Viral load does not correlate with the development of this pathological response, but does with its subsequent severity. Immune recovery is complex, with profound persistent cellular abnormalities correlating with a change in the nature of the inflammatory response, where signatures characteristic of increased oxidative phosphorylation and reactive-oxygen species-associated inflammation replace those driven by TNF and IL-6. These late immunometabolic inflammatory changes and unresolved immune cell defects, if persistent, may contribute to “long COVID”.Funding: We are grateful for the generous support of CVC Capital Partners, the Evelyn Trust (20/75), UKRI COVID Immunology Consortium, Addenbrooke’s Charitable Trust (12/20A) and the NIHR Cambridge Biomedical Research Centre for their financial support. K.G.C.S. is the recipient of a Wellcome Investigator Award (200871/Z/16/Z); M.P.W. is the recipient of Wellcome Senior Clinical Research Fellowship (108070/Z/15/Z); C.H. was funded by a Wellcome COVID-19 Rapid Response DCF and the Fondation Botnar; N.M. was funded by the MRC (CSF MR/P008801/1) and NHSBT (WPA15-02); I.G.G. is a Wellcome Senior Fellow and was supported by funding from the Wellcome (Ref: 207498/Z/17/Z).Conflict of Interest: The authors declare they have no competing interests.

A Prospective Study of Risk Factors Associated with Seroprevalence of SARS-CoV-2 Antibodies in Healthcare Workers at a Large UK Teaching Hospital (preprint)

Background: The COVID-19 pandemic continues to grow at an unprecedented rate. Healthcare workers (HCWs) are at higher risk of SARS-CoV-2 infection than the general population but risk factors for HCW infection are not well described.Methods: We conducted a prospective sero-epidemiological study of HCWs at a UK teaching hospital using a SARS-CoV-2 immunoassay. Risk factors for seropositivity were analysed using multivariate logistic regression.Findings: 410/5,698 (7·2%) staff tested positive for SARS-CoV-2 antibodies. Seroprevalence was higher in those working in designated COVID-19 areas compared with other areas (9·47% versus 6·16%) Healthcare assistants (aOR 2·06 [95%CI 1·14-3·71]; p =0·016) and domestic and portering staff (aOR 3·45 [95% CI 1·07-11·42]; p =0·039) had significantly higher seroprevalence than other staff groups after adjusting for age, sex, ethnicity and COVID-19 working location. Staff working in acute medicine and medical sub-specialities were also at higher risk (aOR 2·07 [95% CI 1·31-3·25]; p <0·002). Staff from Black, Asian and minority ethnic (BAME) backgrounds had an aOR of 1·65 (95% CI 1·32 – 2·07; p <0·001) compared to white staff; this increased risk was independent of COVID-19 area working. The only symptoms significantly associated with seropositivity in a multivariable model were loss of sense of taste or smell, fever and myalgia; 31% of staff testing positive reported no prior symptoms.Interpretation: Risk of SARS-CoV-2 infection amongst HCWs is heterogeneous and influenced by COVID-19 working location, role, age and ethnicity. Increased risk amongst BAME staff cannot be accounted for solely by occupational factors.Funding: Wellcome Trust, Addenbrookes Charitable Trust, National Institute for Health Research, Academy of Medical Sciences, the Health Foundation and the NIHR Cambridge Biomedical Research Centre.Declaration of Interests: None to declare.Ethics Approval Statement: Ethical approval for this study was granted by the East of England – Cambridge Central Research Ethics Committee (IRAS ID: 220277).

Successful treatment of COVID-19 with remdesivir in the absence of humoral immunity (preprint)

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the successful use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. His unusual clinical course identifies a key role for SARS-CoV-2 antibodies in both viral clearance and progression to severe disease. In the absence of these confounders, we took an experimental medicine approach to examine the in vivoutility of remdesivir. Over two independent courses of treatment, we observed a dramatic, temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide unambiguous evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore (preprint)

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to 10 GBP per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

COVID-19 infection dynamics in care homes in the East of England: a retrospective genomic epidemiology study (preprint)

