Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays

The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels

Detection of serum cross-reactive antibodies and memory response to SARS-CoV-2 in pre-pandemic and post-COVID-19 convalescent samples

Background A notable feature of COVID-19 is that children are less susceptible to severe disease. Children are known to experience more infections with endemic human coronaviruses (HCoVs) compared to adults. Little is known whether HCoV infections lead to cross-reactive anti-SARS-CoV-2 antibodies. Methods We investigated the presence of cross-reactive anti-SARS-CoV-2 IgG antibodies (to spike(S)1, S1-receptor-binding receptor (S1-RBD) and nucleocapsid protein(NP)), by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralisation assay, in pre-pandemic sera collected from children(n=50) and adults(n=45), and compared with serum samples from convalescent COVID-19 patients(n=16). Results A significant proportion of children (up to 40%) had detectable cross-reactive antibodies to SARS-CoV-2 S1, S1-RBD and NP antigens, and the anti-S1 and -S1-RBD antibody levels correlated with anti-HCoV-HKU1 and -OC43 S1 antibody titers in pre-pandemic samples(p<0.001). There were marked increases of anti-HCoV-HKU1 and -OC43 S1 (but not anti-NL63 and -229E S-RBD) antibody titres in serum samples from convalescent COVID-19 patients(p<0.001), indicating an activation of cross-reactive immunological memory to β-coronavirus spike. Conclusions We demonstrated cross-reactive anti-SARS-CoV-2 antibodies in pre-pandemic serum samples from children and young adults. Promoting this cross-reactive immunity and memory response derived from common HCoV may be an effective strategy against SARS-COV-2 and future novel coronaviruses

Sequential Infection with Influenza A Virus Followed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads to More Severe Disease and Encephalitis in a Mouse Model of COVID-19

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection (‘twinfection’) is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19

CD4+ and CD8+ T cells and antibodies are associated with protection against Delta vaccine breakthrough infection: a nested case-control study within the PITCH study

Serological correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection after vaccination ("vaccine breakthrough") have been described. However, T cell correlates of protection against breakthrough are incompletely defined, especially the specific contributions of CD4+ and CD8+ T cells. Here, 279 volunteers in the Protective Immunity from T Cells in Healthcare Workers (PITCH) UK cohort study were enrolled in a nested case-control study. Cases were those who tested SARS-CoV-2 PCR or lateral flow device (LFD) positive after two vaccine doses during the Delta-predominant era (n = 32), while controls were those who did not report a positive test or undergo anti-nucleocapsid immunoglobulin G (IgG) seroconversion during this period (n = 247). Previous SARS-CoV-2 infection prior to vaccination was associated with reduced odds of vaccine breakthrough. Using samples from 28 d after the second vaccine dose, before all breakthroughs occurred, we observed future cases had lower ancestral spike (S)- and receptor binding domain-specific IgG titers and S1- and S2-specific T cell interferon gamma (IFNγ) responses compared with controls, although these differences did not persist when individuals were stratified according to previous infection status before vaccination. In a subset of matched infection-naïve cases and controls, vaccine breakthrough cases had lower CD4+ and CD8+ IFNγ and tumor necrosis factor (TNF) responses to Delta S peptides compared with controls. For CD8+ responses, this difference appeared to be driven by reduced responses to Delta compared with ancestral peptides among cases; this reduced response to Delta peptides was not observed in controls. Our findings support a protective role for T cells against Delta breakthrough infection. IMPORTANCE Defining correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine breakthrough infection informs vaccine policy for booster doses and future vaccine designs. Existing studies demonstrate humoral correlates of protection, but the role of T cells in protection is still unclear. In this study, we explore antibody and T cell immune responses associated with protection against Delta variant vaccine breakthrough infection in a well-characterized cohort of UK Healthcare Workers (HCWs). We demonstrate evidence to support a role for CD4+ and CD8+ T cells as well as antibodies against Delta vaccine breakthrough infection. In addition, our results suggest a potential role for cross-reactive T cells in vaccine breakthrough

Clinical characteristics of children and young people admitted to hospital with covid-19 in United Kingdom: prospective multicentre observational cohort study.

