T-Cell Therapeutics Targeting Human Parainfluenza Virus 3 Are Broadly Epitope Specific and Are Cross Reactive With Human Parainfluenza Virus 1

Abstract

Human Parainfluenza Virus-3 (HPIV3) causes severe respiratory illness in immunocompromised patients and lacks approved anti-viral therapies. A phase I study of adoptively transferred virus-specific T-cells (VSTs) targeting HPIV3 following bone marrow transplantation is underway (NCT03180216). We sought to identify immunodominant epitopes within HPIV3 Matrix protein and their cross-reactivity against related viral proteins. VSTs were generated from peripheral blood of healthy donors by ex-vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 matrix protein. Epitope mapping was performed using IFN-γ ELIspot with combinatorial peptide pools. Flow cytometry was used to characterize products with intracellular cytokine staining. In 10 VST products tested, we discovered 12 novel immunodominant epitopes. All products recognized an epitope at the C-terminus. On IFN-γ ELISpot, individual peptides eliciting activity demonstrated mean IFN-γ spot forming units per well (SFU)/1x10 5 cells of 115.5 (range 24.5–247.5). VST products were polyfunctional, releasing IFN-γ and TNF-α in response to identified epitopes, which were primarily HLA Class II restricted. Peptides from Human Parainfluenza Virus-1 corresponding to the HPIV3 epitopes showed cross-reactivity for HPIV1 in 11 of 12 tested epitopes (mean cross reactivity index: 1.19). Characterization of HPIV3 epitopes may enable development of third-party VSTs to treat immune suppressed patients with HPIV infection.

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

“…Without the use of early activation markers, standard FCM demonstrated Actin responses that were 12-27 times higher for CD4+ and 5-30 times higher for CD8+ T cells compared to those measured using A-FCM. Similar range of background IFNγ responses to Actin were also recorded by others [42]. Such high background responses to self-antigens using standard FCM likely represent pre-assay antigen-unrelated responses, (as seen in Figure 4).…”

Transcriptomic analysis reveals optimal cytokine combinations for SARS-CoV-2-specific T cell therapy products

“…To facilitate translation to the clinic, PBMCs from 6 additional SARS-CoV-2 convalescent donors and 1 repeat donor were selected to expand a GMP-compliant product using G-Rex 10 bioreactors, as were used for our other VST products. 36,37 Six out of 7 donor-derived CSTs demonstrated numerically increased expansion in G-Rex 10 bioreactors in the setting of IL-15 + IL-7 compared with IL-4 + IL-7, with a mean fold expansion of 3.2 versus 1.9 (p = 0.08).…”

Pathogen-specific T Cells: Targeting Old Enemies and New Invaders in Transplantation and Beyond

“…106,107 Major advances in synthetic overlapping peptide pools, use of cytokines and culture vessels/bioreactors, along with the in-depth characterization of immunogenic epitopes/antigens presented through human leukocyte antigen (HLA) molecules, have since then facilitated the production process and provided a springboard to extend the breadth of pathogens that can be targeted by T cells. Indeed, over the last years, the pST cell therapy platform has been simplified and adapted to an array of pathogens which complicate immune-deficient states, including viruses such as BK polyomavirus (BKV), [108][109][110] human herpesviruses [varicella zoster virus (VZV), 111 human herpesvirus 6 (HHV6) 112] , respiratory viruses [influenza, 113 human parainfluenza virus-3 (HPIV3), 114,115 human metapneumovirus (hMPV) 116] , herpes simplex virus type 1 (HSV-1), 117 human papillomavirus (HPV), [118][119][120] Zika virus, 121 norovirus, 122 fungi such as AF, Aspergillus flavus, Aspergillus terreus, C. albicans, Candida krusei, Fusarium solani, Fusarium oxysporum, Rhizopus oryzae, Lomentospora prolificans [123][124][125][126] and recently the more complex Mycobacteria 127 (Table 2).…”