Identifying Novel Immunodominant Epitopes within Human Parainfluenza Virus-3 Matrix Protein

Human Parainfluenza Virus-3 (HPIV3) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies. Recent preclinical studies demonstrated T-cell recognition of HPIV3 viral proteins, and a phase I study adoptively transferring virus specific T cells (VSTs) targeting HPIV3 to patients following bone marrow transplantation is underway (NCT03180216). Hence, we sought to identify immunodominant epitopes within the Matrix protein based on the extent of the T-cell responses.

VSTs were generated from the peripheral blood of healthy donors by ex vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 Matrix protein, which has been identified as the HPIV3 antigen eliciting the most robust response (Mclaughlin et al., J Cytotherapy 2016). Epitope mapping was performed using IFN-g ELIspot with combinatorial 15-mer peptide pools (Straathof et al., J Immunology 2005). Flow cytometry was used to characterize the products with intracellular cytokine staining.

Initial mapping with the peptide pools showed activity in 16 of the 19 pools. Pools which elicited activity in 5 or more products had mean spots per well (ASC)/1x10^5 cells range 89 to 217, versus 0.6 ASC/1x10^5 in the negative controls. We identified 13 novel immunodominant epitopes (Table) within Matrix protein identified in 9 tested VST products, with 4 clustered epitopes (peptides 82-85) at the C-terminus, which contains a leucine residue critical for regulating virion release (Ding et al, 2017). Five (55%) donors recognized epitopes in this domain. Individual peptides which elicited activity had mean (ASC)/1x10^5 cells range 13 to 224, versus 0.4 ASC/1x10^5 in the negative controls. VST products were polyfunctional releasing both IFN-γ and TNF-α in response to the newly identified epitopes, and preliminary flow cytometry data showed that the majority of these epitope specific responses were HLA Class II restricted.

In conclusion, we have identified several novel epitopes within the HPIV3 Matrix protein. Comprehensive classification of the breadth and stability of HPIV3 epitopes is critical for the development of third-party "off the shelf" virus specific T cell therapeutics. Therefore, these results will facilitate the development of clinical studies using partially-HLA VSTs as an "off the shelf' treatment for the treatment of immune suppressed patients with severe respiratory infection related to HPIV3.

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