Immunological memory to SARS-CoV-2 assessed for up to eight months after infection

Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥ 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3–5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

cells. The cells were cultured at 37C in a shaker incubator set to 125rpm, 80% humidity and 8% CO2. When cell viability dropped below 80% (typically 4-5 days), media was harvested and centrifuged to remove cells. Biolock reagent was added to the supernatant media to remove any excess biotin. The media was then filtered through a 0.22um filter to remove Biolockedaggregates. Proteins were purified using Streptrap HP 5mL columns (Cytiva) using 100mM Tris, 100mM NaCl as the Wash Buffer and 100mM Tris, 100mM NaCl, 2.5mM d-Desthiobiotin as the Elution Buffer. The eluted fractions for Spike proteins were concentrated on 100kDa Amicon filters while the RBD were concentrated on 10kDa filters. The samples were further purified using S6increase columns for the spike variants and S200increase column for RBD. SARS-CoV-2 ELISAs SARS-CoV-2 ELISAs were performed as previously described (2,5,82). Briefly, Corning 96well half area plates (ThermoFisher 3690) were coated with 1µg/mL of antigen overnight at 4°C. Antigens included recombinant SARS-CoV-2 RBD protein, recombinant Spike protein, and recombinant Nucleocapsid protein (GenScript Z03488) (Recombinant nucleocapsid antigens were also tested from Sino Biological (40588-V07E) and Invivogen (his-sars2-n) and yielded comparable results to GenScript nucleocapsid). The following day, plates were blocked with 3% milk in phosphate buffered saline (PBS) containing 0.05% Tween-20 for 1.5 hours at room temperature. Plasma was heat inactivated at 56°C for 30-60 minutes. Plasma was diluted in 1% milk containing 0.05% Tween-20 in PBS starting at a 1:3 dilution followed by serial dilutions by 3 and incubated for 1.5 hours at room temperature. Plates were washed 5 times with 0.05% PBS-Tween-20. Secondary antibodies were diluted in 1% milk containing 0.05% Tween-20 in PBS. For IgG, anti-human IgG peroxidase antibody produced in goat (Sigma A6029) was used at a 1:5,000 dilution. For IgA, anti-human IgA horseradish peroxidase antibody (Hybridoma Reagent Laboratory HP6123-HRP) was used at a 1:1,000 dilution. The HP6123 monoclonal anti-IgA was used because of its CDC and WHO validated specificity for human IgA1 and IgA2 and lack of crossreactivity with non-IgA isotypes (82).
Endpoint titers were plotted for each sample, using background subtracted data. Negative and positive controls were used to standardize each assay and normalize across experiments. A positive control standard was created by pooling plasma from 6 convalescent COVID-19 donors to normalize between experiments. The limit of detection (LOD) was defined as 1:3 for IgG, 1:10 for IgA. Limit of sensitivity (LOS) for SARS-CoV-2 infected individuals was established based on uninfected subjects, using plasma from normal healthy donors never exposed to SARS-CoV-2. For cross-sectional analyses, modeling for the best fit curve (e.g., one phase decay versus simple linear regression) was performed using GraphPad Prism 8.0. Best curve fit was defined by an extra sum-of-squares F Test, selecting the simpler model unless P < 0.05 (83). Continuous decay (linear regression), one-phased decay, or two-phased decay of log data were assessed in all cases, with the best fitting statistical model chosen based on the F test; in several cases a quadratic equation fit was also considered. To calculate the t1/2, log2 transformed data was utilized. Using the best fit curve, either a one phase decay non-linear fit or a simple linear regression (continuous decay) was utilized. For simple linear regressions, Pearson R was calculated for correlation using log2 transformed data. For one phase decay non-linear fit, R was reported. For longitudinal samples, a simple linear regression was performed, with t1/2 calculated from log2 transformed data for each pair. For gender analyses, modeling and t1/2 was performed similar to cross-sectional analyses; ANCOVA (VassarStats or GraphPad Prism 8.4) was then performed between male and female . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 18, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020 data sets. ANCOVA p-values of the adjusted means were reported and considered significant if the test for homogeneity of regressions was not significant.
