(T1430-10-59) Development of a Broadly Active Influenza Intranasal Vaccine Adjuvanted with Self-Assembled Particles Comprised of Mastoparan-7 and CpG

Purpose: The development of new adjuvant systems for influenza vaccines is needed due to limitations of currently licensed adjuvants¹; mast cells are emerging as a target of a new class of mucosal vaccine adjuvants². Here we developed and characterized the adjuvant activity of nanoparticles (NPs) comprised of a mast cell-agonist adjuvant³ (Mastoparan-7 (M7)) and a TLR-ligand (CpG). This novel NP adjuvant was co-formulated with influenza Computational Optimized Broadly Reactive Antigen COBRA hemagglutinin (HA) that is broadly reactive⁴.

Methods: M7 was combined at different ratios with CpG and tested for in vitro immune responses and cytotoxicity by cytokine ELISA and LDH, respectively. After selecting the most immunogenic ratio in vitro, we characterized the NP size through SEM, TEM, and DLS. Mice were vaccinated intranasally with a prime-boost-boost schedule and serum and mucosal samples were collected. Antibody titer, splenocyte response and hemagglutinin inhibition assay (HAI) were determined for the H3 COBRA HA J4.

Results: M7 and CpG NPs showed a significantly higher in vitro cytokine production in dendritic cells and mast cells with the lowest cytotoxicity when combined at a charge neutralizing ratio of nitrogen/phosphate = 1. The combination showed spherical-NPs around 200 nm with self-assembling capability. Splenocytes isolated from mice vaccinated with COBRA HA and M7-CpG NPs had significantly increased cytokine production with antigen recall. Also, they had significantly higher antigen-specific antibody response (IgG, IgA) in serum and mucosa in mice. The co-immunization of NPs and COBRA HA were able to induce influenza H3N2-specific HAI antibody titers across multiple strains.

Conclusion: M7-CpG self-assemble into NPs that when used as an adjuvant with broadly reactive J4 COBRA HA, significantly increase antigen-specific IgG and IgA antibodies, T cell-responses and virus-specific HAI. This illustrates that this NP adjuvant combination can induce protective immune response with a broadly reactive influenza antigen via mucosal vaccination.

References: 1 Zhu, W., Dong, C., Wei, L. & Wang, B. Z. Promising Adjuvants and Platforms for Influenza Vaccine Development. Pharmaceutics 13, doi:10.3390/pharmaceutics13010068 (2021).
2 Johnson-Weaver, B. T. et al. Nasal Immunization With Small Molecule Mast Cell Activators Enhance Immunity to Co-Administered Subunit Immunogens. Front Immunol 12, 730346, doi:10.3389/fimmu.2021.730346 (2021).
3 St John, A. L. et al. Novel mucosal adjuvant, mastoparan-7, improves cocaine vaccine efficacy. NPJ Vaccines 5, 12, doi:10.1038/s41541-020-0161-1 (2020).
4 Allen, J. D. & Ross, T. M. Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019. J Virol 96, e0165221, doi:10.1128/jvi.01652-21 (2022).

Acknowledgments: We would like to thank to the Chapel Hill Analytical and Nanofabrication Laboratory (CHANL) for helping with the molecular characterization, to the National Institute of Health (NIH) for funding this project with the grant R01AI141333 and to the Eshelman School of Pharmacy at UNC-Chapel Hill for providing funding and the facilities for this project.

Fig. 1. M7 + CpG forms self-assembling NPs. TEM micrographs of Vehicle (a), M7 (b), CpG (c) and M7 + CpG (d).



Fig. 2. M7-CpG NPs in vitro immune response. TNF-α and IL-6 release in supernatants from DC2.4 cells (a,b) and MC-9 mast cells (c,d) stimulated with different concentrations of M7, CpG or M7-CpG particles. (n=6, 2 independent experiments) (*Significant differences for a Two-way ANOVA test with Tukey´s multiple comparison p>0.05).



Fig. 3. M7-CpG NPs in vivo immune response. ELISPOT count of IFN-γ (a) and IL-2 (b) -producing cells in splenocytes from the different mouse groups. Anti-J4 serum IgG (c), BAL IgG (d) and feces IgA (e) titres and serum anti-HK/19 HAI titres (f) from the different mouse groups. (n=5) (*Significant differences for a One-way ANOVA test with Tukey´s multiple comparison p>0.05).