SARS-CoV-2 antibody responses associate with sex, age and disease severity in previously uninfected people admitted to hospital with COVID-19: An ISARIC4C prospective study.

The SARS-CoV-2 pandemic enables the analysis of immune responses induced against a novel coronavirus infecting immunologically naïve individuals. This provides an opportunity for analysis of immune responses and associations with age, sex and disease severity. Here we measured an array of solid-phase binding antibody and viral neutralising Ab (nAb) responses in participants (n=337) of the ISARIC4C cohort and characterised their correlation with peak disease severity during acute infection and early convalescence. Overall, the responses in a Double Antigen Binding Assay (DABA) for antibody to the receptor binding domain (anti-RBD) correlated well with IgM as well as IgG responses against viral spike, S1 and nucleocapsid protein (NP) antigens. DABA reactivity also correlated with nAb. As we and others reported previously, there is greater risk of severe disease and death in older men, whilst the sex ratio was found to be equal within each severity grouping in younger people. In older males with severe disease (mean age 68 years), peak antibody levels were found to be delayed by one to two weeks compared with women, and nAb responses were delayed further. Additionally, we demonstrated that solid-phase binding antibody responses reached higher levels in males as measured via DABA and IgM binding against Spike, NP and S1 antigens. In contrast, this was not observed for nAb responses. When measuring SARS-CoV-2 RNA transcripts (as a surrogate for viral shedding) in nasal swabs at recruitment, we saw no significant differences by sex or disease severity status. However, we have shown higher antibody levels associated with low nasal viral RNA indicating a role of antibody responses in controlling viral replication and shedding in the upper airway. In this study, we have shown discernible differences in the humoral immune responses between males and females and these differences associate with age as well as with resultant disease severity.

Risk factors and vectors for SARS-CoV-2 household transmission: a prospective, longitudinal cohort study.

BACKGROUND: Despite circumstantial evidence for aerosol and fomite spread of SARS-CoV-2, empirical data linking either pathway with transmission are scarce. Here we aimed to assess whether the presence of SARS-CoV-2 on frequently-touched surfaces and residents' hands was a predictor of SARS-CoV-2 household transmission. METHODS: In this longitudinal cohort study, during the pre-alpha (September to December, 2020) and alpha (B.1.1.7; December, 2020, to April, 2021) SARS-CoV-2 variant waves, we prospectively recruited contacts from households exposed to newly diagnosed COVID-19 primary cases, in London, UK. To maximally capture transmission events, contacts were recruited regardless of symptom status and serially tested for SARS-CoV-2 infection by RT-PCR on upper respiratory tract (URT) samples and, in a subcohort, by serial serology. Contacts' hands, primary cases' hands, and frequently-touched surface-samples from communal areas were tested for SARS-CoV-2 RNA. SARS-CoV-2 URT isolates from 25 primary case-contact pairs underwent whole-genome sequencing (WGS). FINDINGS: From Aug 1, 2020, until March 31, 2021, 620 contacts of PCR-confirmed SARS-CoV-2-infected primary cases were recruited. 414 household contacts (from 279 households) with available serial URT PCR results were analysed in the full household contacts' cohort, and of those, 134 contacts with available longitudinal serology data and not vaccinated pre-enrolment were analysed in the serology subcohort. Household infection rate was 28·4% (95% CI 20·8-37·5) for pre-alpha-exposed contacts and 51·8% (42·5-61·0) for alpha-exposed contacts (p=0·0047). Primary cases' URT RNA viral load did not correlate with transmission, but was associated with detection of SARS-CoV-2 RNA on their hands (p=0·031). SARS-CoV-2 detected on primary cases' hands, in turn, predicted contacts' risk of infection (adjusted relative risk [aRR]=1·70 [95% CI 1·24-2·31]), as did SARS-CoV-2 RNA presence on household surfaces (aRR=1·66 [1·09-2·55]) and contacts' hands (aRR=2·06 [1·57-2·69]). In six contacts with an initial negative URT PCR result, hand-swab (n=3) and household surface-swab (n=3) PCR positivity preceded URT PCR positivity. WGS corroborated household transmission. INTERPRETATION: Presence of SARS-CoV-2 RNA on primary cases' and contacts' hands and on frequently-touched household surfaces associates with transmission, identifying these as potential vectors for spread in households. FUNDING: National Institute for Health Research Health Protection Research Unit in Respiratory Infections, Medical Research Council.

