These features, together with their capacity to efficiently adsorb protein Ags, to be readily internalized by APC, and to enhance immune responses to Ag both in vitro and in vivo, make them good potential delivery systems for vaccines, and in particular that of HIV vaccines for the developing world. Manipulation of the YC-wax NP surface charge with surfactants, provides optimal flexibility to adsorb different types of Ag [30]. In this study, Ags as diverse as TT, BSA, and HIV-1 gp140 were efficiently adsorbed to both negatively and positively charged NP. In addition, the surface charge flexibility also facilitated

co-adsorption of more than one molecule onto the NP surface as shown by co-adsorption MLN2238 of Ag with CpGB and PolyI:C. After screening a large range of wax NP, three different types

were selected according to their low toxicity, Ag adsorption efficiency, and cell internalization profile, i.e., YC-SDS, YC-NaMA, and YC-Brij700-chitosan. The first two NP had a net negative charge, whereas the third one was highly positive, a characteristic defined by the presence of the carbohydrate chitosan. We determined adsorption of gp140 to these NP by three different methods: Z potential, Bradford assay, and ELISA. All three methods provided strong evidence of effective Ag adsorption to NP. In addition, the ELISA assay find more suggested that antigenicity was unaffected, which may represent an advantage over Ag encapsulation as reported previously for a form of HIV-gp120 by Singh et al. [31]. Flow cytometry and confocal microscopy studies clearly showed that Ag-adsorbed YC NP were readily internalized by APC, and that these NP were subsequently tracked within endolysosomes, Libraries suggesting that the NP may have the capacity to deliver Ag into the Ag processing Levetiracetam and presentation compartment. Naked YC-wax NP did not induce cytokine/chemokine production or up-regulation of co-stimulatory molecules on DC in vitro, nor induced visible signs of inflammation after both mucosal and systemic administration in vivo (data not shown). This lack of DC activation by naked NP is important especially if used at the urogenital tract,

because such cell activation would induce mucosal inflammation at this level that may facilitate HIV infection. Antigen-adsorbed YC-wax NP (TT in human PBMC and gp140 in mouse splenocytes) enhanced T-cell proliferation responses in vitro. The response to TT by human PBMC was greatly enhanced by co-adsorption with CpGB (Fig. 3B) but not with PolyI:C (data not shown). CpGB on its own enhanced cellular proliferation, and we speculate that CpGB induces non-specific proliferation of PBMC most likely due to polyclonal B cell activation, as has been described previously [32]. Nevertheless, the enhanced proliferation observed with co-adsorption of TT + CpGB particles was significantly greater than the additive effect of TT plus CpGB alone.