Virus Induced Lymphocytes (VIL) as a novel viral antigen-specific T cell therapy for COVID-19 and potential future pandemics

Abstract

The a priori T cell repertoire and immune response against SARS-CoV-2 viral antigens may explain the varying clinical course and prognosis of patients having a mild COVID-19 infection as opposed to those developing more fulminant multisystem organ failure and associated mortality. Using a novel SARS-Cov-2-specific artificial antigen presenting cell (aAPC), coupled with a rapid expansion protocol (REP) as practiced in tumor infiltrating lymphocytes (TIL) therapy, we generate an immune catalytic quantity of Virus Induced Lymphocytes (VIL). Using T cell receptor (TCR)-specific aAPCs carrying co-stimulatory molecules and major histocompatibility complex (MHC) class-I immunodominant SARS-CoV-2 peptide-pentamer complexes, we expand virus-specific VIL derived from peripheral blood mononuclear cells (PBMC) of convalescent COVID-19 patients up to 1000-fold. This is achieved in a clinically relevant 7-day vein-to-vein time-course as a potential adoptive cell therapy (ACT) for COVID-19. We also evaluate this approach for other viral pathogens using Cytomegalovirus (CMV)-specific VIL from donors as a control. Rapidly expanded VIL are enriched in virus antigen-specificity and show an activated, polyfunctional cytokine profile and T effector memory phenotype which may contribute to a robust immune response. Virus-specific T cells can also be delivered allogeneically via MHC-typing and patient human leukocyte antigen (HLA)-matching to provide pragmatic treatment in a large-scale therapeutic setting. These data suggest that VIL may represent a novel therapeutic option that warrants further clinical investigation in the armamentarium against COVID-19 and other possible future pandemics.

Figure 1

Detection and enrichment of antigen-specific Virus Induced Lymphocytes (VIL) in CMV infected individuals. (A) Strategy for the isolation, stimulation and enrichment of CMV antigen-specific T cells from donor PBMCs. (B) Representative flow-cytometric analysis showing proportions of antigen-specific CD8⁺ and CD4⁺ T cells identified by Pentamer staining, 7-days after enrichment and expansion with antigen-specific VIPR Particles. (C) Summary of CD8⁺ and CD4⁺ data obtained in b, (n = 3). (D) Histogram plot showing this VIL enrichment at day 7 is VIPR particle dose-dependent (n = 3). (E) Histogram showing impact on VIL enrichment at day 7 of MHC-I pentamer and anti-CD28 ratio conjugated to VIPR particles (n = 3).

Figure 2

Rapid VIL expansion results in a 1000-fold enrichment of SARS-CoV-2 antigen-specific T cells within 7-days from convalescent donors. (A) Representative flow-cytometric analysis showing proportions of SARS-CoV-2 antigen-specific CD8⁺ T cells 7-days after expansion and enrichment with YLQPRTFLL antigen-specific VIPR Particles. (B) Table describing the clinical presentation of convalescent COVID-19 donors used in this study and the proportions of SARS-CoV-2 specific VIL detectible before enrichment, and fold expansion by VIPR particles. (C) Summary of CD8⁺ SARS-CoV-2 VIL %, cell number and fold change after VIPR expansion (n = 7). (D) Histograms showing total CMV pp65 antigen-specific CD8⁺ T cell numbers expanded by VIPR particles after 7-days and overall fold change in antigen specific cells (n = 3).

Figure 3

The activation and exhaustion phenotype of rapidly-expanded SARS-CoV-2 and CMV antigen-specific VIL. (A) Expression of activation markers, 4-1BB, OX-40, CD25 and HLA-DR, and (B) expression of checkpoint genes PD-1, LAG-3 and TIGIT among enriched and expanded SARS-CoV-2-specific CD8⁺ T cells at day 7 (n = 8). (C) Analysis as in a, for CMV-specific CD8 + T cells and (D) Analysis as in b, for CMV-specific CD8 + T cells (n = 4).

Figure 4

The T cell memory phenotype of rapidly-expanded CMV and SARS-CoV-2 antigen-specific VIL. (A) Representative flow-cytometric analysis showing expression of CD45RO, CD45RA and CD62L among enriched and expanded CMV-specific CD8⁺ T cells at day-7. (B) Summary of naïve, central memory and effector memory T cell subsets from data obtained in a, (n = 4). (C) Analysis as in a, for SARS-CoV-2-specific CD8⁺ T cells, and (D) analysis as in b, for SARS-CoV-2-specific CD8⁺ T cells (n = 8).

Figure 5

Cytokine expression among rapidly-expanded SARS-CoV-2 and CMV antigen-specific VIL. (A) Representative flow-cytometric analysis showing expression of IFN-γ and TNFα among enriched and expanded SARS-COV-2 CD8⁺ T cells at day-7, after 6-h stimulation with specific peptide antigen, and proportions of IFN-γ/TNFα expressing cells also expressing IL-2. (B) Summary of the data obtained as in (A) for each cytokine (n = 7). (C) Representative proportion of SARS-CoV-2 CD8⁺ T cells expressing 1, 2 or 3 cytokines. (D) Extended analysis of SARS-CoV-2 VIL polycytokine function as SPICE representation. (E) Analysis as in a, for CMV-specific CD8⁺ T cells. (F) Histograms analyzed as in b summarizing the data obtained with CMV antigen specific CD8 + T cells (n = 3). (G) Representative proportion of CMV CD8⁺ T cells expressing 1, 2 or 3 cytokines. (H) Extended analysis of CMV-specific VIL polycytokine function as SPICE representation.

Figure 6

Allogeneic VIL Therapy Platform: An adoptive cell therapy for the treatment of individuals suffering from severe symptoms of COVID-19. (A) Schematic for a COVID-19 cell therapy in which PMBCs are collected from the blood of HLA-typed hospitalized patients and total T cells isolated. T cells are stimulated with HLA-matched MHC-I/MHC-II antigen-specific SARS-CoV-2 VIPR beads to enrich and expand CD4⁺ and CD8⁺ T cells with TCRs specific for the SARS-CoV-2 antigen epitopes. These antigen-specific VIL expand at an average of 1000-fold prior to adoptive transfer back to HLA-matched patients to mediate a T cell immune response to support the eradication of the SARS-CoV-2 virus and to engender protective immunity against repeat infection. (B) Estimations of viral-specific T cell numbers generated ex vivo for patient infusion based on the empirical data of VIL expansion by VIPR particles.