Validity of self-testing at home with rapid SARS-CoV-2 antibody detection by lateral flow immunoassay

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow immunoassays (LFIA) can be carried out in the home and have been used as an affordable and practical approach to large-scale antibody prevalence studies. However, assay performance differs from that of high-throughput laboratory-based assays which can be highly sensitive. We explore LFIA performance under field conditions compared to laboratory-based ELISA and assess the potential of LFIAs to identify people who lack functional antibodies following infection or vaccination. MethodsField evaluation of a self-administered LFIA test (Fortress, NI) among 3758 participants from the REal-time Assessment of Community Transmission-2 (REACT-2) study in England selected based on vaccination history and previous LFIA result to ensure a range of antibody titres. In July 2021, participants performed, at home, a self-administered LFIA on finger-prick blood, reported and submitted a photograph of the result, and provided a self-collected capillary blood sample (Tasso-SST) for serological assessment of IgG antibodies to the spike protein using the Roche Elecsys(R) Anti-SARS-CoV-2 assay. We compared the self-administered and reported LFIA result to the quantitative Roche assay and checked the reading of the LFIA result with an automated image analysis (ALFA). In a subsample of 250 participants, we compared the results to live virus neutralisation. ResultsAlmost all participants (3593/3758, 95.6%) had been vaccinated or reported prior infection, with most having received one (862, 22.9%) or two (2430, 64.7%) COVID-19 vaccine doses. Overall, 2777/3758 (73.9%) were positive on self-reported LFIA, 2811/3457 (81.3%) positive by LFIA when ALFA-reported, and 3622/3758 (96.4%) positive on Roche anti-S (using the manufacturer reference standard threshold for positivity of 0.8 U ml-1). Live virus neutralisation was detected in 169 of 250 randomly selected samples (67.6%); 133/169 were positive with self-reported LFIA (sensitivity 78.7%; 95% CI 71.8, 84.6), 142/155 (91.6%; 86.1, 95.5) with ALFA, and 169 (100%; 97.8, 100.0) with Roche anti-S. There were 81 samples with no detectable virus neutralisation; 47/81 were negative with self-reported LFIA (specificity 58.0%; 95% CI 46.5, 68.9), 34/75 (45.3%; 33.8, 57.3) with ALFA, and 0/81 (0%; 0.0, 4.5) with Roche anti-S. All 250 samples remained positive with Roche anti-S when the threshold was increased to 1000U ml-1. ConclusionsSelf-administered LFIA can provide insights into population patterns of infection and vaccine response, and sensitivity can be improved with automated reading of the result. The LFIA is less sensitive than a quantitative antibody test, but the positivity in LFIA correlates better than the quantitative ELISA with virus neutralisation.

Distinct antigenic properties of the SARS-CoV-2 Omicron lineages BA.4 and BA.5

Over the course of the pandemic variants have arisen at a steady rate. The most recent variants to emerge, BA.4 and BA.5, form part of the Omicron lineage and were first found in Southern Africa where they are driving the current wave of infection. In this report, we perform an in-depth characterisation of the antigenicity of the BA.4/BA.5 Spike protein by comparing sera collected post-vaccination, post-BA.1 or BA.2 infection, or post breakthrough infection of vaccinated individuals with the Omicron variant. In addition, we assess sensitivity to neutralisation by commonly used therapeutic monoclonal antibodies. We find sera collected post-vaccination have a similar ability to neutralise BA.1, BA.2 and BA.4/BA.5. In contrast, in the absence of vaccination, prior infection with BA.2 or, in particular, BA.1 results in an antibody response that neutralises BA.4/BA.5 poorly. Breakthrough infection with Omicron in vaccinees leads to a broad neutralising response against the new variants. The sensitivity of BA.4/BA.5 to neutralisation by therapeutic monoclonal antibodies was similar to that of BA.2. These data suggest BA.4/BA.5 are antigenically distinct from BA.1 and, to a lesser extent, BA.2. The enhanced breadth of neutralisation observed following breakthrough infection with Omicron suggests that vaccination with heterologous or multivalent antigens may represent viable strategies for the development of cross-neutralising antibody responses.

