Abstract
Despite decades of research very few vaccine-adjuvants have received FDA approval. Two fundamental challenges plague clinical translation of vaccine-adjuvants: reducing acute toxicities that result from systemic diffusion of many soluble adjuvants, and delivering multiple adjuvants at the same time to mimic the synergistic immune-stimulation of pathogens, while being safe. In order to address these barriers, we evaluated combinations of four clinically relevant immune-agonists, specifically Toll-like receptor (TLR) ligands, using biodegradable, polymer microparticles. We tested them alone and in combinations of 2 or 3, for a total of 10 unique conditions. We evaluated primary bone-marrow-derived Dendritic Cell phenotypes and functionality, and identified several synergistic combinations. We picked a dual and a triple adjuvant combination, TLR4/TLR9 and TLR4/TLR7/TLR9, for further evaluation and found that both combinations promoted antigen cross-presentation in vitro. Studies in mice using the model antigen Ovalbumin, showed that both combinations enhanced lymph node germinal center and T follicular helper cell responses. The triple adjuvant combination showed increased antigen-specific antibody titer with an overall balanced Th1/Th2 response, while the dual combination promoted Th1-polarized IgG responses. Our results show how polymeric particulate-carriers can be adopted to safely deliver combinatorial adjuvants and selectively synergize specific types of immune responses for vaccine applications.
Highlights
Adjuvants operate by engaging innate immune cells, such as dendritic cells (DCs), and shape the subsequent adaptive immunity[6,7,8]
The particles were loaded with Pam3CSK4, Monophosphoryl lipid A (MPLA) and R837, agonists for TLR2, −4 and −7 respectively, by encapsulation, or by surface modifying them with polyethyleneimine (PEI) to generate cationic particles, followed by electrostatic adsorption of CpG, TLR9 agonist, or model antigen Ovalbumin, as described previously and in methods[55, 63, 64]
Most research in this field has focused on using soluble adjuvants, which have several disadvantages; for example, their potential to diffuse systemically can result in serious side effects[50, 54]
Summary
Adjuvants operate by engaging innate immune cells, such as dendritic cells (DCs), and shape the subsequent adaptive immunity[6,7,8]. Simultaneous triggering of multiple PRRs, such as TLRs, with specific agonists has been shown to activate them synergistically, and lead to enhanced cytokine secretion, T cell and antibody responses[24, 43,44,45,46] Such combinatorial agonists that promote defined immune responses can be leveraged for better vaccine design[24, 43, 47, 48]. Combinations of specified dose and ratios of hydrophilic molecules (e.g. CpG) with hydrophobic adjuvants (e.g. MPLA), that have been optimized using in vitro studies in confined tissue culture wells where diffusion away from the application “site” is not an issue, is essentially meaningless in vivo due to their different retention kinetics resulting in altered adjuvant ratios and presentation to immune cells These issues of systemic immunotoxicity and differential retention can be resolved by the use of biomaterial-based controlled delivery systems, especially particulate carriers. Few studies have already shown that biomaterial-based carriers are superior to soluble adjuvants, and might allow effective simultaneous delivery of multiple adjuvants[55,56,57,58]
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