Search

Recent reports highlight a new clinical syndrome in children related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1—multisystem inflammatory syndrome in children (MIS-C)—which comprises multiorgan dysfunction and systemic inflammation2–13. We performed peripheral leukocyte phenotyping in 25 children with MIS-C, in the acute (n = 23; worst illness within 72 h of admission), resolution (n = 14; clinical improvement) and convalescent (n = 10; first outpatient visit) phases of the illness and used samples from seven age-matched healthy controls for comparisons. Among the MIS-C cohort, 17 (68%) children were SARS-CoV-2 seropositive, suggesting previous SARS-CoV-2 infections14,15, and these children had more severe disease. In the acute phase of MIS-C, we observed high levels of interleukin-1β (IL-1β), IL-6, IL-8, IL-10, IL-17, interferon-γ and differential T and B cell subset lymphopenia. High CD64 expression on neutrophils and monocytes, and high HLA-DR expression on γδ and CD4+CCR7+ T cells in the acute phase, suggested that these immune cell populations were activated. Antigen-presenting cells had low HLA-DR and CD86 expression, potentially indicative of impaired antigen presentation. These features normalized over the resolution and convalescence phases. Overall, MIS-C presents as an immunopathogenic illness1 and appears distinct from Kawasaki disease. Characterization of a cohort of children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection provides insights into the immunopathogenic features of the disease.

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

During the first wave of the global COVID-19 pandemic the clinical utility and indications for SARS-CoV-2 serological testing were not clearly defined. The urgency to deploy serological assays required rapid evaluation of their performance characteristics. We undertook an internal validation of a CE marked lateral flow immunoassay (LFIA) (SureScreen Diagnostics) using serum from SARS-CoV-2 RNA positive individuals and pre-pandemic samples. This was followed by the delivery of a same-day named patient SARS-CoV-2 serology service using LFIA on vetted referrals at central London teaching hospital with clinical interpretation of result provided to the direct care team. Assay performance, source and nature of referrals, feasibility and clinical utility of the service, particularly benefit in clinical decision-making, were recorded. Sensitivity and specificity of LFIA were 96.1% and 99.3% respectively. 113 tests were performed on 108 participants during three-week pilot. 44% participants (n = 48) had detectable antibodies. Three main indications were identified for serological testing; new acute presentations potentially triggered by recent COVID-19 e.g. pulmonary embolism (n = 5), potential missed diagnoses in context of a recent COVID-19 compatible illness (n = 40), and making infection control or immunosuppression management decisions in persistently SARS-CoV-2 RNA PCR positive individuals (n = 6). We demonstrate acceptable performance characteristics, feasibility and clinical utility of using a LFIA that detects anti-spike antibodies to deliver SARS-CoV-2 serology service in adults and children. Greatest benefit was seen where there is reasonable pre-test probability and results can be linked with clinical advice or intervention. Experience from this pilot can help inform practicalities and benefits of rapidly implementing new tests such as LFIAs into clinical service as the pandemic evolves.

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10–15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.

There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. Highly specific in-house ELISAs were developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays—a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs)—on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections., Author summary PCR-based throat and nose swab tests for novel coronavirus (SARS-CoV-2) establish if someone is infected with the virus, while antibody tests can determine whether someone has had it in the past. However, for diagnosis later in disease, or in delayed-onset syndromes such as paediatric inflammatory multisystem syndrome (PIMS), antibody tests could form an important part of hospital diagnostic capabilities. They will also be essential for patient management strategies and community seroprevalence studies. We have conducted unbiased, head-to-head comparisons of ten commercial antibody test kits, using blood from patients admitted to hospital with COVID-19 throughout the peak of the epidemic in London, UK. As there was no existing approved diagnostic antibody test, we developed our own sensitive assay and used this to cross-compare the commercial tests. There was a broad range of performance among the tests, but all gave the best results when used 20 days or more after the start of symptoms. Furthermore, antibody levels were higher in individuals with severe illness compared to those with asymptomatic or mild disease. Some of the best-performing tests were rapid lateral flow immunoassays, which are affordable, quick and easy to use, and if they are deployed appropriately could have considerable utility in healthcare settings.

BackgroundUnderstanding 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.MethodsWe 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.FindingsWe demonstrated a seroprevalence of 12% (51participants 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.InterpretationSeroprevalence 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.FundingNIHR BRC, CDRF, ZOE global LTD, RST-UKRI/MRC

We describe the innate and adaptive immune system trajectory in Multi-system inflammatory syndrome of childhood (MIS-C), at acute(within 72 hours of hospitalization), resolution (at clinical improvement) and convalescent phase. In our cohort, in the acute phase, 68% of the children were SARS-CoV-2 seropositive, with hypercytokinenemia (high interleukin(IL)-1beta,IL-6,IL-8,IL-10,IL-17, interferon gamma), procoagulant state, myocardial dysfunction, activated neutrophils and monocytes; differential T and B cell subset lymphopenia; activated chemokine receptor type-7 positive and gamma-delta T cell subsets; antigen presenting cells had reduced HLA-DR expression; and B-cell class-switch responses occurred with illness resolution. MIS-C is an immunopathogenic illness associated with SARS-CoV-2 infections in children.

