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Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients

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Science Immunology  08 Oct 2020:
Vol. 5, Issue 52, eabe5511
DOI: 10.1126/sciimmunol.abe5511
  • Fig. 1 Cross-sectional analysis of IgG and IgA responses to the spike and RBD antigens of SARS-CoV2 in serum.

    (A-F) Indicated immunoglobulins to spike and RBD were profiled by ELISA in cohorts of pre-COVID samples (n=300), hospitalized patients with acute COVID infection (n=132) and convalescent patients (n=364). All data, expressed as ratio-converted ELISA reads to a pool of convalescent samples (relative ratio), were plotted using bean plots. Solid bars denote the median and dotted line represents the median across all samples used in the plot. (G-I) levels of IgG (G), IgA (H) and IgM (I) to the RBD (y-axis) and spike (x-axis) antigens for the indicated patient groups. Spearman correlation coefficient is indicated. Mann-Whitney U test for significance was performed. n.s = not significant, *= p ≤ 0.05, **** = p < 0.0001.

  • Fig. 2

    Persistence of antibodies in the serum of affected individuals. (A-F) Binned ratio-converted ELISA reads (relative ratios to a pool of positive controls) of spike (A-C) and RBD (D-F) to the indicated antibodies, displayed as bean plots. (G) The results of the surrogate neutralization ELISA are also shown, expressed as an integrated score tabulating the area under the curve across the first two points of the dilution series (see Methods). Days PSO are binned in 15-day increments and are compared to pre-COVID samples (neg). Solid bars denote the median and dotted line represents the median across all samples used in the plot. For A–F, the number of samples per bin was: neg=300; 0–15=115; 16–30=41; 31–45=50; 46–60=71; 61–75=62; 76–90=100; 91–105=9. For G, all bins were n=20, with the exception of neg.=19 and 106–115=9 (all available samples).

  • Fig. 3 A longitudinal analysis of IgG and IgA responses to the spike and RBD antigens of SARS-CoV2 in serum.

    Analysis of the changes in the indicated Ig-antigen levels in patients profiled twice, in comparisons to the relative levels in pre-COVID negative controls (left). Dots represent individual serum samples collected at the indicated times, and the samples from the same patients are connected by the lines. A non-parametric loess function is shown as the blue line, with the grey shade representing the 95% confidence interval.

  • Fig. 4 Cross-sectional analysis of antibody responses to the spike and RBD antigens of SARS-CoV-2 in saliva.

    Saliva specimens from the cohort of COVID-19 patients were tested for the presence of IgG, IgA and IgM antibodies to SARS-CoV-2 spike and RBD antigens (Positive), comparing with a mixture of unexposed asymptomatic controls collected locally and pre-COVID era controls (Negative). In these cohort 2 samples collected in Salivettes® we had sufficient material to perform several dilutions and to generate an integrated score for each subject (see Methods). Because the saliva was not diluted during collection, we were able to derive the concentration of antibodies in both negative controls and COVID-19 patients. (A-C) Total IgG, IgA and IgM levels in the saliva. (D-I) Saliva data for negative controls versus COVID-19 patients. Solid bars denote the median and dotted line represents the median across all samples used in the plot. Mann-Whitney U test for significance was performed. **** = p < 0.0001, n.s. = not significant.

  • Fig. 5 A cross-sectional analysis of antibody responses to the spike and RBD antigens of SARS-CoV-2 in saliva correlated with time PSO.

    A second cohort of COVID-19 patients (n=90) was tested for the presence of IgG and IgA antibodies to SARS-CoV-2 spike and RBD antigens in the saliva, comparing with a mixture of unexposed negative controls collected locally and pre-COVID era negative controls. (A-F) Saliva data for all 6 antigen-specific ELISA readouts plotted as time PSO. Spearman correlation coefficients (ρ) and p-value are indicated. In multivariable analysis adjusted for age, sex and severity of illness, there was a significant decline in anti-RBD and anti-spike IgA, but not significant change in the level of anti-RBD or anti-spike IgG.

