ABSTRACT We have previously reported in Nature that patients with newly-diagnosed glioblastoma (GBM) randomized to receiving vaccines against Cytomegalovirus (CMV) using a potent vaccine site preconditioning regimen had a statistically significant increase in progression-free survival (PFS) and overall survival (OS). Half of the patients treated this way were still alive nearly 5 years later despite having no genetic markers predicting long-term survival. These results have been repeated in an additional cohort which showed a median survival of 44.1 months with ~36% of patients alive at 5 years. These results are remarkable because GBM remains uniformly lethal with a median OS of < 21 months despite surgical resection, high dose radiation therapy, chemotherapy, and tumor-treating fields, and only 10% of patients typically live past 5 years. In addition to demonstrating the potential for efficacy, our preliminary clinical and laboratory studies demonstrated that preconditioning the vaccination site with tetanus/diphtheria (Td) recall antigens increased DC migration to the draining lymph nodes (DLNs), which predicted PFS and OS as did the production of polyfunctional, CMV-specific T cells. These T cell responses were enhanced by GM-CSF at the vaccine site and pre-vaccination lymphodepletion with standard of care (SOC) temozolomide (TMZ), but inhibited by subsequent adjuvant doses of TMZ. Moreover, mechanistic studies in mice and humans revealed that efficacy was also dependent on producing high systemic levels of the chemokine (C-C motif) ligand 3 (CCL3). We believe these results warrant confirmation in a larger series of patients. Our Specific Aims are: 1. To conduct a larger Phase 2 trial of CMV pp65-loaded DC vaccination in patients with GBM. Patients with CMV positive, newly diagnosed GBM will receive serial vaccines with CMV pp65-loaded DCs with GM-CSF and Td vaccine site preconditioning. GM-CSF and Td vaccine site preconditioning will be employed because they have been shown to enhance DC migration and increase polyfunctional T cell responses in prior studies. TMZ will be given prior to vaccination to induce homeostatic proliferation of the vaccine-induced T cell responses but not given subsequently to prevent killing of vaccine-induced T cells. 2. To confirm predictors of survival. In our prior studies DC migration to draining lymph nodes, systemic and local CCL3, and CMV pp65-specific polyfunctional T cells predicted PFS and OS. Here we will collect samples to confirm these predictors.