ABSTRACT Hematopoietic stem cell transplantation (HSCT) is a highly effective treatment, but serious virus infections occur in 82% of children undergoing HSCT at our institution and others. Current anti- viral drugs have inadequate response rates, prolong hospitalizations, and are frequently associated with organ toxicity. An alternative cellular therapy approach uses engineered viral specific T-lymphocytes (VSTs) manufactured from blood donated by a patient’s stem cell donor. VST therapy is highly effective when cells are infused in response to viremia, with response rates of over 80%. We seek to make a critical advance in this technology by testing whether scheduled administration of bone marrow donor derived VSTs 21 days after HSCT will be safe and at least as effective as our current pre-emptive treatment approach of only administering VSTs once viral reactivation or infection has occurred. We propose two specific aims. Specific Aim 1: Randomized comparison of donor-derived scheduled vs treatment VSTs in HSCT recipients to prevent viral infections. Hypothesis: Recipients of scheduled VSTs given 21 days after stem cell infusion will have a significantly lower frequency of viremia and invasive viral infections 100 days after HSCT than patients randomized to treatment use of VST’s. Specific Aim 2: Identify product characteristics that predict response to therapy and compare responses in those with and without exposure to viral ligand. Hypothesis: Specific product characteristics can be established that will identify VST products likely to be clinically effective, and VSTs will persist in scheduled VST recipients without stimulation from viral ligand. Aim 2a: We will study the T cell response to adenovirus, EBV, CMV or BK virus, and product persistence by ELISpot testing, TCR clonogram and VST persistence using TCR sequencing to define more and less effective VST products, and to determine if VSTs expand and persist if they are infused on a schedule into a person with no active viral replication to provide ligand. Aim 2b: We will systematically assess HLA restriction of presentation of viral antigens, using peptide mapping and single antigen cell lines (SALs). These data are vital for future “third party” use of VSTs in persons without donor derived product. This clinical trial may change the paradigm of treatment for viral infections if we are able to show that prophylactic VST infusion prevents infections.