(G-269) Optimization of Multi-Adjuvant Loaded Bicontinuous Nanospheres to Elicit Group 1 CD1-Restricted T Cell Response

Introduction:: Tuberculosis (TB) is a highly infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb), which remains a significant global health concern with an estimated 1.6 million deaths in 2020. The only currently available vaccine, live-attenuated Mycobacterium bovis bacillus Calmette–Guérin (BCG) can protect against disseminated TB in young children, but its efficacy against active TB is highly variable. As a result, there has been a growing interest in developing subunit vaccine alternatives, which could also be used in immunocompromised populations and elicit better protection. Most subunit vaccines for TB utilize Mtb protein antigens. However, the Mtb cell wall contains many lipid antigens, including mycolic acid (MA), which makes up approximately 60% of the Mtb cell wall and is the immunodominant lipid antigen. MA can be presented on CD1b molecules and recognized by CD1b-restricted T cells. These subsets of T cells have been detected in human patients infected with Mtb. Unlike major histocompatibility complex (MHC) molecules, CD1 molecules are non-polymorphic and therefore may elicit similar immune responses across various populations. Here, we investigate adjuvant combinations to enhance CD1b-restricted T cell responses using bicontinuous nanosphere (BCN) vehicles, which possess uniquely high stability for loading multiple payloads simultaneously.

Materials and Methods:: We used a human CD1 transgenic mouse (hCD1Tg) that expresses group 1 CD1 molecules, since mice only express group 2 CD1 (CD1d). In order to test MA-specific CD1b-restricted T cells, we used a transgenic mouse strain (DN1Tg) that expresses MA/CD1b-specific T cell receptor (TCR). To better control and optimize immune responses during vaccination, we combined MA with diverse immunostimulants, monophosphoryl lipid A (MPL A) (TLR-4 agonist), CpG (TLR-9 agonist), DMXAA (sting agonist), and trehalose 6,6'-dibehenate (TDB) (Mincle agonist). Stable incorporation of these distinct adjuvants in multiple combinations was achieved using BCNs composed of poly(ethylene glycol)-b-poly(propylene sulfide) (PEG₁₇-b-PPS₇₅) that assembles lyotropic nanostructures possessing internal aqueous channels. The efficacy of the adjuvanted MA BCN formulations was assessed by the production of Th1 and Th17 cytokines (IFN-γ, IL-2, TNF, and IL-17A) from hCD1Tg dendritic cells (DCs) and DN1 T cells co-culture. The production of these cytokines was quantified using cytometric bead array (CBA).

Results, Conclusions, and Discussions:: The BCN nanostructure permits uniquely high loading capacity for both hydrophobic and hydrophilic molecules without compromising particle morphology. We thus hypothesized that the multi-loading of MA and optimized adjuvant combinations into BCNs will activate CD1b-specific DN1 T cells and serve as a subunit vaccine for TB. Our results indicate that all BCN formulations form uniform spherical particles (~100 nm, PDI< 0.1), regardless of the antigen/adjuvant loading. We confirmed that these particles retain their morphology with CryoTEM and Small-Angle X-ray Scattering (SAXS). Our preliminary results show that the encapsulation of antigens and adjuvants can elicit IFN-γ, IL-2, TNF, and IL-17A from DN1 T cells. The combination of all adjuvants outperformed the single adjuvant-antigen loaded BCN. Currently, we are investigating how the concentrations and combination of these adjuvant will modulate the immune response. Our goal is to design a formulation that would have balanced TCR- and cytokine-mediated T cell activation. In general, MA-loaded BCN formulations with or without adjuvants had a better response than unencapsulated free MA. Additionally, adjuvant-only loaded BCN demonstrated no production of cytokines from DN1 T cells which indicates that these adjuvanted BCN formulations do not trigger autoreactivity. We are in the process of testing these formulations in vivo in a humanized mouse model.

Conclusion: Our findings suggest that the combination of MA and selected adjuvant combinations, delivered via PEG₁₇-b-PPS₇₅ BCNs, can activate MA-specific CD1b-restricted T cells. This may lead to the development of the first effective TB subunit vaccine via activation of group 1 CD1-restricted T cells.


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