Cytotoxic T lymphocyte (CTL) epitope peptide-based vaccines are widely used in cancer and infectious disease therapy.We previously generated an immune-tolerant elastin-like polypeptides (iTEPs)-based carrier to deliver a peptide CTL vaccine and enhance the efficiency of the vaccine.To further optimize the vaccine carrier,we intended to potentiate its function by designing an iTEP-based carrier that was able to deliver adjuvant and a vaccine epitope as one molecule.Thus,we fused a 9-mer H100,a peptide derived from the high-mobility group box 1 protein (HMGB1) that could induce activation of dendritic cells (DCs),with an iTEP polymer to generate a new iTEP polymer named H100-iTEP.The H100-iTEP still kept the feature of reversible phase transition of iTEPs and should be able to be used as a polymer carrier to deliver peptide vaccines.The expression levels of CD80/CD86 on DCs were assessed using flow cytometry.The iTEP fusion-stimulated IL-6 secretion by DCs was measured with ELISA.Activation of antigen-specific CD8+ T cells induced by iTEP fusions was examined through a B3Z hybridoma cell activation assay.In vivo CTL activation promoted by iTEP fusions was detected by an IFN-y-based ELISPOT assay.The iTEP fused with H100 could induce maturation of DCs in vitro as evidenced by increased CD80 and CD86 expression.The iTEP fusion also promoted activation of DCs by increasing secretion of a proinflammatory cytokine IL-6.The N-terminus or C-terminus fusion of H100 to iTEP had a similar effect and a reduced form of cysteine in iTEP fusions was required for DC stimulation.iTEP fusions potentiated a co-administrated CTL vaccine by increasing an antigen-specific CTL response in vitro and in vivo.When the H100-iTEP was fused to a CTL epitope to generate a one-molecule vaccine,this self-adjuvanted vaccine elicited a stronger antigen-specific CTL response than a vaccine adjuvanted by Incomplete Freund’s Adjuvant.Thus,we have successfully generated a functional,one-molecule iTEP-based self-adjuvanted vaccine.