OBJECTIVE: To characterise the clinical features of children and young people admitted to hospital with laboratory confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the UK and explore factors associated with admission to critical care, mortality, and development of multisystem inflammatory syndrome in children and adolescents temporarily related to coronavirus disease 2019 (covid-19) (MIS-C). DESIGN: Prospective observational cohort study with rapid data gathering and near real time analysis. SETTING: 260 hospitals in England, Wales, and Scotland between 17 January and 3 July 2020, with a minimum follow-up time of two weeks (to 17 July 2020). PARTICIPANTS: 651 children and young people aged less than 19 years admitted to 138 hospitals and enrolled into the International Severe Acute Respiratory and emergency Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK study with laboratory confirmed SARS-CoV-2. MAIN OUTCOME MEASURES: Admission to critical care (high dependency or intensive care), in-hospital mortality, or meeting the WHO preliminary case definition for MIS-C. RESULTS: Median age was 4.6 (interquartile range 0.3-13.7) years, 35% (225/651) were under 12 months old, and 56% (367/650) were male. 57% (330/576) were white, 12% (67/576) South Asian, and 10% (56/576) black. 42% (276/651) had at least one recorded comorbidity. A systemic mucocutaneous-enteric cluster of symptoms was identified, which encompassed the symptoms for the WHO MIS-C criteria. 18% (116/632) of children were admitted to critical care. On multivariable analysis, this was associated with age under 1 month (odds ratio 3.21, 95% confidence interval 1.36 to 7.66; P=0.008), age 10-14 years (3.23, 1.55 to 6.99; P=0.002), and black ethnicity (2.82, 1.41 to 5.57; P=0.003). Six (1%) of 627 patients died in hospital, all of whom had profound comorbidity. 11% (52/456) met the WHO MIS-C criteria, with the first patient developing symptoms in mid-March. Children meeting MIS-C criteria were older (median age 10.7 (8.3-14.1) v 1.6 (0.2-12.9) years; P<0.001) and more likely to be of non-white ethnicity (64% (29/45) v 42% (148/355); P=0.004). Children with MIS-C were five times more likely to be admitted to critical care (73% (38/52) v 15% (62/404); P<0.001). In addition to the WHO criteria, children with MIS-C were more likely to present with fatigue (51% (24/47) v 28% (86/302); P=0.004), headache (34% (16/47) v 10% (26/263); P<0.001), myalgia (34% (15/44) v 8% (21/270); P<0.001), sore throat (30% (14/47) v (12% (34/284); P=0.003), and lymphadenopathy (20% (9/46) v 3% (10/318); P<0.001) and to have a platelet count of less than 150 × 109/L (32% (16/50) v 11% (38/348); P<0.001) than children who did not have MIS-C. No deaths occurred in the MIS-C group. CONCLUSIONS: Children and young people have less severe acute covid-19 than adults. A systemic mucocutaneous-enteric symptom cluster was also identified in acute cases that shares features with MIS-C. This study provides additional evidence for refining the WHO MIS-C preliminary case definition. Children meeting the MIS-C criteria have different demographic and clinical features depending on whether they have acute SARS-CoV-2 infection (polymerase chain reaction positive) or are post-acute (antibody positive). STUDY REGISTRATION: ISRCTN66726260

Evaluation of eight lateral flow tests for the detection of anti-SARS-CoV-2 antibodies in a vaccinated population

Background: Rapid determination of an individual's antibody status can be beneficial in understanding an individual's immune response to SARS-CoV-2 and for initiation of therapies that are only deemed effective in sero-negative individuals. Antibody lateral flow tests (LFTs) have potential to address this need as a rapid, point of care test. Methods: Here we present a proof-of-concept evaluation of eight LFT brands using sera from 95 vaccinated individuals to determine sensitivity for detecting vaccination generated antibodies. Samples were analysed on eight different brands of antibody LFT and an automated chemiluminescent microparticle immunoassay (CMIA) that identifies anti-spike antibodies which was used as our reference standard. Results: All 95 (100%) participants tested positive for anti-spike antibodies by the chemiluminescent microparticle immunoassay (CMIA) reference standard post-dose two of their SARS-CoV-2 vaccine: BNT162b2 (Pfizer/BioNTech, n = 60), AZD1222 (AstraZeneca, n = 31), mRNA-1273 (Moderna, n = 2) and Undeclared Vaccine Brand (n = 2). Sensitivity increased from dose one to dose two in six out of eight LFTs with three tests achieving 100% sensitivity at dose two in detecting anti-spike antibodies. Conclusions: These tests are demonstrated to be highly sensitive to detect raised antibody levels in vaccinated individuals. RDTs are low cost and rapid alternatives to ELISA based systems

