Evaluating the Effects of Particle-Delivered Combination Adjuvants on Antigen-Presenting Cells
Atalis, Alexandra Maria
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A current vaccine development challenge is to identify adjuvants with well-defined mechanisms of action that selectively induce antigen-presenting cell (APC) functions to modulate the immune response. APCs are activated by various pathogen-associated molecular patterns (PAMPs) that bind to APC-expressed pattern recognition receptors (PRRs). It was hypothesized that polylactic-co-glycolic (PLGA) particles formulated with adjuvant combinations of purified PAMPs provide superior immune protection through complementary and synergistic enhancement of APC responses. Particles mimicking Gram-negative bacteria (TLR4 agonist MPLA plus TLR9 agonist CpG) and viruses (RLR agonist PUUC combined with a TLR agonist) were evaluated in Aims 1 and 2, respectively, on their ability to enhance APC responses compared to particles with single PAMP-based adjuvants. Using microfluidic devices, it was investigated in Aim 1.1 how single and combined MPLA and CpG modulate bone-marrow derived dendritic cell (BMDC) 3D chemotaxis toward gradients of lymphatic chemokines CCL19 and CCL21. Results showed that while there were no synergistic effects observed with MPLA and CpG on BMDC chemotaxis, unformulated MPLA and microparticle (MP)-delivered CpG were separately effective at inducing BMDC migration. Using flow cytometry and near-infrared (NIR) microscopy, it was evaluated in Aim 1.2 how MPLA and CpG nanoparticles (NPs) activated APCs in the muscle and draining lymph nodes, as well as how adjuvants influenced particle transport to the lymph nodes after intramuscular injection. Results showed that there was a synergistic effect by NP-delivered MPLA and CpG on neutrophil recruitment to the muscle, MPLA NPs selectively activated monocytes, macrophages, and CD11b+ dendritic cells (DCs), and CpG NPs selectively activated B cells and plasmacytoid DCs. There were no adjuvant-based differences in NIR-labeled particle transport to the muscle-draining lymph nodes. It was investigated in Aim 2 how combined TLR and RLR agonists on PLGA NPs delivered with stabilized spike protein modulated innate and adaptive immune responses against SARS-CoV-2. TLR-RLR adjuvant combinations were screened in vitro using isogeneic mixed lymphocyte reaction (iso-MLR) assays. TLR-RLR PLGA particles were intranasally administered to mice, and immune responses in the lung were measured acutely with RT-PCR and CITE-Seq analysis and after 4-5 weeks during a prime-boost intranasal vaccination. MPLA-PUUC, CpG-PUUC, and R848-PUUC NPs all induced differential immune responses in vitro with BMDCs as well as in vivo, with MPLA-PUUC NPs inducing the most cellular immunity against SARS-CoV-2 spike protein. Through these aims, it was demonstrated that the success of combination adjuvants depends not only on the adjuvant formulation, but also on the vaccine delivery system (e.g., unformulated versus MP/NP), APC populations present (e.g., macrophages versus DCs), and vaccine route of immunization (e.g., intramuscular versus intranasal).