Enhancing Mucosal Immune Response of Newcastle Disease Virus

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Enhancing mucosal immune response of Newcastle disease virus DNA vaccine using N-2-hydroxypropyl trimethyl ammonium chloride chitosan and N, O-carboxymethyl chitosan nanoparticles as delivery carrier Kai Zhao, Jinyu Han, Yang Zhang, Lin Wei, Shuang Yu, Xiaohua Wang, Zheng Jin, and Yun-Feng Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00826 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 27, 2017

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Molecular Pharmaceutics

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Enhancing mucosal immune response of Newcastle disease virus DNA vaccine using

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N-2-hydroxypropyl trimethyl ammonium chloride chitosan and N, O-carboxymethyl chitosan

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nanoparticles as delivery carrier

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Kai Zhao a, #, *, Jinyu Han b, #, Yang Zhang a, Lin Wei a, Shuang Yu a, Xiaohua Wang a, Zheng Jin b, *, Yunfeng

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Wang c, *

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a

Key Laboratory of Microbiology, School of Life Science, Heilongjiang University, Harbin 150080, China

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b

Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, College

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of Chemistry and Material Sciences, Heilongjiang University, Harbin 150080, China

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c

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Veterinary Research Institute, CAAS, Harbin 150001, China

Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin

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Running title: Quaternized chitosan-based nanodelivery for DNA vaccine

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These authors contributed equally to this work.

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*

Address correspondence to Kai Zhao, [email protected], Zheng Jin, [email protected] and Yunfeng

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Wang, [email protected].

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ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Because mucosal sites are the entry ports of pathogens, immunization via mucosal routes can extremely

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enhance the immunity. To elevate the potential of N-2-hydroxypropyl trimethyl ammonium chloride chitosan

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(N-2-HACC) and N,O-carboxymethyl chitosan (CMC) nanoparticles as a mucosal immune delivery carrier

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for DNA vaccines, we prepared the NDV F gene plasmid DNA with C3d6 molecular adjuvant (pVAX I

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-F(o)-C3d6) encapsulated in the N-2-HACC-CMC nanoparticles (N-2-HACC-CMC/pFDNA-C3d6 NPs).

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The N-2-HACC-CMC/pFDNA-C3d6 NPs had regular spherical morphology and low toxicity with a mean

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diameter of 309.7±6.52 nm, zeta potential of 49.9±4.93 mV, encapsulation efficiency of 92.27±1.48 % and

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loading capacity of 50.75±1.35 %. The N-2-HACC-CMC had high stability and safety. The pVAX I

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-F(o)-C3d6 could be sustainably released from the N-2-HACC-CMC/pFDNA-C3d6 NPs after an initial

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burst release. Immunization intranasally of chickens with N-2-HACC-CMC/pFDNA-C3d6 NPs not only

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produced higher anti-NDV IgG and sIgA antibody than chickens in other groups did, but also significantly

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stimulated lymphocyte proliferation and triggered higher the IL-2, IL-4 and IFN-γ levels. These findings

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indicated that the N-2-HACC-CMC could be used as an efficient and delivery carrier for the mucosal

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immunity of Newcastle disease virus DNA vaccine. The work laid a basis for the quaternized chitosan

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nanoparticles as efficient mucosal immunity delivery carrier for the DNA vaccine, and had immense

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application promise and potential for vaccines and drugs.

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Key words: Newcastle disease; F gene; chitosan derivative nanoparticles; intranasal delivery; mucosal

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immunity

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Introduction Most pathogens access the body through the mucosal membranes. Protective mucosal immune

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responses can be effectively triggered by mucosal administration route such as oral, nasal, rectal or vaginal

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routes. However, despite early success with the live attenuated oral Sabin polio vaccine over 50 years ago,

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only a few new mucosal vaccines in use today have been subsequently launched. Thus, there is an important

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requirement to develop vaccines against many of the pathogens that infect mucosal tissues or have a

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mucosal port of entry. Parenteral vaccination may protect in some instances, but usually a mucosal

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vaccination route is necessary. Mucosal vaccines can prevent both the entrance of the pathogens into the

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body, the inactivation of pathogens and the dissemination of pathogens.1 Additionally, mucosal vaccines also

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have lots of advantages over injectable vaccines by being simpler to administer, having less risk of

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transmitting infections and potentially being easier to manufacture.2 Developing mucosal vaccines based on

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the biomaterial nanoparticles as adjuvant and delivery carrier can circumvent some shortcomings of

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conventional vaccines. The nano vaccines can enhance humoral, cell-mediated and mucosal immune

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response through the sustained release and protection against degradation of loaded antigen.