Risk stratification of patients admitted to hospital with covid-19 using the ISARIC WHO Clinical Characterisation Protocol: development and validation of the 4C Mortality Score.

OBJECTIVE: To develop and validate a pragmatic risk score to predict mortality in patients admitted to hospital with coronavirus disease 2019 (covid-19). DESIGN: Prospective observational cohort study. SETTING: International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) World Health Organization (WHO) Clinical Characterisation Protocol UK (CCP-UK) study (performed by the ISARIC Coronavirus Clinical Characterisation Consortium-ISARIC-4C) in 260 hospitals across England, Scotland, and Wales. Model training was performed on a cohort of patients recruited between 6 February and 20 May 2020, with validation conducted on a second cohort of patients recruited after model development between 21 May and 29 June 2020. PARTICIPANTS: Adults (age ≥18 years) admitted to hospital with covid-19 at least four weeks before final data extraction. MAIN OUTCOME MEASURE: In-hospital mortality. RESULTS: 35 463 patients were included in the derivation dataset (mortality rate 32.2%) and 22 361 in the validation dataset (mortality rate 30.1%). The final 4C Mortality Score included eight variables readily available at initial hospital assessment: age, sex, number of comorbidities, respiratory rate, peripheral oxygen saturation, level of consciousness, urea level, and C reactive protein (score range 0-21 points). The 4C Score showed high discrimination for mortality (derivation cohort: area under the receiver operating characteristic curve 0.79, 95% confidence interval 0.78 to 0.79; validation cohort: 0.77, 0.76 to 0.77) with excellent calibration (validation: calibration-in-the-large=0, slope=1.0). Patients with a score of at least 15 (n=4158, 19%) had a 62% mortality (positive predictive value 62%) compared with 1% mortality for those with a score of 3 or less (n=1650, 7%; negative predictive value 99%). Discriminatory performance was higher than 15 pre-existing risk stratification scores (area under the receiver operating characteristic curve range 0.61-0.76), with scores developed in other covid-19 cohorts often performing poorly (range 0.63-0.73). CONCLUSIONS: An easy-to-use risk stratification score has been developed and validated based on commonly available parameters at hospital presentation. The 4C Mortality Score outperformed existing scores, showed utility to directly inform clinical decision making, and can be used to stratify patients admitted to hospital with covid-19 into different management groups. The score should be further validated to determine its applicability in other populations. STUDY REGISTRATION: ISRCTN66726260

Clinical characteristics of children and young people hospitalised with covid-19 in the United Kingdom: prospective multicentre observational cohort study

