Rapid Generation of Multivirus-Specific T Lymphocytes for the Prevention and Treatment of Respiratory Viral Infections
Authors: Vasileiou Spyridoula , Turney Annie , Kuvalekar Manik , Mukhi Shivani , Watanabe Ayumi , Lulla Premal , Ramos Carlos A. , Vera Juan , Tzannou Ifigeneia , Leen Ann 
Published: October 2019

Abstract

Acute upper and lower respiratory tract infections (RTIs) due to community-acquired respiratory viruses (CARVs) including respiratory syncytial virus (RSV), influenza, parainfluenza virus (PIV) and human metapneumovirus (hMPV) are a leading cause of morbidity and mortality worldwide, with individuals whose immune systems are naïve (e.g. children) or compromised being most vulnerable. In allogeneic hematopoietic stem cell transplant (HSCT) recipients, the incidence of CARV-related respiratory viral infection reaches 29%. Most patients initially present with mild symptoms of upper RTI and in 50% of cases the infection progresses to a lower RTI with severe symptoms including bronchiolitis and pneumonia and mortality rates as high as 50%. Currently there are no approved vaccines nor antiviral drugs for hMPV and PIV, while the preventative vaccine for Influenza is not indicated earlier than 6 months post-HSCT. Aerosolized ribavirin is FDA-approved for the treatment of RSV infections, but it is logistically difficult to administer and comes at a considerable cost. Thus, the lack of approved antiviral agents combined with the high cost of antiviral therapy emphasize the need for alternative treatment strategies for CARVs.
Our group has previously demonstrated the safety and clinical efficacy of using adoptive T-cell transfer for the treatment of both latent [Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), human herpesvirus 6 (HHV6)] and lytic [adenovirus (AdV)] viruses in recipients of allo-HSCT by generation of multivirus-specific T cell (VST) lines. Given that susceptibility to CARVs is highly associated with underlying immune deficiency, we wanted to explore the potential for extending this approach to Influenza, RSV, hMPV and PIV3 infections. In order to do so, we exposed PBMCs from healthy donors to a cocktail of pepmixes (overlapping peptide libraries) spanning immunogenic antigens derived from our target viruses [Influenza - NP1 and MP1; RSV - N and F; hMPV - F, N, M2-1 and M; PIV3 - M, HN, N and F] followed by expansion in the presence of activating cytokines in a G-Rex device. Over 10-13 days we achieved an average 8.5 fold expansion [increase from 0.25x107 PBMCs/cm2 to mean 1.9±0.2x107 cells/cm2; n=12). Cells were comprised almost exclusively of CD3+ T cells (96.2±0.6%; mean±SEM), with a mixture of cytotoxic (CD8+) and helper (CD4+) T cells and a phenotype consistent with immediate effector function and long term memory, as evidenced by upregulation of the activation markers CD25, CD69, and CD28 as well as expression of central (CD45RO+/CD62L+) and effector memory markers (CD45RO+/CD62L−), with minimal PD1 or Tim3 expression.
Anti-viral specificity of multi-R-VSTs was tested in an IFNγ Elispot assay using each of the individual stimulating antigens as an immunogen and all 12 lines screened proved to be reactive against all 4 of the target viruses [Influenza: mean 735±75.6 SFC/2x105, RSV: 758±69.8, hMPV: 526±100.8, PIV3: 391±93.7]. As demonstrated by intracellular cytokine staining, the immune response was mediated by both CD4+ and CD8+ T cell subsets, and the majority of IFNγ-producing cells also produced TNFα. In addition, the cells secreted GM-CSF as measured by Luminex array, with baseline levels of Th2/suppressive cytokines. Furthermore, upon antigenic stimulation our VSTs produced the effector molecule Granzyme B suggesting the cytolytic potential of these expanded cells, which was confirmed in a standard Cr51-release assay against viral pepmix-loaded autologous PHA blasts. Viral antigen-loaded targets were specifically recognized and lysed by our VSTs, while there was no evidence of activity against non-infected autologous or allogeneic targets.
In conclusion, we have shown that it is feasible to rapidly generate a single preparation of polyclonal multi-respiratory (multi-R)-VSTs with specificities directed to Influenza, RSV, hMPV and PIV3 and a total of 12 encoded antigens using GMP-compliant manufacturing methodologies. The expanded cells are Th1-polarized, polyfunctional and selectively able to react to and kill viral antigen-expressing targets with no auto- or alloreactivity, attesting to both their selectivity and their safety for clinical use in HSCT recipients. We anticipate such multi-R-VSTs will provide clinical benefit in preventing or treating CARV infections in the immunocompromised.

Disclosures
Vera: Viracyte: Equity Ownership. Tzannou:Viracyte: Consultancy, Equity Ownership. Leen:Viracyte: Equity Ownership.