Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

Abstract

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.

Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses

“…pDNA was adsorbed onto TMC particles (pDNA/TMC-NPs-0.7) or applied with MDP containing TMC particles (pDNA/TMC-NPs-0.7 + MDP). An immunopotentiating effect in vivo was sought to be investigated, as we have reported in our laboratory a synergistic enhancement of proinflammatory cytokine release in vitro by co-delivery of pDNA with MDP [ 23 ]. As these in vitro experiments did not demonstrate any macrophage activation with MDP only [ 23 ], we excluded this group from the animal experiments.…”

“…An immunopotentiating effect in vivo was sought to be investigated, as we have reported in our laboratory a synergistic enhancement of proinflammatory cytokine release in vitro by co-delivery of pDNA with MDP [ 23 ]. As these in vitro experiments did not demonstrate any macrophage activation with MDP only [ 23 ], we excluded this group from the animal experiments. As shown in Figure 4 A, significantly lower Ag85A-specific total IgG antibody production was detected following vaccination with both adjuvanted pDNA formulations ( p < 0.001), pDNA/TMC-NPs-0.7 and pDNA/TMC-NPs-0.7 + MDP, when compared to vaccination with naked pDNA.…”

Biopolymer‐based Carriers for DNA Vaccine Design

“…[164] Moreover,the polymer backbone itself can be chemically modified to modulate its intrinsic charge.C hitosan, which is positively charged only at acidic pH, can be trimethylated at its primary amines resulting in ap ositive charge over ab roader pH range. [55,[165][166][167][168] In case of proteins such as albumin, methyl esterification of carboxyl groups decreases negative charge and renders the overall protein positively charged and well-suited for DNAc ondensation. [169] Alternatively,hydrophobic attraction can be employed to attach pDNAonto the carrier.T oachieve this,pDNAcan be hydrophobised with cetrimonium bromide [170] or dioleoyl-3trimethylammonium propane.…”

“…Moreover, the polymer backbone itself can be chemically modified to modulate its intrinsic charge. Chitosan, which is positively charged only at acidic pH, can be trimethylated at its primary amines resulting in a positive charge over a broader pH range [55, 165–168] . In case of proteins such as albumin, methyl esterification of carboxyl groups decreases negative charge and renders the overall protein positively charged and well‐suited for DNA condensation [169] …”

Combination of Innate Immune Modulators as Vaccine Adjuvants in Mice

“…In cells, MDP is bound by NOD2, a cytoplasmic receptor belonging to the innate immune system [ 12 ]. MDP induces immune responses by increasing the production of interferon (IFN)-γ and other cytokines [ 13 ], stimulating the differentiation and proliferation of lymphocytes [ 14 ] and it has been shown to influence immune responses with other TLR ligands [ 15 ]. ARC5 is a TLR2 ligand and PAM3CSK4 analogue.…”