Alexandra Atalis 

BME PhD Thesis Defense Presentation 

Date: Friday, February 12th, 2021 

Time: 1:00 – 3:00 PM 

BlueJeans Link: https://bluejeans.com/805978453 

Meeting ID: 805 978 453 

Committee members: 

Krishnendu Roy, Ph.D. (Advisor) 

J. Brandon Dixon, Ph.D. (Co-Advisor) 

Susan Thomas, Ph.D. 

Philip Santangelo, Ph.D. 

Manmohan Singh, Ph.D., FAAPS (Beam Therapeutics) 

Title: Evaluating the Effects of Particle-Delivered Combination Adjuvants on Antigen Presenting Cells 

Vaccination is one of the most successful medical interventions in history and remains the standard method for the global control of communicable diseases. A current vaccine development challenge is to identify adjuvants with well-defined mechanisms of action that induce strong Th1 and CTL-mediated immune responses required to fight cancer and chronic infectious diseases, such as HIV or dengue. Innate immunity is triggered through the recognition of pathogen-associated molecular patterns (PAMPs) that bind to pattern recognition receptors (PRRs) on antigen presenting cells (APCs) and coordinate the elimination of pathogens while directing immune responses to be Th1-, Th2- or Th17-biased. Therefore, natural and synthetic PAMPs have the potential to be effective vaccine adjuvants. We hypothesized that polylactic-co-glycolic (PLGA) micro- and nanoparticles loaded with combinations of PAMPs provide superior protection through complementary and synergistic enhancement of immune responses. Specifically, we evaluated if particles mimicking Gram-negative bacteria (TLR4 agonist MPLA plus TLR9 agonist CpG) or viruses (RLR agonist PUUC combined with a TLR agonist) could enhance immune responses compared to particles with single adjuvants. Aim 1.1 investigated how single and combined MPLA and CpG modulated bone marrow-derived dendritic cell (BMDC) 3D migration toward lymphatic chemokines using a microfluidic platform. Aim 1.2 evaluated how MPLA and CpG affected the recruitment and activation of muscle- and lymph node-resident APC subsets, as well as the kinetics of NIR-fluorescent-adjuvant and particle delivery to various muscle-draining lymph nodes in mice. Aim 2 investigated how combined TLR and RLR agonists on PLGA nanoparticles 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. Through these aims, we demonstrate that the success of combination adjuvants depends not only on the types of adjuvants used but also on the vaccine delivery system, APC subsets present, and the immunization route.