Spatial proteomics identifies a novel CRTC-dependent viral sensing pathway that stimulates production of Interleukin-11 (preprint)

Appropriate cellular recognition of viruses is essential for the generation of effective innate and adaptive antiviral immunity. Viral sensors and their signalling components thus provide a crucial first line of host defence. Many exhibit subcellular relocalisation upon activation, triggering expression of interferon and antiviral genes. To identify novel signalling factors we analysed protein relocalisation on a global scale during viral infection. CREB Regulated Transcription Coactivators-2 and 3 (CRTC2/3) exhibited early cytoplasmic-to-nuclear translocation upon a diversity of viral stimuli, in diverse cell types. This movement was depended on Mitochondrial Antiviral Signalling Protein (MAVS), cyclo-oxygenase proteins and protein kinase A. We identify a key effect of transcription stimulated by CRTC2/3 translocation as production of the pro-fibrogenic cytokine interleukin-11. This may be important clinically in viral infections associated with fibrosis, including SARS-CoV-2.

Proteomic analysis of circulating immune cells identifies novel cellular phenotypes associated with COVID-19 severity (preprint)

Certain serum proteins, including CRP and D-dimer, have prognostic value in patients with SARS-CoV-2 infection. Nonetheless, these factors are non-specific, and provide limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations which drive the pathogenesis of severe COVID-19. To identify novel cellular phenotypes associated with disease progression, we here describe a comprehensive, unbiased analysis of the total and plasma membrane proteomes of PBMCs from a cohort of 40 unvaccinated individuals with SARS-CoV-2 infection, spanning the whole spectrum of disease severity. Combined with RNA-seq and flow cytometry data from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing cumulative immune cell dysregulation in progressive disease. In particular, the cell surface proteins CEACAMs1, 6 and 8, CD177, CD63 and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CD177+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilisation of these markers may facilitate real-time patient assessment by flow cytometry, and identify immune cell populations that could be targeted to ameliorate immunopathology.

Feasibility and efficacy of mass testing for SARS-CoV-2 in a UK university using swab pooling and PCR (preprint)

Transmission of SARS-CoV-2 without symptoms is well described, and may be mitigated by mass testing. Nonetheless, the optimal implementation and quantitative real-world impact of this approach remain unclear. During a period of rising SARS-CoV-2 prevalence, students at the University of Cambridge were enrolled in a voluntary programme of weekly PCR-based asymptomatic screening. Swab pooling by household reduced the total testing capacity required by five-fold, without affecting laboratory workflows or compromising test sensitivity. Participation remained >75% throughout the study period. 299/671 (45%) of students diagnosed with SARS-CoV-2 were either identified or pre-emptively quarantined because of the screening programme. After a negative screening test, the risk of developing COVID-19 over the following 7 days was decreased by 51%. Modelling transmission using parameters from our study suggests a reduction in R0 of up to 31% attributable to weekly screening. We therefore demonstrate the feasibility and efficacy of regular, voluntary mass testing for COVID-19.

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".

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.