Objective To characterise the clinical features of children and young people admitted to hospital with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the UK, and explore factors associated with admission to critical care, mortality, and development of multisystem inflammatory syndrome in children and adolescents temporarily related to covid-19 (MIS-C). Design Prospective observational cohort study with rapid data gathering and near real time analysis. Setting 260 acute care hospitals in England, Wales, and Scotland between 17th January and 5th June 2020, with a minimal follow-up time of two weeks (to 19th June 2020). Participants 451 children and young people aged less than 19 years admitted to 116 hospitals and enrolled into the International Severe Acute Respiratory and emergency Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK study with laboratory-confirmed SARS-CoV-2. Main Outcome Measures Admission to critical care (high dependency or intensive care), in-hospital mortality, or meeting the WHO preliminary case definition for MIS-C. Results Median age was 3.9 years [interquartile range (IQR) 0.3-12.9 years], 36% (162/451) were under 12 months old, and 57% (256/450) were male. 56% (224/401) were White, 12% (49/401) South Asian and 10% (40/401) Black. 43% (195/451) had at least one recorded comorbidity. A muco-enteric cluster of symptoms was identified, closely mirroring the WHO MIS-C criteria. 17% of children (72/431) were admitted to critical care. On multivariable analysis this was associated with age under one month odds ratio 5.05 (95% confidence interval 1.69 to 15.72, p=0.004), age 10 to 14 years OR 3.11 (1.21 to 8.55, p=0.022) and Black ethnicity OR 3.02 (1.30 to 6.84, p=0.008). Three young people died (0.7 %, 3/451) aged 16 to 19 years, all of whom had profound comorbidity. Twelve percent of children (36/303) met the WHO MIS-C criteria, with the first patient developing symptoms in mid-March. Those meeting MIS-C criteria were older, (median age 10.8 years ([IQR 8.4-14.1] vs 2.0 [0.2-12.6]), p [less than] 0.001) and more likely to be of non-White ethnicity (70% (23/33) vs 43% (101/237), p=0.005). Children with MIS-C were four times more likely to be admitted to critical care (61% (22/36) vs 15% (40/267, p [less than] 0.001). In addition to the WHO criteria, children with MIS-C were more likely to present with headache (45% (13/29) vs 11% (19/171), p [less than] 0.001), myalgia (39% (11/28) vs 7% (12/170), p [less than] 0.001), sore throat (37% (10/27) vs (13% (24/183, p = 0.004) and fatigue (57% (17/30) vs 31% (60/192), p =0.012) than children who did not and to have a platelet count of less than 150 x109/L (30% (10/33) vs 10% (24/232), p=0.004). Conclusions Our data confirms less severe covid-19 in children and young people than in adults and we provide additional evidence for refining the MIS-C case definition. The identification of a muco-enteric symptom cluster also raises the suggestion that MIS-C is the severe end of a spectrum of disease

Diagnostic markers of acute encephalitis syndrome and COVID-associated multisystem inflammatory syndrome in children from Southern India.

Acute encephalitis syndrome (AES) in children poses a significant public health challenge in India. This study aims to explore the utility of host inflammatory mediators and neurofilament (NfL) levels in distinguishing etiologies, assessing disease severity, and predicting outcomes in AES. We assessed 12 mediators in serum (n = 58) and 11 in cerebrospinal fluid (CSF) (n = 42) from 62 children with AES due to scrub typhus, viral etiologies, and COVID-associated multisystem inflammatory syndrome (MIS-C) in Southern India. Additionally, NfL levels in serum (n = 20) and CSF (n = 18) were examined. Clinical data, including Glasgow coma scale (GCS) and Liverpool outcome scores, were recorded. Examining serum and CSF markers in the three AES etiology groups revealed notable distinctions, with scrub typhus differing significantly from viral and MIS-C causes. Viral causes had elevated serum CCL11 and CCL2 compared with scrub typhus, while MIS-C cases showed higher HGF levels than scrub typhus. However, CSF analysis showed a distinct pattern with the scrub typhus group exhibiting elevated levels of IL-1RA, IL-1β, and TNF compared with MIS-C, and lower CCL2 levels compared with the viral group. Modeling the characteristic features, we identified that age ≥3 years with serum CCL11 < 180 pg/mL effectively distinguished scrub typhus from other AES causes. Elevated serum CCL11, HGF, and IL-6:IL-10 ratio were associated with poor outcomes (p = 0.038, 0.005, 0.02). Positive CSF and serum NfL correlation, and negative GCS and serum NfL correlation were observed. Median NfL levels were higher in children with abnormal admission GCS and poor outcomes. Measuring immune mediators and brain injury markers in AES provides valuable diagnostic insights, with the potential to facilitate rapid diagnosis and prognosis. The correlation between CSF and serum NfL, along with distinctive serum cytokine profiles across various etiologies, indicates the adequacy of blood samples alone for assessment and monitoring. The association of elevated levels of CCL11, HGF, and an increased IL-6:IL-10 ratio with adverse outcomes suggests promising avenues for therapeutic exploration, warranting further investigation

SARS-CoV-2 RNA detected in blood products from patients with COVID-19 is not associated with infectious virus

Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. Methods: We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. Results: We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. Conclusions: vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19