Research paperThe preparation and characterization of PLG nanoparticles with an entrapped synthetic TLR7 agonist and their preclinical evaluation as adjuvant for an adsorbed DTaP vaccine
Graphical abstract
Introduction
Vaccines based on purified and recombinant antigens retain a better safety profile in comparison with traditional whole-cell vaccines, but generally require the addition of adjuvants to stimulate adequate immune responses [1]. In order to induce more effective protection, modern vaccine research is focused on the development of improved adjuvants and formulations exploiting the progresses within material science and nanotechnology.
Micro- and nanoparticles prepared from biodegradable and biocompatible polymers, such as poly(lactide-co-glycolide) (PLG), have been approved for pharmaceutical and medical device applications [2], [3], [4], [5] and have raised increasing interest over the years as vaccine adjuvants [6], [7], [8], [9]. Resulting in persistent activation of antigen presenting cells at the vaccination site [10], [11], [12], PLG particles have shown comparable adjuvanticity to aluminum-based systems [13], [14], [15]. Notably, the adjuvant effect of PLG particles could be significantly enhanced by the entrapment of Toll-like receptor agonists (TLRa) [16], such as the TLR7/8 agonist resiquimod or the TLR7 agonist imiquimod [17]. TLR7 ligands are recognized as appealing vaccine adjuvant candidates because of the broad expression of their target receptor in human dendritic cells and their capability to stimulate cellular immunity [18], [19], [20]. Therefore, medicinal chemistry has been recently applied to synthetic small molecules immune potentiators (SMIPs), to design optimal adjuvant-active compounds targeting the TLR7 [21]. SMIPs-TLR7a have shown marked immune potentiator activity in combination with aluminum hydroxide, and the present work aimed to expand their evaluation when entrapped within PLG nanoparticles. In particular, the PLG-TLR7a system here described was proposed as alternative adjuvant for an adsorbed acellular pertussis (aP)-containing vaccine. Currently licensed aP vaccines are combined with diphtheria and tetanus toxoids (DTaP) in the cornerstone of childhood immunization and are adjuvanted with aluminum salts, promoting mostly Th2 responses and toxin-neutralizing IgG1 antibodies [22], [23]. Since Th2 responses have been reported to protect against severe pertussis disease but not to prevent bacterial infection and transmission [24], the use of Th1-promoting adjuvants has been suggested to improve aP vaccine protective efficacy [25], [26] and potentially control pertussis resurgence [27], [28], [29].
Previous studies have demonstrated that negatively charged PLG particles enhanced immune responses against adsorbed diphtheria and tetanus toxoids [30], thus fully supporting their evaluation with DTaP combination vaccines. To ensure effective antigen adsorption – a crucial parameter to stimulate potent immune responses [31] – nanoparticles were preferred to microparticles because of the larger surface area and the higher polymer:antigens ratio [32]. Additionally, nanoparticles could offer the advantage of increasing the uptake by antigen presenting cells (APCs) [33], [34], [35], thus resulting in enhanced immunogenicity [12], [36] and stimulation of cell mediated immunity [37], [38].
Following extensive and accurate formulation optimization to co-deliver a TLR7a immune potentiator together with DTaP antigens, the adjuvanticity of the proposed nanosystem was evaluated in the mouse model with the aim of improving vaccine immunogenicity and better tailoring the quality of the immune response against aP antigens.
Section snippets
Materials
RESOMER® RG503 – poly(d,l-lactide-co-glycolide) 50:50 co-polymer, dioctyl sulfosuccinate (DSS), methylene chloride, dimethyl sulfoxide (DMSO), sucrose and polyvinyl alcohol (PVA) were obtained from Sigma–Aldrich (St. Louis, MO). The small-molecule immune potentiator targeting the Toll-like receptor 7 (TLR7a) was synthesized at Genomics Institute of the Novartis Research Foundation (GNF, San Diego, CA) [21]. Aluminum hydroxide (Alum) adjuvant, diphtheria toxoid (DT) and tetanus toxoid (TT) were
Physical characterization of PLG nanoparticles
Negatively charged PLG nanoparticles were prepared as a single population of particles with mean size 350 nm and, as reported in Table 1, particle size and net surface charge remained basically unvaried upon TLR7a encapsulation. TLR7a encapsulation into PLG nanoparticles was shown to be highly efficient at 90% of the loading. The use of 0.5% w/v DSS as stabilizer for the first W/O emulsion controlled the distribution of the TLR7a into the inner aqueous phase of the nanoparticles, significantly
Discussion
Appropriate adjuvant selection has a fundamental role in the development of effective vaccines. Hence, one of the major objectives of current vaccine research is the design of novel adjuvants and delivery systems to be potentially used with various antigens. The combination of PLG particles with immune potentiators of the TLR agonist group represents an attractive approach to enhance antigens immunogenicity. Different groups have showed that the TLR4 agonist monophosphoryl lipid A (MPL) [49],
Acknowledgments
The authors are grateful to Padma Malyala, Simone Vecchi and Daniela Proietti for scientific discussion, to Marco Tortoli and the animal facility personnel for the precious help with the in vivo studies, and to Ilaria Ferlenghi and Eugenio Paccagnini for SEM analysis.
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