OBJECTIVEDetermine indications and clinical utility of SARS-CoV-2 serology testing in adults and children.DESIGNProspective evaluation of initial three weeks of a daily Monday to Friday pilot SARS-CoV-2 serology service for patients.SETTINGEarly post “first-wave” SARS-CoV-2 transmission period at single centre London teaching hospital that provides care to the local community, as well as regional and national referral pathways for specialist services.PARTICIPANTS110 (72 adults, 38 children, age range 0-83 years, 52.7% female (n=58)).INTERVENTIONSPatient serum from vetted referrals tested on CE marked and internally validated lateral flow immunoassay (LFIA) (SureScreen Diagnostics) detecting antibodies to SARS-CoV-2 spike proteins, with result and clinical interpretation provided to the direct care team.MAIN OUTCOME MEASURESPerformance characteristics, source and nature of referrals, feasibility and clinical utility of the service, particularly the benefit for clinical decision-making.RESULTSThe LFIA was deemed suitable for clinical advice and decision making following evaluation with 310 serum samples from SARS-CoV-2 PCR positive patients and 300 pre-pandemic samples, giving a sensitivity and specificity of 96.1% and 99.3% respectively. For the pilot, 115 referrals were received leading to 113 tests performed on 108 participants (sample not available for two participants); paediatrics (n=35), medicine (n=69), surgery (n=2) and general practice (n=2). 43.4% participants (n=49) had detectable antibodies to SARS-CoV-2. There were three main indications for serology; new acute presentations potentially triggered by recent COVID-19 infection e.g. PIMS-TS (n=26) and pulmonary embolism (n=5), potential missed diagnoses in context of a recent compatible illness (n=40), and making infection control and immunosuppression treatment decisions in persistently SARS-CoV-2 RNA PCR positive individuals (n=6).CONCLUSIONSThis study shows acceptable performance characteristics, feasibility and clinical utility of a SARS-CoV-2 serology service using a rapid, inexpensive and portable assay for adults and children presenting with a range of clinical indications. Results correlated closely with a confirmatory in-house ELISA. The study showed the benefit of introducing a serology service where there is a reasonable pre-test probability, and the result can be linked with clinical advice or intervention. Experience thus far is that the volume of requests from hospital referral routes are manageable within existing clinical and laboratory services; however, the demand from community referrals has not yet been assessed. Given recent evidence for a rapid decline in antibodies, particularly following mild infection, there is likely a limited window of opportunity to realise the benefit of serology testing for individuals infected during the “first-wave” before they potentially fall below a measurable threshold. Rapidly expanding availability of serology services for NHS patients will also help understand the long-term implications of serostatus and prior infection in different patient groups, particularly before emergence of any “second-wave” outbreak or introduction of a vaccination programme.SUMMARY BOXWHAT IS ALREADY KNOWN ON THIS TOPICThe mechanisms and utility of providing a SARS-CoV-2 (COVID-19) serology service is under evaluation. There are different technologies detecting antibodies against different SARS-CoV-2 proteins. Antibodies are known to appear from about 10 days after symptom onset but it is unclear how long they persist.WHAT THIS STUDY ADDSA SARS-CoV-2 serology service using a validated lateral flow immunoassay measuring antibodies against spike protein can be rapidly introduced with clinical benefit demonstrated for a broad range of individuals. Indications include ‘missed’ diagnoses where COVID-19 infection has been suspected but SARS-COV-2 RNA tests were either negative or not performed, conditions potentially triggered by COVID-19 such as pulmonary embolism, and predicting infectivity or immunity in patients with persistently detectable SARS-CoV-2 RNA. Testing is quick, simple to perform and inexpensive, however emerging evidence that antibodies fall rapidly particularly in mild disease, and the observed breadth of emerging indications highlight the urgent need for targeted testing with clinical interpretation provided on a case-by-case basis.

Antibody (Ab) responses to SARS-CoV-2 can be detected in most infected individuals 10-15 days following the onset of COVID-19 symptoms. However, due to the recent emergence of this virus in the human population it is not yet known how long these Ab responses will be maintained or whether they will provide protection from re-infection. Using sequential serum samples collected up to 94 days post onset of symptoms (POS) from 65 RT-qPCR confirmed SARS-CoV-2-infected individuals, we show seroconversion in >95% of cases and neutralizing antibody (nAb) responses when sampled beyond 8 days POS. We demonstrate that the magnitude of the nAb response is dependent upon the disease severity, but this does not affect the kinetics of the nAb response. Declining nAb titres were observed during the follow up period. Whilst some individuals with high peak ID50 (>10,000) maintained titres >1,000 at >60 days POS, some with lower peak ID50 had titres approaching baseline within the follow up period. A similar decline in nAb titres was also observed in a cohort of seropositive healthcare workers from Guy’s and St Thomas’ Hospitals. We suggest that this transient nAb response is a feature shared by both a SARS-CoV-2 infection that causes low disease severity and the circulating seasonal coronaviruses that are associated with common colds. This study has important implications when considering widespread serological testing, Ab protection against re-infection with SARS-CoV-2 and the durability of vaccine protection.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants., Graphical abstract, The impact of mutations arising in SARS-CoV-2 Spike on antigenicity is still not known. Graham et al. isolate potent neutralizing monoclonal antibodies from individuals experiencing a range of COVID-19 disease severity that target RBD, NTD, and non-S1 epitopes. The B.1.1.7 variant of concern was most resistant to NTD-specific nAbs whereas RBD-specific nAbs retained potent neutralization.