Background COVID-19 poses a major challenge to infection control in care homes. SARS-CoV-2 is readily transmitted between people in close contact and causes disproportionately severe disease in older people. Methods Data and SARS-CoV-2 samples were collected from patients in the East of England (EoE) between 26th February and 10th May 2020. Care home residents were identified using address search terms and Care Quality Commission registration information. Samples were sequenced at the University of Cambridge or the Wellcome Sanger Institute and viral clusters defined based on genomic and time differences between cases. Findings 7,406 SARS-CoV-2 positive samples from 6,600 patients were identified, of which 1,167 (18.2%) were residents from 337 care homes. 30/71 (42.3%) care home residents tested at Cambridge University Hospitals NHS Foundation Trust (CUH) died. Genomes were available for 700/1,167 (60%) residents from 292 care homes, and 409 distinct viral clusters were defined. We identified several probable transmissions between care home residents and healthcare workers (HCW). Interpretation Care home residents had a significant burden of COVID-19 infections and high mortality. Larger viral clusters were consistent with within-care home transmission, while multiple clusters per care home suggested independent acquisitions.

Combined point of care nucleic acid and antibody testing for SARS-CoV-2: a prospective cohort study in suspected moderate to severe COVID-19 disease. (preprint)

BackgroundRapid COVID-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. The diagnosis of infection is complicated by 30-50% of COVID-19 hospital admissions with nose/throat swabs testing negative for SARS-CoV-2 nucleic acid, frequently after the first week of illness when SARS-CoV-2 antibody responses become detectable. We assessed the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease in the emergency department. MethodsWe developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike (S) protein and (ii) an ELISA test to detect IgG antibodies to nucleocapsid (N) and S proteins from SARS-CoV-2. We tested two lateral flow rapid fingerprick tests with bands for IgG and IgM. We then prospectively recruited participants with suspected moderate to severe COVID-19 and tested for SARS-CoV-2 nucleic acid in a combined nasal/throat swab using the standard laboratory RT-PCR and a validated rapid POC nucleic acid amplification (NAAT) test. Additionally, serum collected at admission was retrospectively tested by in vitro neutralisation, ELISA and the candidate POC antibody tests. We evaluated the performance of the individual and combined rapid POC diagnostic tests against a composite reference standard of neutralisation and standard laboratory based RT-PCR. Results45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. Using the composite reference standard, prevalence of COVID-19 disease was 53.3% (24/45). Median age was 73.5 (IQR 54.0-86.5) years in those with COVID-19 disease by our reference standard and 63.0 (IQR 41.0-72.0) years in those without disease. The overall detection rate by rapid NAAT was 79.2% (95CI 57.8-92.9%), decreasing from 100% (95% CI 65.3-98.6%) in days 1-4 to 50.0% (95% CI 11.8-88.2) for days 9-28 post symptom onset. Correct identification of COVID-19 with combined rapid POC diagnostic tests was 100% (95CI 85.8-100%) with a false positive rate of 5.3-14.3%, driven by POC LFA antibody tests. ConclusionsCombined POC tests have the potential to transform our management of COVID-19, including inflammatory manifestations later in disease where nucleic acid test results are negative. A rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used.

A thermostable, closed, SARS-CoV-2 spike protein trimer. (preprint)

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission (preprint)

Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3-week period (April 2020), 1,032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19) >7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B{middle dot}1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff.

Rapid implementation of real-time SARS-CoV-2 sequencing to investigate healthcare-associated COVID-19 infections (preprint)

Background The burden and impact of healthcare-associated COVID-19 infections is unknown. We aimed to examine the utility of rapid sequencing of SARS-CoV-2 combined with detailed epidemiological analysis to investigate healthcare-associated COVID-19 infections and to inform infection control measures. Methods We set up rapid viral sequencing of SARS-CoV-2 from PCR-positive diagnostic samples using nanopore sequencing, enabling sample-to-sequence in less than 24 hours. We established a rapid review and reporting system with integration of genomic and epidemiological data to investigate suspected cases of healthcare-associated COVID-19. Results Between 13 March and 24 April 2020 we collected clinical data and samples from 5191 COVID-19 patients in the East of England. We sequenced 1000 samples, producing 747 complete viral genomes. We conducted combined epidemiological and genomic analysis of 299 patients at our hospital and identified 26 genomic clusters involving 114 patients. 66 cases (57.9%) had a strong epidemiological link and 15 cases (13.2%) had a plausible epidemiological link. These results were fed back clinical, infection control and hospital management teams, resulting in infection control interventions and informing patient safety reporting. Conclusions We established real-time genomic surveillance of SARS-CoV-2 in a UK hospital and demonstrated the benefit of combined genomic and epidemiological analysis for the investigation of healthcare-associated COVID-19 infections. This approach enabled us to detect cryptic transmission events and identify opportunities to target infection control interventions to reduce further healthcare-associated infections.