(B,C) Corresponding average Log IgG2b/Log IgG1 and Log IgG2c/Log IgG1 ratios are indicative for the quality of the immune response, where values higher than 1 (dotted line) characterize Th1 biased immune responses. TMC nanoparticle adjuvanted pDNA delivery induced a clear bias of Th activation towards type 1, which was indicated by detection of ratios of serum IgG2b/IgG1 (Physique 2B) and IgG2c/IgG1 (Physique 2C) titers above unity. mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used. (experiment in mice we compared TMC nanoparticles, SWE06, and Cationorm? as pDNA delivery systems to increase Th1 related immune Faropenem sodium responses against Ag85A. Following these investigations, we then exploited the potential of concurrent activation of two non-redundant PRR pathways with the aim of further optimizing immunogenicity of pDNA. Our results show that cationic TMC nanoparticles are promising carriers for pDNA and co-delivery with MDP can be used to further increase immunogenicity of this DNA vaccine formulation. 2. Results and Discussion 2.1. Nanoparticle Characterization The formulations were characterized for their size by differential laser light scattering (DLS) expressed as 0.001) and both nanoemulsions ( 0.0001), while the zeta potential decreased drastically to 7 mV (TMC nanoparticles), ?14 mV (SWE06) and ?39 mV (Cationorm?). The addition of MDP did not have any influence on size and Rabbit Polyclonal to OAZ1 zeta potential of TMC nanoparticles and SWE06. However, size increase and higher PDI values of pDNA loaded Cationorm? with MDP indicated aggregation tendencies of this formulation. PDIs between 0.1 and 0.5 were observed for all particles, corresponding to systems of mid-range polydispersity [29]. Only small amounts of pDNA were found in the supernatant, Faropenem sodium having measured pDNA adsorption of 99.8% to TMC nanoparticles, 95% to SWE06, and 93% to Cationorm? of initially added 50 g/mL pDNA to the cationic nanocomplexes. Table 1 Physicochemical properties of trimethyl chitosan (TMC) nanoparticles, a cationic squalene-in-water nanoemulsion (SWE06) and Cationorm?, either unloaded or loaded with pDNA, muramyl dipeptide (MDP), or both. To determine size in nm, polydispersity index (PDI), and zeta potential () in mV, samples were prepared in water and diluted with 1 mM NaCl prior to measurements. antigen Ag85A encoding pDNA for their potential to increase antigen-specific Th1 related immune responses of a tuberculosis DNA vaccine candidate. Ag85A possess enzymatic mycolyltransferase activity involved in cell wall synthesis Faropenem sodium and belongs to the key immunodominant antigens of Mtb. We decided to apply the same formulation preparations as described above but with a higher quantity of pDNA applied (50 g per dose) to ensure a detectable magnitude of antigen-specific antibodies. The influence of the nature of the nanocomplexes on the outcome of elicited immune responses in mice, dependent on the nature of the delivery systems was evaluated. The loading efficiency of pDNA to the nanoparticles within these formulations was 43% to 44%, while surplus pDNA remained in suspension. Antigen specific total IgG responses to pDNA in the adjuvanted groups were higher than those observed for naked pDNA. In TMC nanoparticle vaccinated mice significantly increased titers of total IgG were observed in comparison with pDNA alone, as shown in Figure 2A ( 0.05). Oil-in-water emulsions based on squalene or mineral oils reportedly induce Th2 responses in protein vaccines [31,32]. Formulated with DNA both nanoemulsions tested promoted increase in Ag85A specific antibodies to pDNA without altering the balanced Th1/Th2 responses observed with naked pDNA (Figure 2). Open in a separate window Figure 2 Immune responses in mice to pDNA (50 g per dose) with/without TMC nanoparticles one week after the second booster injection (i.m.). Ag85A-specific serum immunoglobulin G (IgG) titers were analyzed by endpoint enzyme-linked immunosorbent assay (ELISA). (A) Bars represent mean = 4 SEM, * 0.05, compared to pDNA alone. (B,C) Corresponding average Log IgG2b/Log IgG1 and Log IgG2c/Log IgG1 ratios are indicative for the quality of the immune response, where values higher than 1 (dotted line) characterize Th1 biased immune responses. TMC nanoparticle adjuvanted pDNA delivery induced a clear bias of Th activation towards type 1, which was indicated by detection of ratios of serum IgG2b/IgG1 (Figure 2B) and IgG2c/IgG1 (Figure 2C) titers above unity. According to the.
