Durability of Functional SARS-CoV-2-Specific Immunological Memory and T Cell Response up to 8-9 Months Postrecovery From COVID-19

Abstract

Research on long-term follow-up in individuals who have recovered from coronavirus disease-19 (COVID-19) would yield insights regarding their immunity status and identify those who need booster vaccinations. This study evaluated the longevity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific cellular and humoral memory responses, as well as T cell effector functionalities, at 1-2 months (n = 40), 8-9 months (n = 40), and 12 months/1 year (n = 27) following recovery from SARS-CoV-2 infection. CTL response by enzyme-linked immunospot (ELISPOT); levels of cytokine by Bio-Plex, natural killer (NK), CD4+ helper, and CD8+ cytotoxic T cell functionalities using flow cytometry; anti-SARS-CoV-2 IgG by ELISA; and levels of neutralizing antibodies (NAbs) by surrogate virus NAb assay were assessed. The levels of SARS-CoV-2-specific IgG and NAb at 1-2 and 8-9 months postrecovery were hand in hand and appeared declining. SARS-CoV-2-specific B, memory B and plasma cells, and T cells sustained up to 8-9 months. Increased expression of CD107a/IFN-γ by NK cells and cytotoxic T cells at 8-9 months could be indicative of SARS-CoV-2-specific effector functions. Recovered individuals with positive and negative IgG antibody status displayed T cell response up to 1 year and 8-9 months, respectively, emphasizing the durabilty of effector immunity up to 8-9 months regardless of IgG antibody status. Overall, the recovered individuals exhibited robust immunological memory, sustained T cell response with effector functionality against SARS-CoV-2 that persists for at least 8-9 months.

Keywords: COVID-19; ELISPOT; T cell response; antibody; flow cytometry; memory B and T cells; recovered individuals.

Figure 1

Gating strategy to distinguish different lymphocyte populations by flow cytometry. The peripheral blood mononuclear cells (PBMCs) from the study participants were stained with panels of fluorochrome-labeled antibodies to assess the frequency and immune profile. The numbers in the histogram are the mean of the cell population representing for the study group. (A) Lymphocytes and natural killer (NK cells [CD3–CD56+]), natural killer-like T (NKT-like [CD3+CD56+]) cell profile. (B) B (CD19+), IgG+ B cells, and memory B (CD19+CD27+) cell profile. (C) Helper T (CD3+CD4+), cytotoxic T (CD3+CD8+) cell profile, memory Th and Tc cells, CD4+ naïve cells, CD4+ T central memory cell effector cells, and terminally differentiated effector memory cells (TEMRA) cells.

Figure 2

Gating strategy to distinguish different lymphocyte populations and intracellular cytokine expression by flowcytometry. IFN-γ and CD107a produced by CD4+ T cells and CD8+ T cells. T cells were derived from the isolated peripheral blood mononuclear cells (PBMCs) gated by forward and side scatter. The numbers in the histogram are the mean of the cell population representing the study group. (A) Lymphocytes and T helper (CD3+CD4+), T cytotoxic (CD3+CD8+) cell profile. (B) Percentage of IFN-γ produced by CD4+ T and CD8+T cells according to the fluorescence of each cytokine in unstimulated and stimulated cells. (C) Percentage of CD107a produced by CD4+ T and CD8+ T cells according to the fluorescence of each cytokine in unstimulated and stimulated cells.

Figure 3

Peripheral blood mononuclear cells (PBMCs) from recovered individuals R1: post 1–2 months (n = 40), R2: 8–9 months (n = 40) and R3:12 months/1 year post recovery (n = 22) and HC: healthy controls (n = 17) were stained and acquired on flowcytometer. Vertical scatter plots denote the comparisons of frequencies of immune cells and their subpopulation among different study groups: (A) natural killer (NK) cells (B) natural killer T (NKT) cells (C) CD4+ T helper cells (D) CD8+ T cytotoxic cells profile (E) B cells (F) Memory B cells (G) IgG+ B cells post 8–9 months and 12 months/1 year (H) IgG+ memory B cells post 8–9 months and 12 months/1 year. Data are presented as the percentage of immune cells out of lymphocytes. The dots represent individual values and bars represent mean + SD values. (⁣^∗p-value <0.05, ⁣^∗∗p-value <0.005, and ⁣^∗∗∗p-value <0.0001).

