Viral infections remain a major cause of morbidity and mortality post-transplant. To address this issue, and with NHLBI-PACT support, we have made virus-specific T-cell lines (VSTs) with activity against 5 common post-transplant viruses (EBV, CMV, Adv, BK, HHV6), using a simplified 10-day. To date 48 clinical-grade multivirus (m)VSTs have been generated. By exposing 30x10ˆ6 PBMCs to overlapping peptide libraries spanning Adv (Hexon, Penton), CMV (pp65, IE1), EBV (LMP2, EBNA1, BZLF1), BK (Large T, VP1) and HHV6 (U11, U14, U90) antigens we expanded a median of 35.7x10ˆ7 polyclonal cells over 9-11 days. pVST specificity was dependent on the donor's prior viral exposure; 45/48 lines had Adv activity (Hexon: 470±71; Penton: 366±86 SFC/2x10ˆ5), 26/48 against CMV (IE1: 356±157; pp65: 1048±446), 37/48 against EBV (LMP2: 137±76; EBNA1: 123±52; BZLF1: 99±7), 28/48 against BK (Large T: 123±61; VP1: 208±89) and 29/48 against HHV6 (U90: 109±78; U11: 37±17; U14: 84±26). None of the lines reacted against recipient cells. To date 11 allogeneic HSCT recipients have received 0.5-2x10ˆ7 pVSTs/m2 without adverse events. Three patients were infused prophylactically while 8 were treated for one or more active infections. A single infusion successfully controlled active infections associated with all our targeted viruses: CMV (2 CR, 1 PR); EBV (5 CR); Adv (1 CR); HHV6 (2 CR) and BK (5 CR, 1 PR, 1 NR). Of note, all 3 patients with BK hemorrhagic cystitis had marked improvement/disappearance of hematuria post-mVSTs. Our only “mixed” responder cleared EBV and HHV6 but not BK following the infusion of a line that lacked specificity for this virus, likely reflecting the seronegative status of the donor. Thus, infusion of mVSTs has been safe and clinically effective against up to four simultaneous/sequential infections in a single HSCT recipient. We are planning to assess the activity of “off the shelf” 3rd party pVSTs for broader implementation.