Figure legends
Fig 1. Activation Induced Marker (AIM) assay of circulating memory Tfh revealed that cTfh cells of both infection naïve vaccinees and convalescent vaccinees recognized spike proteins of both wild type and delta strain. Representative flow cytometry plot showing negative unstimulated control (A) . Representative flow cytometry plot showing cTfh response in infection naïve vaccinees to the Spike protein of Wild type strain of SARS-CoV2 (SWT) (B) . Representative flow cytometry plot showing cTfh response in infection naïve vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT) (C) Representative flow cytometry plot showing cTfh response in convalescent vaccinees to the Spike protein of Wild type strain of SARS-CoV2 (SWT) (D) . Representative flow cytometry plot showing cTfh response in infection convalescent vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT) (E) Representative flow cytometry plot showing stimulated with α-CD3 and α-CD28 (F) . Cumulative graphical representation of AIM assay indicated by CD25+OX40+cTfh cells responding to SWT and SDT in infection naïve vaccinees; (n=20) (G ) Cumulative graphical representation of AIM assay indicated by CD25+OX40+ cTfh cells responding to SWT and SDT in convalescent vaccinees; (n=20)(H ). Cumulative graphical representation of comparison of the AIM assay indicated by CD25+OX40+ cTfh cell response to SWT between infection naïve vaccinees (n=20) and convalescent vaccinees (n=20) (I) Cumulative graphical representation of comparison of AIM assay indicated by CD25+OX40+cTfh cell response to SDT between infection naïve vaccinees (n=20) and convalescent vaccinees (n=20) (J,) . Representative flow cytometry plot showing different T helper memory compartment response in infection naïve vaccinees to the Spike protein of Wild type strain of SARS-CoV2 (SWT) (K) , Representative flow cytometry plot showing different T helper memory compartment response in infection naïve vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT) (L). Representative flow cytometry plot showing different T helper memory compartment response in convalescent vaccinees to the Spike protein of Wild type strain of SARS-CoV2 (SWT) (M).Representative flow cytometry plot showing different T helper memory compartment response in convalescent vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT) (N). Cumulative graphical representation of T helper central memory cells indicated by CCR7+CD45RA- responding to SWT and SDT in naive vaccinees; (n=20) (O).Cumulative graphical representation of T helper central memory cells indicated by CCR7+CD45RA- responding to SWT and SDT in convalescent vaccinees; (n=20) (P). Error bar indicates SEM. Wilcoxon Test was used to determine statistical significance of immunological response to the wild type and delta spike proteins between same individuals of naïve vaccinees and convalescent vaccinees groups. Mann-Whitney U test was used to compare statistics between naïve vaccinees and convalescent vaccinees. (*), p<0.01 was considered to be very significant (**), P < 0.001 was considered to highly significant (***), P < 0.0001 was considered extremely significant (****) ns, not significant.
Fig 2. Spike specific cTfh cytokine response revealed that cTfh response is damped in convalescent vaccinees as compared to infection naïve vaccinees and spike specific IL-21 secretion from cTfh positive correlated with neutralizing antibody in infection naïve vaccinees .Representative flow cytometry plot showing negative unstimulated control(A) . Representative flow cytometry plot showing cTfh cytokine response in infection naïve vaccinees to the Spike protein of Wild type strain of SARS-CoV2 (SWT) (B) . Representative flow cytometry plot showing cTfh cytokine response in infection naïve vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT) (C).Representative flow cytometry plot showing cTfh cytokine response in convalescent vaccinees to the Spike protein of Wild type strain of SARS-CoV2 (SWT) (D) . Representative flow cytometry plot showing cTfh cytokine response in infection convalescent vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT) (E). Representative flow cytometry plot showing stimulated with PMA/Ionomycin (F) . Cumulative graphical representation of cytokine assay indicated by IL-21+ cTfh cells responding to SWT and SDT in infection naïve vaccinees; (n=20) (G ). Cumulative graphical representation of cytokine assay indicated by IL-21+ cTfh cells responding to SWT and SDT in convalescent vaccinees; (n=20)(H ). Cumulative graphical representation of comparison of cytokine assay indicated by IL-21+ cTfh cell response to SWT between infection naïve vaccinees(n=20) and convalescent vaccinees (n=20) (I). Cumulative graphical representation of comparison of the cytokine assay indicated by IL-21+cTfh cell response to SDT between infection naïve vaccinees (n=20) and convalescent vaccinees (n=20) (J) . Cumulative graphical representation of % Neutralization between infection naïve vaccinees and convalescent vaccinees (K). Graphical representation of co-relation analysis between % IL-21+ spike specific cTfh cells and % neutralizing activity in Infection naïve vaccinees(L). Graphical representation of co-relation analysis between % IL-21+ spike specific cTfh cells and % neutralizing activity in Infection convalescent vaccinees (M).Representative flow cytometry plot showing different cTfh subsets based on chemokine markers CXCR3 and CCR6 in infection naïve vaccinees(N) Representative flow cytometry plot showing different cTfh subsets based on chemokine markers CXCR3 and CCR6 in convalescent vaccinees (O). Cumulative graphical representation of T helper central memory cells indicated by CCR7+CD45RA- responding to SWT and SDT in naive vaccinees; (n=20) (O). Error bars indicate SEM. Wilcoxon Test was used to determine statistical significance of immunological response to wild type and delta spike proteins between same individuals of naïve vaccinees and convalescent vaccinees groups. Mann-Whitney U test was used to compare statistics between naïve vaccinees and convalescent vaccinees including the neutralisation assay. The spearman’s rank correlation coefficient was used to measure the correlation between two different variables (*), p<0.01 was considered to be very significant (**), P < 0.001 was considered to highly significant (***), P < 0.0001 was considered extremely significant (****) and ns, not significant.
Fig 3. Cytokines analyses from plasma of and healthy controls. Representative figures from T cell cytokines analyzed in infection naïve vaccinees (n=20) and convalescent vaccinees (n=20) are represented as dot plots. cTfh promoting cytokines IL-21 (A), IL-10 (B), IL-4 (C). were elevated in infection naïve vaccinees as compared to convalescent vaccinees. Amongst anti-viral memory inducing cytokines IFN-γ (D) , IL-15(E), IL-12 (F) and IL-17 (G) were also elevated in infection naïve vaccinees as compared to convalescent while inflammatory cytokines IL-1β (H) and IL-6 (I) were elevated in convalescent vaccinees as compared to infection naïve vaccinees. Error bar indicates SEM. Mann-Whitney U Test was performed to compare between the two groups, p<0.05 was considered statistically significant(*), p<0.01 was considered to be very significant (**), P < 0.001 was considered to highly significant (***), P < 0.0001 was considered extremely significant (****) ns, not significant.
Numbers between the brackets indicate range of the data set