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Currently, DNA vaccines have displayed a huge potential for the development of new vaccines, and

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increasing attention has been focused on DNA vaccines because of their easy production, superior stability

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in ambient temperature, non-requirement for cold chain, and their ability to generate antigen-specific

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immune responses.3, 4 However, many studies have indicated that DNA vaccines usually administered via

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intramuscular injection can fail to reach the antigen-presenting cells (APCs) and therefore fail to induce

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immune responses because of the difficulty for them to pass through cell membranes.5, 6 Also, weak immune

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responses and low levels of DNA vaccine expression will be induced, especially in large animals.7 At present, 3



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in order to improve the efficacy of DNA vaccines, several effective strategies are desirable, including

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optimization of plasmid DNA, delivery methods, selection of suitable target for effective antigen

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presentation, and the use of a powerful adjuvant to enhance immunogenicity.8-10

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When it comes to gene delivery, the two major gene delivery methods are viral and non-viral

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(nanoparticles). Each delivery system has its own set of advantages and disadvantages. Although viral

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delivery of plasmid DNA is extremely efficient, nanoparticles (NPs) technologies have also been proved to

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significantly improve effectiveness in the delivery of plasmid DNA compared to viral-based delivery

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systems, and the NPs approach is desirable.11 Various NPs have been developed, such as iron oxide NPs, gold

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NPs, cerium oxide NPs, carbon-based materials and polymeric NPs.12-14 In all potential NPs delivery systems,

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polymeric NPs when used as nanosized particulate carriers can either encapsulate or entrap the DNA

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vaccines, antigens, proteins and drugs and deliver it to the desired site of action.2 NPs have lower

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cytotoxicity and can protect the antigens from being damaged under unfavorable conditions after systemic or

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mucosal administration, and the uptake of nanoparticles by APCs can increase and induce potent immune

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responses;15, 16 Additionally, nanomaterials are widely used as vaccine delivery systems because they cause

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the vaccine antigen to be released slowly.17 Hence, the disadvantages of DNA vaccines can be avoided when

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biomaterials-based nanoparticles are used as delivery carrier of vaccine antigens.18 NPs have great

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advantages and are attracting much attention, especially in the field of vaccines and drugs delivery.19, 20

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In all of the natural polymers, dextran and chitosan are the two most popular polysaccharides for the

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formulation of NPs for the delivery of plasmid DNA. Dextran is neutral at physiological pH and therefore

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can not effectively bind DNA, thus chitosan NPs are significantly more popular due to their cationic nature.

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Chitosan also known as a linear amino polysaccharide, is rich in chitin material from exoskeleton of 4


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crustaceans and insects, or the endoskeletons of cephalopods, or the cuticles of arthropods and fungal cell

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walls.12 Chitosan which consists of both 2-amino-2-deoxy-β-D-glucan units and

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2-acetamido-2-deoxy-β-D-glucan, has great potential in biomedicine because of its interesting biological and

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physicochemical properties. Chitosan as a drug delivery system has been extensively evaluated. Presently,

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chitosan and its derivatives have drawn much attention as both adjuvant and delivery carrier for vaccines.21

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Chitosan can bind with negatively charged protein or plasmid DNA by electrostatic interaction and protect

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them from degradation,22 hence it is widely used as an delivery carrier in the field of vaccines.22 However,

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chitosan can be dissolved in acidic solution, although not easily dissolved in the condition of physiological

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potential of hydrogen (pH).23 This poor solubility becomes the largest limitation of chitosan for medical

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application, since it must be dissolved in aqueous solutions with a positive charge for transporting a target

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vaccine into cells.25 The abundant hydroxyl and amino groups on the chitosan skeleton make it easy for

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chemical modification. Chitosan derivatives with certain functional groups can be obtained by chemical

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modification of chitosan. Compared with chitosan, the performance of derivatives is often better than

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chitosan.26 Thus, nanoparticles based on water-soluble chitosan derivatives have become new drug carriers

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because of their stability and biocompatibility in biological solutions (pH 7.4). Nowadays, there are many

