# Genetically engineered M13 phage-mediated H9N2 DNA vaccine with enhanced mucosal and systemic immune responses in mice

## TL;DR

A new DNA vaccine delivery system using engineered M13 phage and nanoparticles boosts immune responses and protection against H9N2 bird flu in mice.

## Contribution

A novel DNA vaccine platform combining genetically engineered M13 phage and chitosan-based nanoparticles is developed for enhanced immune responses.

## Key findings

- The composite nanoparticles effectively protect and deliver the HA gene while promoting immune cell maturation.
- Mice immunized with the new vaccine showed higher antibody levels and longer protection than a commercial vaccine.
- The vaccine enhanced both mucosal and systemic immune responses, including increased cytokine and T cell levels.

## Abstract

Vaccine adjuvants and delivery systems have long been used in DNA vaccines to enhance immunogenicity. In this study, we developed a DNA vaccine delivery platform by combining N-2-hydroxypropyl trimethyl ammonium chloride chitosan/carboxymethyl chitosan nanoparticles (N-2-HACC/CMCS NPs) with a genetically engineered M13 phage containing the HA gene of H9N2 AIV (HA-M13). The composite NPs (HA-M13/N-2-HACC/CMCS) had an average particle size of 135.24 ± 4.36 nm, and the HA gene encapsulated in the composite NPs could be expressed in vitro. Additionally, the N-2-HACC/CMCS NPs exhibited high stability and effectively protected the HA gene and M13 phage from degradation while sustaining antigen release. Furthermore, the N-2-HACC/CMCS NPs promoted the maturation of DC2.4, enhanced MHC I and MHC II pathways and improved cellular, humoral, and mucosal immune responses. Mice immunized with HA-M13/N-2-HACC/CMCS via nasal and intramuscular injections presented higher anti-H9N2 AIV antibody titers than those given the commercial vaccine. Lymphocyte proliferation, as well as the levels of the cytokines IL-2, IL-4, IFN-γ, CD4+, and CD8+ T lymphocyte levels, also significantly increased. The nanovaccine provided effective protection against H9N2 AIV infection for 154 days postimmunization, surpassing the 120-day protection provided by the commercial vaccine. Consequently, the N-2-HACC/CMCS NPs loaded with M13 phages exhibit significant potential as vaccine adjuvants and mucosal immune delivery system.