SARS-CoV-2-specific T cells are rapidly expanded for therapeutic use and target conserved regions of the membrane protein

Abstract

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.

Graphical abstract

Figure 1.

T-cell recognition of SARS-CoV-2 viral antigens. Specificity of the expanded cells in response to SARS-CoV-2 antigens from convalescent patients (n = 46) and unexposed controls (n = 15) was assayed by IFN-γ ELISpot assay (bars = median). Unstimulated T cells (control [CTL] only) and stimulation with actin were used as negative controls. Results are presented as spot-forming units (SFC) per 1 × 10⁵ cells. Specificity was defined as ≥20 spots per well with significance above background (actin) via 2-tailed Student t test. *P = .0008, **P = 6.24 × 10⁻⁶.

Figure 2.

Specificity of ex vivo–expanded CST. Following 10 to 12 days of culture, specificity of CD4 and CD8 T-cell populations for membrane, spike, and nucleocapsid proteins was assessed by intracellular cytokine staining for IFN-γ and TNF-α. (A) Subject 2 demonstrates a CD4-predominant response targeting structural proteins. (B-C) Summary data of the response of expanded CD4⁺ T cells (B) and CD8⁺ T cells (C) in response to membrane, spike, and nucleocapsid proteins by intracellular cytokine staining was analyzed in convalescent donors (n = 11), and the percentage of T cells was compared with actin-stimulated controls via 2-tailed Student t test. *P < .05, **P < .01.

Figure 3.

T-cell specificity of seropositive vs seronegative patients. Comparison of IFN-γ ELISpot results from postexpansion CSTs from SARS-CoV-2 seropositive vs seronegative convalescent patients was performed via Student t test. *P = .0015, **P = .00075.

Figure 4.

SARS-CoV-2 epitope mapping of CSTs. T-cell epitope mapping of structural proteins was performed using minipools containing 8 to 24 peptides each, with responses measured via IFN-γ ELISpot (SFC per 1 × 10⁵ cells). (A) Epitopes within membrane protein were identified within the C terminus at AA 144-163 and 173-192, which were recognized by 8 and 6 donors, respectively. (B) Mapping of spike epitopes demonstrated three regions at AA 57-75, 205-224, and 449-463, which were recognized by 3 donors. (C) Mapping of nucleocapsid epitopes showed 2 regions at AA 357-271 and 313-335 were recognized by 3 donors.

Figure 5.

T-cell restrictions of SARS-CoV-2 epitopes. Identification of the T cells responding to each identified epitope was performed via intracellular cytokine staining on expanded CSTs, with percentages of TNF-a⁺/IFN-γ⁺ populations depicted. Intracellular cytokine staining demonstrated a predominant CD4-mediated response to membrane peptides 37-38 (A), membrane peptides 44-45 (B), nucleocapsid peptide 65 (C), and spike proteins 15-16 (D), and a predominant CD8-mediated response to nucleocapsid peptide 81 (E). SEB, staphylococcal enterotoxin β.

Figure 6.

Epitope locations within SARS-CoV-2 structural proteins. (A) Epitopes within membrane protein were identified at the C-terminal intravirion domain. TMD, transmembrane domains. (B) Within spike proteins, epitopes were found within the S1 region, including 1 epitope within the receptor-binding domain (RBD). (C) In nucleocapsid protein, epitopes were identified in the region of the dimerization domain (DD).