Obesity Differs from Diabetes Mellitus in Antibody and T Cell Responses Post COVID-19 Recovery (preprint)

Objective: Obesity and type 2 diabetes (DM) are risk factors for severe COVID-19 outcomes, which disproportionately affect South Asian populations. This study aims to investigate the humoral and cellular immune responses to SARS-CoV-2 in adult COVID-19 survivors with obesity and DM in Bangladesh. Methods: In this cross-sectional study, SARS-CoV-2-specific antibody and T cell responses were investigated in 63 healthy and 75 PCR-confirmed COVID-19 recovered individuals in Bangladesh, during the pre-vaccination first wave of the COVID-19 pandemic in 2020. Results: In COVID-19 survivors, SARS-CoV-2 infection induced robust antibody and T cell responses, which correlated with disease severity. After adjusting for age, sex, DM status, disease severity, and time since onset of symptoms, obesity was associated with decreased neutralising antibody titers, and increased SARS-CoV-2 spike-specific IFN-{gamma} response along with increased proliferation and IL-2 production by CD8+ T cells. In contrast, DM was not associated with SARS-CoV-2-specific antibody and T cell responses after adjustment for obesity and other confounders. Conclusions: Obesity is associated with lower neutralising antibody levels and higher T cell responses to SARS-CoV-2 post COVID-19 recovery, while antibody or T cell responses remain unaltered in DM.

Large scale phenotyping of long COVID inflammation reveals mechanistic subtypes of disease (preprint)

One in ten SARS-CoV-2 infections result in prolonged symptoms termed "long COVID", yet disease phenotypes and mechanisms are poorly understood. We studied the blood proteome of 719 adults, grouped by long COVID symptoms. Elevated markers of monocytic inflammation and complement activation were associated with increased likelihood of all symptoms. Elevated IL1R2, MATN2 and COLEC12 associated with cardiorespiratory symptoms, fatigue, and anxiety/depression, while elevated MATN2 and DPP10 associated with gastrointestinal (GI) symptoms, and elevated C1QA was associated with cognitive impairment (the proteome of those with cognitive impairment and GI symptoms being most distinct). Markers of neuroinflammation distinguished cognitive impairment whilst elevated SCG3, indicative of brain-gut axis disturbance, distinguished those with GI symptoms. Women had a higher incidence of long COVID and higher inflammatory markers. Symptoms did not associate with respiratory inflammation or persistent virus in sputum. Thus, persistent inflammation is evident in long COVID, distinct profiles being associated with specific symptoms.

CD4+ and CD8+ T cell and antibody correlates of protection against Delta vaccine breakthrough infection: A nested case-control study within the PITCH study (preprint)

T cell correlates of protection against SARS-CoV-2 infection after vaccination ('vaccine breakthrough') are incompletely defined, especially the specific contributions of CD4+ and CD8+ T cells. We studied 279 volunteers in the Protective Immunity from T Cells in Healthcare Workers (PITCH) UK study, including 32 cases (with SARS-CoV-2 positive testing after two vaccine doses during the Delta-dominant era) and 247 controls (no positive test nor anti-nucleocapsid seroconversion during this period). 28 days after second vaccination, before all breakthroughs occurred, cases had lower ancestral S- and RBD-specific immunoglobulin G titres and S1- and S2-specific T cell interferon gamma (IFN{gamma}) responses compared with controls. In a subset of matched cases and controls, cases had lower CD4+ and CD8+ IFN{gamma} and tumour necrosis factor responses to Delta S peptides with reduced CD8+ responses to Delta versus ancestral peptides compared with controls. Our findings support a protective role for T cells against Delta breakthrough infection.

Omicron BA.1/BA.2 infections in triple-vaccinated individuals enhance a diverse repertoire of mucosal and blood immune responses (preprint)

Pronounced immune escape by the SARS-CoV-2 Omicron variant has resulted in large numbers of individuals with hybrid immunity, generated through a combination of vaccination and infection. Based primarily on circulating neutralizing antibody (NAb) data, concerns have been raised that omicron breakthrough infections in triple-vaccinated individuals result in poor induction of omicron-specific immunity, and that a history of prior SARS-CoV-2 in particular is associated with profound immune dampening. Taking a broader and comprehensive approach, we characterized mucosal and blood immunity to both spike and non-spike antigens following BA.1/BA.2 infections in triple mRNA-vaccinated individuals, with and without a history of previous SARS-CoV-2 infection. We find that the majority of individuals increase BA.1/BA.2/BA.5-specific NAb following infection, but confirm that the magnitude of increase and post-omicron titres are indeed higher in those who were infection-naive. In contrast, significant increases in nasal antibody responses are seen regardless of prior infection history, including neutralizing activity against BA.5 spike. Spike-specific T cells increase only in infection-naive vaccinees; however, post-omicron T cell responses are still significantly higher in previously-infected individuals, who appear to have maximally induced responses with a CD8+ phenotype of high cytotoxic potential after their 3rd mRNA vaccine dose. Antibody and T cell responses to non-spike antigens also increase significantly regardless of prior infection status, with a boost seen in previously-infected individuals to immunity primed by their first infection. These findings suggest that hybrid immunity induced by omicron breakthrough infections is highly dynamic, complex, and compartmentalised, with significant immune enhancement that can help protect against COVID-19 caused by future omicron variants.

Nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination. (preprint)

Background Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. Methods Plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. Findings Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months. Nasal and plasma anti-S IgG remained elevated for at least 12 months with high plasma neutralising titres against all variants. Of 180 with complete data, 160 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal. Samples 12 months after admission showed no association between nasal IgA and plasma IgG responses, indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. Interpretation The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity.

Evolution of long-term hybrid immunity in healthcare workers after different COVID-19 vaccination regimens: a longitudinal observational cohort study (preprint)

Both infection and vaccination, alone or in combination, generate antibody and T cell responses against SARS-CoV-2. However, the maintenance of such responses - and hence protection from disease - requires careful characterisation. In a large prospective study of UK healthcare workers (PITCH, within the larger SIREN study) we previously observed that prior infection impacted strongly on subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination. Here, we report longer follow up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZ1222 (Oxford/AstraZeneca) vaccination and following a subsequent BNT162b2 booster vaccination. We make three important observations: Firstly, the dynamics of humoral and cellular responses differ; binding and neutralising antibodies declined whereas T and B cell responses were better maintained after the second vaccine dose. Secondly, vaccine boosting restored IgG levels to post second dose levels and broadened neutralising activity against variants of concern including omicron BA.1, alongside further boosting of T cell responses. Thirdly, prior infection maintained its impact driving larger T cell responses compared to never infected people, including after the third dose. In conclusion, the maintenance of T cell responses in time and against variants of concern may account for continued protection against severe disease.

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2 (preprint)

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, detection of seroconversion after vaccination, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests have a long history in blood typing, and general serology through linkage of reporter molecules to the red cell surface. They do not require special equipment, are read by eye, have short development times, low cost and can be applied as a Point of Care Test (POCT). We describe a red cell agglutination test for the detection of antibodies to the SARS-CoV-2 receptor binding domain (RBD). We show that the Haemagglutination Test (HAT) has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. The HAT can be titrated, detects rising titres in the first five days of hospital admission, correlates well with a commercial test that detects antibodies to the RBD, and can be applied as a point of care test. The developing reagent is composed of a previously described nanobody to a conserved glycophorin A epitope on red cells, linked to the RBD from SARS-CoV-2. It can be lyophilised for ease of shipping. We have scaled up production of this reagent to one gram, which is sufficient for ten million tests, at a cost of ~0.27 UK pence per test well. Aliquots of this reagent are ready to be supplied to qualified groups anywhere in the world that need to detect antibodies to SARS-CoV-2, but do not have the facilities for high throughput commercial tests.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses (preprint)

A major issue in identification of protective T cell responses against SARS-CoV-2 lies in distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity generated by exposure to other coronaviruses. We characterised SARS-CoV-2 T cell immune responses in 168 PCR-confirmed SARS-CoV-2 infected subjects and 118 seronegative subjects without known SARS-CoV-2 exposure using a range of T cell assays that differentially capture immune cell function. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) were found in those who had been infected by SARS-CoV-2 but were rare in pre-pandemic and unexposed seronegative subjects. However, seronegative doctors with high occupational exposure and recent COVID-19 compatible illness showed patterns of T cell responses characteristic of infection, indicating that these readouts are highly sensitive. By contrast, over 90% of convalescent or unexposed people showed proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on the choice of assay and antigen. Memory responses to specific non-spike proteins provides a method to distinguish recent infection from pre-existing immunity in exposed populations.

De Novo Discovery of High Affinity Peptide Binders for the SARS-CoV-2 Spike Protein (preprint)

The beta-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein, the most exposed surface structure of the virus, are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants of 80 to 970 nM) for RBD and selectivity over human serum proteins. Picomolar RBD concentrations in biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides might associate with the SARS-CoV-2-spike-RBD at a site unrelated to ACE2 binding, making them potential orthogonal reagents for sandwich immunoassays. We envision our discovery as a robust starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus directed delivery of therapeutics.

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients. (preprint)

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN{gamma} based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

Evaluation of antibody testing for SARS-Cov-2 using ELISA and lateral flow immunoassays (preprint)

BackgroundThe COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. MethodsWe tested plasma for COVID (SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). ResultsELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested [≥]10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. ConclusionsCurrently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.

Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19 (preprint)

COVID-19 is a complex disease phenotype where the underlying microbiome could influence morbidity and mortality. Amplicon and metagenomic MinION based sequencing was used to rapidly (within 8 hours) identify SARS-CoV-2 and assess the microbiome in nasopharyngeal swabs obtained from patients with COVID-19 by the ISARIC 4C consortium.