Detectable SARS-CoV-2 specific immune responses in recovered unvaccinated individuals 250 days post wild type infection

Abstract

Memory T cells play an important role in mediating long-lasting adaptive immune responses to viral infections, such as SARS-CoV-2. In the context of the latter, much of our current knowledge stems from studies in vaccinated individuals or repeatedly infected individuals. However, limited knowledge is available on these responses in fully naive individuals in German communities. We performed immunophenotyping of a previously naive SARS-CoV-2 cohort in convalescent individuals after asymptomatic to moderate COVID-19. The samples were collected median 250 days post infection during the first wave of the COVID pandemic in Germany (March - May 2020). In this cohort of 174 individuals, we phenotyped different leukocyte cell populations in peripheral blood (B, T and Natural Killer cells). We then assessed the serostatus against the SARS-CoV-2 antigens Nucleocapsid (N) and Spike subunit (S1) with its receptor binding domain (RBD), as these are important correlates of protection, by testing for presence of immunoglobulin G (IgG) antibodies. We also measured IgG antibody responses against the N antigen of the common cold coronaviruses HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E, to determine possible cross-reactivity. In a subset of the cohort (n = 76), we performed intracellular staining assays (ICS) after stimulation with SARS-CoV-2 and HCoV antigens. Key findings are significant differences in frequency of CD4+ memory T cell populations, notably CD4+ TEM and CD4+ TEMRA cells, between the group of SARS-CoV-2 positive individuals and the control group. These differences correlated with cytokine production (TNFα, IFNγ) after stimulation with SARS-CoV-2 peptides, indicating a specific T cell immune response. In conclusion, a clear memory T cell and humoral response can be detected up to 250 days post mild to moderate COVID-19 disease. Our results underline findings reported by others indicating a lasting cellular immune response even in a population which previously had not been exposed to SARS-CoV-2.

Fig 1. Overview of symptoms reported.

(A) shows the distribution of symptoms reported by the participants in the SARS-CoV-2 positive (red, n = 105) and the exposed (blue, n = 36) group. Members of the control group did not report any symptoms. The dendrogram on the left side of the heatmap depicts two clusters of symptoms. The top cluster features symptoms commonly associated with COVID-19 disease, such as loss of smell/taste and fatigue; the bottom cluster indicates nonspecific symptoms of common respiratory infections like fever, cough, shivering or muscular pain. (B) illustrates the number of participants reporting a specific symptom. The symptoms are grouped according to the affected functional system, represented by different colours.

Fig 2. Coronavirus serostatus clustering.

Shown are the results of the serological tests for SARS-CoV-2 and HCoVs clustered by the strength of response (seropositivity). The exposed (blue, n2 = 36) and control (yellow, n3 = 33) groups did – by definition – not show a serological answer against SARS-CoV-2. The positive (red, n1 = 105) group prominently features the bottom right cluster of samples that had a strong serological answer against the N, RBD and S1 SARS-CoV-2 antigens. This cluster is subdivided into smaller clusters separated by their answer to the different SARS-CoV-2-antigens.

Fig 3. CD4+ T cell memory subset alterations after SARS-CoV-2 infection.

Shown are differences in frequency of T cell subsets between the positive (red, n = 50), exposed (blue, n = 16) and control (yellow, n = 10) groups, generated by analyzing the phenotype-FACS-data. (A) to (E) depict five different subsets of effector memory CD4+ T cells: (A) CD4+ T_EM (CD45RA-/CCR7-) with its subsets CD4+ T_EM CD27+ (B) and CD4+ T_EM CD127+ (C) and (D) CD4+ T_EMRA (CD45RA+ /CCR7-) T cells with its subset CD4+ T_EMRA CD27+ (E). Statistics were performed using the Kruskal-Wallis-test and Dunne’s post hoc test and adjusted using the Benjamini-Hochberg-correction.

Fig 4. Correlation between CD4+ T_EMRA subsets and SARS-CoV-2-specific CD4+ T cell responses.

Shown are correlations between frequency of CD4+ T_EMRA, CD4+ T_EMRA CD27+ T cell subsets and SARS-CoV-2 specific CD4+ T cells responses after stimulation with one of the following peptides: S1 (Spike glycoprotein subunit 1), S2 (Spike glycoprotein subunit 2), N, M within all positive samples in the ICS sub cohort (n = 50). Antigen-specific responses are shown for: interleukin 2 and 4 (IL-2, IL-4), IFNγ, TNFα, double positive TNFα/IFNγ and the sum of TNFα and IFNγ responses (“reactive”). The upper part of both subsets shows the spearman correlation coefficient, the lower part visualizes the strength of said spearman correlation coefficient. The empty boxes for CD4+ T_EM cells had a correlation coefficient of 0.00 and were removed for better readability. The boxes bordered in red highlight a significant correlation coefficient (p < 0.05).