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

BackgroundMost 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. MethodsPlasma 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. FindingsStrong 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. InterpretationThe 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. Research in contextO_ST_ABSEvidence before the studyC_ST_ABSWhile systemic immunity to SARS-CoV-2 is important in preventing severe disease, mucosal immunity prevents viral replication at the point of entry and reduces onward transmission. We searched PubMed with search terms "mucosal", "nasal", "antibody", "IgA", "COVID-19", "SARS-CoV-2", "convalescent" and "vaccination" for studies published in English before 20th July 2022, identifying three previous studies examining the durability of nasal responses that generally show nasal antibody to persist for 3 to 9 months. However, these studies were small or included individuals with mild COVID-19. One study of 107 care-home residents demonstrated increased salivary IgG (but not IgA) after two doses of mRNA vaccine, and another examined nasal antibody responses after infection and subsequent vaccination in 20 cases, demonstrating rises in both nasal IgA and IgG 7 to 10 days after vaccination. Added value of this studyStudying 446 people hospitalised for COVID-19, we show durable nasal and plasma IgG responses to ancestral (B.1 lineage) SARS-CoV-2, Delta and Omicron (BA.1) variants up to 12 months after infection. Nasal antibody induced by infection with pre-Omicron variants, bind Omicron virus in vitro better than plasma antibody. Although nasal and plasma IgG responses were enhanced by vaccination, Omicron binding responses did not reach levels equivalent to responses for ancestral SARS-CoV-2. Using paired plasma and nasal samples collected approximately 12 months after infection, we show that nasal IgA declines and shows a minimal response to vaccination whilst plasma antibody responses to S antigen are well maintained and boosted by vaccination. Implications of all the available evidenceAfter COVID-19 and subsequent vaccination, Omicron binding plasma and nasal antibody responses are only moderately enhanced, supporting the need for booster vaccinations to maintain immunity against SARS-CoV-2 variants. Notably, there is distinct compartmentalisation between nasal IgA and plasma IgA and IgG responses after vaccination. These findings highlight the need for vaccines that induce robust and durable mucosal immunity.

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

Both infection and vaccination, alone or in combination, generate antibody and T cell responses against SARSCoV2. However, the maintenance of such responses, and hence protection from disease, requires careful characterisation. In a large prospective study of UK healthcare workers (Protective immunity from T cells in Healthcare workers (PITCH), within the larger SARSCoV2 immunity and reinfection evaluation (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 AZD1222 (Oxford/AstraZeneca) vaccination and up to 6 months following a subsequent mRNA booster vaccination. We make three observations: Firstly, the dynamics of humoral and cellular responses differ; binding and neutralising antibodies declined whereas T and memory B cell responses were maintained after the second vaccine dose. Secondly, vaccine boosting restored IgG levels, broadened neutralising activity against variants of concern including omicron BA.1, BA.2 and BA.5, and boosted T cell responses above the 6 month level post dose 2. Thirdly, prior infection maintained its impact driving larger as well as broader T cell responses compared with never-infected people, a feature maintained until 6 months after the third dose. In conclusion, broadly cross-reactive T cell responses are well maintained over time, especially in those with combined vaccine and infection-induced immunity (hybrid immunity), and may contribute to continued protection against severe disease.

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

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

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.

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

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

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

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.