Molecular Medicine Israel

Zeolitic imidazolate frameworks activate endosomal Toll-like receptors and potentiate immunogenicity of SARS-CoV-2 spike protein trimer


Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework–8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.


Subunit protein antigens are safer and easier to manufacture than traditional whole pathogen vaccines (13), but they often require coadministration with an adjuvant to potentiate antigen presentation and produce effective immune outcomes (45). Rationally designed vaccine adjuvants aim to trigger specific receptors on antigen-presenting cells (APCs) to direct the immune system to respond in a T helper 1, T helper 2, or T helper 17 manner (67). Pattern recognition receptors (PRRs), particularly Toll-like receptors (TLRs), expressed mainly on APCs play a critical role in the modulation of these immune responses (810). PRR agonists help generate greater immune response magnitude and long-lasting memory response against vaccine targets when coadministered with subunit protein antigens (1112). Nevertheless, only a few PRR adjuvants are licensed for use in U.S. Food and Drug Administration–approved vaccines owing to safety and tolerability concerns. However, these concerns can be potentially mitigated by novel delivery approaches including nanoparticle (NP)–based methods (1314).

Biomimetic NP delivery strategies have been shown to enhance antibody titers even at ultralow antigen doses (4). A range of NPs have demonstrated adjuvant activity through enhancing the codelivery of antigens and adjuvants to APCs and lymph nodes (LNs) (415). NPs allow for passive vaccine drainage to LNs (1214), create multivalent antigen interactions with B cells, and facilitate antigen presentation onto class I major histocompatibility complex (MHC I) molecules that enhance CD8+ T cell responses (16). Several studies have revealed that polymeric NP antigens (1718) and liposomes (19) activate stimulatory immune pathways such as TLRs or inflammasomes in the absence of adjuvants and antigens. 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’ immunostimulatory properties offers an 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.

Zeolitic imidazolate framework-8 (ZIF-8)—a subclass of metal organic frameworks—encapsulates antigen through a biomimetic mineralization process, affording exceptional protection from biological, thermal, and chemical degradation while maintaining bioavailability (2022). ZIF-8 is also established as an efficient vaccine delivery platform (2325). Preclinical studies demonstrated that ZIF-8 modulates vaccine-induced immune responses and stimulates high antibody responses through the codelivery of antigen and adjuvant to APCs (2324). However, the contribution of ZIF-8’s composition to its adjuvant properties is poorly characterized. Imidazole, the basic building block of ZIF-8, is the smallest moiety in synthetic TLR agonists and can be modified to produce TLR-specific agonist or antagonist molecules, especially TLR-7 and TLR-8 (2627). Here, we investigated how ZIF-8 NPs can modulate innate immune activation to enhance adaptive immune responses to protein antigens, using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD) trimer as a model antigen. To better understand how ZIF-8 improves immunogenicity, we used biodistribution studies, immunological techniques, and transcriptome sequencing.

We found that ZIF-8 can passively drain to the draining LN (dLN) while also activating innate immune responses at the injection site. After uptake, ZIF-8 NPs undergo pH-responsive degradation in the endosome, allowing them to simultaneously release their payload, while the imidazole produced by their degradation activates TLRs in endosomes (TLRe). Analysis of gene expression in the dLN suggests a MyD88-dependent response and the activation of nuclear factor κB (NFB), leading to the production of interleukin-6 (IL-6) and type I interferon β (IFN-β) associated with antiviral responses. This coincided with increased expression of CCR-7 and costimulatory molecules such as CD80 on TLR-responsive macrophage and dendritic cell (DC) populations. When administered to mice while encapsulating RBD, ZIF-8 allows for dose sparing compared to free RBD and the ability to coencapsulate with a TLR-7 agonist adjuvant, gardiquimod (Gdq). ZIF-8 also improves the stability of RBD in storage.


ZIF-8 encapsulates Gdq and RBD trimer and exhibits pH-responsive release

ZIF-8 was prepared as described previously (22). Transmission electron microscopy (TEM) revealed cubic crystals of ZIF-8 encapsulating Gdq and RBD trimer (GR-ZIF) with an average size of 150 nm that is similar to that of ZIF-8 (Fig. 1, A and B). Likewise, the powder x-ray diffraction (PXRD) pattern of GR-ZIF showed a similar pattern to that of simulated ZIF-8 (Fig. 1C). The Fourier transform infrared (FTIR) spectrum of GR-ZIF spectrum depicted obvious stretching vibrations of C═O at 1606 cm−1 and O─H at 3451 cm−1, which were attributed to RBD trimer (Fig. 1D). FTIR analysis demonstrated that RBD trimer is not only encapsulated within ZIF-8 but also adsorbed on the surface (Fig. 1D), which allows for the enhanced presentation of RBD trimer. The uniformity and intercalation of Gdq and RBD trimer in ZIF-8 were also validated (text S1 and fig. S1).

The acidic environment of lysosomes and endosomes in APCs is expected to induce the release of RBD trimer and Gdq from ZIF-8 (Fig. 1, E and F). The pH-responsive RBD trimer release from ZIF-8 was monitored by enzyme-linked immunosorbent assay (ELISA) at physiological (pH 7.4) and lyso/endosomal acidic (pH 5.3) conditions. Under physiological conditions, less than 3% of RBD trimer was released over 24 hours in solution (Fig. 1E). Screening R-ZIF in a proteinaceous environment [Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (pH 7.2)] indicated that 6% of RBD trimer was released from ZIF-8 in 4 hours with no further release observed after 24 hours. However, a total of 43 and 60% of RBD trimer were released at acidic pH after 20 and 120 min, respectively (Fig. 1E). TEM images confirmed that low pH triggers the degradation process for GR-ZIF. Its complete decomposition after 24 hours (Fig. 1, F and G) agrees with previously reported data (22).

ZIF-8 potentiates innate immunity through the biomimetic presentation of RBD trimer

We first assessed ZIF-8 accumulation in dLN and subsequent immune activation using a series of experiments to characterize ZIF-8 biodistribution. A model cargo, XenoLight DiR, was loaded in ZIF-8 (D-ZIF) to assess the temporal localization of ZIF-8 in the dLN. Measuring Zn2+ in dLNs using inductively coupled plasma mass spectrometry (ICP-MS) confirmed a significant increase (P < 0.01) in Zn2+ 24 hours after injection (Fig. 2A). Flow cytometry analysis of APCs in dLN demonstrates that levels of CD45+ CD11c+ associated with D-ZIF continuously increased over time (Fig. 2B). The accumulation of D-ZIF in CD45+ CD11c+ DCs within 1 hour indicates intercellular drainage, while the significant increase in the proportion of CD45+ CD11c+ DCs associated with D-ZIF signal at 24 hours indicates intracellular drainage. This trafficking was also observed in pathogens (28) and other adjuvants such as MF59 adjuvant (29) and in agreement with other reports for submicron particles (1430)…

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