Immune Memory in Mild COVID-19 Patients and Unexposed Donors Reveals Persistent T Cell Responses After SARS-CoV-2 Infection

Abstract

Understanding the causes of the diverse outcome of COVID-19 pandemic in different geographical locations is important for the worldwide vaccine implementation and pandemic control responses. We analyzed 42 unexposed healthy donors and 28 mild COVID-19 subjects up to 5 months from the recovery for SARS-CoV-2 specific immunological memory. Using HLA class II predicted peptide megapools, we identified SARS-CoV-2 cross-reactive CD4⁺ T cells in around 66% of the unexposed individuals. Moreover, we found detectable immune memory in mild COVID-19 patients several months after recovery in the crucial arms of protective adaptive immunity; CD4⁺ T cells and B cells, with a minimal contribution from CD8⁺ T cells. Interestingly, the persistent immune memory in COVID-19 patients is predominantly targeted towards the Spike glycoprotein of the SARS-CoV-2. This study provides the evidence of both high magnitude pre-existing and persistent immune memory in Indian population. By providing the knowledge on cellular immune responses to SARS-CoV-2, our work has implication for the development and implementation of vaccines against COVID-19.

Keywords: B cells; CD4+ T cells; human coronavirus; neutralizing antibody; pre-existing immunity.

Figure 1

SARS-CoV-2 IgG response in pre-pandemic unexposed donors and individuals recovered from mild COVID-19. The IgG titre was measured in plasma sample from unexposed donors collected prior to pandemic and the COVID-19 patients up to 5 months of recovery by ELISA using the full length Spike protein and Nucleoprotein. The ELISA curves in serially diluted samples are shown from 6 representative unexposed donors (grey line) and COVID-19 cases (red line) for (A) Spike protein, (B) Nucleoprotein. Area under the curve (AUC) for ELISA quantitation of the IgG binding to (C) Spike protein, (D) Nucleoprotein for 42 unexposed donors and 28 COVID-19 cases inclusive of 6 representative donors of each group shown in panel A and B. (E) Neutralizing antibody quantitation in unexposed donors (n=8) and COVID-19 patients (n=12) after >4 months recovery measured using the SARS-CoV-2 surrogate virus neutralization test. (F) HCoVs Nucleoprotein antigen binding (expressed as OD) assessed by ELISA in unexposed donors (n=42) and COVID-19 recovered patients (n=28). Black bars indicate the geometric mean. Dotted line in panels A-E represent the cut-off of positivity. Statistical comparisons were performed by two-tail Mann-Whitney test. ns: non-significant.

Figure 2

SARS-CoV-2-specific CD4⁺ T cells response in unexposed donors and recovered COVID-19 patients. The magnitude of SARS-CoV-2 specific CD4⁺ T cells was determined in PBMCs collected from unexposed donors (“Unexposed”, n=32) prior to pandemic and in COVID-19 patients (“COVID-19”, n=28) up to 5 months of recovery. The PBMCs were stimulated with the peptide megapool specific to Spike glycoprotein (Spike) or to the remainder of the SARS-CoV-2 polyprotein (Non-spike). DMSO was used as the negative control, and CMV peptide megapool and SEB were used for positive stimulation controls. (A) Representative FACS contour plots of unexposed and COVID-19 patient in stimulation conditions of DMSO, Spike peptide megapool, Non-spike peptide megapool, CMV and SEB. Paired graphs depicting the reactivity of AIM⁺ (OX40⁺CD137⁺) CD4⁺ T cells between the negative control (DMSO) and antigen-specific stimulation in (B) Unexposed donors (C) COVID-19 patients. (D) Frequency of responders to Spike and Non-spike peptide pools in unexposed and COVID-19 recovered subjects as determined by the Fischer’s exact test. The value on bars denote the number of responders/total number of donors tested. (E) Stimulation index quantitation of the AIM⁺ (OX40⁺CD137⁺) CD4⁺ T cells in Unexposed versus COVID-19 cases analysed in the same samples as in panel B and (C) Black bars indicate the geometric mean. Dotted line in panels B, C and E represent the limit of detection. Statistical comparisons were performed by (B, C) Wilcoxon paired t-test and (E) two-tail Mann-Whitney test. ns: non-significant.

Figure 3

SARS-CoV-2-specific memory B cells in recovered COVID-19 patients. The frequency and isotype distribution of antibody secreting B cells (ASC) was measured in the unexposed subjects (“Unexposed”, n=28) prior to pandemic and in patients (“COVID-19”, n=18) up to 5 months of recovery from mild COVID-19. The memory B cells in PBMCs were polyclonally stimulated before measuring the frequency of SARS-CoV-2 Spike glycoprotein- and Nucleoprotein-specific IgG, IgM and IgA antibody secreting cells in Fluorospot assay. (A) Representative images of IgG, IgM and IgA secreting B cells in Unexposed subject and recovered COVID-19 patient. Graphs depicting the magnitude of antibody secreting B cells specific to the SARS-CoV-2 Spike glycoprotein and Nucleoprotein (expressed as spot forming cells (SFC) in 10⁶ PBMCs) for (B) IgG-ASC (C) IgM-ASC and (D) IgA-ASC, in Unexposed subjects (grey circle) and COVID-19 patients (red circle). For log scale, the spot count of less than one is depicted as 1. Black bars indicate the geometric mean. Statistical comparisons were performed by two-tail Mann-Whitney test. ns: non-significant.