Background Adoptive immunotherapy using banked virus-specific T-lymphocytes (VSTs) derived from partially HLA-matched donors has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT), although broad availability of this therapy is currently limited, and best methods for donor-recipient matching remain unclear. Objective To determine the feasibility and efficacy of third-party VSTs for the treatment of immunocompromised pediatric patients with refractory viral infections in a multicenter phase I/II consortium study. Design/Methods There are two arms to this study. Arm A are pediatric subjects who had infections with CMV, EBV, and/or adenovirus after hematopoietic stem cell transplant (HSCT) and had failed antiviral therapy, or who had toxicities related to anti-viral drug-related. Arm B enrolls patients with primary immunodeficiency disorders with refractory viral infection prior to HSCT. A VST bank containing products generated from 40 donors at Children's National was utilized, and antiviral HLA mapping was applied to identify the most suitable product for each patient referred. Patients received a dose of 2 × 10^7 VSTs/m2, with optional additional doses given 28 days after the initial VST infusion. Viral PCRs were monitored for efficacy, and VST persistence and expansion in vivo was measured via IFN-g ELISpot and flow cytometry. Results Twenty-three patients were enrolled from 11 study sites to date. Twenty-two were treated in Arm A (after HSCT), at a median time of 95 days post-HSCT (range 16-476). Fourteen patients were treated for adenovirus and 9 for CMV. Patients received a median of 1 infusion (range 1-3). Infused VSTs were matched at a median of 3/10 HLA alleles (range 2-5), and 22 had confirmation of antiviral activity through at least one shared HLA allele. Of 18 evaluable patients, 14 had antiviral responses at 1-month post-infusion (4/5 with CMV, and 10/13 with adenovirus, Figure 1), including 7 who had complete resolution of viremia. No GVHD was observed after infusion, and 17 of 23 (74%) are virus free up to 15 months post-infusion. Virus-specific T-cell activity was higher by day 45 (mean 177 IFN-g SPW) post-infusion than at baseline (mean 13 IFN-g SPW, p=0.045). VSTs remained detectable for up to 3 months via flow cytometry staining of discordant HLA alleles (Figure 2). Conclusions Third-party VSTs utilized from a single bank can be distributed and administered in a multicenter consortium setting, and are safe and feasible for the treatment of refractory viral infections in pediatric patients. Preliminary efficacy data shows high rates of response and long-term survival. Studies of targeting strategies and characterization of T-cell epitopes are ongoing.