CoVITEST: A Fast and Reliable Method to Monitor Anti-SARS-CoV-2 Specific T Cells From Whole Blood

Abstract

Cellular and humoral immune responses are essential for COVID-19 recovery and protection against SARS-CoV-2 reinfection. To date, the evaluation of SARS-CoV-2 immune protection has mainly focused on antibody detection, generally disregarding the cellular response, or placing it in a secondary position. This phenomenon may be explained by the complex nature of the assays needed to analyze cellular immunity compared with the technically simple and automated detection of antibodies. Nevertheless, a large body of evidence supports the relevance of the T cell's role in protection against SARS-CoV-2, especially in vulnerable individuals with a weakened immune system (such as the population over 65 and patients with immunodeficiencies). Here we propose to use CoVITEST (Covid19 anti-Viral Immunity based on T cells for Evaluation in a Simple Test), a fast, affordable and accessible in-house assay that, together with a diagnostic matrix, allows us to determine those patients who might be protected with SARS-CoV-2-reactive T cells. The method was established using healthy SARS-CoV-2-naïve donors pre- and post-vaccination (n=30), and further validated with convalescent COVID-19 donors (n=51) in a side-by-side comparison with the gold standard IFN-γ ELISpot. We demonstrated that our CoVITEST presented reliable and comparable results to those obtained with the ELISpot technique in a considerably shorter time (less than 8 hours). In conclusion, we present a simple but reliable assay to determine cellular immunity against SARS-CoV-2 that can be used routinely during this pandemic to monitor the immune status in vulnerable patients and thereby adjust their therapeutic approaches. This method might indeed help to optimize and improve decision-making protocols for re-vaccination against SARS-CoV-2, at least for some population subsets.

Keywords: COVID-19; SARS-CoV-2; T cells; blood test; cellular immune response.

Figure 1

Schematic representation of the workflow for direct peptide stimulation of whole peripheral blood by CoVITEST, IFN-γELISpot or antibody quantification by Luminex^® (Created withBioRender.com).

Figure 2

Representative flow cytometry analysis for CoVITEST. The flow cytometry gating strategy was performed sequentially; lymphocyte complexity (SSC vs. FSC), single cell, CD3+ and finally either CD4+ or CD8+ (A). From there, 40,000 CD4+ or CD8+ T cells were further evaluated for CD69 and IFN- γ expression in S and N peptide pool, negative control (vehicle) and positive control (SEB) samples to determine the T cell reactivity for each donor (B).

Figure 3

Correlation of SARS-CoV-2-specific T cell response from fresh whole blood CoVITEST with classic T cell IFN-γELISpot Convalescent COVID-19 donors at two weeks, three and six months after the first positive RT-PCR test. Linear regression analysis by comparing the number of CD4+ IFN-γ+ CD69+ T cells from whole blood with specific T cells quantified by IFN-γELISpot (n = 51).

Figure 4

Distribution of the cellular immune response to SARS-CoV-2 by IFN-γELISpot, CoVITEST and antibodies against the Receptor Binding Domain (RBD) of the spike glycoprotein of SARS-CoV-2 (IgG, IgA or IgM) by Luminex in 51 COVID-19 patients.

Figure 5

SARS-CoV-2-specific T cell response at baseline and two weeks after the second vaccine dose in healthy unexposed donors. SARS-CoV-2 specific T cells (CD4+ IFN-γ+ CD69+) after stimulation with spike and nucleocapsid SARS-CoV-2 peptide pools at baseline and two weeks after the second vaccine dose. Each dot represents an individual subject. *Statistical comparison at baseline and post-vaccination was performed with the Wilcoxon test.