Third primary SARS-CoV-2 prophylactic mRNA vaccines enhances antibody responses in the majority of patients with haematological malignancies: Results from the MARCH study (preprint)

SARS-CoV-2 infection, and resulting disease, COVID-19, has a high mortality amongst patients with haematological malignancies. Global vaccine rollouts have successfully reduced hospitalisations and deaths, but the efficacy of vaccination in patients with haematological malignancies is known to be reduced. The UK-strategy offered a third, mRNA-based, vaccine as an extension to the primary course in these patients. Here we quantify serological responses following these vaccines in a cohort of 381 patients with haematological malignancies attending routine haematology outpatient clinics. By comparison with healthy controls, we report suboptimal responses following two primary vaccines, with significantly enhanced responses following the third primary dose. These responses however are heterogeneous and determined by haematological malignancy sub-type and therapy. We identify a group of patients with continued sub-optimal vaccine responses who may benefit from additional doses, as well as early intervention with monoclonal therapies in the event of developing SARS-CoV-2 infection.

Comparison of vaccine effectiveness against the Omicron (B.1.1.529) variant in patients receiving haemodialysis (preprint)

Background Emerging data suggest a reduction in SARS-CoV-2 vaccine effectiveness against Omicron SARS-CoV-2 infection. There is also evidence to show that Omicron is less pathogenic than previous variants. For clinically vulnerable populations, a less pathogenic variant may still have significant impact on morbidity and mortality. Herein we assess the clinical impact of Omicron infection, and vaccine effectiveness, in an in-centre haemodialysis (IC-HD) population. Methods One thousand, one hundred and twenty-one IC-HD patients were included in the analysis, all patients underwent weekly screening for SARS-CoV-2 infection via RT-PCR testing between 1 st December 2021 and 16 th January 2022. Screening for infection via weekly RT-PCR testing and 3-monthly serological assessment started prior to the vaccine roll out in 2020. Results Omicron infection was diagnosed in 145/1121 (12.9%) patients over the study period, equating to an infection rate of 3.1 per 1000 patient days. Vaccine effectiveness (VE) against Omicron infection in patients who had received a booster vaccine was 58 (23-75)%, p=0.002; VE was seen in patients who received either ChAdOx1, VE of 47(2-70)%, p=0.034, or BNT162b2, VE of 66 (36-81)%, p=0.0005, as their first two doses. No protection against infection was seen in patients who were partially vaccinated (2-doses), p=0.83. Prior infection was associated with reduced likelihood of Omicron infection, HR 0.69 (0.50-0.96), p=0.0289. Four (2.8%) patients died within 28 days of infection diagnosis, with no excess mortality was seen in patients with infection. Conclusion 3-doses of SARS-CoV-2 vaccines are required in ICHD to provide protection against Omicron infection.

SARS-CoV-2 Antibody Lateral Flow Assay for antibody prevalence studies following vaccine roll out: a Diagnostic Accuracy Study (preprint)

Background Lateral flow immunoassays (LFIAs) have the potential to deliver affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of SARS-CoV-2 vaccine. Methods This is a prospective diagnostic accuracy study. Setting Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Laboratory analyses were performed across Imperial College London sites and university facilities. Participants Two cohorts of patients were recruited; the first was a cohort of 108 renal transplant patients attending clinic following SARS-CoV-2 vaccine booster, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination, and 21 day follow up. A total of 186 paired samples were collected. Interventions During the participants visit, capillary blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG were detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. Main outcome measures The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott Assay. Results Using the threshold value for positivity on serological testing of ≥7.10 BAU/ml, the overall performance of the test produces an estimate of sensitivity of 91.94% (95% CI 85.67% to 96.06%) and specificity of 93.55% (95% CI 84.30% to 98.21%) using the Abbott assay as reference standard. Conclusions Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveys, but does not meet criteria for individual testing.

