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.

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.

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.

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.

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.

Estimates of the rate of infection and asymptomatic COVID-19 disease in a population sample from SE England.

BACKGROUND: Understanding of the true asymptomatic rate of infection of SARS-CoV-2 is currently limited, as is understanding of the population-based seroprevalence after the first wave of COVID-19 within the UK. The majority of data thus far come from hospitalised patients, with little focus on general population cases, or their symptoms. METHODS: We undertook enzyme linked immunosorbent assay characterisation of IgM and IgG responses against SARS-CoV-2 spike glycoprotein and nucleocapsid protein of 431 unselected general-population participants of the TwinsUK cohort from South-East England, aged 19-86 (median age 48; 85% female). 382 participants completed prospective logging of 14 COVID-19 related symptoms via the COVID Symptom Study App, allowing consideration of serology alongside individual symptoms, and a predictive algorithm for estimated COVID-19 previously modelled on PCR positive individuals from a dataset of over 2 million. FINDINGS: We demonstrated a seroprevalence of 12% (51 participants of 431). Of 48 seropositive individuals with full symptom data, nine (19%) were fully asymptomatic, and 16 (27%) were asymptomatic for core COVID-19 symptoms: fever, cough or anosmia. Specificity of anosmia for seropositivity was 95%, compared to 88% for fever cough and anosmia combined. 34 individuals in the cohort were predicted to be Covid-19 positive using the App algorithm, and of those, 18 (52%) were seropositive. INTERPRETATION: Seroprevalence amongst adults from London and South-East England was 12%, and 19% of seropositive individuals with prospective symptom logging were fully asymptomatic throughout the study. Anosmia demonstrated the highest symptom specificity for SARS-CoV-2 antibody response. FUNDING: NIHR BRC, CDRF, ZOE global LTD, RST-UKRI/MRC.