Intranasal Immunization With O-2′-Hydroxypropyl Trimethyl Ammonium Chloride Chitosan Nanoparticles Loaded With Newcastle Disease Virus DNA Vaccine Enhance Mucosal Immune Response in Chickens


 Background: There is a great interest to develop strategies for enhancing antigen delivery to mucosal immune system as well as to identify mucosal active immunostimulating agents. To elevate the potential of O -2′-Hydroxypropyl trimethyl ammonium chloride chitosan (O-2′-HACC) nanoparticles as adjuvant and mucosal immune delivery carrier for DNA vaccine, we prepared the O-2′-HACC nanoparticles loaded with Newcastle disease virus F gene plasmid DNA with C3d6 molecular adjuvant (O-2′-HACC/pFDNA). Results: The O-2′-HACC/pFDNA had regular spherical morphology with a particle size of 202.3±0.52 nm, zeta potential of 50.8±8.21 mV, encapsulation efficiency of 90.74±1.10 %, and loading capacity of 49.84±1.20 %. The plasmid DNA could be sustainably released from the O-2′-HACC/pFDNA after an initial burst release. Intranasal vaccination of chickens immunized with O-2′-HACC/pFDNA not only induced higher anti-NDV IgG and sIgA antibody titers, but also significantly promoted lymphocyte proliferation and produced the higher levels of IL-2, IL-4, IFN-γ, CD4+ and CD8+ T lymphocytes than the NDV commercial attenuated live vaccine. Intranasal delivery of the O-2′-HACC/pFDNA enhanced humoral, cellular and mucosal immune responses, and protected chickens from the infection of highly virulent NDV than intramuscular delivery. Conclusions: This study indicated that the O-2′-HACC nanoparticles could be used as vaccine adjuvant and delivery system for mucosal immunity and have an immense application promise.

2 Background 41 Mucosal immune system is an important part of the body's entire immune network, and it plays an active 42 and important role in fighting infection [1]. Mucosal immune response can be improved by selecting the 43 optimal immunization route, vaccine adjuvant and delivery system etc. 3 Viral vectors and non-viral vectors have been used as carrier to deliver gene safely and effectively.

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Although viral vector has many advantages for the delivery of plasmid DNA, one of the most important 64 issues is able to ensure that plasmid DNA is not degraded by lysosomes during transport to the host cell.

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And the viral vector must be non-pathogenic to the human body and will not cause proliferation and spread 66 in the environment, high titer production and high immunogenicity safety [12]. Compared to viral vector, 67 non-viral vector has some advantages, including no infectivity, low immunogenic response, safety, high 68 gene capacity, stability, no carrier capacity limitation, and are easy to prepare in large quantities [13,14].

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Non-viral gene delivery system generally consists of the naked DNA delivery, lipid-based delivery and 70 polymer-based delivery etc. Cationic polymer, which electrostatically interact with plasmid DNA to 71 neutralize its negative charge and condense the plasmid DNA into nanosized particles, is generally served 72 as gene delivery systems. Cationic polymer nanoparticles can protect the plasmid DNA from enzymatic 73 degradation and facilitate cellular uptake. Intramuscularly administered polyvinyl alcohol/plasmid DNA 74 formulation resulted in a significant increasing in the number and distribution of the reporter-gene 75 expressing cells in rat, compared to naked plasmid DNA [15]. Biodegradable, non-antigenic 76 polymer-based microspheres/nanoparticles have many advantages as vaccine adjuvant and delivery system.

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Our previous studies have shown that cellular, humoral and mucosal immune responses can be elicited to 78 antigens encapsulated in, or conjugated onto polymer-based microspheres/nanoparticles [16, 17].

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Since the particle size of nanoparticles is comparable to that of the pathogen, nanoparticles can pass 80 through the interstitial space and capillaries to reach a site that is difficult to administer, and has the many 81 advantages, including controlling drug release, protecting drug from degradation or leakage, and targeting 82 administration etc., thus, nanoparticles can significantly improve the delivery efficiency of plasmid DNA.               Serum IgG antibody titers (A) and IL-2 (B), IL-4 (C), IFN-γ (D) levels in the supernatant of splenocytes harvested from the immunized SPF chickens after challenge with the highly virulent NDV strain F48E9. IFN-γ, IL-2, and IL-4 levels in the supernatant were analyzed in a chicken IFN-γ, IL-2, and IL-4 enzymelinked immunosorbent assay. Results are represented as mean ± SD of three separate experiments. *P<0.05.

Figure 8
Histopathological analyses of glandular stomach, duodenum, and myocardium obtained from healthy chickens and those challenged with the highly virulent NDV strain F48E9. Tissues of the glandular stomach, duodenum, and myocardium from the PBS i.m., blank O-2'-HACC nanoparticles i.n., attenuated live ND vaccine i.m., and O-2'-HACC/pFDNA i.m. and i.n. groups.

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