  • Fig. 6

    Correlation of IgG, IgA and IgM responses to the spike and RBD antigens in serum and saliva. (A-F) A subset of serum and saliva sample pairs (n=71) collected from the same patient within 4 days were analyzed for correlations in levels of anti-spike and anti-RBD IgG, IgA and IgM antibodies. For serum, data are presented as ratio-normalized ELISA reads, while the saliva results are expressed as an integrated score, as in previous figures. The data are presented on a logarithmic scale. Spearman correlation coefficient (ρ) and p-value are indicated.

  • Table 1

    Cohorts of patients and negative controls.

    SALIVABLOOD
    No. patientsNo. samplesMedian AgeSex No. patientsNo. samplesMedian AgeSex
    No. MNo.
    F
    No. MNo. F
    All samples 247263-141106All samples739796-379360
    Patients with COVID-19Cohort 14754612819Patients with COVID-1943949658229210
    Cohort 28190584833
    Pre-COVID Negative ControlsCohort 100000Pre-COVID Negative Controls30030054.5150150
    Cohort 22727431215
    Unexposed Negative Controls Collected in 2020Cohort 14242602418
    Cohort 25050582921
    Matched saliva-serum samples 7171583338

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/52/eabe5511/DC1

    Fig. S1. Development and validation of manual colorimetric and automated chemiluminescent assays for monitoring RBD and spike trimers antibodies in serum or plasma.

    Fig. S2. Correlations between antibody levels and day of symptom onset to sample collection.

    Fig. S3. IgG and IgA responses to the Nucleocapsid antigen of SARS-CoV-2 in serum.

    Fig. S4. Effect of heat versus detergent inactivation of saliva samples on the detection of anti-RBD antibodies in a manual, colorimetric ELISA.

    Fig. S5. IgG and IgA levels against SARS-CoV-2 antigens in the saliva of cohort 1.

    Fig. S6. Strategy for calculating an integrated score in antigen-specific saliva assay.

    Fig. S7. Validation of manual colorimetric assays for monitoring RBD and spike trimers antibodies in saliva.

    Fig. S8. Dilution curves of positive control ELISAs for serum assays.

    Fig. S9. Distribution of the intensity values across serum assays for negative controls and samples from patients with COVID-19 infection.

    Table S1. ROC statistics table for ELISAs conducted on serum/plasma derived samples.

    Table S2. Testing effect of Triton-X treatment on saliva samples.

    Table S3. ROC statistics table for ELISAs conducted on saliva samples.

    Table S4. Raw data file (Excel spreadsheet).

  • The PDF file includes:

    • Fig. S1. Development and validation of manual colorimetric and automated chemiluminescent assays for monitoring RBD and spike trimers antibodies in serum or plasma.
    • Fig. S2. Correlations between antibody levels and day of symptom onset to sample collection.
    • Fig. S3. IgG and IgA responses to the Nucleocapsid antigen of SARS-CoV-2 in serum.
    • Fig. S4. Effect of heat versus detergent inactivation of saliva samples on the detection of anti-RBD antibodies in a manual, colorimetric ELISA.
    • Fig. S5. IgG and IgA levels against SARS-CoV-2 antigens in the saliva of cohort 1.
    • Fig. S6. Strategy for calculating an integrated score in antigen-specific saliva assay.
    • Fig. S7. Validation of manual colorimetric assays for monitoring RBD and spike trimers antibodies in saliva.
    • Fig. S8. Dilution curves of positive control ELISAs for serum assays.
    • Fig. S9. Distribution of the intensity values across serum assays for negative controls and samples from patients with COVID-19 infection.
    • Table S1. ROC statistics table for ELISAs conducted on serum/plasma derived samples.
    • Table S2. Testing effect of Triton-X treatment on saliva samples.
    • Table S3. ROC statistics table for ELISAs conducted on saliva samples.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4. Raw data file (Excel spreadsheet).

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