Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19

Abstract

In order to demonstrate the feasibility of preparing clinical-grade SARS-CoV-2-specific T-cells from convalescent donors and the ability of these cells to neutralize the virus in vitro, we used blood collected from two COVID-19 convalescent donors (before and after vaccination) that was stimulated with specific SARS-CoV-2 peptides followed by automated T-cell isolation using the CliniMacs Prodigy medical device. To determine cytotoxic activity, HEK 293T cells were transfected to express the SARS-CoV-2 M protein, mimicking SARS-CoV-2 infection. We were able to quickly and efficiently isolate SARS-CoV-2-specific T lymphocytes from both donors before and after they received the Pfizer-BioNTech vaccine. Althoughbefore vaccination, the final product contained up to 7.42% and 30.19% of IFN-γ+ CD3+ T-cells from donor 1 and donor 2, respectively, we observed an enrichment of the IFN-γ+ CD3+ T-cells after vaccination, reaching 70.47% and 42.59%, respectively. At pre-vaccination, the isolated SARS-CoV-2-specific T-cells exhibited cytotoxic activity that was significantly higher than that of unstimulated controls (donor 2: 15.41%, p-value 3.27 × 10^-3). The cytotoxic activity of the isolated SARS-CoV-2-specific T-cells also significantly increased after vaccination (donor 1: 32.71%, p-value 1.44 × 10^-5; donor 2: 33.38%, p-value 3.13 × 10^-6). In conclusion, we demonstrated that SARS-CoV-2-specific T-cells can quickly and efficiently be stimulated from the blood of convalescent donors using SARS-CoV-2-specific peptides followed by automated isolation. Vaccinated convalescent donors have a higher percentage of SARS-CoV-2-specific T-cells and may be more suitable as donors. Although further studies are needed to assess the clinical utility of the functional isolated SARS-CoV-2-specific T-cells in patients, previous studies using the same stimulation and isolation methods applied to other pathologies support this idea.

Keywords: M protein; SARS-CoV-2; T-lymphocytes; cytotoxicity.

Figure 1

SARS-CoV-2 antigen-specific T-cell production and reactivity werefirst tested using 10 mL of whole peripheral blood collected from each donor in EDTA tubes. (A) Flowcytometry analysis was based on doublet exclusion before selection of the population of interest (lymphocytes) and CD3+ T-cells. A minimum of 100,000 events within the population of interest were analyzed. (B) The percentage of CD3+ IFN-γ+ cells was quantified upon stimulation with the different SARS-CoV-2 peptides (M, N, and S). For each experiment, an unstimulated sample was used as negative control and a non-specific stimulation was used as positive control. Triplicate experiments were performed for both donors before vaccination (BV) and after vaccination (AV).

Figure 2

A total of 1 × 10⁹ leukocytes were used to purify and enrich M-protein SARS-CoV-2-specific T-cells from both donors before and after vaccination. Enrichment of IFNγ+ CD3+ T-cells in the final product was analyzed by flow cytometry for donor 1 (A) and donor 2 (B). Percentage of enrichment was quantified by establishing the quality control before purification as baseline (not purified).

Figure 3

HEK 293Tcell model to test M-protein-mediated specific cytotoxicity activity. (A) Representative image of the transfected cells. Nuclei are shown in blue (Hoechst 33342), while transfected cells are visible in green (GFP). (B) Expression levels of SARS-CoV-2 mRNA for M protein were measured 48 h post-transfection of HEK 293T cells using RT-qPCR. The obtained results were normalized to the mRNA levels of the constitutive gene GAPDH. The asterisk (*) indicates that levels of SARS-CoV-2 mRNA for M protein were significantly higher (p-value ≤ 0.05, tailed t-student test) in HEK 293T cells transfected with the plasmid pcDNATM3.1/myc-His containing the M protein coding gene than the levels obtained after transfecting the HEK 293T cells with the control plasmid (pcDNATM3.1). (C) Percentage of cytotoxic activity of the stimulated and non-stimulated T-cells against M protein of SARS-CoV-2 expressed in HEK 293Tcells in the membrane. T-cells isolated from two convalescent donors before and after vaccination were isolated for the cytotoxicity assay. All the experiments were performed at least in triplicate at both time points (before and after vaccination).