In non-depleted mice, RFP+ cells were detected in the lymph node, while DC-depleted mice contained no detectable RFP+ cells above the level of background fluoresence (Fig 3B). levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node comparable to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. strong class=”kwd-title” Keywords: Adjuvant, dendritic cell, replicon, alphavirus INTRODUCTION Vaccines have been tremendously successful at limiting and even eradicating many diseases, yet techniques that were successfully used to develop existing vaccines have often been inadequate to induce protective immunity with new vaccine candidates. For this reason, new tools and approaches are necessary for the next generation of vaccines. In the development of new vaccines, dendritic cell (DC) activation and antigen uptake are major goals, for these cells are central mediators of the link between innate and adaptive immunity and thus have the capacity to strongly amplify the magnitude of an immune response [1, 2]. A past example of a highly effective DC-targeting vaccine is the yellow fever vaccine, PDGFRA in which an attenuated yellow fever virus infects DCs in humans, inducing robust immunity [3]. To achieve activation of DCs that will potently enhance an immune response one of the most promising approaches is to utilize novel adjuvants. Adjuvants are compounds that strengthen and expand the scope of the immune response to a co-delivered antigen, often by activation of pattern recognition receptors in DCs and other APCs [4]. Adjuvant selection is critical, as many adjuvants generate a skewed immune response [5], in contrast to live-attenuated vaccines such as the smallpox and yellow-fever vaccines which produce a more balanced response [6]. A highly effective adjuvant may therefore be one that resembles a live virus and is able to target and activate dendritic cells. Alphaviruses show promise as the basis for such an adjuvant, for many alphaviruses have a tropism for DCs [7] and the alphavirus Venezuelan equine encephalitis virus (VEE) can enhance the immune response to antigen delivered after contamination [8, 9]. A promising VEE-based adjuvant is usually VEE replicon particles (VRP) [10C12], which consist of the wildtype VEE capsid and envelope encapsulating a truncated VEE genome encoding only the nonstructural protein sequence [13]. Because their genome lacks the structural protein sequences, VRP are able to infect and replicate within cells but cannot form new particles and propagate, rendering them safe for use in humans [14, 15]. VRP were originally conceived as antigen expression vectors in which a vaccine antigen was encoded in the VRP genome [13, 16C19]. While VRP expression vectors have been used successfully to induce immunity, it is possible that innate immune shutdown of VRP replication will limit production of antigenic mass, FR194738 free base FR194738 free base whereas use of VRP as an adjuvant allows antigen dose to be controlled independently of the VRP dose. VRP have the further advantage that they can be rapidly combined with any antigen. When injected into mice with a soluble antigen, VRP increase the level of antigen-specific serum IgG and mucosal IgA, and also augment the CD8 T cell response to antigen [10C12]. VRP adjuvant activity has also been exhibited in rats (unpublished data) and FR194738 free base non-human primates [20]. When included in an immunization, VRP improve vaccine-mediated protection to challenge by FR194738 free base several pathogens including norovirus and influenza [20, 21]. Blocking mucosal entry of pathogens is key to protection from many diseases [22, 23], so the ability of VRP to establish a mucosal immune response to antigen is an outstanding feature of this adjuvant. Furthermore, mucosal immunity is usually rarely generated in response to parenteral delivery of antigen and adjuvant, and this non-classical mucosal immune induction may be a valuable tool to circumvent some of the obstacles encountered in mucosal antigen delivery [22]. These various qualities of VRP make them a promising candidate for use as an adjuvant in human vaccines. DCs have been hypothesized to play a FR194738 free base role in VRP adjuvant.