SARS-CoV-2-Specific T-Cell as a Potent Therapeutic Strategy against Immune Evasion of Emerging COVID-19 Variants

Abstract

Despite advances in vaccination and therapies for coronavirus disease, challenges remain due to reduced antibody longevity and the emergence of virulent variants like Omicron (BA.1) and its subvariants (BA.1.1, BA.2, BA.3, and BA.5). This study explored the potential of adoptive immunotherapy and harnessing the protective abilities using virus-specific T cells (VSTs). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VSTs were generated by stimulating donor-derived peripheral blood mononuclear cells with spike, nucleocapsid, and membrane protein peptide mixtures. Phenotypic characterization, including T-cell receptor (TCR) vβ and pentamer analyses, was performed on the ex vivo-expanded cells. We infected human leukocyte antigen (HLA)-partially matched human Calu-3 cells with various authentic SARS-CoV-2 strains in a Biosafety Level 3 facility and co-cultured them with VSTs. VSTs exhibited a diverse TCR vβ repertoire, confirming their ability to target a broad range of SARS-CoV-2 antigens from both the ancestral and mutant strains, including Omicron BA.1 and BA.5. These ex vivo-expanded cells exhibited robust cytotoxicity and low alloreactivity against HLA-partially matched SARS-CoV-2-infected cells. Their cytotoxic effects were consistent across variants, targeting conserved spike and nucleocapsid epitopes. Our findings suggest that third-party partial HLA-matching VSTs could counter immune-escape mechanisms posed by emerging variants of concern.

Keywords: coronavirus disease; immunotherapy; severe acute respiratory syndrome coronavirus 2; viral immunity; virus-specific T cells.

Figure 1

Enhancing SARS-CoV-2-specific T-cell (VST) responses for adoptive immunotherapy: phenotypic characterization and TCR diversity. (A) Representation of the 21-day VST expansion process, where PBMC or leukapheresis samples were stimulated with S, M, and N peptide pools. (B) Post-expansion fold increase in total cell count. (C) Post-expansion VST response against SARS-CoV-2 antigens. (D) Phenotypic distribution of expanded cell population, showing predominance of CD3⁺ T cells, with helper (CD3⁺CD4⁺), cytotoxic (CD3⁺CD8⁺), and NKT (CD3⁺CD56⁺) cell subsets. (E) Expression levels of T-cell memory markers, including central memory (CD45RA⁻/CD62L⁺), effector memory (CD45RA⁻/CD62L⁻), terminally differentiated effector memory (CD45RA⁺/CD62L⁻), and naïve markers (CD45RA⁺/CD62L⁺). (F) Multichannel flow-cytometric analysis of TCR vβ repertoire diversity in expanded cells, capturing >70% of all vβ chains and confirming the presence of all measurable vβ family members; representative donor (left) and summary data are shown as means ± SEMs (right). (G) Intracellular cytokine staining depicting antigen-specific IFNγ, IL-2, and TNF-α production in CD3⁺CD4⁺, CD3⁺CD8⁺, and CD3⁺CD56⁺ T-cell subsets in response to spike (S), nucleocapsid (N), and membrane (M) antigens. Minimal response in the absence of peptide stimulation underscores VST-activation specificity against SARS-CoV-2. Data are shown as SFC ± standard error of mean (SEM). (H) VST cytolytic activity toward carboxyfluorescein succinimidyl (CFSE)-labeled SARS-CoV-2 peptide-loaded autologous phytohemagglutinin-activated (PHA) blasts. Results show targeted lysis at an effector–target ratio of 50:1. (I) HLA-mismatched allogenic PBMC experiments confirm the absence of nonspecific autotargeting and alloreactivity that could lead to graft-versus-host disease; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale and error bars indicate median and range, respectively. IFNγ: interferon gamma; M: membrane; N: nucleocapsid; PBMC: peripheral blood mononuclear cells; S: spike; SARS-CoV-2: severe acute respiratory syndrome coronavirus-2; SC: spike complete, SFC: spot-forming cell; SI, spike immunodominant; Tem: effector memory T cells; Tcm: central memory T cells; Temra: terminally differentiated effector memory T cells; TCR: T-cell receptor; VST: virus-specific T cell.