National and regional prevalence of SARS-CoV-2 antibodies in primary and secondary school children in England: the School Infection Survey, a national open cohort study, November 2021SARS-CoV-2 antibody prevalence in school children.

BACKGROUND: SARS-CoV-2 infection rates are likely to be underestimated in children because of asymptomatic or mild infections. We aim to estimate national and regional prevalence of SARS-CoV-2 antibodies in primary (4-11 years old) and secondary (11-18 years old) school children between 10 November and 10 December 2021. METHODS: Cross-sectional surveillance in England using two-stage sampling, firstly stratifying into regions and selecting local authorities, then selecting schools according to a stratified sample within selected local authorities. Participants were sampled using a novel oral fluid-validated assay for SARS-CoV-2 spike and nucleocapsid IgG antibodies. RESULTS: 4980 students from 117 state-funded schools (2706 from 83 primary schools, 2274 from 34 secondary schools) provided a valid sample. After weighting for age, sex, and ethnicity, and adjusting for assay accuracy, the national prevalence of SARS-CoV-2 antibodies in primary school students, who were all unvaccinated, was 40.1% (95% CI 37.3-43.0). Antibody prevalence increased with age (p < 0.001) and was higher in urban than rural schools (p = 0.01). In secondary school students, the adjusted, weighted national prevalence of SARS-CoV-2 antibodies was 82.4% (95% CI 79.5-85.1); including 71.5% (95% CI 65.7-76.8) in unvaccinated and 97.5% (95% CI 96.1-98.5) in vaccinated students. Antibody prevalence increased with age (p < 0.001), and was not significantly different in urban versus rural students (p = 0.1). CONCLUSIONS: In November 2021, using a validated oral fluid assay, national SARS-CoV-2 seroprevalence was estimated to be 40.1% in primary school students and 82.4% in secondary school students. In unvaccinated children, this was approximately threefold higher than confirmed infections highlighting the importance of seroprevalence studies to estimate prior exposure. DATA AVAILABILITY: Deidentified study data are available for access by accredited researchers in the ONS Secure Research Service (SRS) for accredited research purposes under part 5, chapter 5 of the Digital Economy Act 2017. For further information about accreditation, contact Research.support@ons.gov.uk or visit the SRS website.

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Simple, sensitive, specific self-sampling assay secures SARS-CoV-2 antibody signals in sero-prevalence and post-vaccine studies.

At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or 'invalid'. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.

Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts.

Cross-reactive immune responses to SARS-CoV-2 have been observed in pre-pandemic cohorts and proposed to contribute to host protection. Here we assess 52 COVID-19 household contacts to capture immune responses at the earliest timepoints after SARS-CoV-2 exposure. Using a dual cytokine FLISpot assay on peripheral blood mononuclear cells, we enumerate the frequency of T cells specific for spike, nucleocapsid, membrane, envelope and ORF1 SARS-CoV-2 epitopes that cross-react with human endemic coronaviruses. We observe higher frequencies of cross-reactive (p = 0.0139), and nucleocapsid-specific (p = 0.0355) IL-2-secreting memory T cells in contacts who remained PCR-negative despite exposure (n = 26), when compared with those who convert to PCR-positive (n = 26); no significant difference in the frequency of responses to spike is observed, hinting at a limited protective function of spike-cross-reactive T cells. Our results are thus consistent with pre-existing non-spike cross-reactive memory T cells protecting SARS-CoV-2-naïve contacts from infection, thereby supporting the inclusion of non-spike antigens in second-generation vaccines.

Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR.

