Longitudinal analysis of clinical serology assay performance and neutralising antibody levels in COVID19 convalescents

Abstract

Objectives: To investigate longitudinal trajectory of SARS-CoV-2 neutralising antibodies and the performance of serological assays in diagnosing prior infection and predicting serum neutralisation titres with time Design Retrospective longitudinal analysis of a COVID19 case cohort . Setting NHS outpatient clinics Participants Individuals with RT-PCR diagnosed SARS-CoV-2 infection that did not require hospitalization Main outcome measures The sensitivity with which prior infection was detected and quantitative antibody titres were assessed using four SARS-CoV-2 serologic assay platforms. Two platforms employed SARS-CoV-2 spike (S) based antigens and two employed nucleocapsid (N) based antigens. Serum neutralising antibody titres were measured using a validated pseudotyped virus SARS-CoV-2 neutralisation assay. The ability of the serological assays to predict neutralisation titres at various times after PCR diagnosis was assessed. Results The three of the four serological assays had sensitivities of 95 to100% at 21-40 days post PCR-diagnosis, while a fourth assay had a lower sensitivity of 85%. The relative sensitivities of the assays changed with time and the sensitivity of one assay that had an initial sensitivity of >95% declined to 85% at 61-80 post PCR diagnosis, and to 71% at 81-100 days post diagnosis. Median antibody titres decreased in one serologic assay but were maintained over the observation period in other assays. The trajectories of median antibody titres measured in serologic assays over this time period were not dependent on whether the SARS-CoV-2 N or S proteins were used as antigen source. A broad range of SARS-CoV-2 neutralising titres were evident in individual sera, that decreased over time in the majority of participants; the median neutralisation titre in the cohort decreased by 45% over 4 weeks. Each of the serological assays gave quantitative measurements of antibody titres that correlated with SARS-CoV-2 neutralisation titres, but, the S-based serological assay measurements better predicted serum neutralisation potency. The strength of correlation between serologic assay results and neutralisation titres deteriorated with time and decreases in neutralisation titres in individual participants were not well predicted by changes in antibody titres measured using serologic assays.

Conclusions: SARS-CoV-2 serologic assays differed in their comparative diagnostic performance over time. Different assays are more or less well suited for surveillance of populations for prior infection versus prediction of serum neutralisation potency. Continued monitoring of declining neutralisation titres during extended follow up should facilitate the establishment of appropriate serologic correlates of protection against SARS-CoV-2 reinfection.

Fig 1.. Longitudinal analysis of COVID-19 participant sera.

(A) Sensitivity of the Abbott, Diasorin, Siemens and Roche serological assays (as indicated) measured in samples collected at four different timepoints, as indicated, post PCR test and 95% confidence interval. (B) Relative antibody titres for the Diasorin, Siemens, Abbott and Roche, assays at visits 1–3, normalized to visit 1. Horizontal line indicates median value with 95% confidence interval. Statistical significance was assessed with the Wilcoxon test. (C) Values for Diasorin, Siemens, Abbott and Roche serological assays for each participant plotted over time (each line represents one participant). Assay thresholds are indicated by a dotted horizontal line.

Fig 2 –. Neutralisation activity in COVID-19 participant sera.

(A) Half-maximal neutralisation titres (NT50s) for each individual participant measured in serum samples collected at three different visits, as indicated by color. Inserts show correlation of NT50 values for samples collected at each visit, the spearman r is indicated (p<0.0001). (B) Relative NT50 values in sera obtained at visit 1 to 3, normalized to visit 1. Horizontal line represents median with 95% confidence interval. Statistical significance was assessed with the Wilcoxon test. (C) Frequency of sera with NT50 values falling to various quantitative categories at each visit. (D) NT50 values for each participant plotted over time (each line represents one participant). The limit of detection (LOD) is indicated by a dotted horizontal line.

Fig 3 –. Correlation of serology results with neutralisation titres.

(A-D) Serological assay values for the Diasorin (A), Siemens (B), Abbott (C) and Roche (D) assays versus NT50 values. Samples collected at each visit are indicated by color and are plotted individually as well as on a composite graph. Spearman r for all visits (black) and individual visits are indicated (p<0.0001). (E-H) Fold-change (visit 1 to visit 3) in serological assay values for the Diasorin (E), Siemens (F), Abbott (G) and Roche (H) assay versus fold-change in NT₅₀ values. Spearman r and p-value are indicated.