Omicron breakthrough infections in vaccinated or previously infected hamsters

The second and third years of the SARS-CoV-2 pandemic have been marked by the repeated emergence and replacement of variants with genetic and phenotypic distance from the ancestral strains, the most recent examples being Delta and Omicron. Here we describe a hamster contact exposure challenge model to assess protection conferred by vaccination or prior infection against re-infection. We found that 2-doses of self-amplifying RNA vaccine based on the ancestral spike ameliorated weight loss following Delta infection and decreased viral loads, but had minimal effect on Omicron/BA.1 infection. Prior infection with ancestral or Alpha variant was partially protective against Omicron/BA.1 infection, whereas all animals previously infected with Delta and exposed to Omicron became infected, although shed less virus. We further tested whether prior infection with Omicron/BA.1 protected from re-infection with Delta or Omicron/BA.2. Omicron/BA.1 was protective against Omicron/BA.2, but not Delta reinfection, again showing Delta and Omicron have a very large antigenic distance. Indeed, cross-neutralisation assays with human antisera from otherwise immunonaive individuals (unvaccinated and no known prior infection), confirmed a large antigenic distance between Delta and Omicron. Prior vaccination followed by Omicron or Delta breakthrough infection led to a higher degree of cross-reactivity to all tested variants. To conclude, cohorts whose only immune experience of COVID is Omicron/BA.1 infection may be particularly vulnerable to future circulation of Delta or Delta-like derivatives. In contrast, repeated exposure to antigenically distinct spikes, via infection and or vaccination drives a more cross-reactive immune response, both in hamsters and people. One Sentence SummaryInfection with the Delta and Omicron SARS-CoV-2 variants do not provide cross-protective immunity against reinfection with one another in hamsters.

Variant-specific symptoms of COVID-19 among 1,542,510 people in England

Infection with SARS-CoV-2 virus is associated with a wide range of symptoms. The REal-time Assessment of Community Transmission -1 (REACT-1) study has been monitoring the spread and clinical manifestation of SARS-CoV-2 among random samples of the population in England from 1 May 2020 to 31 March 2022. We show changing symptom profiles associated with the different variants over that period, with lower reporting of loss of sense of smell and taste for Omicron compared to previous variants, and higher reporting of cold-like and influenza-like symptoms, controlling for vaccination status. Contrary to the perception that recent variants have become successively milder, Omicron BA.2 was associated with reporting more symptoms, with greater disruption to daily activities, than BA.1. With restrictions lifted and routine testing limited in many countries, monitoring the changing symptom profiles associated with SARS-CoV-2 infection and induced changes in daily activities will become increasingly important.

Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature upon SARS-CoV-2 sensing by monocytes in COVID-19

Alterations in the myeloid immune compartment have been observed in COVID-19, but the specific mechanisms underlying these impairments are not completely understood. Here we examined the functionality of classical CD14+ monocytes as a main myeloid cell component in well-defined cohorts of patients with mild and moderate COVID-19 during the acute phase of infection and compared them to that of healthy individuals. We found that ex vivo isolated CD14+ monocytes from mild and moderate COVID-19 patients display specific patterns of costimulatory and inhibitory receptors that clearly distinguish them from healthy monocytes, as well as altered expression of histone marks and a dysfunctional metabolic profile. Decreased NF{kappa}B activation in COVID-19 monocytes ex vivo is accompanied by an intact type I IFN antiviral response. Subsequent pathogen sensing ex vivo led to a state of functional unresponsiveness characterized by a defect in pro-inflammatory cytokine expression, NF{kappa}B-driven cytokine responses and defective type I IFN response in moderate COVID-19 monocytes. Transcriptionally, COVID-19 monocytes switched their gene expression signature from canonical innate immune functions to a pro-thrombotic phenotype characterized by increased expression of pathways involved in hemostasis and immunothrombosis. In response to SARS-CoV-2 or other viral or bacterial components, monocytes displayed defects in the epigenetic remodelling and metabolic reprogramming that usually occurs upon pathogen sensing in innate immune cells. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 may contribute to disease pathology.