SARS-CoV-2 lineage B.1.1.7 is associated with greater disease severity among hospitalised women but not men (preprint)

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented. Methods We collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16/11/2020 - 10/01/2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity. Results Sequences were obtained from 2341 inpatients (HOCI cases = 786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The hazard ratio (HR) for mortality of B.1.1.7 compared to other lineages was 1.01 (95% CI 0.79-1.28, P=0.94) and for ITU admission was 1.01 (95% CI 0.75-1.37, P=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95-1.78) and ITU admission (HR 1.82, 95% CI 1.15-2.90) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61-1.10; ITU HR 0.74, 95% CI 0.52-1.04). Conclusions In common with smaller studies of patients hospitalised with SARS-CoV-2 we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared to other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.

Longevity of SARS-CoV-2 immune responses in haemodialysis patients and protection against reinfection (preprint)

Background Patients with end stage kidney disease (ESKD) receiving in-centre haemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, ICHD patients frequently develop serological evidence of infection, even with asymptomatic disease. The aim of this study is to investigate the durability and functionality of immune responses to SARS-CoV-2 infection in ICHD patients. Methods Three hundred and fifty-six ICHD patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies. Patients who became seronegative at 6 months were investigated for SARS-CoV-2 specific T-cell responses. Results One hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at Time 0, of which 127(98.4%) also had detectable anti-RBD. At 6 months, of 111 patients tested, 71(64.0%) and 97 (87.4%) remained anti-NP and anti-RBD seropositive respectively, p<0.001. For patients who retained antibody, both anti-NP and anti-RBD levels reduced significantly after 6 months. Ten patients who were anti-NP and anti-RBD seropositive at Time 0, had no detectable antibody at 6 months; of which 8 were found to have SARS-CoV-2 antigen specific T cell responses. Independent of antibody status at 6 months, patients with serological evidence of SARS-CoV-2 antibodies at Time 0, were at significantly lower risk of being diagnosed with infection Conclusions ICHD patients mount durable immune responses 6 months post SARS-CoV-2 infection, with <3% of patients showing no evidence of humoral or cellular immunity. These immune responses are associated with a reduced risk of subsequent reinfection.

Detection and Quantification of Antibody to SARS-CoV-2 Receptor Binding Domain Provides Enhanced Sensitivity, Specificity and Utility (preprint)

Background: Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity.Methods: The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA) provides the most sensitive format. It has been exploited in a novel hybrid manner employing an S1 solid-phase preferentially presenting RBD once solid-phase bound, coupled with a labelled RBD conjugate, used in a two-step sequential assay.Findings: This assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine. The early response at presentation with illness, elevated responsiveness with disease severity, detection of asymptomatic seroconversion and persistence after the loss of antibody to the nucleoprotein (anti-NP) are all documented.Trial Registration: The ISARIC WHO CCP-UK study was registered at https://www.isrctn.com/ISRCTN66726260 and designated an Urgent Public Health Research Study by NIHR.Interpretation: The hybrid DABA displays the attributes necessary for an antibody test to be used in both clinical and reference serology. It allows the neutralising antibody response to be inferred early in infection and potentially in vaccine recipients. It is also of sufficient sensitivity to be used to provide serological confirmation of prior infection and provides a more secure measure for seroprevalence studies in the population generally than does anti-NP based on the Architect platform.Funding: This work is variously supported by grants from: the National Institute for Health Research (NIHR; award CO-CIN-01), the Medical Research Council (MRC; grant MC_PC_19059 and MC_PC_19078), MRC NIHR (grant CV220-111) and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (award 200907), NIHR HPRU in Respiratory Infections at Imperial College London with PHE (award 200927), Wellcome Trust and Department for International Development (DID; 215091/Z/18/Z), the Bill and Melinda Gates Foundation (OPP1209135), Liverpool Experimental Cancer Medicine Centre (grant reference C18616/A25153), NIHR Biomedical Research Centre at Imperial College London (IS-BRC-1215-20013), EU Platform for European Preparedness Against (Re-)emerging Epidemics (PREPARE; FP7 project 602525), and NIHR Clinical Research Network for providing infrastructure support for this research.Declaration of Interests: RST, MOM and PC report patent pending (Patent Application No. 2011047.4 for “SARS-CoV-2 antibody detection assay). All other authors declare no competing interests.Ethics Approval Statement: The use of tissues was approved by the CDRTB Steering Committee in accordance with the responsibility delegated by the National Research Ethics Service (South Central Ethics Committee – C, NRES reference 15/SC/0089).Written informed consent was obtained from all patients. Ethical approval was given by the South Central–Oxford C Research Ethics Committee in England (reference: 13/SC/0149), Scotland A Research Ethics Committee (reference: 20/SS/0028) and World Health Organization Ethics Review Committee (RPC571 and RPC572l; 25 April 2013)

