Background: Recent data show clinical activity of cancer vaccines containing a defined cancer antigen, and a peptide vaccine for melanoma. However, immune responses to peptide vaccines are often transient and of low magnitude. The most common adjuvant for peptide vaccines for melanoma has been an incomplete Freund’s adjuvant (IFA), which may have suboptimal adjuvant properties. Toll-like receptor (TLR) agonists offer the potential to improve the magnitude and persistence of antitumor T cell responses, either in combination with IFA or alone. CD40 ligation at the vaccine site microenvironment (VSME) may also improve adjuvant activity of TLR agonists and may be provided by CD4 T cell activation. We report a clinical trial of a multipeptide vaccine using TLR agonists and IFA, with correlative studies in 3 immunologic compartments. Methods: This trial is enrolling patients with resected stage IIB-IV melanoma (n=48) and is designed to evaluate the safety and immunogenicity of vaccination with peptides and either of 2 toll-like receptor agonists (TLR3 agonist polyICLC; TLR4 agonist endotoxin), with or without IFA. Patients are vaccinated 6 times over 12 weeks with 12 Class I MHC-restricted nonamer peptides. An immunogenic tetanus helper peptide is included to activate CD4 T cells in the VSME and secondarily to ligate CD40. Goals include safety assessment, measures of CD8 T cell responses, and characterization of cellular and molecular events induced in the blood, VSME and vaccine-draining node (sentinel immunized node, SIN), as well as a preliminary assessment of whether vaccination with TLR agonists improves the persistence of CD8 and CD4 T cell responses to melanoma antigens compared to prior studies with IFA. This includes a first-in-humans evaluation of the safety and immunogenicity of LPS, a classic TLR4 agonist, as a vaccine adjuvant. Thus, there is a novel dose-escalation phase with this adjuvant, which has been safely administered in other settings by intravenous and inhalation routes. An aim of this study is to identify an improved adjuvant for use in future trials combining peptides with other immune therapies. Clinical trial information: NCT01585350.