Impact of ageing on homologous and human-coronavirus-reactive antibodies after SARS-CoV-2 vaccination or … – Nature.com

Study design and participants

We conducted a prospective cohort study of adults receiving pandemic COVID-19 vaccine (BNT162b2, Pfizer BioNTech) at Eidsvg general practice and Haukeland University Hospital in Bergen, Norway. All participants received the first two doses of BNT162b2 vaccine at a 3-week interval in JanuaryFebruary 2021, and the third dose of BNT162b2 vaccine 1011 months later in NovemberDecember 2021. No vaccinee had tested rt-PCR positive for SARS-CoV-2 or had any COVID-19 symptom before receiving the 1st dose mRNA vaccine.

The COVID-19 patients were participants of a larger case-ascertained study conducted in Bergen, Norway. All patients tested rt-PCR positive for SARS-CoV-2 from nasopharyngeal swabs during March and April 2020. None of the infected patients received any COVID-19 vaccine within 12 months post diagnosis.

The study was approved by the regional ethics committee (Regional Committee for Medical Research Ethics, Western Norway (REK Vest number 118664) and Northern Norway (REK Nord number 218629)) and is registered in the National Institute for Health database Clinical trials.gov (NCT04706390). All participants provided written informed consent before inclusion in the study.

Electronic case report forms (eCRF) were used to collect demographics, comorbidities, infection history (rt-PCR test results and presence of COVID-19 symptoms), vaccination data and side reactions.

The vaccine used in the study was a monovalent COVID-19 mRNA vaccine BNT162b2 embedded in lipid nanoparticles contained 30g of a purified single-stranded, 5-capped messenger RNA (mRNA), encoding the viral spike protein of SARS-CoV-2 from the founder Wuhan-Hu1 strain (pre-alpha). The vaccine was supplied as a multidose vial reconstituted in sodium chloride 9mg/mL (0.9%) containing 0.45ml per dose, 5 doses per vial, and administered by intramuscular injection.

Serum samples were collected pre-, post 1st (day 21) and 2nd doses (2 months), and pre- (9 months) and post 3rd (12 months) vaccination from all COVID-19 vaccinees, and during the acute (08 days post diagnosis), convalescent phase (1676 days post diagnosis) and 12 months (334387 days post diagnosis) after infection from all COVID-19 patients. Sera were separated, aliquoted and stored at 80C until use.

The hCoV-19/Norway/Bergen-01/2020 (GISAID accession ID EPI_ISL_541970, termed as Bergen-1 hereafter) virus was isolated in-house from an rt-PCR-confirmed patient in March 2020 and propagated in Vero cells in a certified Biosafety Level-3 Laboratory.

The human coronavirus (HCoV) strain NL63 (GenBank: AY567487) was obtained from BEI Resources (Cat. NR-470) and propagated in LLC-MK2 cells (ATCC CCL-7) in biosafety level-2 laboratory. The HCoV strain OC43 (GenBank: AY585228) was obtained from BEI Resources (Cat. NR-52725) and propagated in HCT-8 cells (ATCC CCL-244) in biosafety level-2 laboratory.

The full-length spike proteins from SARS-CoV-2 Wuhan-1 isolate (GenBank: QHD43416), HCoV 229E strain (GenBank: A0G74783), NL63 strain (GenBank: AFV53148), HKU1 strain (UniProtKB/Swiss-Prot: Q0ZME7), and OC43 strain (GenBank: AIL49484) were produced in-house in Expi293F cells (Thermo Fisher Scientific) using the constructs provided by Prof. Barney Graham. The S1 and S2 domains of spike protein from SARS-CoV-2 Wuhan-Hu-1 isolate were obtained commercially (Sino Biological Cat. 40591-V08H and 40590-V08B, respectively).

To quantify the SARS-CoV-2 and HCoV spike specific binding IgG, Maxi Sorp 96-well plates (Thermo Fisher) were coated with in-house prepared full-length spike proteins (Wuhan-Hu-1 spike 0.05g/well; 229E, HKU1 and OC43 spikes 0.1g/well; NL63 spike 0.3g/well) or commercial spike proteins (Wuhan-Hu-1 S1 and S2 domains, 0.05g/well) at 4C overnight. Sera were 5-fold serially diluted from 1:100 and tested in duplicates. Biotin labelled anti-human IgG (1:1000, Sigma-Aldrich Cat. B-1140); horseradish peroxidase (HRP) labelled streptavidin (1:1400, Southern Biotech Cat. 7105-05) were added followed by o-Phenylenediamine dihydrochloride (OPD, 0.05mg/well, Sigma-Aldrich Cat. P-8287). The chromogenic reaction was stopped by sulfuric acid. Optical density (OP) values were read at 490nm using a synergy H1 plate reader (BioTek). Immunoglobulin concentrations were interpolated as binding antibody unit (BAU)/ml from the standard curve with purified human IgG (Sigma-Aldrich Cat. I-4506).