Evaluation of the Abbott Architect, Roche Elecsys and Virtus S1 SARS-CoV-2 antibody tests in community-managed COVID-19 cases (preprint)

Background. Antibody testing can help define how protective immunity to SARS-CoV-2 is and how long this immunity lasts. Many antibody tests have been evaluated in hospitalised rather than community based COVID-19 cases. Virtus Respiratory Research Ltd (Virtus) has developed its own quantitative IgM and IgG SARS CoV-2 antibody assay. We report its validation and performance characteristics and compare its performance with the Abbott Architect and Roche Elecsys assays in community COVID cases. Methods We developed a quantitative antibody test to detect IgM and IgG to the SARS-CoV-2 S1 spike protein (the Virtus test) and validated this test in 107 true positive sera from 106 community-managed and 1 hospitalised COVID-19 cases and 208 true negative serum samples. We validated the Virtus test against a neutralising antibody test. We determined sensitivities of the Abbott test in the 107 true positive samples and the Roche test in a subset of 75 true positive samples. Results The Virtus quantitative test was positive in 93 of 107 (87%) community cases of COVID-19 and both IgM and IgG levels correlated strongly with neutralising antibody titres (r=0.75 for IgM, r=0.71 for IgG, P<0.0001 for both antibodies). The specificity of the Virtus test was 98.6% for low level antibody positives, 99.5% for moderate positives and 100% for high or very high positives. The Abbott test had a sensitivity of 68%. In the 75 sample subset, the Virtus test was positive in 91%, the Roche test in 69%. Conclusions The Abbott and Roche tests had sensitives of 68% and 69% respectively in this community set of COVID-19 sera, while the Virtus test had sensitivities of 87% and 91% in the same sample sets. The strong positive correlation with virus neutralization suggests a positive Virtus quantitative antibody test is likely predictive of protective against recurrent COVID-19.

CovidNudge: diagnostic accuracy of a novel lab-free point-of-care diagnostic for SARS-CoV-2 (preprint)

Background Access to rapid diagnosis is key to the control and management of SARS-CoV-2. Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) testing usually requires a centralised laboratory and significant infrastructure. We describe the development and diagnostic accuracy assessment of a novel, rapid point-of-care RT-PCR test, the DnaNudge platform CovidNudge test, which requires no laboratory handling or sample pre-processing. Methods Nasopharyngeal swabs are inserted directly into a cartridge which contains all reagents and components required for RT-PCR reactions, including multiple technical replicates of seven SARS-CoV-2 gene targets (rdrp1, rdrp2, e-gene, n-gene, n1, n2 and n3) and human ribonuclease P (RNaseP) as a positive control. Between April and May 2020, swab samples were tested in parallel using the CovidNudge direct-to-cartridge platform and standard laboratory RT-PCR using swabs in viral transport medium. Samples were collected from three groups: self-referred healthcare workers with suspected COVID-19 (Group 1, n=280/386; 73%); patients attending the emergency department with suspected COVID-19 (Group 2, n=15/386; 4%) and hospital inpatient admissions with or without suspected COVID-19 (Group 3, n=91/386; 23%). Results Of 386 paired samples tested across all groups, 67 tested positive on the CovidNudge platform and 71 with standard laboratory RT-PCR. The sensitivity of the test varied by group (Group 1 93% [84-98%], Group 2 100% [48-100%] and Group 3 100% [29-100%], giving an average sensitivity of 94.4% (95% confidence interval 86-98%) and an overall specificity of 100% (95%CI 99-100%; Group 1 100% [98-100%]; Group 2 100% [69-100%] and Group 3 100% [96-100%]). Point of care testing performance was comparable during a period of high (25%) and low (3%) background prevalence. Amplification of the viral nucleocapsid (n1, n2, n3) targets were most sensitive for detection of SARS-CoV2, with the assay able to detect 1x104 viral particles in a single swab. Conclusions The CovidNudge platform offers a sensitive, specific and rapid point of care test for the presence of SARS-CoV-2 without laboratory handling or sample pre-processing. The implementation of such a device could be used to enable rapid decisions for clinical care and testing programs.

