Gut microbiome remains stable following COVID-19 vaccination in healthy and immuno-compromised individuals (preprint)

The bidirectional interaction between the immune system and the gut microbiota is a key contributor to various host physiological functions. Immune-associated diseases such as cancer and autoimmunity, as well as the efficacy of immunomodulatory therapies, have been linked to microbiome variation. Here, we investigate the temporal impact of COVID-19 vaccination on the gut microbiome in healthy and immuno-compromised individuals; the latter included patients with primary immunodeficiency and cancer patients on immunosuppressive therapy. We find that the gut microbiome, assessed using shotgun metagenomics, remained stable post-vaccination irrespective of diverse immune status, vaccine response, and microbial composition spanned by the cohort. The stability is evident at all tested levels including phylum, species, and functional capacity. Our results show the resilience of the gut microbiome to host immune changes triggered by COVID-19 vaccination and suggest minimal, if any, impact on microbiome-mediated processes.

Age-Associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade (preprint)

The effect of immune checkpoint blockade on COVID-19 immunity is unclear. In this study, we determine whether immune checkpoint blockade expanded age-associated B cells (ABCs) are similar to those present in other conditions, and whether they enhance or detract from the COVID-19 vaccine responses. First, we use single cell RNA sequencing (scRNAseq) to show that ABCs arising from distinct aetiologies have common transcriptional profiles and may be further subdivided according to expression of genes associated with different immune functions, including the autoimmune regulator (AIRE). Next, we perform detailed longitudinal profiling of the COVID-19 vaccination response in patients. Finally, we show that high pre-vaccination ABC frequency correlates with decreased levels of antigen-specific memory B cells, and reduced magnitude and longevity of neutralising capacity against authentic SARS-CoV-2 virus. Expansion of ABCs is a biomarker for individuals with cancer requiring additional or more frequent booster immunisation against COVID-19.

Hospitalisation for COVID-19 predicts long lasting cerebrovascular impairment: A prospective observational cohort study (preprint)

Human coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has multiple neurological consequences, but its long-term effect on brain health is still uncertain. The cerebrovascular consequences of COVID-19 may also affect brain health. Here we assess cerebrovascular health in 45 hospitalised patients using the resting state fluctuation amplitudes (RSFA) from functional magnetic resonance imaging, in relation to disease severity and in contrast with 42 controls. Widespread changes in frontoparietal RSFA were related to the severity of the acute COVID-19 episode, as indexed by COVID-19 WHO Progression Scale, inflammatory and coagulatory biomarkers. This relationship was not explained by chronic cardiorespiratory dysfunction, age, or sex. Exploratory analysis suggests that the level of cerebrovascular dysfunction is associated with cognitive, mental, and physical health at follow-up. The principal findings were consistent across univariate and multivariate approaches. The results indicate chronic cerebrovascular impairment following severe acute COVID-19, with the potential for long-term consequences on cognitive function and mental wellbeing.

Brain Injury in COVID-19 is Associated with Autoinflammation and Autoimmunity (preprint)

COVID-19 has been associated with many neurological complications including stroke, delirium and encephalitis. Furthermore, many individuals experience a protracted post-viral syndrome which is dominated by neuropsychiatric symptoms, and is seemingly unrelated to COVID-19 severity. The true frequency and underlying mechanisms of neurological injury are unknown, but exaggerated host inflammatory responses appear to be a key driver of severe COVID-19 more broadly. We sought to investigate the dynamics of, and relationship between, serum markers of brain injury (neurofilament light [NfL], Glial Fibrillary Acidic Protein [GFAP] and total Tau) and markers of dysregulated host response including measures of autoinflammation (proinflammatory cytokines) and autoimmunity. Brain injury biomarkers were measured using the Quanterix Simoa HDx platform, cytokine profiling by Luminex (R&D) and autoantibodies by a custom protein microarray. During hospitalisation, patients with COVID-19 demonstrated elevations of NfL and GFAP in a severity-dependant manner, and there was evidence of ongoing active brain injury at follow-up 4 months later. Raised NfL and GFAP were associated with both elevations of pro-inflammatory cytokines and the presence of autoantibodies; autoantibodies were commonly seen against lung surfactant proteins as well as brain proteins such as myelin associated glycoprotein, but reactivity was seen to a large number of different antigens. Furthermore, a distinct process characterised by elevation of serum total Tau was seen in patients at follow-up, which appeared to be independent of initial disease severity and was not associated with dysregulated immune responses in the same manner as NfL and GFAP.

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)

Background: 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. 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.Methods: We did a prospective cohort study of individuals presenting for first dose vaccination. Following the first and second doses of the BNT162b2 vaccine, we measured IFNγ T cell responses, as well as binding antibody (IgA, IgG and IgG1-4) responses to Spike and Spike RBD. We also measured neutralising antibody responses to Spike in sera using a lentiviral pseudotyping system. We correlated age with immune responses and compared responses after the first and second doses.Findings: Median age was 63.5 years amongst 42 participants. Three weeks after the first dose a lower proportion of participants over 80 years old achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (8/17 versus 19/24, p=0.03). Geometric mean neutralisation titres in this age group after the first dose were lower than in younger individuals (p<0.001). Binding IgA and IgG1 and 3 responses developed post vaccination, as observed in natural infection. T- cell responses were not different in those above or below 80 years. Following the second dose, 50% neutralising antibody titres were above 1:20 in all individuals and there was no longer a difference by age grouping.Interpretation: A high proportion of individuals above the age of 80 have suboptimal neutralising antibody responses following first dose vaccination with BNT162b2, cautioning against extending the dosing interval in this high risk population.Funding Statement: RKG is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). DAC is supported by a Wellcome Trust Clinical PhD Research Fellowship. KGCS is the recipient of a Wellcome Investigator Award (200871/Z/16/Z). This research was supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, the Cambridge Clinical Trials Unit (CCTU), the NIHR BioResource and Addenbrooke’s Charitable Trust. JAGB is supported by the Medical Research Council (MC_UP_1201/16). IATM is funded by a SANTHE award.Declaration of Interests: None to declare. Ethics Approval Statement: The study was approved by the East of England – Cambridge Central Research Ethics Committee (17/EE/0025).

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.

SARS-CoV-2 B.1.1.7 escape from mRNA vaccine-elicited neutralizing antibodies (preprint)

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (<1:4 to 3449) that were reduced against B.1.1.7 variant by 3.85 fold (IQR 2.68-5.28). This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralization was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 outof 29), but not in neutralizing mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background led to a further loss of neutralizing activity by vaccine-elicited antibodies over that conferred by the B.1.1.7 mutations alone. Further work is needed to establish the impact of these observations on protective vaccine efficacy in the context of the evolving B.1.1.7 lineage that will likely acquire E484K.

Impact of SARS-CoV-2 B.1.1.7 Spike variant on neutralisation potency of sera from individuals vaccinated with Pfizer vaccine BNT162b2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

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".

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.