Despite recent advances, there is a great need for more potent formulations to enhance immunogenicity of vaccines. Pro-active induction and/ or harnessing of beneficial innate immune responses may be the mechanism underlying the effectiveness of certain adjuvants to significantly contribute to the ability of vaccines to generate adaptive immune responses. Several classes of immune receptors have been targeted for novel adjuvant development. An important emerging class of immune receptors is the SLAM family of receptors. We endeavored to develop a strategy to improve the efficacy of vaccines by incorporation of proteins known to be important in SLAM mediated signaling. In this dissertation, we described that coexpression of the SLAMꞌs adaptor, EAT-2, along with pathogen derived antigens facilitates induction of beneficial innate immune responses, resulting in improved induction of antigen-specific adaptive immune responses. We utilized recombinant Adenovirus-based vaccines expressing murine EAT-2 along with HIV-1/Gag or Plasmodium falciparum derived circumsporozoite (CS) protein. As compared to appropriate controls, rAd5 vectors expressing EAT-2 facilitated bystander activation of NK-, NKT-, B-, and T-cells early after their administration into animals. EAT-2 expression also augments the expression of surface maturation markers of antigen presenting cells (CD40, CD80, CD86, MHC-II, and CCR7). Indeed, this multi-tiered activation of the innate immune system by vaccine mediated EAT-2 expression enhanced the induction of antigen specific cellular immune responses. We also compared the utility of vaccine mediated expression of EAT-2 relative to another innate immune stimulator, a recombinant TLR agonist derived from Eimeria tenella, referred to as rEA. We confirmed that rEA activates multiple immune cell types and elicits the induction of pleiotropic pro-inflammatory cytokines in a MyD88-dependent manner. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. However, we uncovered a potent immunosuppressive activity inherent to the combined expression of the CS protein and rEA, in contrast to continued enhancement of CS specific adaptive immune responses by use of the EAT-2 adjuvant. We subsequently went on to partially unveil the mechanism underlying the ability of the EAT-2 adaptor protein to regulate the induction of adaptive immune responses. We demonstrate that EAT-2 expression specifically prevents vaccine induced CRACC upregulation on APCs in a MAPK-dependent mechanism. Confirming these results, utilization of a mutated SH-2 domain form of EAT-2 adaptor (EAT-2(R31Q) failed to prevent CRACC upregulation. We have also demonstrated that EAT-2 expression triggers the production of several cytokines and chemokines from macrophages in a MAPK-dependent and independent mechanisms. Future studies will further expand upon these findings. We leave our readers with the view that, understanding the molecular mechanism and the role of SLAM/ EAT-2 and rEA adjuvant activity, will allow for the development of next generation vaccine platforms for use in a number of immunotherapy approaches targeting HIV-1 and Malaria derived antigens, as well as numerous other vaccine targets in general.
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