Background Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences. Methods Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome. Results We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates. Conclusions Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.

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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.

Mycobacterial Infections can be severe in patients with T-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. Restoration of T-cell immunity via stem cell transplantation facilitates control of mycobacterial infections, but presence of active infections during transplantation is associated with a higher risk of mortality. Adoptive T cell immunotherapy has been successful in targeting viruses, but has not been attempted to treat mycobacterial infections. We sought to expand and characterize mycobacterial-specific T-cells derived from healthy donors in order to determine suitability for adoptive immunotherapy. Mycobacteria-specific T-cells (MSTs) were generated from 10 healthy donors using a rapid ex vivo expansion protocol targeting five known mycobacterial target proteins (AG85B, PPE68, ESXA, ESXB, and ADK). MSTs were compared to T-cells expanded from the same donors using lysate from M. tuberculosis or purified protein derivative from M. avium (sensitin). MST expansion from seven patients with primary immunodeficiency disorders (PID) and two patients with IFN-γ autoantibodies and invasive M. avium infections. MSTs expanded from healthy donors recognized a median of 3 of 5 antigens, with production of IFN-γ, TNF, and GM-CSF in CD4 + T cells. Comparison of donors who received BCG vaccine ( n = 6) to those who did not ( n = 4) showed differential responses to PPE68 ( p = 0.028) and ADK ( p = 0.015) by IFN-γ ELISpot. MSTs expanded from lysate or sensitin also recognized multiple mycobacterial antigens, with a statistically significant differences noted only in the response to PPE68 ( p = 0.016). MSTs expanded from patients with primary immunodeficiency (PID) and invasive mycobacterial infections showed activity against mycobacterial antigens in only two of seven subjects, whereas both patients with IFN-γ autoantibodies recognized mycobacterial antigens. Thus, MSTs can be generated from donors using a rapid expansion protocol regardless of history of BCG immunization. Most tested PID patients had no detectable T-cell immunity to mycobacteria despite history of infection. MSTs may have clinical utility for adoptive immunotherapy in T-cell deficient patients with invasive mycobacterial infections.