(E-199) Zeolitic Imidazolate Frameworks Activate Endosomal Toll-like Receptors and Potentiate Immunogenicity of SARS-CoV-2 Spike Protein Trimer

Introduction:: Nanoparticles (NPs) modulate vaccine biological activity by a pathogen-mimetic approach. The delivery of vaccine formulation, containing antigen and adjuvant, in a pathogen-mimetic fashion enhance antibody titers even at ultra-low antigen doses. A range of NPs have demonstrated adjuvant activity through enhancing the delivery of antigens to APCs and LNs. However, several studies reported the activation of stimulatory immune pathways such as, TLRs and inflammasomes in the absence of antigens in response to NPs. The intrinsic immune characteristics of NPs should be explored to avoid potential alteration in immune signal processing by APCs that result from the excessive activation of innate immune responses. Investigating NPs’ intrinsic immunostimulatory properties offer opportunity to study novel carriers that not only tune the spatiotemporal distribution of vaccine antigen, but also directly engage with innate signaling pathways and enable selective activation of immunologic pathways.

Materials and Methods:: We investigated how the biomimetic presentation achieved through the encapsulation and adsorption of an antigen on nanoscale ZIF-8 can modulate innate immune activation in vivo to enhance adaptive immune responses to protein antigens. The model protein antigen SARS-CoV-2 spike protein receptor binging domain trimer (RBD trimer) was encapsulated within ZIF-8 (R-ZIF, Fig. 1A). We have also incorporated Gadiquimod (Gdq, TLR7 agonist, GR-ZIF) to assess the versatility of ZIF-8 in delivering both antigen and adjuvant to APCs.

Results, Conclusions, and Discussions:: Three doses of 3 μg of soluble RBD trimer were required to elicit similar titers to 0.9 μg of RBD trimer delivered in R-ZIF and GR-ZIF NPs. Additionally, GR-ZIF produced rapid SARS-CoV-2 IgG after a prime dose delivering 0.9 μg of RBD trimer, and R-ZIF elicited significantly higher titers than soluble RBD trimer after the first booster using 1/3 the dose. To better understand the mechanism of this enhanced ZIF-8 immunogenicity, we employed immunological techniques and transcriptome sequencing. Upon injection, ZIF-8 triggers monocyte-derived dendritic cells, plasmacytoid dendritic cells, CD8+ DC, MHC II+ DC, mimicking pathogens in activating innate immune responses. Our study reveals for the first time the activation of endosomal TLRs in response to ZIF-8. ZIF-8 accumulation in endosomes initiated MyD88-dependent responses, which enhanced both the innate and adaptive signaling pathways. This resulted in the activation of multiple TLR-responsive macrophage and DC populations.

Acknowledgements (Optional): : S.K.A. and S.N thank King Abdulaziz City of Science and Technology for Ibn Khaldun fellowship support. We thank the Koch Institute Swanson Biotechnology Center for technical support, specifically Microscopy, Histology, Flow cytometry, and Animal Imaging & Preclinical Testing facilities.

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