To measure serum neutralizing antibody titres, the microneutralization assay was performed in a certified Biosafety Level 3 Laboratory against the infectious hCoV-19/Norway/Bergen-01/2020 (GISAID accession ID. EPI_ISL_541970). Briefly, sera were heat inactivated at 56C for 60min, analysed in serial dilutions (duplicated, starting from 1:20), and mixed with 100 50% tissue culture infectious doses (TCID50) viruses in 96-well plates (ThermoFisher). After one hour incubation, the sera-virus mixtures were added to Vero cells and further incubated at 37C for 24h. Cells were fixed and permeabilized with methanol (Sigma-Aldrich) and 0.6% H2O2 (Sigma-Aldrich) and incubated with rabbit monoclonal IgG against SARS-CoV-2 NP (1:2000, Sino Biological Cat. 40143-R019). Cells were further incubated with biotinylated goat anti-rabbit IgG (H+L) (1:2500, Southern Biotech Cat. 4050-08), and Streptavidin-HRP (1:1400, Southern Biotech Cat. 7105-05). The reactions were developed with OPD (0.05mg/well, Sigma-Aldrich Cat. P-8287). The neutralizing (IC50) titer was determined as the reciprocal of the serum dilution giving 50% inhibition of virus infectivity. Negative samples were assigned a value of 10 for calculation purpose.

A virus neutralizing assay against the HCoV NL63 strain was developed. Serum samples were heat inactivated and 2-fold serially diluted (starting from 1:10) in DMEM supplemented with 2% heat-inactivated fetal bovine serum, 1% non-essential amino acid (Sigma-Aldrich) and 1.5g/L sodium bicarbonate (NaHCO3), then incubated with 100 TCID50 NL63 virus at 3334C for 60min. The mixture was then added into 96-well plates (ThermoFisher) pre-seeded with LLC-MK2 cells (7000 cells/well). The virus neutralization (VN) endpoint titer against NL63 virus was determined as the highest sera dilution giving 100% inhibition of cytopathic effect on LLC-MK2 cells 7 days after infection. Negative samples were assigned a value of 5 for calculation purpose.

When testing for virus neutralizing antibodies against the HCoV OC43 strain, serum samples were heat inactivated and 2-fold serially diluted (starting from 1:10) in RPMI-1640 supplemented with 2% heat-inactivated horse serum, then incubated with 100 TCID50 OC43 virus at 3334C for 60min. The mixture was then added into 96-well plates (ThermoFisher) pre-seeded with HCT-8 cells (15,000 cells/well). After 13 days incubation, 100l/well supernatant were mixed with 50l human O erythrocytes (0.7% v/v). The VN endpoint titer against OC43 virus was determined as the highest sera dilution giving 100% inhibition of hemagglutination. Negative samples were assigned a value of 5 for calculation purpose.

The spike protein amino acid sequences from HCoVs and SARS-CoV-2 used in ELISA and micro-/virus neutralization assays were obtained from NCBI database. Phylogenetic analyses were performed at ngPhylogeny.fr35 using MAFFT (Multiple Alignment using Fast Fourier Transform, default settings), BMGE (Block Mapping and Gathering with Entropy, default settings), and PhyML (Phylogeny software based on the Maximum-likelihood, default settings).

Biological replicates were used in all experiments. Antibody titers and fold-inductions were Ln transformed prior to all statistical analyses. RM one-way or two-way ANOVA with the Geisser-Greenhouse correction and Turkeys multiple comparisons were performed among time points within the same vaccinee or patient group. To compare adult and elderly vaccinees the unpaired t test was performed at each time point. The one-way ANOVA and Bunnetts multiple comparisons were used to compare between COVID-19 patients and vaccinees at different time points. All statistical analyses were performed with GraphPad Prism 9.

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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