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studies on chitosan derivatives, such as acylation, alkylation, sulfation, hydroxylation, quaternization and

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carboxymethylation.27-30 Recently, our group has synthesized O-2′-hydroxypropyl trimethyl ammonium

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chloride chitosan (O-2′-HACC),31 N-2-hydroxypropyl trimethyl ammonium chloride chitosan

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(N-2-HACC),32 N-2-hydroxypropyl dimethyl ethyl ammonium chloride chitosan (N-2-HFCC) 33 and

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N,O-carboxymethyl chitosan (CMC).32 O-2′-HACC, N-2-HACC, N-2-HFCC and CMC have the higher

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water solubility than chitosan,31-33 and N-2-HACC has higher water solubility and a more suitable 5



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nanoparticles size than O-2′-HACC and N-2-HFCC.34 Additionally, the preparation process of N-2-HACC

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was easier, convenient and lower cost than that of O-2′-HACC and N-2-HFCC, and there are no organic

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solvents in the preparation process, so antigen immunogenicity can’t be destroyed. Moreover, the synthesis

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of N-2-HACC was optimized, and the relationship between nanoparticles size and degree of N-2-HACC

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substitution has also been studied by our group.35 N,O-carboxymethyl chitosan (CMC) is a

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carboxymethylated product of chitosan, with better solubilityin water compared to N-2-HACC. However,

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CMC has lower encapsulation efficiency and drug loading than O-2′-HACC, N-2-HACC and N-2-HFCC.

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Therefore, to further improve water solubility, encapsulation efficiency and loading capacity, we developed

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two chitosan derivatives, positively charged N-2-HACC and negatively charged CMC. In the present study,

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to prove whether the biodegradable polymers N-2-HACC and CMC nanoparticles can also be used as a

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delivery carrier to realize sustained release and induce desired mucosal immunity for DNA vaccines, we

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prepared the NDV F gene plasmid DNA with C3d6 molecular adjuvant (pVAX I -F(o)-C3d6) encapsulated

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in the N-2-HACC-CMC nanoparticles (N-2-HACC-CMC/pFDNA-C3d6 NPs) by the polyelectrolyte

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complex method, and their characteristics for the intranasal delivery of NDV DNA vaccine were studied. In

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vitro and in vivo experiments were performed to systematically evaluate the adjuvant effect of

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N-2-HACC-CMC nanoparticles. This study has provided foundation for the further development and

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immense application potential of mucosal vaccines and drugs encapsulated in biodegradable polymeric

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nanoparticles.

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Materials and methods

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Synthesis of N-2-hydroxypropyl trimethyl ammonium chloride chitosan and N,O-carboxymethyl

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chitosan 6


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The two water soluble chitosan derivatives with positive charge, N-2-hydroxypropyltrimethyl

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ammonium chloride chitosan (N-2-HACC) and N,O-carboxymethyl chitosan (CMC), are synthesized by our

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laboratory as the nanomaterials for the delivery of vaccine antigen. Synthesis of N-2-HACC and CMC were

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carried out as described previously.32

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Preparation of the N-2-HACC-CMC nanoparticles encapsulating plasmid pVAX I -F(o)-C3d6

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The eukaryotic expression plasmid pVAX-optiF that carries and drives the expression of F gene of

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NDV was provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences

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(Harbin, China). The eukaryotic expression plasmid pVAX-optiF with C3d6 molecular adjuvant (pVAX I

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-F(o)-C3d6) was constructed by our Laboratory in School of Life Science of Heilongjiang University, China.

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The pVAX I -F(o)-C3d6 was encapsulated in N-2-HACC-CMC nanoparticles

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(N-2-HACC-CMC/pFDNA-C3d6 NPs) by the polyelectrolyte complex method as follows. 2 ml of the 100

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mg/ml pVAX I -F(o)-C3d6 solution was slowly added to 5 ml of 1.0 mg/ml N-2-HACC solution under

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stirring at 300 r/min for 3 min, followed by stirring again but at 1200 r/min for 1 min. Next, 2 ml of the 0.85

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mg/ml CMC solution was slowly added to the mixture and stirring continued at 1200 r/min for 40 min.

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Finally, after stirring at 4o C for 20 min at 12000 r/min, the supernatant was removed, and the precipitate was

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resuspended in ddH2O at 4o C. The process was repeated three times, and the final precipitate dissolved in 2

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ml of ddH2O to obtain N-2-HACC-CMC/pFDNA-C3d6 NPs.

