Pre-existing T-cell immunity to SARS-CoV-2 in unexposed healthy controls in Ecuador, as detected with a COVID-19 Interferon-Gamma Release Assay

Abstract

Background: Studies of T-cell immune responses against SARS-CoV-2 are important in understanding the immune status of individuals or populations. Here, we use a simple, cheap, and rapid whole blood stimulation assay - an Interferon-Gamma Release Assay (IGRA) - to study T-cell immunity to SARS-CoV-2 in convalescent COVID-19 patients and in unexposed healthy contacts from Quito, Ecuador.

Methods: Interferon-gamma (INF-γ) production was measured in the heparinized blood of convalescent and unexposed subjects after stimulation for 24 h with the SARS-CoV-2 Spike S1 protein, the Receptor Binding Domain (RBD) protein or the Nucleocapsid (NP) protein, respectively. The presence of IgG-RBD protein antibodies in both study groups was determined with an "in-house" ELISA.

Results: As measured with INF-γ production, 80% of the convalescent COVID-19 patients, all IgG-RBD seropositive, had a strong T-cell response. However, unexpectedly, 44% of unexposed healthy controls, all IgG-RBD seronegative, had a strong virus-specific T-cell response with the COVID-19 IGRA, probably because of prior exposure to common cold-causing coronaviruses or other viral or microbial antigens.

Conclusion and discussion: The high percentage of unexposed healthy subjects with a pre-existing immunity suggests that a part of the Ecuadorian population is likely to have SARS-CoV-2 reactive T-cells. Given that the IGRA technique is simple and can be easily scaled up for investigations where high numbers of patients are needed, this COVID-19 IGRA may serve to determine if the T-cell only response represents protective immunity to SARS-CoV-2 infection in a population-based study.

Keywords: COVID-19; ELISPOT; Interferon-Gamma (IFN-γ) release assay (IGRA); Nucleocapsid protein; Pre-existing immunity; SARS-CoV-2; Spike protein; T cells.

Figure 1

IFN-γ production after stimulating whole blood of convalescent COVID-19 patients and healthy unexposed controls with RBD, N, or S1 protein. The results of stimulation of convalescent patients and controls with, respectively, the RBD, N, or S1 protein are shown in Fig. 1A, B, and C. In Fig. 1D, the INF-γ responses of each patient and to the three antigens are grouped together. Patients with confirmed COVID-19 infection are numbered 1–15, of which patients 14 and 15 were asymptomatic. Subjects 16–33 are COVID-19 naïve patients. After 24 h of stimulation, IFN-γ concentrations in plasma were determined using a commercially available human IFN-γ enzyme-linked immunosorbent assay–based kit (Biolegend cat. No. 430,101). The magnitude of the stimulation was expressed as the optical density at 450 nm (OD₄₅₀) for blood stimulated with antigen minus the OD₄₅₀ for blood without antigen stimulation. An OD of 1.0 corresponded with approximately 400 pg/mL IFN-γ. Phytohemagglutinin (PHA) was used as the mitogen at a final concentration of 5 μg/mL in the IGRA to assess the quality and response capacity of the blood. All patients and controls showed a maximum production of the cytokine with this mitogen with a mean optical density (OD₄₅₀) of 2.54 (SD = 0.13), representing approximately 1000 pg/mL of IFN-γ. There was no difference in the median IFN-γ level in the unstimulated blood of convalescent patients and healthy controls (OD₄₅₀ = 0.38, SD = 0.14 versus OD₄₅₀ = 0.32. SD = 0.16). These results are not shown. A horizontal line has been drawn in each graph to represent the cut-off value for stimulation. This value was determined by calculating the mean plus two standard deviations of the response of nine subjects (patients 16, 18, 19, 22, 27–31) of the control group with the lowest INF–γ production after stimulation.