BACKGROUND: The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in many parts of the world. To help guide public health policies it is important to monitor efficiently its spread. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. OBJECTIVE: To develop and evaluate a rapid, simple and inexpensive alternative to sequencing for Delta identification. METHODS: A double-mismatch allele-specific RT-PCR (DMAS-RT-PCR) was developed. The technique exploits allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer was enhanced by an additional mismatch located at the fourth nucleotide from the 3' end. Specificity was assessed by testing well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. RESULTS: In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants (Alpha and Beta), and 'wild-type' SARS-CoV-2. Influenza A and RSV were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay. Several samples that could not be sequenced due to insufficient virus were successfully genotyped by DMAS-RT-PCR. CONCLUSION: The method we describe would be simple to establish in any laboratory that can conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant globally.

Broadening symptom criteria improves early case identification in SARS-CoV-2 contacts.

BACKGROUND: The success of case isolation and contact tracing for the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission depends on the accuracy and speed of case identification. We assessed whether inclusion of additional symptoms alongside three canonical symptoms (CS), i.e. fever, cough and loss or change in smell or taste, could improve case definitions and accelerate case identification in SARS-CoV-2 contacts. METHODS: Two prospective longitudinal London (UK)-based cohorts of community SARS-CoV-2 contacts, recruited within 5 days of exposure, provided independent training and test datasets. Infected and uninfected contacts completed daily symptom diaries from the earliest possible time-points. Diagnostic information gained by adding symptoms to the CS was quantified using likelihood ratios and area under the receiver operating characteristic curve. Improvements in sensitivity and time to detection were compared with penalties in terms of specificity and number needed to test. RESULTS: Of 529 contacts within two cohorts, 164 (31%) developed PCR-confirmed infection and 365 (69%) remained uninfected. In the training dataset (n=168), 29% of infected contacts did not report the CS. Four symptoms (sore throat, muscle aches, headache and appetite loss) were identified as early-predictors (EP) which added diagnostic value to the CS. The broadened symptom criterion "≥1 of the CS, or ≥2 of the EP" identified PCR-positive contacts in the test dataset on average 2 days earlier after exposure (p=0.07) than "≥1 of the CS", with only modest reduction in specificity (5.7%). CONCLUSIONS: Broadening symptom criteria to include individuals with at least two of muscle aches, headache, appetite loss and sore throat identifies more infections and reduces time to detection, providing greater opportunities to prevent SARS-CoV-2 transmission.

Upregulation of Human Endogenous Retroviruses in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Severe COVID-19 pneumonia has been associated with the development of intense inflammatory responses during the course of infections with SARS-CoV-2. Given that human endogenous retroviruses (HERVs) are known to be activated during and participate in inflammatory processes, we examined whether HERV dysregulation signatures are present in COVID-19 patients. By comparing transcriptomes of bronchoalveolar lavage fluid (BALF) of COVID-19 patients and healthy controls, and peripheral blood monocytes (PBMCs) from patients and controls, we have shown that HERVs are intensely dysregulated in BALF of COVID-19 patients compared to those in BALF of healthy control patients but not in PBMCs. In particular, upregulation in the expression of specific HERV families was detected in BALF samples of COVID-19 patients, with HERV-FRD being the most highly upregulated family among the families analyzed. In addition, we compared the expression of HERVs in human bronchial epithelial cells (HBECs) without and after senescence induction in an oncogene-induced senescence model in order to quantitatively measure changes in the expression of HERVs in bronchial cells during the process of cellular senescence. This apparent difference of HERV dysregulation between PBMCs and BALF warrants further studies in the involvement of HERVs in inflammatory pathogenetic mechanisms as well as exploration of HERVs as potential biomarkers for disease progression. Furthermore, the increase in the expression of HERVs in senescent HBECs in comparison to that in noninduced HBECs provides a potential link for increased COVID-19 severity and mortality in aged populations. IMPORTANCE SARS-CoV-2 emerged in late 2019 in China, causing a global pandemic. Severe COVID-19 is characterized by intensive inflammatory responses, and older age is an important risk factor for unfavorable outcomes. HERVs are remnants of ancient infections whose expression is upregulated in multiple conditions, including cancer and inflammation, and their expression is increased with increasing age. The significance of this work is that we were able to recognize dysregulated expression of endogenous retroviral elements in BALF samples but not in PBMCs of COVID-19 patients. At the same time, we were able to identify upregulated expression of multiple HERV families in senescence-induced HBECs in comparison to that in noninduced HBECs, a fact that could possibly explain the differences in disease severity among age groups. These results indicate that HERV expression might play a pathophysiological role in local inflammatory pathways in lungs afflicted by SARS-CoV-2 and their expression could be a potential therapeutic target.

SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

Longevity of SARS-CoV-2 immune responses in hemodialysis patients and protection against reinfection.

Patients with end stage kidney disease receiving in-center hemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, patients receiving ICHD frequently develop circulating antibodies to SARS-CoV-2, even with asymptomatic infection. Here, we investigated the durability and functionality of the immune responses to SARS-CoV-2 infection in patients receiving ICHD. Three hundred and fifty-six such patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were regularly screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies, and those who became seronegative at six months were screened for SARS-CoV-2 specific T-cell responses. One hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at time zero, of whom 127 also had detectable anti-RBD. Significantly, at six months, 71/111 (64.0%) and 99/116 (85.3%) remained anti-NP and anti-RBD seropositive, respectively. For patients who retained antibody, both anti-NP and anti-RBD levels were reduced significantly after six months. Eleven patients who were anti-NP seropositive at time zero, had no detectable antibody at six months; of whom eight were found to have SARS-CoV-2 antigen specific T cell responses. Independent of antibody status at six months, patients with baseline positive SARS-CoV-2 serology were significantly less likely to have PCR confirmed infection over the following six months. Thus, patients receiving ICHD mount durable immune responses six months post SARS-CoV-2 infection, with fewer than 3% of patients showing no evidence of humoral or cellular immunity.

The Association Between Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Post-COVID-19 Syndrome in Healthcare Workers.

It is currently unknown how post-COVID-19 syndrome (PCS) may affect those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This longitudinal study includes healthcare staff who tested positive for SARS-CoV-2 between March and April 2020, with follow-up of their antibody titers and symptoms. More than half (21 of 38) had PCS after 7-8 months. There was no statistically significant difference between initial reverse-transcription polymerase chain reaction titers or serial antibody levels between those who did and those who did not develop PCS. This study highlights the relative commonality of PCS in healthcare workers and this should be considered in vaccination scheduling and workforce planning to allow adequate frontline staffing numbers.

3 Clinical implications of the differential antibody response in mild-moderate SARS-CoV-2: a prospective multi-centre cross-sectional study