The origins and molecular evolution of SARS-CoV-2 lineage B.1.1.7 in the UK

The first SARS-CoV-2 variant of concern (VOC) to be designated was lineage B.1.1.7, later labelled by the World Health Organisation (WHO) as Alpha. Originating in early Autumn but discovered in December 2020, it spread rapidly and caused large waves of infections worldwide. The Alpha variant is notable for being defined by a long ancestral phylogenetic branch with an increased evolutionary rate, along which only two sequences have been sampled. Alpha genomes comprise a well-supported monophyletic clade within which the evolutionary rate is more typical of SARS-CoV-2. The Alpha epidemic continued to grow despite the continued restrictions on social mixing across the UK, and the imposition of new restrictions, in particular the English national lockdown in November 2020. While these interventions succeeded in reducing the absolute number of cases, the impact of these non-pharmaceutical interventions was predominantly to drive the decline of the SARS-CoV-2 lineages which preceded Alpha. We investigate the only two sampled sequences that fall on the branch ancestral to Alpha. We find that one is likely to be a true intermediate sequence, providing information about the order of mutational events that led to Alpha. We explore alternate hypotheses that can explain how Alpha acquired a large number of mutations yet remained largely unobserved in a region of high genomic surveillance: an under-sampled geographical location, a non-human animal population, or a chronically-infected individual. We conclude that the last hypothesis provides the best explanation of the observed behaviour and dynamics of the variant, although we find that the individual need not be immunocompromised, as persistently-infected immunocompetent hosts also display a higher within-host rate of evolution. Finally, we compare the ancestral branches and mutation profiles of other VOCs to each other, and identify that Delta appears to be an outlier both in terms of the genomic locations of its defining mutations, and its lack of rapid evolutionary rate on the ancestral branch. As new variants, such as Omicron, continue to evolve (potentially through similar mechanisms) it remains important to investigate the origins of other variants to identify ways to potentially disrupt their evolution and emergence.

The SARS-CoV-2 variant, Omicron, shows rapid replication in human primary nasal epithelial cultures and efficiently uses the endosomal route of entry.

The SARS-CoV-2 Omicron/BA.1 lineage emerged in late 2021 and rapidly displaced the Delta variant before being overtaken itself globally by, the Omicron/BA.2 lineage in early 2022. Here, we describe how Omicron BA.1 and BA.2 show a lower severity phenotype in a hamster model of pathogenicity which maps specifically to the spike gene. We further show that Omicron is attenuated in a lung cell line but replicates more rapidly, albeit to lower peak titres, in human primary nasal cells. This replication phenotype also maps to the spike gene. Omicron spike (including the emerging Omicron lineage BA.4) shows attenuated fusogenicity and a preference for cell entry via the endosomal route. We map the altered Omicron spike entry route and partially map the lower fusogenicity to the S2 domain, particularly the substitution N969K. Finally, we show that pseudovirus with Omicron spike, engineered in the S2 domain to confer a more Delta-like cell entry route retains the antigenic properties of Omicron. This shows a distinct separation between the genetic determinants of these two key Omicron phenotypes, raising the concerning possibility that future variants with large antigenic distance from currently circulating and vaccine strains will not necessarily display the lower intrinsic severity seen during Omicron infection.

Neutralising antibody activity against SARS-CoV-2 variants, including Omicron, in an elderly cohort vaccinated with BNT162b2

SARS-CoV-2 variants threaten the effectiveness of tools we have developed to mitigate against serious COVID-19. This is especially true in clinically vulnerable sections of society including the elderly. Using sera from BNT162b2 (Pfizer-BioNTech) vaccinated individuals aged between 70 and 89 (vaccinated with two doses 3-weeks apart) we examined the neutralising antibody (nAb) response to wildtype SARS-CoV-2. Between 3 and 20-weeks post 2nd dose, nAb titres dropped 4.9-fold to a median titre of 21.3 (ND80) with 21.6% of individuals having no detectable nAbs at the later time point. Experiments examining the neutralisation of twenty-one different SARS-CoV-2 variant spike proteins confirmed a significant potential for antigenic escape, especially for the Omicron (BA.1), Beta (B.1.351), Delta (B.1.617.2), Theta (P.3), C.1.2 and B.1.638 variants. Interestingly, however, the recently-emerged sub-lineage AY.4.2 was more efficiently neutralised than parental Delta pseudotypes. Combining pseudotype neutralisation with specific receptor binding domain (RBD) ELISAs we confirmed that changes to position 484 in the spike RBD were predominantly responsible for SARS-CoV-2 nAb escape, although the effect of spike mutations is both combinatorial and additive. Lastly, using sera from the same individuals boosted with a 3rd dose of BNT162b2 we showed that high overall levels of neutralising antibody titre can provide significant levels of cross-protection against Omicron. These data provide evidence that SARS-CoV-2 neutralising antibodies wane over time and that antigenically variable SARS-CoV-2 variants are circulating, highlighting the importance of ongoing surveillance and booster programmes. Furthermore, they provide important data to inform risk assessment of new SARS-CoV-2 variants, such as Omicron, as they emerge.

