Background: Poly-ICLC (Hiltonol, Oncovir Inc) is a synthetic dsRNA complex which directly activates DCs and also triggers NK cells to kill tumor cells. Compared to LPS and R848, Poly-ICLC is the most potent TLR adjuvant due to its induction of cytokines such as IL-12 in the absence of IL-10, and maintenance of high levels of CD80 and CD86 in DCs. This study evaluated the therapeutic potential of TLR3 activation by adding Poly-ICLC to an NY-ESO-1 protein vaccine with and without Montanide in surgically resected stage IIB-IV melanoma patients. Methods: In Phase I of the study, patients received subcutaneous injections of 100μg NY-ESO-1 protein and 1.1mL Montanide emulsified in escalating doses of Poly-ICLC: 0.35mg (cohort 1; N = 3), 0.70mg (cohort 2; N = 3), or 1.4mg (cohort 3; N = 3). Vaccinations were repeated every 3 weeks for a total of 4 cycles. In Phase II of the study, patients were randomized to subcutaneous vaccination of 100μg NY-ESO-1 protein with 1.4 mg Poly-ICLC, the dose established in the Phase I of the study, (Arm A; N = 12) or with 100μg NY-ESO-1 protein, 1.4mg Poly-ICLC and 1.1ml Montanide (Arm B; N = 12). Vaccinations were repeated every 3 weeks for a total of 4 cycles. Blood samples were collected at baseline, one week after each cycle of vaccination, and at follow-up visit for the assessment of NY-ESO-1-specific humoral and cellular immune responses. Results: The vaccine combination was generally well tolerated, with no grade 3 or 4 adverse events or study-related deaths. Most patients experienced mild grade 1 or 2 (CTCAE criteria v4) adverse reactions. The most common reactions were influenza-like symptoms and injection site reactions. Five patients did not complete the study due to disease recurrence or personal choice (unrelated medical issue). Analysis of T cell responses shows induction of NY-ESO-1-specific humoral and cellular immune responses. Conclusions: This study demonstrates the safety and immunogenicity of Poly-ICLC as an adjuvant for NY-ESO-1 protein vaccination. Clinical trial information: NCT01079741