Formulation dependent and nonsynergistic effects of TLR4 and TLR7 agonists in a liposomal LdAIRK leishmaniasis vaccine

Visceral leishmaniasis remains a neglected tropical disease for which no licensed human vaccine is available. Although whole-cell vaccines can induce durable Th1 responses, purified subunit vaccines often require optimized delivery and adjuvant systems. Here, we evaluated recombinant Leishmania donovani Aurora-like kinase (LdAIRK), a conserved cell-cycle-associated antigen, as a candidate subunit vaccine. NetMHCpan-based analysis predicted multiple MHC class I and II binding peptides within LdAIRK, supporting its immunogenic potential. LdAIRK was incorporated into DSPC-based cationic liposomes containing individual or combined Toll-like receptor agonists, namely MPLA, R848 and 3M052, and tested in a murine model of visceral leishmaniasis. Among the formulations evaluated, MPLA-adjuvanted liposomal LdAIRK produced the most consistent short-term protective efficacy, with elevated IFN-γ and IL-12 secretion, measurable delayed-type hypersensitivity responses, Th1-associated antibody profiles, and reduced liver and splenic parasite burdens for up to 3 months post-infection. Antigen entrapment in liposomes improved protection compared with empty liposomes, but maximal protection required MPLA incorporation. In contrast, R848- and 3M-052-containing formulations induced mixed Th1/Th2-associated responses and did not improve protection beyond MPLA under the present formulation and dosing conditions. The combined use of TLR4 and TLR7/8 agonists also failed to provide additive protection. Overall, this comparative preclinical study identifies MPLA-adjuvanted liposomal LdAIRK as the most favourable formulation under the experimental conditions evaluated and provides a basis for future studies examining durability and long-term memory in addition to highlighting how adjuvant identity and delivery context shape protective immunity against visceral leishmaniasis.