Context-specific emergence and growth of the SARS-CoV-2 Delta variant

The Delta variant of concern of SARS-CoV-2 has spread globally causing large outbreaks and resurgences of COVID-19 cases1-3. The emergence of Delta in the UK occurred on the background of a heterogeneous landscape of immunity and relaxation of non-pharmaceutical interventions4,5. Here we analyse 52,992 Delta genomes from England in combination with 93,649 global genomes to reconstruct the emergence of Delta, and quantify its introduction to and regional dissemination across England, in the context of changing travel and social restrictions. Through analysis of human movement, contact tracing, and virus genomic data, we find that the focus of geographic expansion of Delta shifted from India to a more global pattern in early May 2021. In England, Delta lineages were introduced >1,000 times and spread nationally as non-pharmaceutical interventions were relaxed. We find that hotel quarantine for travellers from India reduced onward transmission from importations; however the transmission chains that later dominated the Delta wave in England had been already seeded before restrictions were introduced. In England, increasing inter-regional travel drove Deltas nationwide dissemination, with some cities receiving >2,000 observable lineage introductions from other regions. Subsequently, increased levels of local population mixing, not the number of importations, was associated with faster relative growth of Delta. Among US states, we find that regions that previously experienced large waves also had faster Delta growth rates, and a model including interactions between immunity and human behaviour could accurately predict the rise of Delta there. Deltas invasion dynamics depended on fine scale spatial heterogeneity in immunity and contact patterns and our findings will inform optimal spatial interventions to reduce transmission of current and future VOCs such as Omicron.

SARS-CoV-2 Omicron-B.1.1.529 Variant leads to less severe disease than Pango B and Delta variants strains in a mouse model of severe COVID-19

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The B.1.1.529 Omicron variant is rapidly emerging and has been designated a Variant of Concern (VOC). The variant is highly transmissible and partially or fully evades a spectrum of neutralising antibodies due to a high number of substitutions in the spike glycoprotein. A major question is the relative severity of disease caused by the Omicron variant compared with previous and currently circulating variants of SARS-CoV-2. To address this, a mouse model of infection that recapitulates severe disease in humans, K18-hACE2 mice, were infected with either a Pango B, Delta or Omicron variant of SARS-CoV-2 and their relative pathogenesis compared. In contrast to mice infected with Pango B and Delta variant viruses, those infected with the Omicron variant had less severe clinical signs (weight loss), showed recovery and had a lower virus load in both the lower and upper respiratory tract. This is also reflected by less extensive inflammatory processes in the lungs. Although T cell epitopes may be conserved, the antigenic diversity of Omicron from previous variants would suggest that a change in vaccine may be required to mitigate against the higher transmissibility and global disease burden. However, the lead time to develop such a response may be too late to mitigate the spread and effects of Omicron. These animal model data suggest the clinical consequences of infection with the Omicron variant may be less severe but the higher transmissibility could still place huge burden upon healthcare systems even if a lower proportion of infected patients are hospitalised.

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

Vaccine uptake and SARS-CoV-2 antibody prevalence among 207,337 adults during May 2021 in England: REACT-2 study

