= not significant

= not significant. malignant B16-F10 melanoma tumor. This study provides the first evidence that a combination of antigen-mRNA and -GalCer can be used as an effective antitumor vaccine, inducing strong innate and adaptive immune responses. 1.?Introduction Cancer immunotherapy is a therapeutic strategy that exploits the natural ability of the immune system to recognize and kill cancer cells.1 Several cancer immunotherapies have been proposed such as vaccines and antibody- and cell-based therapies.2?4 Vaccines, compared to other immunotherapies, offer the advantage of promoting both humoral and cell-mediated immune responses and, more importantly, they can create immunological memory, which could ensure the promotion of an immunological response of greater magnitude and faster kinetics in the case of tumor recurrence.5 Nucleic acids have been extensively investigated in the context of vaccine development for the priming of tumor-specific cytotoxic T lymphocytes (CTLs).6?8 The use of nucleic acid-based vaccines is a novel approach developed to address the issues associated with more traditional antigen forms, such as proteins and peptides. They are easily produced, provide opportunities for molecular engineering, and have the potential to promote both innate and adaptive immune responses.7,9 Moreover, unlike peptide-based vaccines, nucleic acid-based vaccines expressing complete genes are not a priori human leucocyte antigen (HLA)-restricted.7,9 Particularly, mRNA-based vaccines are attractive because they retain the same appealing characteristics of DNA-based vaccines and also offer additional benefits, such as superior immunogenicity, and, unlike DNA, mRNA only needs to gain entry to the cytoplasm, where translation occurs, in order to achieve cell transfection.8?10 Moreover, mRNA does not have any risk of integration into the genome and therefore has no oncogenic potential and can be easily synthesized.10 Finally, mRNA can act not only as a source for antigen but also as an adjuvant by providing costimulatory signals, for example, via toll-like receptor (TLR) 3, TLR7, and TLR8, to amplify the immune response.8,10 The main challenge associated with the use of mRNA for vaccine development is its Z-VAD(OH)-FMK sensitivity toward catalytic hydrolysis by omnipresent ribonucleases.9,10 Unprotected mRNA is highly unstable under physiological conditions and hence unsuitable for broad therapeutic applications. Because of the potential advantages of mRNA-based vaccines, intense efforts have been made toward the stabilization of mRNA in vivo.9,11 Several strategies have been proposed for mRNA delivery, including viral vectors and nonviral vectors.12?19 Nonviral vectors represent a simple, flexible, and, more importantly, safer alternative to viral vectors. Thanks to their Goat polyclonal to IgG (H+L)(HRPO) relatively simple quantitative production and their low host immunogenicity, nonviral vectors are Z-VAD(OH)-FMK attractive tools in vaccine strategies. With the development of new biocompatible materials and innovative fabrication approaches, nonviral vectors are becoming the preferred vehicle to deliver mRNA. A variety of nonviral vectors have been explored as platforms for mRNA-based vaccine development, focusing mainly on the use of lipids and polymers.20?26 An additional advantage associated with the use of nonviral vectors for vaccine development is that they can facilitate codelivery of antigens with adjuvants, enhancing significantly the therapeutic efficacy of the vaccine.27?30 The codelivery of antigens with adjuvants has been extensively investigated for peptide-based vaccines, but little Z-VAD(OH)-FMK is known whether mRNA-based vaccines may benefit from its combination with vaccine adjuvants. In this framework, -GalCer represents an optimal new class of vaccine adjuvant as it acts as an effective link between innate and adaptive antitumor immunity. Indeed, -GalCer, also known as KRN7000, is an invariant natural killer T (iNKT) cell antigen presented on CD1d of antigen-presenting cells (APCs).31?33 Previous studies have reported that -GalCer shows stronger ability to generate antigen-specific CD8+ T cells, compared to TLR ligands.29,34,35 In response to -GalCer, invariant iNKT cells rapidly produce immunostimulatory cytokines, particularly interferon- (IFN-), and elicit the induction of several costimulatory molecules.31?33 These events promote the activation APCs, which release key Th1 cytokines (e.g., interleukin (IL)-12), and the downstream activation of CD4+ and CD8+ T lymphocytes, as well as NK cells and B cells, with important effects on the magnitude and effectiveness of the immune responses.31?33 Despite this, there is a limitation for the clinical application of -GalCer; its poor hydrophilicity makes it essential to incorporate -GalCer into a vector in order to improve its solubility, uptake, and immunogenicity.29,36,37 In addition, it has been demonstrated that vectorization of -GalCer can prevent iNKT cell anergy, which usually occurs after repeated injections of -GalCer.29,35,36,38 In Z-VAD(OH)-FMK this study, we have developed a lipopolyplex vector termed multi-LP, which consists of a poly-(-amino ester) polymer (PbAE)/mRNA polyplex core entrapped into a lipid Z-VAD(OH)-FMK shell composed of multivalent cationic lipid (MLV5), 1,2-dioleoyl-for 5 min, treated with.