Potential participants at the identified long-term care facility (LTCF) will be informed about the study and provided with the participant information leaflet and consent form. Suitable time will be afforded to consider this information before consent is sought. Clear direction will be provided that their decision to not participate will in no way negatively affect their care, the offer of a vaccine, or involvement in any future studies should they wish to partake.
Sample: 2x5ml vial of whole blood will be drawn. One vial will be sent within 1 working day to the centralised laboratory for further testing as per the study method. The second sample will be spun down and frozen, marked with the participant’s anonymous identification number and subject ID number and stored on site at -80oC in keeping with all local regulations and approvals required for the long-term storage of blood samples in case of requirements to repeat the sample testing procedures.
Phase 1 (Initial testing): All eligible participants will undergo venepuncture for serological ELISA testing, pseudovirus neutralisation and anti-receptor binding domain antibody avidity testing prior to SARS-CoV-2 vaccination. These results will be fed-back to the medical team at the LCTF for onward distribution to participants.
Individuals who have only received a single dose of the Pfizer-BioNTech COVID-19 Vaccine will go on for repeat testing at one month. All participants who have received two doses of the Pfizer-BioNTech COVID-19 Vaccine at the point of initial testing will exit the study after phase 1. Any individual who is unable to receive, or chooses not to receive, the second dose of the Pfizer-BioNTech COVID-19 Vaccine will exit the study after Phase 1.
Phase 2 (Post vaccination testing):
All participants recruited in phase 1 and since initial testing having received their second dose of the Pfizer-BioNTech COVID-19 Vaccine will undergo a second round of venepuncture repeat testing as listed above 4 weeks after the second dose.
SARS-CoV-2 serological testing
Participants were invited to undertake blood draw for assessment of SARS-CoV-2 antibodies at three intervals, namely (i) as part of an initial outbreak investigation and coinciding with the end of the UK’s first wave of infection and relaxation of initial social distancing regulations (June 2020) and (ii) prior to receipt of the Pfizer BioNTech BNT162b2 vaccine (December 2020) and (iii) four weeks after the second dose of Pfizer BioNTech BNT162b2 vaccine (April 2021). Antibody wane over eight months in older adults (using the same assay, see below)10 informed the decision to test at least once, but not more than, every 6 months for evidence of new seroconversion, while not unnecessarily increasing sampling burden to participants. Likewise, in order to reduce test burden on participants, maximise study retention and in line with UK vaccine strategy for an initial two-vaccine regimen, a separate blood draw after a single vaccine dose was not carried out.
Antibodies to the SARS-CoV-2 nucleocapsid protein (anti-NP) may be detected in serum following infection but not in response to immunisation. Assessment for evidence of seroconversion following SARS-CoV-2 infection (symptomatic or asymptomatic) was via an anti-nucleocapsid (anti-NP) IgG chemiluminescent microparticle immunoassay (CMIA) as per manufacture instructions (Abbot Laboratories, Lake Bluff, IL, USA). Anti-receptor binding domain (anti-RBD) antibodies have been previously shown to be predictive of neutralising response.11 In order to further characterise the response to prior SARS-CoV-2 infection and vaccination, paired serum samples pre- and post-vaccine were measured using a quantitative hybrid in-house anti-RBD double antigen binding assay (hybrid-DABA) (Imperial College, London, UK) as previously described.11 The hybrid-DABA utilises S1 antigen manipulated to express stably RBD epitopes and enable highly specific capture of anti-RBD antibody, detected by labelled fluid phase horseradish peroxidase-coupled RBD. Hybrid-DABA binding ratios are converted and reported in binding antibody units/ml (BAU/ml), allowing correlation with World Health Organisation (WHO) international standards for SARS-CoV-2 IgG binding antibody units. Samples were also tested on the commercial quantitative Abbott Architect IgG Quant II CMIA (Abbot Laboratories, Lake Bluff, IL, USA) in order to observe comparison with a commercially available assay marketed to assess SARS-CoV-2 antibodies, including neutralizing antibodies. The Abbott CMIA is a two-step automated immunoassay for qualitative and quantitative assessment of total SARS-CoV-2 IgG antibodies, including those to the S1 subunit. Abbott arbitrary units (AU/ml) are multiplied by a factor of 0.142, giving a binding antibody unit (BAU)/ml result, providing correlation with the WHO international standards.12
Pseudotype virus neutralisation
Post-vaccine in vitro antibody neutralisation activity was further explored by pseudotype virus assay, with comparison of results between SARS-CoV-2 infection naïve individuals and those with prior evidence of SARS-CoV-2 infection (both asymptomatic and symptomatic). A lentivirus that encodes for luciferase and pseudo-typed with the spike of SARS-CoV-2 was utilised. In brief, 16µl participant sera was heat inactivated (56oC, 30 mins) and initial dilutions made with 5µl HI serum and 2% growth medium (DMEM, penicillin/streptomycin, 2% FBS). Following this, a total of eight serial dilutions were made and mixed with PsV (0.5-1 x 10^6 RLU/well) and incubated at 37oC for 48 hrs. Luciferase was then measured at 48 hrs post-infection (BrightGlo, Promega). An initial 1:40 dilution was carried out and data expressed as a percentage neutralisation e.g. 100% meaning serum neutralised 100% of the PsV, as previously described.13
Competitive ELISA
An inhouse competitive anti-RBD antibody competitive enzyme linked immunosorbent assay was utilised to assess antibody binding avidity maturation. As part of the consent process, participants were asked for permission to utilise any remaining serum taken for serological and pseudovirus neutralisation testing on additional SARS-CoV-2 antibody assays as they became available. Those individuals that had sufficient paired sample remaining from pre- and post-vaccine sampling and had given permission were assessed for antibody binding avidity at these time points. Assay methodology has previously been made available.14 Solid phase 96-microwells plates (NUNC Immunomodule, U8 Maxisorp wells) were coated with 100 μl of S1 antigen at a concentration of 5 μg/mL (MicroImmune Coating Buffer; ClinTech, Guildford, UK) overnight at 2−8 °C, followed by 3 h at 35−37 °C (under moist conditions) and 1 h at room temperature. Plates were washed once with 0.05 % Tween 20/PBS, blocked with MicroImmune Blocking Solution (3−4 hours at 37 °C in a moist box), aspirated, dried overnight at 37 °C and stored dry at 4 °C in sealed pouches with desiccant. The assay was carried out by simultaneous addition of 25μl of sera and 75ul of recombinant anti-RBD neutralizing monoclonal antibody (MAB12444; Native Antigen, Oxford, UK) conjugated with HRP, diluted in conjugant buffer (Conjugant Diluent; ClinTech, Guildford, UK) supplemented with 10% FBS and 10% Normal Human Plasma to each well. Plates were incubated for 1 h at 37 °C then washed five times with wash buffer (ClinTech, Guildford, UK). One hundred microlitres of TMB substrate was then added (ClinTech, Guildford, UK), incubated for 30 min at 37 °C, after which the reaction was stopped and read spectrometrically at 450−630 nm. Results were calculated as a percentage of the average of optical density (OD) obtained for three negative controls assayed in each run.