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)/1x105 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.

Keywords: T-cell; anti-viral; bone marrow transplant; immunotherapy; parainfluenza.

Copyright © 2020 Harris, Horn, Grant, Lang, Sani, Jensen-Wachspress, Kankate, Datar, Lazarski, Bollard and Keller.

Figures

Figure 1

(A) Virus-specific T-cells (VST) Product Phenotyping. Phenotyping data of the hexaviral-specific T cell products specific for cytomegalovirus (CMV), Epstein Barr virus (EBV), AdV, HHV6, BKV, and HPIV3 (product #s 1-4, 6, 8-10) and monoviral-specific T cell products specific only for HPIV3 (product #s 5, 7) was obtained using flow cytometry. Phenotypes, including memory differentiation status panel, are reported as % of total CD3+ cells. Product #5 was unavailable for memory/differentiation phenotyping. Definitions of phenotypes are as follows: Naïve: CD45RA+CD45RO-CCR7+CD62L+CD95- Stem Cell Memory: CD45RA+CD45RO-CCR7+CD62L+CD95+ Central Memory: CD45RA-CD45RO+CCR7+CD62L+ Effector Memory: CD45RA-CD45RO+CCR7-CD62L- Terminal Effector Memory: CD45RA-CD45RO-CCR7-CD62L- (B) Overall HPIV3 Matrix specificity by product. All 10 viral-specific T cell products were stimulated with HPIV3 pepmix. Response was measured as spot forming units per well (SFU/1x105 cells) by anti-IFN-γ ELISpot assay. Unstimulated T-cells (CTL only) and stimulation with actin pepmix (irrelevant antigen) were used as negative controls.

Figure 2

Peptide Pool specificity by product. Virus-specific T-cells (VSTs) were stimulated with combinatorial HPIV3 peptide pools. Response was measured as spots per well (SFU/1x105 cells) by anti-IFN-γ ELISpot assay. Unstimulated T-cells (CTL only) and stimulation with actin (irrelevant antigen) were used as negative controls.

Figure 3

(A) Cross reactivity between HPIV3 and HPIV1. Virus-specific T-cells (VSTs) were stimulated with individual HPIV3 peptides and corresponding HPIV1 peptides. Response was measured as spots per well (SFU/1x105 cells) by anti-IFN-γ ELISpot assay, and mean responses were calculated among responding products. Black bars represent HPIV3 peptides and gray bars represent HPIV1 peptides. (B) CD4+ T Cell Responses against HPIV3 and HPIV1 peptides. Intracellular flow cytometry was performed on VSTs following stimulation with Actin, SEB, HPIV3 or HPIV1 peptides and anti-CD28/CD49. After 2 h stimulation with peptides, Brefeldin A was added for an additional 4 h following which cells were labeled with dead cell exclusion dye, Fc Receptor block and antibodies against CD3, CD4, CD8, TNF-α, IFN-γ, and CD95. Following fixation and permeabilization cells were labeled intracellularly with antibodies targeting TNF-α, IFN-γ, Percentage of CD4+ and CD8+ T-cells producing both IFNγ+ and/or TNFα+ were measured.

Figure 4

(A) HLA Restriction Mapping. Two phytohemagglutinin (PHA) blast lines (each partially matched to a Virus-specific T-cell (VST) product at HLA-DR*15:01 and HLA-DQ*03:01, respectively) were pulsed with HPIV3 peptides 82, 83, and 84 for 60 min, triple washed, and then presented to VSTs. T cell activity was detected using anti-IFN-γ ELISpot. (B) HPIV3 Matrix diagram with epitopes and C-terminal domain. HPIV3 Matrix protein contains 353 amino acids. Diamonds represent the novel epitopes identified during mapping. The oligomerization domain at the C-terminus contains 7 out of 12 epitopes, including the three most immunodominant peptides, 82–84. The black arrow denotes the L302 residue, which is a target of ubiquitination during virion production.