Figure 2

Comparative analysis of VST production and proliferation responses to viral peptides among groups based on SARS-CoV-2 exposure and vaccination status. (A,B) Evaluation of IFN-γ ELISPOT responses to spike, membrane, and nucleocapsid peptides using VSTs produced from donors in groups classified by SARS-CoV-2 exposure and vaccination status: unvaccinated (no prior infection), vaccinated (no prior infection), and recovered (post-infection). (C) IFN-γ response distribution in the unvaccinated group: Spike (49.62%), Nucleocapsid (46.68%), and Membrane (3.7%). IFN-γ response distribution in the vaccinated group: Spike (92.65%). IFN-γ response distribution in the recovered group: Spike (44.8%), Membrane (20.84%), and Nucleocapsid (34.36%). (D) Trends in proliferation rates and fold expansion after 10 and 17 days of incubation. IFN: interferon, SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; VST: virus-specific T cell.

Figure 3

Evaluation of virus-specific T cell (VST) cytolytic capacity across SARS-CoV-2 spike variants. (A) An illustrative overview of the experimental setup and methodology including the generation and infection process of SARS-CoV-2 spike-pseudotyped lentivirus. (B) Flow cytometric characterization, highlighting pronounced expressions of ACE2, TMPRSS2, HLA *A02, and HLA-A/B/C in both HEK293 wild-type (293T-WT) and HEK293T-hACE2-TMPRSS2-mCherry (293T-ACE2) cells. (C) Bioluminescence analysis demonstrating a dose-dependent VST-induced cytotoxic response against cells infected by different variants of SARS-CoV-2 spike-pseudotyped lentiviruses. Induction of bioluminescence through luciferase is indicative of lentiviral infection, and a decrease in bioluminescence signals signifies significant VST-induced cytotoxicity toward the infected target cells. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Figure 4

Evaluation of virus-specific T-cell (VST) reactivity and cytotoxicity across SARS-CoV-2 spike variants. (A) Fluorescence microscopy after 48 h co-culture of VST with HEK293T-hACE2-TMPRSS2-mCherry cells infected with SARS-CoV-2 spike-pseudotyped lentivirus. Green fluorescent protein (GFP) expression indicates lentiviral infection, whereas observations indicate significant VST-induced cytotoxicity toward infected, but not uninfected, target cells. (B) Flow cytometric analysis after VST co-culture shows a reduction in viable GFP-expressing cells, with an increase in the number of fixed-viability dye (FVD)-positive necrotic cells, which emphasizes the potent cytolytic capacity of VSTs. (C) Mutation sites within the spike protein for the Ancestral (D614), Delta (B.1.617.2), and Omicron (B.1.1.529/BA.1) variants are indicated by red arrows. (D) Fluorescence microscopic images of VST-induced cytotoxic effects against cells infected by different variants of SARS-CoV-2 spike-pseudotyped lentiviruses. The green color represents viral infection-induced GFP-expressing cells, whereas arrows indicate cells undergoing VST-induced cytolytic effects. (E) Flow cytometric analysis demonstrating dose-dependent VST-induced cytotoxic response against all evaluated spike variants. * p < 0.05; *** p < 0.001. HLA: human leukocyte antigen; IL: interleukin; INF: interferon; PBMC: peripheral blood mononuclear cell; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SFC: spot-forming cell; TNF: tumor necrosis factor; RBD: receptor-binding domain; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; VST: virus-specific T cell.

Figure 5

VSTs educated by the ancestral SARS-CoV-2 strain recognize conserved epitopes in the Omicron variant. The expected response frequency for the spike-mutation domain of Omicron BA.5, as inferred from the Immune Epitope Database, emphasizes the maintained high-response frequency even with the 34 present mutations. Analysis of spike-protein epitopes in the Omicron subvariant (BA.5) (A) targeted by CD4⁺ T cells (conservation rate: 82%), and (B) recognized by CD8⁺ T cells (conservation rate: 85%). (C) Representative data showcasing the pronounced reactivity of VSTs against seven specific Omicron BA.5 spike-protein immunogenic epitopes (indicated by green triangles in (B)), determined using pentamer analysis. A bar graph consolidates the results from 10 donors. FMO: fluorescence minus one; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; VST: virus-specific T cell.

