Broad T Cell Targeting of Structural Proteins After SARS-CoV-2 Infection: High Throughput Assessment of T Cell Reactivity Using an Automated Interferon Gamma Release Assay

Abstract

Background: Adaptive immune responses to structural proteins of the virion play a crucial role in protection against coronavirus disease 2019 (COVID-19). We therefore studied T cell responses against multiple SARS-CoV-2 structural proteins in a large cohort using a simple, fast, and high-throughput approach.

Methods: An automated interferon gamma release assay (IGRA) for the Nucleocapsid (NC)-, Membrane (M)-, Spike-C-terminus (SCT)-, and N-terminus-protein (SNT)-specific T cell responses was performed using fresh whole blood from study subjects with convalescent, confirmed COVID-19 (n = 177, more than 200 days post infection), exposed household members (n = 145), and unexposed controls (n = 85). SARS-CoV-2-specific antibodies were assessed using Elecsys^® Anti-SARS-CoV-2 (Ro-N-Ig) and Anti-SARS-CoV-2-ELISA (IgG) (EI-S1-IgG).

Results: 156 of 177 (88%) previously PCR confirmed cases were still positive by Ro-N-Ig more than 200 days after infection. In T cells, most frequently the M-protein was targeted by 88% seropositive, PCR confirmed cases, followed by SCT (85%), NC (82%), and SNT (73%), whereas each of these antigens was recognized by less than 14% of non-exposed control subjects. Broad targeting of these structural virion proteins was characteristic of convalescent SARS-CoV-2 infection; 68% of all seropositive individuals targeted all four tested antigens. Indeed, anti-NC antibody titer correlated loosely, but significantly with the magnitude and breadth of the SARS-CoV-2-specific T cell response. Age, sex, and body mass index were comparable between the different groups.

Conclusion: SARS-CoV-2 seropositivity correlates with broad T cell reactivity of the structural virus proteins at 200 days after infection and beyond. The SARS-CoV-2-IGRA can facilitate large scale determination of SARS-CoV-2-specific T cell responses with high accuracy against multiple targets.

Keywords: COVID-19; SARS-CoV-2; T cell response; high through put; interferon gamma release assay (IGRA).

Figure 1

Flowchart of study subject groups.

Figure 2

Sensitive and specific detection of T cell responses to four SARS-CoV-2 antigenic regions. Concentration of IFNγ in stimulated whole blood supernatants (y-axis) is shown as mIU/ml for the Nucleocapsid (NC), Membrane protein (M), Spike-C-Terminus (SCT) and Spike-N-Terminus (SNT). The numbers of subjects tested are indicated for each antigenic region and group. The black number at the bottom indicates overall number of study subjects in each group, numbers in the middle and the top show the number of subjects with IFNγ concentration of or above 0 mlU/ml, respectively. Cutoff of 40 mIU/ml IFNγ for T cell reactivity to an antigenic region is indicated as dashed line. Thick black lines mark median values. Each dot represents one study subject. Due to low blood volume, not all participants underwent the same analysis regarding the stimulation with the main three tested antigenic regions (NC, SCT and M). Therefore, sample sizes at each group between Antigens differ (see black sample size n below). 232 study subjects were also stimulated with SNT. (A) Ro-N-Ig seropositive subjects with PCR confirmed convalescent COVID-19 (green dots) were compared to negative controls from unexposed households (orange dots). (B) T cell recognition to the four tested structural antigens was compared for subjects with serological and/or PCR confirmed convalescent COVID-19. The p-values were calculated using Wilcoxon signed rank test. ^***p ≤ 0.001, ^****p ≤ 0.0001.

Figure 3

Broad T cell recognition of structural SARS-CoV-2 proteins in subjects with convalescent infection. Percentages of subjects (y-axis) who responded to 0, 1, 2 or 3 of 3 tested antigenic regions are shown in (A) for 5 groups delineated by PCR, Ro-N-Ig serostatus and SARS-CoV-2 exposure status. The number of study subjects in each group and the breadth of T cell recognition from 0 to 3 structural proteins are indicated for each group. A subgroup of 208 study subjects was tested with a fourth antigen - the Spike-N-Terminus (B). The breadth of T cell recognition of the four tested antigens is shown for each of the 5 groups. Every dot represents an individual. Reactivity to Nucleocapsid Protein (NC), Spike-C-Terminus (SCT), and Membrane protein (M) was tested in stimulated whole blood supernatants.

Figure 4

(A) Correlation of SARS-CoV-2-specific T cell reactivity to different antigenic regions and Roche-N-Ig titer. Shown are individuals of the convalescent group in different colors for each subgroup. Strong humoral immune response correlates with cellular reactivity to SARS-CoV-2 specific antigens measured in IFNγ (y-axis). Cutoffs for seropositivity and T cell reactivity to an antigenic region are indicated as dashed lines. Each dot represents one study subject. (B) Correlation of Roche-N-Ig and breadth reactivity of detected antigens. The plot shows Roche-N-Ig values for each individual recognizing 0, 1, 2, 3 or 4 antigenic regions. Only individuals which were tested for all four antigenic regions (Nucleocapsid NC, Spike-C-Terminus SCT, Spike-N-Terminus SNT and Membrane M) are shown. Cutoff for seropositivity is indicated by a dashed line. The p- value of non-zero correlation from all groups combined is shown in black. A low p-value means that the correlation is unlikely to be non-zero due to chance. Each dot represents one study subject.