Lung tissue-resident memory T (T_RM) cells induced by influenza vaccination are crucial for heterosubtypic immunity upon re-exposure to the influenza virus, enabling rapid and robust responses upon reactivation. To enhance the efficacy of influenza vaccines, we induce the generation of lung T_RM cells following intranasal vaccination with a commercial influenza vaccine adjuvanted with NexaVant (NVT), a TLR3 agonist-based adjuvant. We demonstrate that intranasal immunization with the NVT-adjuvanted vaccine provides improved protection against influenza virus infections by inducing the generation of CD4⁺ T_RM cells in the lungs in a type I interferon-dependent manner. These pulmonary CD4⁺ T_RM cells provide potent mucosal immunity and cross-protection against heterosubtypic infections in both mouse and ferret models. This vaccine platform has the potential to significantly improve conventional intramuscular influenza vaccines by providing broader protection.

Lung-resident memory T (T_RM) cells respond rapidly and effectively to respiratory pathogen invasion, suppressing pathogen proliferation. Previously, we identified a defined TLR3 agonist called Nexavant (NVT) and developed a vaccine platform that utilizes it to induce lung T_RM. In this study, we aimed to determine whether the protective effect of T_RM cells is observed in tuberculosis (TB), a chronic bacterial respiratory disease. We synthesized a peptide vaccine by elongating the CD4⁺ T cell epitopes from Mycobacterium tuberculosis antigens ESAT-6, CFP-10, and HspX, adjuvanted it with NVT and administered the vaccine intranasally or intramuscularly to mice. We demonstrated that intranasal administration of an NVT-formulated peptide vaccine induced the generation of CD4⁺ T_RM cells in the lungs, and that our vaccine platform, containing a limited number of CD4 epitopes, provided protective efficacy comparable to that of the BCG vaccine, which contains multiple T cell epitopes. Furthermore, the peptides used in the vaccine were reactive in 23 out of 24 (95.8%) human PBMCs, indicating that they contain promiscuous epitopes. Our results suggest a straightforward approach to controlling pulmonary TB more effectively through the induction of lung CD4⁺ T_RM cells, even when using the same target antigen. Additionally, this study supports a theoretical basis for developing an inhalable TB vaccine using synthetic peptides.

Nexavant was reported as an alternative to the TLR3 agonist of Poly(I:C) and its derivatives. The physicochemical properties, signaling pathways, anti-cancer effects, and mechanisms of Nexavant were investigated. The distinctive characteristics of Nexavant compared to that of Poly(I:C) were demonstrated by precise quantification, enhanced thermostability, and increased resistance to RNase A. Unlike Poly(I:C), which activates TLR3, RIG-I, and MDA5, Nexavant stimulates signaling through TLR3 and RIG-I but not through MDA5. Compared to Poly(I:C), an intratumoral Nexavant treatment led to a unique immune response, immune cell infiltration, and suppression of tumor growth in various animal cancer models. Nexavant therapy outperformed anti-PD-1 antibody treatment in all the tested models and showed a synergistic effect in combinational therapy, especially in well-defined cold tumor models. The effect was similar to that of nivolumab in a humanized mouse model. Intranasal instillation of Nexavant led to the recruitment of immune cells (NK, CD4+ T, and CD8+ T) to the lungs, suppressing lung metastasis and improving animal survival. Our study highlighted Nexavant’s defined nature for clinical use and unique signaling pathways and its potential as a standalone anti-cancer agent or in combination with anti-PD-1 antibodies.

Potent cellular immune responses are crucial for the development of effective vaccines against cancer and chronic infectious diseases. Here, we formulate Nexavant (NVT), a well-characterized TLR3 agonist, into lipid nanoparticles (LNPs) using either the ionizable lipid SM-102 or the cationic lipid DOTAP, and characterize their physicochemical properties and adjuvant potential. Both formulations achieve high encapsulation efficiency and enhance cellular uptake. In contrast to the stronger in vitro potency of DOTAP-based NVT/LNPs, SM-102–based NVT/LNPs (NVT/SM-LNPs) induce greater dendritic cell activation, cytokine production, and systemic T cell responses in vivo, likely due to more efficient delivery of NVT to the spleen. As an adjuvant for peptide vaccines, NVT/SM-LNP enhances antigen-specific CD4⁺ and CD8⁺ T cell responses and demonstrates potent therapeutic efficacy across subcutaneous, orthotopic, and metastatic TC-1 and B16-OVA tumor models, while also reducing viral titers in a chronic LCMV infection model. Compared to conventional adjuvants (poly(I:C), CpG, GM-CSF, IFA) and current mRNA vaccine platforms at clinically relevant doses, NVT/SM-LNP elicits stronger T cell immunity and enables effective neoantigen responses without requiring peptide-carrier conjugation. These findings establish NVT/SM-LNP as a potent adjuvant for T cell–targeted vaccines, with the lipid composition critically influencing immune targeting and efficacy, thereby guiding the design of next-generation vaccines.

Mucosal vaccines offer the advantage of inducing immunity at pathogen entry sites; however, concerns about safety and limited efficacy have hindered the widespread use of viral-vectored intranasal vaccines. Nexavant (NVT), a well-defined TLR3 agonist, was investigated as a non-viral mucosal adjuvant. NVT was formulated with commercial subunit and polysaccharide–protein conjugate antigens and administered intranasally to mice. NVT-adjuvanted vaccines elicited robust mucosal IgA and systemic IgG responses, enhanced antigen-specific CD4 ⁺ T cell activation, and conferred strong protection against high-dose influenza virus challenge. Antigen-specific mucosal IgA was detected not only in nasal washes but also in distal mucosal sites such as saliva, vaginal washes, and feces, indicating broad mucosal immune crosstalk. These immune responses were abolished in IFNAR1 ^- / ^- mice, demonstrating a critical role for type I interferon signaling in NVT’s mechanism of action. The adjuvant was effective across diverse antigen types and demonstrated a favorable safety profile. These findings support NVT as a promising mucosal adjuvant platform for next-generation intranasal vaccines.