Figure 6

Antigen-specific activation and targeted cytotoxicity toward cells expressing SARS-CoV-2 spike and nucleocapsid proteins. (A) Flow cytometric analyses of SARS-CoV-2 spike (S) and nucleocapsid (N) protein expression in engineered cell lines. (B) Confocal imaging visually confirms S and N protein expressions. (C) Western blotting validates S and N protein expressions. (D) Flow cytometric characterization showing pronounced HLA-A/B/C and HLA *A02 expressions across HEK293 wild-type (WT), HEK293 S, and HEK293 N cells. (E) Effector VSTs from a donor with matching HLA class I allele relative to target 293T cells. (F) Cell Tox Green assay demonstrating increased cytotoxic susceptibility in S protein-expressing HEK293 cells, with cytotoxicity against N protein-transduced HEK293 cells. (G) Post-administration tumor-growth pattern in a xenograft mouse model inoculated with HEK293-WT cells. Green arrows indicate the time points of VST injection. VSTs neither recognize nor exert cytotoxic effects on SARS-CoV-2 antigen-unmodified WT cells, leading to continued tumor growth. (H) With HEK293-N cells expressing the SARS-CoV-2 N protein in the xenograft model, green arrows indicate the time points of VST injection. VSTs exhibit specific recognition and potent cytotoxicity, resulting in significant suppression of tumor growth. ** p < 0.01; *** p < 0.001. HLA: human leukocyte antigen; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; VST: virus-specific T cell.

Figure 7

Cytotoxic activity of VST against SARS-CoV-2 Omicron mutant-infected Calu-3 cells. (A) Comparison of SARS-CoV-2 infectivity in Calu-3 cells at 24 and 48 h post-infection. Data indicate increased infectivity and significant cytopathic effects at the 48 h timepoint. (B) Antiviral activity of remdesivir against SARS-CoV-2 in Calu-3 cells, serving as a positive control for the suppression of viral replication. (C) Visualization of SARS-CoV-2 N protein in Calu-3 cells after a 24 h co-culture period with VSTs, indicating viral infection. (D) Dose-dependent cytotoxic activity of VSTs against SARS-CoV-2-infected Calu-3 cells, showcasing the ability of VSTs to selectively target and eliminate virus-infected cells. (E) Efficacy of VSTs derived from four different donors against the Omicron mutant strains, NCCP43408 and NCCP43426, revealing a consistent and potent dose-dependent cytotoxic response. (F) Reflective comparison of the potency of VSTs against the ancestral SARS-CoV-2 strain, emphasizing the consistent therapeutic potential across virus variants. ** p < 0.01; *** p < 0.001. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; VST: virus-specific T cell.

Figure 8

Cytotoxic activity of VST on cells infected with authentic SARS-CoV-2 Omicron mutants. (A) Dose-dependent response against the ancestral SARS-CoV-2 strain. (B) Dose-dependent response against the SARS-CoV-2 BA.1 variant. (C) Dose-dependent response against the SARS-CoV-2 BA.5 variant. N, nucleocapsid; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; VST: virus-specific T cell.

Figure 9

VSTs against SARS-CoV-2 infected cells: In response to SARS-CoV-2 infection, virus-specific T cells (VSTs) specifically target cells including epithelial cells and macrophages that become infected and express the ACE2 receptor. These cells, once infected, process and present viral antigens, which VSTs recognize through TCR-MHC I interactions. This detection triggers VSTs to release cytotoxic granules containing interferon-gamma (IFN-γ) and granzyme B, leading to the effective elimination of the infected cells and impeding the virus’s replication process. Notably, VSTs demonstrate specificity in their response: (A) they do not show cytotoxicity toward normal, uninfected cells, as these do not present SARS-CoV-2 antigens on their surface, and (B) they actively exhibit cytotoxicity against cells expressing virus antigens, thereby effectively inhibiting the viral replication cycle. IL: interleukin; MHC: major histocompatibility complex; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TCR: T cell receptor; TNF: tumor necrosis factor.