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Characterization of the N-2-HACC-CMC/pFDNA-C3d6 NPs

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The N-2-HACC-CMC/pFDNA-C3d6 NPs were observed under transmission electron microscopy

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(JEM-200EX, Hitachi Ltd, Japan) to assess their surface characteristics and morphology. The zeta potential

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and particle size of N-2-HACC-CMC/pFDNA-C3d6 NPs were measured by a Zeta Sizer ZS90 (Malvern 7



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Instruments Ltd., Southborough, MA, USA). Loading capacity (LC) and Encapsulation efficiency (EE) were

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measured as described previously.31

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DNase protection of the N-2-HACC-CMC/pFDNA-C3d6 NPs

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To test stability of the N-2-HACC-CMC/pFDNA-C3d6 NPs, 100 µl of

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N-2-HACC-CMC/pFDNA-C3d6 NPs suspension were incubated with 25 µl of DNase (TaKaRa, Dalian,

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China) at 37º C for 30, 60, 120 and 180 min, respectively. The final concentration of the DNase is 1.0 U/ml.

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Next, 100 µl of termination solutions (400 mmol/l NaCl, 100 mmol/l EDTA, pH 8.0) was added into the

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reaction system at 65º C for 10 min to terminate the reaction. The plasmid pVAX I -F(o)-C3d6 as the

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negative control was incubated at 37º C for 30 min. The integrity of plasmid pVAX I -F(o)-C3d6 was

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analyzed by using 0.8 % agarose gel electrophoresis at 100 V for 30 min.

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In vitro release of the N-2-HACC-CMC/pFDNA-C3d6 NPs

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To determine the release of pVAX I -F(o)-C3d6 from the nanoparticles, the

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N-2-HACC-CMC/pFDNA-C3d6 NPs suspension was centrifugalized at 4º C at 16000 r/min for 30 min and

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the precipitate was re-suspended in 2 ml of PBS buffer (pH 7.4) and stirred at 100 r/min at 4º C. Samples

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(100 µl) were withdrawn at the different time intervals, and the same volume of fresh PBS was added. The

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sample was centrifuged at 4º C at 12,000 r/min for 20 min. The concentration of the released pVAX I

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-F(o)-C3d6 in the supernatant was determined by UV spectrophotometry. All the experiments were repeated

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five times.

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Safety of the N-2-HACC-CMC/pFDNA-C3d6 NPs

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In vitro cytotoxicity assay

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To test safety of the N-2-HACC-CMC NPs as adjuvant and plasmid DNA delivery carrier for mucosal 8


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immunity, in vitro cytotoxicity was evaluated by the Cell Counting Kit-8 (CCK-8) reagent (Dojindo Ltd,

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Kumamoto, Japan), and the survival rate of chicken embryonic fibroblast (CEF) cells was determined by

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measuring OD450 according to the reference.31

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In vivo safety assay

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To test in vivo biological safety, , thirty 4-week-old SPF chickens obtained from Animal Center of

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Harbin Veterinary Research Institute Laboratory were randomly assigned into three groups, were inoculated

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with ten times the dose. Chicken in Group 1 were adminisered i.m. with N-2-HACC-CMC/pFDNA-C3d6

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NPs suspension; Chicken in Group 2 were administered i.n. with N-2-HACC-CMC/pFDNA-C3d6 NPs

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suspension; and chicken in Group 3 were administered i.m. with 0.2 ml of PBS (pH 7.4). The three groups

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were monitored continuously for 14 days, and any abnormal changes including feeding, drinking, mental

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state and body weight were recorded.

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In vitro expression of the N-2-HACC-CMC/pFDNA-C3d6 NPs

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An in vitro transfection assay was performed according to the instructions from the LipofectamineTM

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2000 reagent kit (Invitrogen, USA), and the expression of pVAX I -F(o)-C3d6 in the transfected cells was

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detected by using an indirect immunofluorescent test. This transfection experiment was divided into 4

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groups. Group 1 was N-2-HACC-CMC/pFDNA-C3d6 NPs containing 4 µg of plasmid pVAX I -F(o)-C3d6

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which was transfected into 293T cells; Group 2 was 4 µg of plasmid pVAX I -F(o)-C3d6; Group 3 was blank

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N-2-HACC-CMC nanoparticles included as a negative control; and Group 4 was cell control. The NDV

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positive serum and fluorescein isothiocyanate-labeled goat-anti-chicken IgG from Sigma was diluted at

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1:100 and 1:5, 000, respectively. Epifluorescence images were obtained using an Axio observer Z1

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microscope (Zeiss). 9



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Immunization of the N-2-HACC-CMC/pFDNA-C3d6 NPs One hundred 1-day-old healthy SPF chickens obtained from Animal Center of Harbin Veterinary

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Research Institute Laboratory were randomly assigned into five groups with 20 chickens in each group.

