Biomaterial vaccines offer new capabilities that can be exploited for both infectious disease and cancer. We recently developed a novel vaccine platform based on self-assembly of immune signals into immune polyelectrolyte multilayers (iPEMs). These iPEM vaccines are electrostatically assembled from peptide antigens and nucleic acid-based toll-like receptor agonists (TLRas) that serve as molecular adjuvants. Gold nanoparticles (AuNPs) coated with iPEMs stimulate effector cytokine secretion in vitro and expand antigen-specific T cells in mice. Here we investigated how the dose, injection route, and choice of molecular adjuvant impacts the ability of iPEMs to generate T cell immunity and anti-tumor response in mice. Three injection routesintradermal, subcutaneous, and intramuscularand three iPEM dosing levels were employed. Intradermal injection induced the most potent antigen-specific T cell responses and, for all routes, the level of response was dose-dependent. We further discovered that these vaccines generate durable memory, indicated by potent, antigen-specific CD8(+) T cell recall responses in mice challenged with vaccine 49 days after a prime-boost immunization regimen. In a common exogenous antigen melanoma model, iPEM vaccines slowed or stopped tumor growth more effectively than equivalent ad-mixed formulations. Further, iPEMs containing CpGa TLR9awere more potent compared with iPEMs containing polyIC, a TLR3a. These findings demonstrate the ability of iPEMs to enhance response to several different classes of vaccine cargos, supporting iPEMs as a simple vaccine platform that mimics attractive features of other nanoparticles using immune signals that can be self-assembled or coated on substrates. Biotechnol. Bioeng. 2017;114: 423-431. (c) 2016 Wiley Periodicals, Inc.