BackgroundThe programme to vaccinate adults in England has been rapidly implemented since it began in December 2020. The community prevalence of SARS-CoV-2 anti-spike protein antibodies provides an estimate of total cumulative response to natural infection and vaccination. We describe the distribution of SARS-CoV-2 IgG antibodies in adults in England in May 2021 at a time when approximately 7 in 10 adults had received at least one dose of vaccine. MethodsSixth round of REACT-2 (REal-time Assessment of Community Transmission-2), a cross-sectional random community survey of adults in England, from 12 to 25 May 2021; 207,337 participants completed questionnaires and self-administered a lateral flow immunoassay test producing a positive or negative result. ResultsVaccine coverage with one or more doses, weighted to the adult population in England, was 72.9% (95% confidence interval 72.7-73.0), varying by age from 25.1% (24.5-25.6) of those aged 18 to 24 years, to 99.2% (99.1-99.3) of those 75 years and older. In adjusted models, odds of vaccination were lower in men (odds ratio [OR] 0.89 [0.85-0.94]) than women, and in people of Black (0.41 [0.34-0.49]) compared to white ethnicity. There was higher vaccine coverage in the least deprived and highest income households. People who reported a history of COVID-19 were less likely to be vaccinated (OR 0.61 [0.55-0.67]). There was high coverage among health workers (OR 9.84 [8.79-11.02] and care workers (OR 4.17 [3.20-5.43]) compared to non-key workers, but lower in hospitality and retail workers (OR 0.73 [0.64-0.82] and 0.77 [0.70-0.85] respectively) after adjusting for age and key covariates. The prevalence of antibodies (weighted to the adult population of England and adjusted for test characteristics) was 61.1% (95% CI 60.9-61.4), up from 6.6% (5.4-5.7) in round 4 (27 October to 10 November 2020) and 13.9% (13.7-14.1) in round 5 (26 January to 8 February 2021). Prevalence (adjusted and weighted) increased with age, from 35.8% (35.1-36.5) in those aged 18 to 24 years, to 95.3% (94.6-95.9) in people 75 and over. Antibodies were 30% less likely to be detected in men than women (adjusted OR 0.69, 0.68-0.70), and were higher in people of Asian (OR 1.67 [1.58-1.77]), Black (1.55 [1.41-1.69]), mixed 1.17 [1.06-1.29] and other (1.37 [1.23-1.51]) ethnicities compared with white ethnicity. Workers in hospitality (OR 0.69 [0.63-0.74]) and retail (0.71 [0.67-0.75]) were less likely to have antibodies. Following two doses of Pfizer-BioNTech vaccine, antibody positivity (adjusted for test performance) was 100% (100-100) at all ages except 80 years and older when it was 97.8% (95.9-99.6). For AstraZeneca positivity was over 90% up to age 69, and then 89.2% (88.5-89.9) in 70-79 year olds and 83.6% (78.5-88.3) in those aged 80 and over. Following a single dose of Pfizer-BioNTech positivity ranged from 100.0% (91.1-100.0) in those aged 18-29 to 32.2% (18.2-51.1) in those aged 70-79 years. For AstraZeneca this was 72.2% (68.5-75.9) in the youngest and 46.2% (40.0-52.7) in the oldest age group. DiscussionThe successful roll out of the vaccination programme in England has led to a high proportion of individuals having detectable antibodies, particularly in older age groups and those who have had two doses of vaccine. This is likely to be associated with high levels of protection from severe disease, and possibly from infection. Nonetheless, there remain some key groups with a lower prevalence of antibody, notably unvaccinated younger people, certain minority ethnic groups, those living in deprived areas and workers in some public facing employment. Obtaining improved rates of vaccination in these groups is essential to achieving high levels of protection against the virus through population immunity. FundingDepartment of Health and Social Care in England.

The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 B.1.351 and other variants of concern in preclinical studies.

There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine.

Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination

Vaccines are proving to be highly effective in controlling hospitalisation and deaths associated with SARS-CoV-2 infection but the emergence of viral variants with novel antigenic profiles threatens to diminish their efficacy. Assessment of the ability of sera from vaccine recipients to neutralise SARS-CoV-2 variants will inform the success of strategies for minimising COVID19 cases and the design of effective antigenic formulations. Here, we examine the sensitivity of variants of concern (VOCs) representative of the B.1.617.1 and B.1.617.2 (first associated with infections in India) and B.1.351 (first associated with infection in South Africa) lineages of SARS-CoV-2 to neutralisation by sera from individuals vaccinated with the BNT162b2 (Pfizer/BioNTech) and ChAdOx1 (Oxford/AstraZeneca) vaccines. Across all vaccinated individuals, the spike glycoproteins from B.1.617.1 and B.1.617.2 conferred reductions in neutralisation of 4.31 and 5.11-fold respectively. The reduction seen with the B.1.617.2 lineage approached that conferred by the glycoprotein from B.1.351 (South African) variant (6.29-fold reduction) that is known to be associated with reduced vaccine efficacy. Neutralising antibody titres elicited by vaccination with two doses of BNT162b2 were significantly higher than those elicited by vaccination with two doses of ChAdOx1. Fold decreases in the magnitude of neutralisation titre following two doses of BNT162b2, conferred reductions in titre of 7.77, 11.30 and 9.56-fold respectively to B.1.617.1, B.1.617.2 and B.1.351 pseudoviruses, the reduction in neutralisation of the delta variant B.1.617.2 surpassing that of B.1.351. Fold changes in those vaccinated with two doses of ChAdOx1 were 0.69, 4.01 and 1.48 respectively. The accumulation of mutations in these VOCs, and others, demonstrate the quantifiable risk of antigenic drift and subsequent reduction in vaccine efficacy. Accordingly, booster vaccines based on updated variants are likely to be required over time to prevent productive infection. This study also suggests that two dose regimes of vaccine are required for maximal BNT162b2 and ChAdOx1-induced immunity.

The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin

The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence 681PRRAR/S686. This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.

Children develop strong and sustained cross-reactive immune responses against spike protein following SARS-CoV-2 infection

SARS-CoV-2 infection is generally mild or asymptomatic in children but the biological basis for this is unclear. We studied the profile of antibody and cellular immunity in children aged 3-11 years in comparison with adults. Antibody responses against spike and receptor binding domain (RBD) were high in children and seroconversion boosted antibody responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Seroneutralisation assays against alpha, beta and delta SARS-CoV-2 variants demonstrated comparable neutralising activity between children and adults. T cell responses against spike were >2-fold higher in children compared to adults and displayed a TH1 cytokine profile. SARS-CoV-2 spike-specific T cells were also detected in many seronegative children, revealing pre-existing responses that were cross-reactive with seasonal Alpha and Beta-coronaviruses. Importantly, all children retained high antibody titres and cellular responses at 6 months after infection whilst relative antibody waning was seen in adults. Spike-specific responses in children also remained broadly stable beyond 12 months. Children thus distinctly generate robust, cross-reactive and sustained immune responses after SARS-CoV-2 infection with focussed specificity against spike protein. These observations demonstrate novel features of SARS-CoV-2-specific immune responses in children and may provide insight into their relative clinical protection. Furthermore, this information will help to guide the introduction of vaccination regimens in the paediatric population.

Inactivation of SARS-CoV-2 in chlorinated swimming pool water

SARS-CoV-2 transmission remains a global problem which exerts a significant direct cost to public health. Additionally, other aspects of physical and mental health can be affected by limited access to social and exercise venues as a result of lockdowns in the community or personal reluctance due to safety concerns. Swimming pools have reopened in the UK as of April 12th, but the effect of swimming pool water on inactivation of SARS-CoV-2 has not yet been directly demonstrated. Here we demonstrate that water which adheres to UK swimming pool guidelines is sufficient to reduce SARS-CoV-2 infectious titre by at least 3 orders of magnitude.

Ultrastructural insight into SARS-CoV-2 attachment, entry and budding in human airway epithelium

Ultrastructural studies of SARS-CoV-2 infected cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Many studies are limited by the lack of access to appropriate cellular models. As the airway epithelium is the primary site of infection it is essential to study SARS-CoV-2 infection of these cells. Here, we examined human airway epithelium, grown as highly differentiated air-liquid interface cultures and infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant (Variant of Concern 202012/01) by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Two key SARS-CoV-2 entry molecules, ACE2 and TMPRSS2, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistent with these observations, extracellular virions were frequently seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion at the apical plasma membrane demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein. Profiles strongly suggesting viral budding from the membrane was observed in these compartments and this may explain how virions gain their S glycoprotein containing envelope.