Figure 4

Flow cytometric analysis of T and memory T cell subsets in SARS-CoV-2-recovered individuals. Peripheral blood mononuclear cells (PBMCs) from recovered individuals R2, 8–9 months (n = 40); R3, 12 months/1 year postrecovery (n = 22); and healthy controls (HCs) (n = 17) were stained and acquired on flowcytometer. Vertical scatter plots denote the comparisons of frequencies of immune cells and their subpopulation among different study groups: (A–D) CD4+ memory T cell subsets and (E–H) CD8+ memory T cell subsets, namely, naive, central, terminally differentiated effector memory cells (TEMRA), and effector memory cells post 8–9 months and 12 months/1 year. Data are presented as percentage of immune cells out of lymphocytes. The dots represent individual values, and the bars represent mean + SD values (⁣^∗p value <0.05, ⁣^∗∗p value <0.005, and ⁣^∗∗∗p value <0.0001).

Figure 5

Flowcytometric analysis of SARS-CoV-2 S1-stimulated memory T, B, and IgG+ B cells in SARS-CoV-2-recovered individuals. Peripheral blood mononuclear cells (PBMCs) from the recovered individuals R2, 8–9 months (n = 40), and R3, 12 months/1 year postrecovery (n = 22), were stimulated with SARS-CoV-2 S1antigen for 48 h and then stained and acquired on flowcytometer. Vertical scatter plots denote the comparisons of frequencies of immune cells and their subpopulation among different study groups: (A) CD4+ helper T/Th and CD8+ cytotoxic/Tc memory T cell profile, (B) B cells and memory B cell subsets, (C) CD4+ helper memory T cell profile, and (D) CD8+ cytotoxic memory T cell profile post 8–9 months and 12 months/1 year. Data are presented as percentage of immune cells out of lymphocytes. The dots represent individual values, and the bars represent mean + SD values (⁣^∗p value <0.05, ⁣^∗∗p value <0.005, and ⁣^∗∗∗p value <0.0001). ns, nonsignificant.

Figure 6

Flowcytometric analysis intracellular expression of SARS-CoV-2 S1-stimulated IFN-γ and CD107a in the recovered individuals. Peripheral blood mononuclear cells (PBMCs) from the recovered individuals R2, 8–9 months (n = 24), and R3, 12 months/1 year postrecovery (n = 13), were stimulated with SARS-CoV-2 S1 antigen for 6 h and then stained and acquired on flowcytometer. Vertical scatter plots denote the comparisons of frequencies of immune cells and their subpopulation among different study groups: (A) CD4+ T helper cells, CD8+ T cytotoxic cells, and natural killer (NK) cell profile with CD107a expression. (B) CD4+ T helper cells, CD8+ T cytotoxic cells, and NK cell profile with IFN-γ expression post 8–9 months and 12 months/1 year. Data are presented as the percentage of immune cells out of lymphocytes. The dots represent individual values, and the bars represent mean + SD values (⁣^∗p value <0.05, ⁣^∗∗p value <0.005, and ⁣^∗∗∗p value <0.0001).

Figure 7

Strength and magnitude of SARS-CoV-2 S1-specific IFN-γ-producing T cell response in SARS-CoV-2-recovered individuals. Strength and magnitude of SARS-CoV-2 S1-specific IFN-γ-producing T cell response in different groups post 1–2 months (R1, n = 34), 8–9 months (R2, n = 39), and 1 year of recovery (R3, n = 22). Peripheral blood mononuclear cells (PBMCs) were isolated from all subjects mentioned and were cultured with SARS-CoV-2 S1 antigen protein in vitro. IFN-γ-secreting cell frequencies were determined by ELISPOT assay. (A) Magnitude and (B) strength of the SARS-CoV-2 S1-specific IFN-γ-producing T cell response in terms of percentage.

Figure 8

Surrogate neutralization antibody activities against SARS-CoV-2 infection in recovered individuals from SARS-CoV-2 infection. Surrogate neutralization antibody activities against SARS-CoV-2 infection in recovered individuals (1 year postrecovery) from SARS-CoV-2 infection were detected using surrogate virus neutralization (sVNT) assay (cPass, GenScript USA) in plasma samples of recovered individuals R1 (n = 40) at 1–2 months, R2 (n = 40) at 8–9 months, and R3 (n = 22) at 12 months/1 year of postrecovery. The Y-axis represents the percentage of signal inhibition (⁣^∗p value <0.05, ⁣^∗∗p value <0.005, and ⁣^∗∗∗p value <0.0001).