Quantitative temporal analysis of modified vaccinia Ankara, the monkeypox and smallpox vaccine (preprint)

Modified vaccinia Ankara (MVA) immunisation is being deployed to curb the current outbreak of monkeypox in multiple countries1. Originally authorized for vaccination against smallpox, MVA is a vaccinia virus (VACV) strain that does not replicate in human cells or cause serious adverse events. Here, we conducted a highly multiplexed proteomic analysis2 to quantify ~7,500 cellular proteins and ~80% of viral proteins at five time points throughout MVA infection of human cells3. >380 human proteins were down-regulated >2-fold by MVA, revealing a profound remodelling of the host proteome. >25% of these MVA targets, including multiple components of the nuclear pore complex (NPC), were not shared with VACV-Western Reserve4, which is derived from a first generation smallpox vaccine associated with serious adverse events. Using pharmacological inhibition of viral DNA replication and killed virions, we discovered that post-replicative gene expression is necessary for the downregulation of NPC proteins and for elements of MVA antagonism of innate immune sensing. Our approach thus provides the first global view of the impact of MVA infection on the host proteome, offers insights into how MVA interacts with the antiviral defences and identifies cellular mechanisms that may underpin the abortive infection of human cells. These discoveries will prove vital to the rational design of future generations of vaccines.

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.

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission (preprint)

Understanding the drivers for spread of SARS-CoV-2 in higher education settings is important to limit transmission between students, and onward spread into at-risk populations. In this study, we prospectively sequenced 482 SARS-CoV-2 isolates derived from asymptomatic student screening and symptomatic testing of students and staff at the University of Cambridge from 5 October to 6 December 2020. We performed a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. After a limited number of viral introductions into the university, the majority of student cases were linked to a single genetic cluster, likely dispersed across the university following social gatherings at a venue outside the university. We identified considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and dramatically reduced following a national lockdown. We observed that transmission clusters were largely segregated within the university or within the community. This study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.

Factor V is an immune inhibitor that is expressed at increased levels in leukocytes of patients with severe Covid-19 (preprint)

Severe Covid-19 is associated with elevated plasma Factor V (FV) and increased risk of thromboembolism. We report that neutrophils, T regulatory cells (Tregs), and monocytes from patients with severe Covid-19 express FV, and expression correlates with T cell lymphopenia. In vitro full length FV, but not FV activated by thrombin cleavage, suppresses T cell proliferation. Increased and prolonged FV expression by cells of the innate and adaptive immune systems may contribute to lymphopenia in severe Covid-19. Activation by thrombin destroys the immunosuppressive properties of FV. Anticoagulation in Covid-19 patients may have the unintended consequence of suppressing the adaptive immune system.

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).

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<0.0001) 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.

A2B-COVID: A method for evaluating potential SARS-CoV-2 transmission events (preprint)

Identifying linked cases of infection is a key part of the public health response to viral infectious disease. Viral genome sequence data is of great value in this task, but requires careful analysis, and may need to be complemented by additional types of data. The Covid-19 pandemic has highlighted the urgent need for analytical methods which bring together sources of data to inform epidemiological investigations. We here describe A2B-COVID, an approach for the rapid identification of linked cases of coronavirus infection. Our method combines knowledge about infection dynamics, data describing the movements of individuals, and novel approaches to genome sequence data to assess whether or not cases of infection are consistent or inconsistent with linkage via transmission. We apply our method to analyse and compare data collected from two wards at Cambridge University Hospitals, showing qualitatively different patterns of linkage between cases on designated Covid-19 and non-Covid-19 wards. Our method is suitable for the rapid analysis of data from clinical or other potential outbreak settings.

A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities (preprint)

The COVID-19 pandemic is expanding at an unprecedented rate. As a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. Academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. Here, we describe the process of developing a SARS-Cov2 diagnostic workflow in a conventional academic Containment Level 2 (CL2) laboratory. Our outline includes simple SARS-Cov2 deactivation upon contact, the methods for a quantitative real-time reverse transcriptase PCR (qRT-PCR) detecting SARS-Cov2, a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. This was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. Within 14 days, we created a validated COVID-19 diagnostics service for healthcare workers in our local hospital. The described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame.