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.

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.

Tracking the incidence and risk factors for SARS-CoV-2 infection using historical maternal booking serum samples.

The early transmission dynamics of SARS-CoV-2 in the UK are unknown but their investigation is critical to aid future pandemic planning. We tested over 11,000 anonymised, stored historic antenatal serum samples, given at two north-west London NHS trusts in 2019 and 2020, for total antibody to SARS-CoV-2 receptor binding domain (anti-RBD). Estimated prevalence of seroreactivity increased from 1% prior to mid-February 2020 to 17% in September 2020. Our results show higher prevalence of seroreactivity to SARS-CoV-2 in younger, non-white ethnicity, and more deprived groups. We found no significant interaction between the effects of ethnicity and deprivation. Derived from prevalence, the estimated incidence of seroreactivity reflects the trends observed in daily hospitalisations and deaths in London that followed 10 and 13 days later, respectively. We quantified community transmission of SARS-CoV-2 in London, which peaked in late March / early April 2020 with no evidence of community transmission until after January 2020. Our study was not able to determine the date of introduction of the SARS-CoV-2 virus but demonstrates the value of stored antenatal serum samples as a resource for serosurveillance during future outbreaks.

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.

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

Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

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.

Asymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in a Rehabilitation Facility: Evolution of the Presence of Nasopharyngeal SARS-CoV-2 and Serological Antibody Responses.

At the start of the UK coronavirus disease 2019 epidemic, this rare point prevalence study revealed that one-third of patients (15 of 45) in a London inpatient rehabilitation unit were found to be infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but asymptomatic. We report on 8 patients in detail, including their clinical stability, the evolution of their nasopharyngeal viral reverse-transcription polymerase chain reaction (RT-PCR) burden, and their antibody levels over time, revealing the infection dynamics by RT-PCR and serology during the acute phase. Notably, a novel serological test for antibodies against the receptor binding domain of SARS-CoV-2 showed that 100% of our asymptomatic cohort remained seropositive 3-6 weeks after diagnosis.

Severe Acute Respiratory Syndrome Coronavirus-2 Infections in Critical Care Staff: Beware the Risks Beyond the Bedside.

OBJECTIVES: Critical care workers were considered to be at high risk of severe acute respiratory syndrome coronavirus-2 infection from patients during the first wave of the pandemic. Staff symptoms, previous swab testing, and antibody prevalence were correlated with patient admissions to investigate this assumption. DESIGN: Cross-sectional study. SETTING: A large critical care department in a tertiary-care teaching hospital in London, United Kingdom. SUBJECTS: Staff working in critical care. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Participants completed a questionnaire and provided a serum sample for severe acute respiratory syndrome coronavirus-2 antibody testing over a 3-day period in April 2020. We compared the timing of symptoms in staff to the coronavirus disease 2019 patient admissions to critical care. We also identified factors associated with antibody detection. Of 625 staff 384 (61.4%) reported previous symptoms and 124 (19.8%) had sent a swab for testing. Severe acute respiratory syndrome coronavirus-2 infection had been confirmed in 37 of those swabbed (29.8%). Overall, 21% (131/625) had detectable severe acute respiratory syndrome coronavirus-2 antibody, of whom 9.9% (13/131) had been asymptomatic. The peak onset of symptoms among staff occurred 2 weeks before the peak in coronavirus disease 2019 patient admissions. Staff who worked in multiple departments across the hospital were more likely to be seropositive. Staff with a symptomatic household contact were also more likely to be seropositive at 31.3%, compared with 16.2% in those without (p < 0.0001). CONCLUSIONS: Staff who developed coronavirus disease 2019 were less likely to have caught it from their patients in critical care. Other staff, other areas of the hospital, and the wider community are more likely sources of infection. These findings indicate that personal protective equipment was effective at preventing transmission from patients. However, staff also need to maintain protective measures away from the bedside.