Longitudinal variation in SARS-CoV-2 antibody levels and emergence of viral variants: a serological analysis

Abstract

Background: Serological assays are being used to monitor antibody responses in individuals who had SARS-CoV-2 infection and those who received a COVID-19 vaccine. We aimed to determine whether such assays can predict neutralising antibody titres as antibody levels wane and viral variants emerge.

Methods: We measured antibody levels in serum samples from a cohort of 112 participants with SARS-CoV-2 infection using ten high-throughput serological tests and functional neutralisation assays. Serum samples were taken at baseline and at up to four subsequent visits. We assessed the effects of time and spike protein sequence variation on the performance and predictive value of the various assays. We did correlation analyses for individual timepoints using non-parametric Spearman correlation, and differences between timepoints were determined by use of a two-tailed Wilcoxon matched-pairs signed rank test.

Findings: Neutralising antibody titres decreased over the first few months post-infection but stabilised thereafter, at about 30% of the level observed shortly after infection. Serological assays commonly used to measure antibodies against SARS-CoV-2 displayed a range of sensitivities that declined to varying extents over time. Quantitative measurements generated by serological assays based on the spike protein were better at predicting neutralising antibody titres than those based on nucleocapsid, but performance was variable, and manufacturer positivity thresholds were not able to predict the presence or absence of detectable neutralising activity. Although we observed some deterioration in correlation between serological measurements and functional neutralisation activity, some assays maintained an ability to predict neutralising titres, even against variants of concern.

Interpretation: The ability of high-throughput serological assays to predict neutralising antibody titres is likely to be crucial for evaluation of immunity at the population scale. These data can facilitate the selection of the most suitable assays as surrogates of functional neutralising activity and suggest that such measurements might be useful in clinical practice.

Funding: US National Institutes of Health and National Health Service Research Scotland BioResource.

Figure 1

Neutralisation activity in longitudinal serum samples from patients with COVID-19 (A) NT₅₀s for each sample collected at the visit indicated. (B) Relative NT₅₀ values in serum samples obtained at visits 1 to 5, normalised to visit 1; coloured horizontal bars indicate median values with 95% CIs; statistical significance was determined with the two-tailed Wilcoxon matched-pairs signed rank test. (C) Relative decay of NT₅₀ per day between visits 1 and 3 and relative decay of NT₅₀ per day between visits 3 and 5; red horizontal bars indicate the median; statistical significance was assessed with the Wilcoxon test. NT₅₀=half-maximal neutralisation titre.

Figure 2

Serological analysis of longitudinal serum samples from patients with COVID-19 (A) Sensitivity of the indicated serological assays in samples collected at three different time intervals after the PCR test; mean and 95% CIs are shown. (B) Relative serological results at visits 1 to 5, normalised to visit 1 for the indicated serological assays; horizontal bars indicate median with 95% CIs, and statistical significance was determined with the two-tailed Wilcoxon matched-pairs signed rank test. N=nucleocapsid. RBD=receptor binding domain. S=spike protein.

Figure 3

Correlation of neutralisation titres and serology assays (A) Correlation of NT₅₀ (x-axis) with serological assay values (y-axis) obtained at visit 1 and visit 5 for each participant; statistical significance was determined with the Spearman correlation test for samples obtained at visit 1 and visit 5 independently, with Spearman's r and respective 95% CIs as indicated; dotted lines indicate serological assay thresholds. (B) Correlation of fold change (visit 1 to visit 5) of NT₅₀s with corresponding fold change in serological assay values for indicated serology assays. Statistical significance was determined using the Spearman correlation test, with Spearman's r and respective 95% CIs as indicated. Dotted lines at x=1 and y=1 indicate unchanged assay results over time. For the cPass assay, the serum dilution at which 50% signal inhibition was achieved was defined as 50% surrogate virus neutralisation titre (sVNT₅₀). AU=arbitrary unit. COI=confidence interval. N=nucleocapsid. NT₅₀=half-maximal neutralisation titre. RBD=receptor binding domain. S=spike protein. S/C=ratio over threshold value. U=unit.

Figure 4

Neutralisation of variants of concern (A) NT₅₀ values for variant pseudoviruses alpha (B.1.1.7), alpha with a Glu484Lys, beta (B.1.351) version 1, beta version 2, or delta (B.1.617.2) measured at indicated timepoints, normalised to wild type NT₅₀; analysis done in a cohort of 58 participants; statistical significance was determined with Wilcoxon test; dotted line indicates a ratio of 1 (equal NT₅₀). (B) Correlation coefficients (Spearman's r values) of NT₅₀ values between original and variant viruses and each serological assay, measured at visit 1 and visit 5; analysis done in 58 participants for all groups shown, except for DiaSorin S1/S2 (n=54). N=nucleocapsid. NT₅₀=half-maximal neutralisation titre. RBD=receptor binding domain. S=spike protein. sVNT₅₀=50% surrogate virus neutralisation titer.