A handheld point-of-care system for rapid detection of SARS-CoV-2 in under 20 minutes (preprint)

The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (< 20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 90.55% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 {+/-} 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n=40), showing average detection times of 12.89 {+/-} 2.59 min for positive samples (n=34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geo-localization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.

SARS-CoV-2 infection, clinical features and outcome of COVID-19 in United Kingdom nursing homes (preprint)

Objectives: To understand SARS-Co-V-2 infection and transmission in UK nursing homes in order to develop preventive strategies for protecting the frail elderly residents. Design: An outbreak investigation. Setting: 4 nursing homes affected by COVID-19 outbreaks in central London. Participants: 394 residents and 70 staff in nursing homes. Interventions: Two point-prevalence surveys one week apart where residents underwent SARS-CoV-2 testing and had relevant symptoms documented. Asymptomatic staff from three of the four homes were also offered SARS-CoV-2 testing. Main outcome measures: All-cause mortality, and mortality attributed to COVID-19 on death certificates. Prevalence of SARS-CoV-2 infection and symptoms in residents and staff. Results: Overall, 26% (95% confidence interval 22 to 31) of residents died over the two-month period. All-cause mortality increased by 203% (95% CI 70 to 336). Systematic testing identified 40% (95% CI 35 to 46) of residents, of whom 43% (95% CI 34 to 52) were asymptomatic and 18% (95% CI 11 to 24) had atypical symptoms, as well as 4% (95% CI -1 to 9) of asymptomatic staff who tested positive for SARS-CoV-2. Conclusions: The SARS-CoV-2 outbreak was associated with a very high mortality rate in residents of nursing homes. Systematic testing of all residents and a representative sample of staff identified high rates of SARS-CoV-2 positivity across the four nursing homes, highlighting a potential for regular screening to prevent future outbreaks.

A new role for Biofoundries in rapid prototyping, development, and validation of automated clinical diagnostic tests for SARS-CoV-2 (preprint)

The SARS-CoV-2 pandemic has shown how the rapid rise in demand for patient and community sample testing, required for tracing and containing a highly infectious disease, has quickly overwhelmed testing capability globally. With most diagnostic infrastructure dependent on specialised instruments, their exclusive reagent supplies quickly become bottlenecks in times of peak demand, creating an urgent need for novel approaches to boost testing capacity. We address this challenge by refocusing the full synthetic biology stack available at the London Biofoundry onto the development of alternative patient sample testing pipelines. We present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled, and that accepts a diverse range of reagents. Using an in-house-generated, open-source, MS2-virus-like-particle-SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two novel detection assays based on CRISPR-Cas and Loop-mediated isothermal Amplification (LAMP) approaches. In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and benchmark SARS-CoV-2 detection in patient samples via RT-qPCR, CRISPR-Cas, and LAMP against clinical test sets. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs with a testing capacity of 1000 samples per day and now contributes to increased patient sample processing in the UK while we continue to refine and develop novel high-throughput diagnostic methods. Finally, our workflows and protocols can be quickly implemented and adapted by members of the Global Biofoundry Alliance and the wider scientific and medical diagnostics community.