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.

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.

Attenuated humoral responses in HIV infection after SARS-CoV-2 vaccination are linked to global B cell defects and cellular immune profiles (preprint)

People living with HIV (PLWH) on suppressive antiretroviral therapy (ART) can have residual immune dysfunction and often display poorer responses to vaccination. We assessed in a cohort of PLWH (n=110) and HIV negative controls (n=64) the humoral and spike-specific B-cell responses following 1, 2 or 3 SARS-CoV-2 vaccine doses. PLWH had significantly lower neutralizing antibody (nAb) titers than HIV-negative controls at all studied timepoints. Moreover, their neutralization breadth was reduced with fewer individuals developing a neutralizing response against the Omicron variant (BA.1) relative to controls. We also observed a delayed development of neutralization in PLWH that was underpinned by a reduced frequency of spike-specific memory B cells (MBCs) and pronounced B cell dysfunction. Improved neutralization breadth was seen after the third vaccine dose in PLWH but lower nAb responses persisted and were associated with global, but not spike-specific, MBC dysfunction. In contrast to the inferior antibody responses, SARS-CoV-2 vaccination induced robust T cell responses that cross-recognized variants in PLWH. Strikingly, a subset of PLWH with low or absent neutralization had detectable functional T cell responses. These individuals had reduced numbers of circulating T follicular helper cells and an enriched population of CXCR3+CD127+CD8+ T cells after two doses of SARS-CoV-2 vaccination, which may compensate for sub-optimal serological responses in the event of infection. Therefore, normalisation of B cell homeostasis could improve serological responses to vaccines in PLWH and evaluating T cell immunity could provide a more comprehensive immune status profile in these individuals and others with B cell imbalances.

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.

Accelerated waning of the humoral response to SARS-CoV-2 vaccines in obesity (preprint)

Obesity is associated with an increased risk of severe Covid-19. However, the effectiveness of SARS-CoV-2 vaccines in people with obesity is unknown. Here we studied the relationship between body mass index (BMI), hospitalization and mortality due to Covid-19 amongst 3.5 million people in Scotland. Vaccinated people with severe obesity (BMI>40 kg/m2) were significantly more likely to experience hospitalization or death from Covid-19. Excess risk increased with time since vaccination. To investigate the underlying mechanisms, we conducted a prospective longitudinal study of the immune response in a clinical cohort of vaccinated people with severe obesity. Compared with normal weight controls, six months after their second vaccine dose, significantly more people with severe obesity had unquantifiable titres of neutralizing antibody against authentic SARS-CoV-2 virus, reduced frequencies of antigen-experienced SARS-CoV-2 Spike-binding B cells, and a dissociation between anti-Spike antibody levels and neutralizing capacity. Neutralizing capacity was restored by a third dose of vaccine, but again declined more rapidly in people with severe obesity. We demonstrate that waning of SARS-CoV-2 vaccine-induced humoral immunity is accelerated in people with severe obesity and associated with increased hospitalization and mortality from breakthrough infections. Given the prevalence of obesity, our findings have significant implications for global public health.

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.

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.

Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays (preprint)

The world is in the grip of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and there is an urgent unmet clinical need for effective antiviral therapies. Many inhibitors of viral enzymes identified in vitro have limited efficacy against viral replication in cells, but conventional plaque assays are impractical for high-throughput screens. In this study, we therefore engineer cell-based biosensors of SARS-CoV-2 infection. Our assays exploit the cleavage of specific oligopeptide linkers by SARS-CoV-2 Main or Papain-like proteases, leading to the activation of green fluorescent protein (GFP) or firefly luciferase-based reporters. First, we characterise these biosensors in cells using recombinant viral proteases. Next, we confirm their ability to detect endogenous viral protease expression during infection with wildtype SARS-CoV-2. Finally, we develop a sensitive luminescent reporter cell line, confirm that it accurately quantitates infectious SARS-CoV-2 virus, and demonstrate its utility for drug screening and titration of neutralising antibodies.

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.