O23 - Programmable B Cell Interactions Using Metal–Organic Framework–Hydrogel Depots

Introduction: : Developing long-lasting, potent vaccines remains a challenge, particularly as emerging variants undermine the efficacy of existing platforms.¹ Protein-based vaccines offer a unique opportunity to broaden raised immune responses because they contain every possible peptide immune cells can process and present, and their spatial arrangement enables engagement with B cell receptors (BCRs) through conformational epitopes.² However, free proteins naturally degrade in transit and are unstable in commonly employed alternative vaccine platforms (e.g. lipid nanoparticles). We overcome this limitation by protecting proteins within biocompatible porous metal–organic framework (MOF) nanocarriers, which enhance the stability of encapsulated cargo and enable prolonged release.³ To further encourage multivalent B cell–protein interactions, we leverage a hydrogel depot where immune cells can engage with protein–encapsulated MOFs. Herein, we illustrate how the design of protein@MOF hydrogel structures fosters controlled interactions between proteins and immune cells to heighten B cell signaling and activation.

Materials and

Methods: : Antigen protein (i.e., ovalbumin (OVA), receptor binding domain (RBD)) was encapsulated into zirconium-based 150-200 nm NU-1000 MOFs. Encapsulation efficiency was determined via bicinchoninic acid (BCA) assay. Agarose hydrogel depots were formed by mixing antigen-encapsulated MOFs in a solution of agarose and media, heated until the agarose dissolved, and then cast into molds. OVA release was measured over time via BCA assay. Immortal RAMOS B lymphocytes or mortal murine splenic B cells were incubated with the hydrogel-MOF complex and immune cell permeation was analyzed with flow cytometry. Viability and localized interaction with OVA@NU-1000 nanoparticles were measured via immunofluorescent (IF) staining and confocal microscopy. In vitro activation via surface markers (e.g. CD86, CD69) on permeated mortal murine splenic B cells were analyzed via flow cytometry as well as IF staining and confocal microscopy. Antibody generation was assessed via ELISA, BCR clustering was measured via IF staining and confocal microscopy, and antibody-secreting cells were quantified via ELISpot.

Results, Conclusions, and Discussions:: OVA-loaded NU-1000 nanoparticles sustained the release of OVA antigen for >3 days. To ensure immune cell compatibility with the depot setup, we quantified the permeation and viability of RAMOS B lymphocytes and splenic B cells after 24 h. 3% agarose gels resulted in 2-fold higher permeation than 3.5% gels and 3-fold higher than 4% gels. Splenic B cells showed similar permeation profiles (Fig. 1A). 3% gels also maintained the highest permeated cell viability at ca. 80%. RAMOS permeation into OVA@NU-1000 3% agarose depots enhanced localized interactions with the OVA@NU-1000 nanoparticles, illustrated by 40% of RAMOS containing nanoparticle signal at 48 h. To analyze immune responses elicited by the depot platform, we measured early activation marker CD69 and costimulatory marker CD86. We observed a ca. 1.5-fold increase in both CD69 and CD86 expression on splenic B cells interacting with OVA@NU-1000 3% agarose depots where nearly 50% of the population exhibited CD86, compared to depots containing solely free OVA (Fig. 1B, C). Notably, we found increased CD86 expression in splenic B cells containing NU-1000 signal (ca. 20% of CD19+ population), highlighting that costimulatory expression was heightened by OVA delivery mediated specifically through the NU-1000 nanocarrier. The utilization of OVA as a model protein subsequently enabled studies employing clinically relevant antigen (RBD from SARS-CoV-2 spike protein and influenza-based Hemagglutinin A), where we observed retained B cell permeation and antigen interaction through the MOF nanoparticle, and increased antibody generation. Incubation of the hydrogel-MOF complex with human B cells exhibited robust BCR clustering and antigen-specific antibody-secreting cell counts, illustrating the translatability of this design.
The protein@MOF hydrogel vaccine architecture offers a tunable approach to multivalent antigen interactions and release for robust immune cell activation. Moreover, the stabilization afforded by MOFs permits the use of “off-the-shelf” native proteins, eliminating the need for genetic engineering or other modifications to stabilize protein structure. With these combined advantages, our platform presents a solution that maintains the fragile conformation of proteins as they traverse the body, eliciting heightened immunity that is broadly protective against mutated pathogens. Our work can be expanded to explore various antigens relevant to emerging infectious diseases.

Acknowledgements and/or References (Optional):: 1. Zaeck LM, et al. The Lancet Resp. Med. 2023, 11:395-396; 2. Scheiblhofer S, et al. Exp. Rev. of Vacc. 2017, 16:479-489; 3. Teplensky MH, et al. Chem. 2019, 5:2926-2941.