A common TMPRSS2 variant protects against severe COVID-19

Infection with SARS-CoV-2 has a wide range of clinical presentations, from asymptomatic to life-threatening. Old age is the strongest factor associated with increased COVID19-related mortality, followed by sex and pre-existing conditions. The importance of genetic and immunological factors on COVID19 outcome is also starting to emerge, as demonstrated by population studies and the discovery of damaging variants in genes controlling type I IFN immunity and of autoantibodies that neutralize type I IFNs. The human protein transmembrane protease serine type 2 (TMPRSS2) plays a key role in SARS-CoV-2 infection, as it is required to activate the virus spike protein, facilitating entry into target cells. We focused on the only common TMPRSS2 non-synonymous variant predicted to be damaging (rs12329760), which has a minor allele frequency of [~]25% in the population. In a large population of SARS-CoV-2 positive patients, we show that this variant is associated with a reduced likelihood of developing severe COVID19 (OR 0.87, 95%CI:0.79-0.97, p=0.01). This association was stronger in homozygous individuals when compared to the general population (OR 0.65, 95%CI:0.50-0.84, p=1.3x10-3). We demonstrate in vitro that this variant, which causes the amino acid substitution valine to methionine, impacts the catalytic activity of TMPRSS2 and is less able to support SARS-CoV-2 spike-mediated entry into cells. TMPRSS2 rs12329760 is a common variant associated with a significantly decreased risk of severe COVID19. Further studies are needed to assess the expression of the TMPRSS2 across different age groups. Moreover, our results identify TMPRSS2 as a promising drug target, with a potential role for camostat mesilate, a drug approved for the treatment of chronic pancreatitis and postoperative reflux esophagitis, in the treatment of COVID19. Clinical trials are needed to confirm this.

REACT-2 Round 5: increasing prevalence of SARS-CoV-2 antibodies demonstrate impact of the second wave and of vaccine roll-out in England

BackgroundEngland has experienced high rates of SARS-CoV-2 infection during the COVID-19 pandemic, affecting in particular minority ethnic groups and more deprived communities. A vaccination programme began in England in December 2020, with priority given to administering the first dose to the largest number of older individuals, healthcare and care home workers. MethodsA cross-sectional community survey in England undertaken between 26 January and 8 February 2021 as the fifth round of the REal-time Assessment of Community Transmission-2 (REACT-2) programme. Participants completed questionnaires, including demographic details and clinical and COVID-19 vaccination histories, and self-administered a lateral flow immunoassay (LFIA) test to detect IgG against SARS-CoV-2 spike protein. There were sufficient numbers of participants to analyse antibody positivity after 21 days from vaccination with the PfizerBioNTech but not the AstraZeneca/Oxford vaccine which was introduced slightly later. ResultsThe survey comprised 172,099 people, with valid IgG antibody results from 155,172. The overall prevalence of antibodies (weighted to be representative of the population of England and adjusted for test sensitivity and specificity) in England was 13.9% (95% CI 13.7, 14.1) overall, 37.9% (37.2, 38.7) in vaccinated and 9.8% (9.6, 10.0) in unvaccinated people. The prevalence of antibodies (weighted) in unvaccinated people was highest in London at 16.9% (16.3, 17.5), and higher in people of Black (22.4%, 20.8, 24.1) and Asian (20.0%, 19.0, 21.0) ethnicity compared to white (8.5%, 8.3, 8.7) people. The uptake of vaccination by age was highest in those aged 80 years or older (93.5%). Vaccine confidence was high with 92.0% (91.9, 92.1) of people saying that they had accepted or intended to accept the offer. Vaccine confidence varied by age and ethnicity, with lower confidence in young people and those of Black ethnicity. Particular concerns were identified around pregnancy, fertility and allergies. In 971 individuals who received two doses of the Pfizer-BioNTech vaccine, the proportion testing positive was high across all age groups. Following a single dose of Pfizer-BioNTech vaccine after 21 days or more, 84.1% (82.2, 85.9) of people under 60 years tested positive (unadjusted) with a decreasing trend with increasing age, but high responses to a single dose in those with confirmed or suspected prior COVID at 90.1% (87.2, 92.4) across all age groups. ConclusionsThere is uneven distribution of SARS-CoV-2 antibodies in the population with a higher burden in key workers and some minority ethnic groups, similar to the pattern in the first wave. Confidence in the vaccine programme is high overall although it was lower in some of the higher prevalence groups which suggests the need for improved communication about specific perceived risks. Two doses of Pfizer-BioNTech vaccine, or a single dose following previous infection, confers high levels of antibody positivity across all ages. Further work is needed to understand the relationship between antibody positivity, clinical outcomes such as hospitalisation, and transmission.