Neutralizing antibody assays The pseudovirus neutralizing antibody assay was performed as previously described (5). Briefly, Vero cells were seeded in 96-well plates to produce a monolayer at the time of infection. Pretitrated amounts of rVSV-SARS-Cov-2 (phCMV3-SARS-CoV-2 Spike SARS-CoV-2pseduotyped VSV-ΔG-GFP were generated by transfecting HEK293T cells, ATCC CRL-3216) were incubated with serially diluted human plasma at 37°C for 1 hour before addition to confluent Vero cell monolayers (ATCC CCL-81) in 96-well plates. Cells were incubated for 12-16 hours at 37°C in 5% CO2. Cells were then fixed in 4% paraformaldehyde, stained with 1µg/mL Hoechst, and imaged using a CellInsight CX5 imager to quantify the total number of cells expressing GFP. Infection was normalized to the average number of cells infected with rVSV-SARS-CoV-2 incubated with normal human plasma. The limit of detection (LOD) was established as < 1:20 based on plasma samples from a series of unexposed control subjects. Negative signals were set to 1:19. Neutralization IC50 titers were calculated using One-Site Fit LogIC50 regression in GraphPad Prism 8.0.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10. 1101/2020 The frequency of antigen-specific memory B cells was expressed as a percentage of total B cells (CD19 + CD20 + CD38 int/-, CD3 -, CD14 -, CD16 -, CD56 -, LIVE/DEAD -, lymphocytes), or as number per 10 6 PBMC (LIVE/DEADcells). LOD was set based on median + 2× standard deviation (SD) of [1 / (number of total B cells recorded)] or median + 2×SD of [10 6 / (number of PBMC recorded)]. LOS was set as the median + 2×SD of the results in unexposed donors. Phenotype analysis of antigen-specific B cells was performed only in subjects with at least 10 cells detected in the respective antigen-specific memory B cell gate. In each experiment, PBMC from a known positive control (COVID-19 convalescent subject) and unexposed subjects were included to ensure consistent sensitivity and specificity of the assay. For each data set, second order polynomial, simple linear regression, and pseudo-first order kinetic models were considered. The model with a lower Akaike's Information Criterion value was determined to be a better-fit and visualized.
Activation induced markers (AIM) T cell assay Antigen-specific CD4 + T cells were measured as a percentage of AIM + (OX40 + CD137 + ) CD4 + T and (CD69 + CD137 + ) CD8 + T cells after stimulation of PBMC with overlapping peptide megapools (MP) spanning the entire SARS-CoV-2 ORFeome, as previously described (2). Cells were cultured for 24 hours in the presence of SARS-CoV-2 specific MPs [1 µg/mL] or 5 µg/mL phytohemagglutinin (PHA, Roche) in 96-wells U-bottom plates at 1x10 6 PBMC per well. A stimulation with an equimolar amount of DMSO was performed as a negative control, PHA, and stimulation with a combined CD4 + and CD8 + cytomegalovirus epitope MP (CMV, 1 µg/mL) were included as positive controls. Any sample with low PHA signal was excluded as a quality control.
Antigen-specific CD4 + and CD8 + T cells were measured as background (DMSO) subtracted data, with a minimal DMSO level set to 0.005%. All positive ORFs (> 0.02% for CD4 + , > 0.05% for CD8 + ) were then aggregated into a combined sum of SARS-CoV-2-specific CD4 + or CD8 + T cells. The threshold for positivity for antigen-specific CD4 + T cell responses (0.03%) and antigen-specific CD8 + T cell responses (0.12%) was calculated using the median two-fold standard deviation of all negative controls measured (>150). The antibody panel utilized in the (OX40 + CD137 + ) CD4 + T and (CD69 + CD137 + ) CD8 + T cells AIM staining is shown in Table S2. A consistency analysis was performed for multiple measurements of AIM T cell assays by two different operators. Before merging, we compared the protein immunodominance, total SARS-CoV-2-specific CD4 + and CD8 + T cell responses, and half-life calculations between the two groups of experimental data. In longitudinal analyses, half-life calculations excluded any samples that were negative at both timepoints (since a half-life could not be calculated), though all data were included in the graphs.
