Development of an Intranasally- and Intramuscularly-Administrable Replicon Vaccine Efficacious Against H5N1 Influenza Virus

McClary, W. D.; Brandt, D. S.; Jennewein, M. F.; Singh, J.; Beaver, S.; Ykema, M. R.; Press, C.; Melief, E.; Bakken, J.; Fusco, P.; Lo, E.; Battisti, P.; Cross, N.; Kasal, D. N.; Hartwig, A. T.; Casper, C.; Bowen, R. A.; Gerhardt, A.; Voigt, E. A.

doi:10.1101/2025.03.31.646478

bioRxiv · 10.1101/2025.03.31.646478

Development of an Intranasally- and Intramuscularly-Administrable Replicon Vaccine Efficacious Against H5N1 Influenza Virus

McClary, W. D.·Brandt, D. S.·Jennewein, M. F.·Singh, J.·Beaver, S.·Ykema, M. R.·Press, C.·Melief, E.·Bakken, J.·Fusco, P.·Lo, E.·Battisti, P.·Cross, N.·Kasal, D. N.·Hartwig, A. T.·Casper, C.·Bowen, R. A.·Gerhardt, A.·Voigt, E. A.

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Abstract

The risk of a respiratory viral pandemic is significant, including from the now widespread panzootic H5N1 influenza virus, highlighting the need for effective, stable, and inexpensive vaccine technologies that elicit strongly protective immunity. Intranasal vaccines can stimulate local immune responses at the site of natural respiratory viral infection, a key characteristic that can not only reduce morbidity and mortality caused by respiratory viruses but also potentially reduce viral transmissibility to limit outbreaks. Nucleic acid vaccines are now a valuable tool in pandemic responses, with high potency and rapid adaptability to target circulating or emerging viral strains; however, data are limited on which vaccine attributes are needed for efficient transmucosal delivery and immune stimulation following intranasal delivery. To demonstrate proof of concept, here we have developed a replicon vaccine expressing an H5 influenza antigen that uses a nanostructured lipid carrier (NLC) delivery system. A relationship was established between the molar ratio of positive charges on the NLC to the negative charges on the nucleic acid (N:P ratio) and the immunogenicity of the vaccine formulations, with higher N:P ratios resulting in an increase in vaccine immunogenicity. We demonstrated the ability of this replicon vaccine to be administered via intramuscular and intranasal routes with a singular vaccine formulation. The vaccine induced systemic immunity when dosed intramuscularly or intranasally in an immunocompetent mouse model, whereas intranasal dosing uniquely stimulated a strong mucosal immune response. Moreover, a mixed intramuscular/intranasal dosing strategy using this unified formulation stimulated a balanced systemic and mucosal immune response. Finally, we demonstrated the protective efficacy of this intranasally and intramuscularly/intranasally delivered H5 replicon-NLC vaccine against morbidity and mortality in a lethal H5N1 influenza challenge ferret model. This work establishes the replicon-NLC vaccine platform as a potential novel intranasal technology for rapid pandemic response.

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McClary, W. D., Brandt, D. S., Jennewein, M. F., Singh, J., Beaver, S., Ykema, M. R., Press, C., Melief, E., Bakken, J., Fusco, P., Lo, E., Battisti, P., Cross, N., Kasal, D. N., Hartwig, A. T., Casper, C., Bowen, R. A., Gerhardt, A., Voigt, E. A.. 2025-04-05. Development of an Intranasally- and Intramuscularly-Administrable Replicon Vaccine Efficacious Against H5N1 Influenza Virus. https://doi.org/10.1101/2025.03.31.646478

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