Mycolic acid nanoparticle vaccination leads to lipid antigen persistence and unique differentiation of mycobacterial-specific T cells

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the properties of vaccine-induced Mtb lipid-specific memory T cells remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to T cells. MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized bicontinuous nanospheres (BCN) to efficiently encapsulate MA and deliver it to MA-specific T cells both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary delivery of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Interestingly, pulmonary vaccination with MA-Ag85B-BCN led to the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated to have a T follicular helper-like phenotype. Overall, BCN platform allows for the dual encapsulation of lipid and protein antigen and leads to persistent lipid depots that could offer long-lasting immune response and protection. Bone marrow cells from DN1Tg/hCD1Tg/Rag-/- (CD45.2) and hCD1Tg mice (CD45.1) were depleted of mature T cells using anti-Thy-1.2 (AT83.A-6) plus rabbit complement (Cedarlane Laboratories). Equal numbers of cells (5x106) were adoptively transferred into hCD1Tg (CD45.1) mice irradiated with 900 rads. After 6 weeks, mixed BM chimeric mice were intratracheally vaccinated with either 2.5 micrograms of MA in MA-BCN or equivalent volume of PBS. CD44+CD62L+ and CD44-CD62L+ DN1 T cells were sorted from lymph nodes of DN1-hCD1Tg BM chimera mice vaccinated with MA-BCN at 6 weeks post vaccination by BD FACS Aria.