IntroductionSerological testing can augment delayed case identification programmes for Severe Acute Respiratory Syndrome coronoravirus-2 (SARS-CoV-2). Immunoassays employ anti-nucleocapsid (anti-NP;the majority) or potentially neutralising anti-spike (including anti-receptor binding domain;anti-RBD) antibody targets, yet correlation between assays and variability arising from disease symptomatology remains unclear. We explore these possibly differential immune responses across the disease spectrum.MethodsA multicentre prospective study was undertaken via a SARS-CoV-2 delayed case identification programme (08 May-11 July 2020). Matched samples were tested for anti-NP and anti-RBD (utilising an ‘inhouse’ double-antigen bridged assay), reactivity expressed as test/cut-off binding ratios (BR) and results compared. A multivariate linear regression model analysed age, sex, symptomatology, PCR positivity, anti-NP, and anti-RBD BRs. Participants were followed up for possible reinfection.Results902 individuals underwent matched testing;109 were SARS-CoV-2 PCR swab positive. Anti-NP, anti-RBD immunoassay agreement was 87.5% (95% CI 85.3–89.6), with BRs strongly correlated (R=0.75). PCR confirmed cases were more frequently identified by anti-RBD (sensitivity 108/109, 99.1%, 95% CI 95.0–100.0) than anti-NP (102/109, 93.6%, 95% CI 87.2–97.4). Anti-RBD identified an additional 83/325 (25.5%) cases in those seronegative for anti-NP. Presence of anti-NP (p<0.0001), fever (p=0.005), or anosmia (p=0.002) were all significantly associated with an increased anti-RBD BR. Age was associated with reduced anti-RBD BR (p=0.052). Three cases with evidence of seroconversion (anti-RBD seropositive) presented with subsequent reactive PCR results, two of which coincided with first time onset of Public Heath England SARS-CoV-2 symptoms.ConclusionsSARS-CoV-2 anti-RBD shows significant correlation with anti-NP for absolute seroconversion, and BRs. Higher BRs are seen in symptomatic individuals with significantly higher levels seen in those with fever and anosmia. The degree of discordant results (12.5%) limits the use of anti-NP as a stand-alone for delayed case finding programmes. Similarly, this discordance limits the utility of non-neutralising anti-NP assays in place of potentially neutralising anti-RBD to infer possible immunity.** this abstract presentation was awarded an Honourable Mention

SARS-CoV-2 lateral flow assays for possible use in national covid-19 seroprevalence surveys (React 2): diagnostic accuracy study.

OBJECTIVE: To evaluate the performance of new lateral flow immunoassays (LFIAs) suitable for use in a national coronavirus disease 2019 (covid-19) seroprevalence programme (real time assessment of community transmission 2-React 2). DESIGN: Diagnostic accuracy study. SETTING: Laboratory analyses were performed in the United Kingdom at Imperial College, London and university facilities in London. Research clinics for finger prick sampling were run in two affiliated NHS trusts. PARTICIPANTS: Sensitivity analyses were performed on sera stored from 320 previous participants in the React 2 programme with confirmed previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Specificity analyses were performed on 1000 prepandemic serum samples. 100 new participants with confirmed previous SARS-CoV-2 infection attended study clinics for finger prick testing. INTERVENTIONS: Laboratory sensitivity and specificity analyses were performed for seven LFIAs on a minimum of 200 serum samples from participants with confirmed SARS-CoV-2 infection and 500 prepandemic serum samples, respectively. Three LFIAs were found to have a laboratory sensitivity superior to the finger prick sensitivity of the LFIA currently used in React 2 seroprevalence studies (84%). These LFIAs were then further evaluated through finger prick testing on participants with confirmed previous SARS-CoV-2 infection: two LFIAs (Surescreen, Panbio) were evaluated in clinics in June-July 2020 and the third LFIA (AbC-19) in September 2020. A spike protein enzyme linked immunoassay and hybrid double antigen binding assay were used as laboratory reference standards. MAIN OUTCOME MEASURES: The accuracy of LFIAs in detecting immunoglobulin G (IgG) antibodies to SARS-CoV-2 compared with two reference standards. RESULTS: The sensitivity and specificity of seven new LFIAs that were analysed using sera varied from 69% to 100%, and from 98.6% to 100%, respectively (compared with the two reference standards). Sensitivity on finger prick testing was 77% (95% confidence interval 61.4% to 88.2%) for Panbio, 86% (72.7% to 94.8%) for Surescreen, and 69% (53.8% to 81.3%) for AbC-19 compared with the reference standards. Sensitivity for sera from matched clinical samples performed on AbC-19 was significantly higher with serum than finger prick at 92% (80.0% to 97.7%, P=0.01). Antibody titres varied considerably among cohorts. The numbers of positive samples identified by finger prick in the lowest antibody titre quarter varied among LFIAs. CONCLUSIONS: One new LFIA was identified with clinical performance suitable for potential inclusion in seroprevalence studies. However, none of the LFIAs tested had clearly superior performance to the LFIA currently used in React 2 seroprevalence surveys, and none showed sufficient sensitivity and specificity to be considered for routine clinical use.