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.

Age-related heterogeneity in immune responses to SARS-CoV-2 following BNT162b2 vaccination (preprint)

Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks and other countries are likely to follow suit given the demand for mRNA vaccines and ongoing uncontrolled transmission. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2. Median age was 81 years amongst 101 participants after the first dose of the BNT162b2 vaccine. Geometric mean neutralisation titres in participants over 80 years old after the first dose were lower than in younger individuals [83.4 (95% CI 52.0-133.7) vs 46.6 (95% CI 33.5-64.8) p 0.01]. A lower proportion of participants 80 years and older achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (21% vs 51%, p 0.003). Binding IgG responses correlated with neutralisation. Sera from participants in both age groups showed significantly lower neutralisation potency against B.1.1.7 Spike pseudotyped viruses as compared to wild type. The adjusted ORs for inadequate neutralisation in the 80 years and above age group were 3.7 (95% CI 1.2-11.2) and 4.4 (95% CI 1.5-12.6) against wild type and B.1.1.7 pseudotyped viruses. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T- cell IFN𝛾 and IL-2 responses were impaired in the older age group after 1 dose and although IFN𝛾 increased between vaccine doses, IL-2 responses did not significantly increase. There was a significantly higher risk of suboptimal neutralising antibody and T cell response following first dose vaccination with BNT162b2 in half of participants above the age of 80, persisting up to 12 weeks. These high risk populations warrant specific measures in order to mitigate against vaccine failure, particularly where SARS-CoV-2 variants of concern are circulating.

Age-related heterogeneity in Neutralising antibody responses to SARS-CoV-2 following BNT162b2 vaccination (preprint)

Two dose mRNA vaccination provides excellent protection against SARS-CoV-2. However, there are few data on vaccine efficacy in elderly individuals above the age of 801. Additionally, new variants of concern (VOC) with reduced sensitivity to neutralising antibodies have raised fears for vulnerable groups. Here we assessed humoral and cellular immune responses following vaccination with mRNA vaccine BNT162b22 in elderly participants prospectively recruited from the community and younger health care workers. Median age was 72 years and 51% were females amongst 140 participants. Neutralising antibody responses after the first vaccine dose diminished with increasing age, with a marked drop in participants over 80 years old. Sera from participants below and above 80 showed significantly lower neutralisation potency against B.1.1.7, B.1.351 and P.1. variants of concern as compared to wild type. Those over 80 were more likely to lack any neutralisation against VOC compared to younger participants following first dose. The adjusted odds ratio for inadequate neutralisation activity against the B.1.1.7, P.1 and B.1.351 variant in the older versus younger age group was 4.3 (95% CI 2.0-9.3, p<0.001), 6.7 (95% CI 1.7-26.3, p=0.008) and 1.7 (95% CI 0.5-5.7, p=0.41). Binding IgG and IgA antibodies were lower in the elderly, as was the frequency of SARS-CoV-2 Spike specific B-memory cells. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T-cell IFN{gamma} and IL-2 responses fell with increasing age, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high risk population that warrant specific measures in order to mitigate against vaccine failure, particularly where variants of concern are circulating.

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".

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.