We describe here the development of nanoparticles made from poly(lactic-co-glycolic acid) (PLGA) able to deliver an encapsulated antigen with a Toll-Like Receptor-7 (TLR-7) agonist as immunostimulatory signal and coated with a muco-adhesive chitosan-derivate layer. The potential to stimulate an immune response of these vaccine formulations in the absence or presence of the TLR-7 agonist at the systemic and mucosal level were evaluated in mice following subcutaneous or nasal administrations. Intranasally immunized mice developed a high systemic immune response equivalent to mice injected subcutaneously. However, mucosal immune responses were only induced at local and distal sites in mucosally immunized animals. The adjuvant effect of imiquimod on the polarization of the immune response was only detected at local sites, which tends to increase safety of this vaccine delivery system.
The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP.
Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, OPEN ACCESSNanomaterials 2015, 5 2318 dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.
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