Antibody correlates of protection from SARS-CoV-2 reinfection prior to vaccination: A nested case-control within the SIREN study

Abstract

Objectives: To investigate serological differences between SARS-CoV-2 reinfection cases and contemporary controls, to identify antibody correlates of protection against reinfection.

Methods: We performed a case-control study, comparing reinfection cases with singly infected individuals pre-vaccination, matched by gender, age, region and timing of first infection. Serum samples were tested for anti-SARS-CoV-2 spike (anti-S), anti-SARS-CoV-2 nucleocapsid (anti-N), live virus microneutralisation (LV-N) and pseudovirus microneutralisation (PV-N). Results were analysed using fixed effect linear regression and fitted into conditional logistic regression models.

Results: We identified 23 cases and 92 controls. First infections occurred before November 2020; reinfections occurred before February 2021, pre-vaccination. Anti-S levels, LV-N and PV-N titres were significantly lower among cases; no difference was found for anti-N levels. Increasing anti-S levels were associated with reduced risk of reinfection (OR 0·63, CI 0·47-0·85), but no association for anti-N levels (OR 0·88, CI 0·73-1·05). Titres >40 were correlated with protection against reinfection for LV-N Wuhan (OR 0·02, CI 0·001-0·31) and LV-N Alpha (OR 0·07, CI 0·009-0·62). For PV-N, titres >100 were associated with protection against Wuhan (OR 0·14, CI 0·03-0·64) and Alpha (0·06, CI 0·008-0·40).

Conclusions: Before vaccination, protection against SARS-CoV-2 reinfection was directly correlated with anti-S levels, PV-N and LV-N titres, but not with anti-N levels. Detectable LV-N titres were sufficient for protection, whilst PV-N titres >100 were required for a protective effect.

Trial registration number: ISRCTN11041050.

Keywords: Immunity; Neutralising antibodies; Reinfection; SARS-CoV-2; SARS-CoV-2 serology.

Fig. 1

Trajectories of antibody levels and neutralisation titres in cases before and after reinfection. The vertical red line at Time=0 is the date of the PCR test detecting reinfection. Points with a plus (+) sign refer to samples collected after vaccination. Dashed lines indicate detection thresholds of assays, except the upper dashed lines in panels E and F that indicate the upper end of the quantitative range of the LV-N assay. Same colour used for same participant across panels, but panels A and B have 3 more participants.

Fig. 2

Comparison of antibody levels and neutralisation titres before and after reinfection for cases. Top and middle rows: antibody levels and neutralisation titres after reinfection (AR, black) are significantly higher than before reinfection (BR, red) for anti-S (p < 10⁻⁴, paired t-test), anti-N (=10⁻⁴, Wilcoxon signed-rank), anti-PV-N Wuhan (p < 10⁻⁴, paired t-test) and anti-PV-N Alpha (p < 10⁻⁴, random effect tobit model). The same effects and similar significance levels are obtained when considering only samples after reinfection but before vaccination (ARBV, blue). Bottom row: among cases, the fraction of LV-N with nAb titres >40 is significantly higher (McNemar's test) after reinfection than before, for LV-N Wuhan (p = 0.001) and LV-N Alpha (p < 10⁻⁴). Dashed lines indicate positivity threshold of the assay.

Fig. 3

Serological status of single infection controls and reinfection cases (A-C). Supervised heatmaps with pre-reinfection sera from cases and temporally matched samples from controls. For (A), Log₂ anti-S and log₂ anti-N are shown. Log₂ PV-N and log₂ LV-N are shown in (B) and (C), respectively.

Fig. 4

Comparison between case and control antibody levels and neutralisation titres in last sample before reinfection (cases) and the closest corresponding sample in calendar time (controls), with p-values obtained from fixed effect linear regression. Top row: geometric mean of anti-S levels is significantly higher in cases (p = 0.001) than in controls, while no significant difference is observed in geometric means of anti-N levels (p = 0.29). Middle row: geometric means of PV-N titres are significantly higher in cases than in controls for Wuhan (p = 0.01) and Alpha (p = 0.0044, random effect tobit model). Bottom row: among participant nAb titres > 40 with LV-N Wuhan and LV-N Alpha, the proportion of controls is higher than that of cases, with disjoint confidence intervals. Dashed lines indicate positivity threshold of the assay.

Fig. 5

Correlation between neutralisation assays and binding anti-S levels. (A) PV-N titres against Wuhan and Alpha in pre-reinfection sera and temporally matched control samples, plotted against binding anti-S antibodies. (B) LV- titres, reported as IC₅₀, plotted against binding anti-S antibodies. (C) PV-N titres against Wuhan and Alpha in pre-reinfection sera and temporally matched control samples, plotted against LV-N titres, reported as IC₅₀. In (A) and (B), binding antibodies are plotted as log₂, PV-N titres as log₂(x+1) and LV-N titres as log₂, after assigning 5, 10 or 5120 as no, weak or complete inhibition, respectively. In (A) correlation coefficient and P value are from Spearman's correlation, and a regression line is shown using all data. In (B) and (C), all data are used for Spearman's correlation, whereas the regression line uses only data within the quantifiable range (40-2560). Dashed lines indicate an anti-S level of >0.8U/mL (considered “positive” by the manufacturer), and a PV-N or LV-N titre of 100 or 40 respectively, as described in the Results section.