For surface CD40L + OX40 + CD4 + T cell AIM assays, experiments were performed as previously described (5), with the following modifications. Cells were cultured in complete RPMI containing 5% human AB serum (Gemini Bioproducts), beta-mercaptoethanol, Penicillin/Streptomycin, sodium pyruvate (NaPy), and non-essential amino acids. Prior to addition of peptide MPs, cells were blocked at 37°C for 15 minutes with 0.5µg/mL anti-CD40 mAb (Miltenyi Biotec). A stimulation with an equimolar amount of DMSO was performed to determine background subtraction, and activation from Staphylococcal enterotoxin B (SEB) at 1 µg/mL was used as (positive) quality control. LOD for antigen-specific cTFH among CD4 + T cells was based on the LOD for antigen-specific CD4 + T cells (described above) multiplied by the average % cTFH in the bulk CD4 T cells among control samples. An inclusion threshold of ten events after the cTFH . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10.1101/2020.11.15.383323 doi: bioRxiv preprint CXCR5 + gate was used for PD-1 hi and CCR6 + calculations, and Mann-Whitney nonparametric and Wilcoxon signed-rank statistical tests were applied for the respective comparisons.
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Fig. S7. Serological memory and disease severity. (A)
Cross-sectional analysis of Spike IgG, as per Figure 1A, color coded based on subject COVID-19 disease severity (white: asymptomatic, gray: mild, blue: moderate, red: severe). Letters indicate donors that were sampled at multiple timepoints after the onset of symptoms. One letter per donor. (B) Cross-sectional analysis of RBD IgG, as per Figure 1C, color coded based on subject COVID-19 disease severity. (C) Distribution of timepoints of COVID-19 convalescent subjects (120+ days PSO) analyzed in Figure 5B. Line indicates median. For subjects with multiple sample timepoints, only the final timepoint was used for these analyses. p = 0.40, Mann-Whitney test.
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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10.1101/2020.11.15.383323 doi: bioRxiv preprint Fig. S10. Immune memory relationships. (A) Percentage dot plots showing frequencies (normalized to 100%) of subjects with indicated immune memory components during the early (1-2 mo) or late (5-8 mo) phase. "PSV", PSVneutralizing antibodies. "B", RBD-specific memory B cells. "4", SARS-CoV-2 specific CD4 + T cells. "8", SARS-CoV-2 specific CD8 + T cells. "A", Spike-specific IgA. n = 78 (1-2 mo), n = 44 (5-8 mo). (B) The ratio of SARS-CoV-2 specific CD4 + T cell frequency relative to SARS-CoV-2 specific CD8 + T cell frequency (best-fit simple linear regression line, |R| = 0.11). Three data points are outside the axis limits. (C) The ratio of RBD-specific memory B cell frequency (percentage) relative to RBD-specific IgG (pseudo-first order kinetic model, |R| = 0.60). Three data points are outside the axis limits. (D) The ratio of RBD-specific memory B cell frequency (percentage) relative to Spike IgA antibodies (pseudo-first order kinetic model, |R| = 0.55). One data point is outside the axis limits. (E) The ratio of SARS-CoV-2 specific CD4 + T cell frequency relative to RBD IgG antibodies (best-fit simple linear regression line, R = 0.046). Three data points are outside the axis limits. (F) The ratio of RBD-specific memory B cell frequency (percentage) relative to total SARS-CoV-2 specific CD4 + T cell frequency (best-fit simple linear regression line, |R| = 0.48). One data point is outside the axis limits. For Figure 5H: The ratio of RBD-specific memory B cell frequency (percentage) relative to Spike IgA antibodies (blue curve; best-fit pseudo-first order kinetic curve transformed by ×10 6 ), RBD IgG antibodies (orange; best-fit pseudo-first order kinetic curve transformed by ×10 5 ) and total SARS-CoV-2 specific CD4 + T cell frequency purple; best-fit simple linear regression line transformed by ×10 2 ), or the ratio of SARS-CoV-2 specific CD4 + T cell frequency relative to SARS-CoV-2 specific CD8 + T cell frequency (teal; best-fit simple linear regression line) and RBD IgG antibodies (black; best-fit simple linear regression line transformed by ×10 3 ).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10.1101/2020.11.15.383323 doi: bioRxiv preprint Table S1. Memory B cell flow cytometry panel. Table S2.

Reagents Source Identifier Dilution
Mouse anti-human CD45RA BV421 (clone HI100) . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 18, 2020. ; https://doi.org/10. 1101/2020