We observed significant increases in the numbers of antigen-specific IFN-producing cells in mice receiving either g

We observed significant increases in the numbers of antigen-specific IFN-producing cells in mice receiving either g.g. protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes. Keywords: adaptive immunity, DNA adjuvant, Bromperidol flagellin, NLRC4, TLR5 1. Introduction DNA-vaccines are promising tools with great potential for combating infectious disease. Non-living/replicating DNA vaccines have several advantages over living viral delivery vectors, such as lower production costs, increased stability, a higher overall safety profile, and recent evidence indicates that they can provide humans with protective immunity to viral infection [1]. However, living viral vectors used CD24 in DNA vaccine settings (such as Adenovirus) can still elicit stronger immune responses in humans than naked DNA. Yet in the case of adenovirus, evidence suggests that they may not promote the desired immune responses to the recombinant antigen. As results from clinical trials show, the use of a viral vector can, possibly as a consequence of the anti-vector immunity, potentially even enhance the risk of infection with certain pathogens [2]. These observations emphasize the critical need to continue research on methods for adjuvanting minimal, non-living/replicating DNA vaccines. There are many approaches to improving the efficacy of plasmid DNA vaccines such as choice of delivery method, modifications of antigen location/stability/presentation, and the use of immunopotentiators [3]. Here, we investigate a formulation-compatible immunopotentiating adjuvant, with the potential to activate innate and adaptive immune responses through Toll-like Receptor Bromperidol 5 (TLR5) and/or possibly Nod-like Receptor (NLR) family members NLRC4 and Naip5 [4]. This approach employs plasmid DNA encoding a secreted form of flagellin (FliC) from as an adjuvant in DNA vaccinations. This adjuvant allows mammalian cells to create an environment of sterile-inflammation, thus mimicking natural infection in a Bromperidol safe manner and promoting adaptive immune responses to co-delivered DNA-encoded antigens [5]. This approach is unique in that it uses a plasmid-encoded agonist of innate immune receptors to activate a large variety of molecules capable of promoting adaptive immunity, unlike many other approaches which use single cytokines or chemokines [3]. A major benefit of this system is that it works without physically linking the antigen to flagellin. This ensures that the antigen is properly folded and processed and constitutes a major practical advantage as the system is flexible and can be applied with ease to various antigens without the need for time-consuming development of fusion-constructs. Recombinant flagellin produced in bacteria is currently being used by many as an experimental adjuvant to promote humoral and cellular immunity against microbial pathogens [6,7,8]. However, the use of flagellin in protein-form presents formulation and stability issues with non-living/replicating DNA vaccines such as plasmids. In previous work, we vaccinated mice epidermally, using a gene-gun, with a transmembrane-anchored form of flagellin (pFliC-Tm) and secreted ovalbumin (pOVA). We observed significant increases in antigen-specific serum IgG levels compared to pOVA alone as well as strong antigen-specific CD4+/8+ cellular immune responses [5]. Importantly, we also showed that the pFliC-Tm adjuvant delivered with a DNA-encoded nucleoprotein gene from Influenza A resulted in a strong antigen-specific CD4+/8+ cellular immune response which correlated with protection from lethal virus infection [5]. This work demonstrated that pFliC-Tm acts as an adjuvant when delivered dermally however it is not clear whether this is the optimal route for eliciting the broadest or Bromperidol strongest immune responses. Additionally, not all DNA vaccination approaches are applied dermally therefore further studies of adjuvant effects induced by various delivery routes are warranted. The HIV-1 pandemic has been estimated to have according with WHO/UNAIDS reports been spread globally and infected individuals exist in all countries in the world. So far, only a few experimental vaccine studies have shown promising and protective results in clinical trials. Thus there is a continued need to find more efficient vaccination strategies.

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