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Chickens in Group 1 were immunized i.m. with PBS buffer as a negative control; Chickens in Group 2 were

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immunized i.m. with blank N-2-HACC-CMC-NPs; Chickens in Group 3 were immunized i.m. with 0.1 ml

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of pVAX I -F(o)-C3d6 (200 µg); Chickens in Groups 4 and 5 were immunized with 0.1 ml of

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N-2-HACC-CMC/pFDNA-C3d6 NPs containing 200 µg of pVAX I -F(o)-C3d6 i.m. and i.n., respectively. At

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two weeks post first immunization, booster immunization was performed with the same dosages and routes

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as the first immunization. The experimental protocol was approved by the Animal Ethics Committee as

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stipulated in the guide to the care and use of experimental animals of the Harbin Veterinary Research

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Institute of the Chinese Academy of Agricultural Sciences. The chickens were euthanized by intravenous

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injection of pentobarbital.

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Detection of serum IgG antibody

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Blood samples were collected via the wing veins from three chickens in each of the five groups post

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immunization per week. The serum samples were obtained by centrifugation at 4º C at 3000 r/min for 10

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min. The titers of anti-NDV IgG antibody were detected by hemagglutination inhibition (HI) test (n=3).

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Detection of IgA antibody

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To detect the mucosal immune response, serum, tracheal fluid, bile and Harderian glands were

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collected from three chickens at 1 to 10 week post the immunization. Mucosal extracts were obtained by

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centrifugation and the supernatant was collected. The titers of IgA antibody were detected by using the NDV

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IgA ELISA Kit (Rapidbio Co. Ltd, Beijing, China) (n=3). 10


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Lymphocyte proliferation response The cellular-mediated immune responses of immunized chickens were assessed at 1, 2, 3, 4, 5, 6, 7, 8 9,

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and 10 weeks post immunization, and the MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3,

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5-diphenytetrazoliumromide) colorimetric assay was performed to evaluate lymphocyte proliferation as

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described previously.31 Splenic lymphocytes were prepared from all the experimental chickens using the

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standard protocol.

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Detection of IL-2, IL-4 and IFN-γ

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For cytokines assays, serum samples were collected from three chickens at 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10

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weeks post immunization. The concentration of IFN-γ, IL-2 and IL-4 in spleen cell culture supernatants

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were detected by using a chicken IFN-γ, IL-4 and IL-2 ELISA (enzyme-linked immunosorbent assay) Kit

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(Thermo Fisher Scientific Inc, MA, USA) in accordance with the manufacturer’s instructions, respectively.

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All the operations were performed according to the procedures described for the cytokine ELISA kits.

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Protection against NDV strain F48E9

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The protection conferred by N-2-HACC-CMC/pFDNA-C3d6 NPs on chickens against NDV infection

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was investigated using a virulent strain of NDV (NDV strain F48E9) with genotype IX (Harbin

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Pharmaceutical Group Bio-vaccine Co. Ltd). When the levels of NDV-specific antibody in serum of every

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immune group increased to 6.0 log2 post first immunization, seven chickens were selected randomly from

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each of the five groups and were infected i.m. with 100 µl of strain F48E9 at a viral titer of 104.5 EID50/0.1

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ml for challenge studies. Meanwhile, feed, water, mental state, clinical symptoms and mortality of chickens

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were continuously observed and recorded for 35 d. Serum samples were collected from three chickens at 1, 2,

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3, 4, and 5 weeks after challenge, the contents of IgG, IL-2, IFN-γ and IL-4 were determined. The negative 11



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control chickens and infected chickens were euthanized, and the glandular duodenum, stomach and

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myocardium were collected for histological staining assay.

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Statistical analysis

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The immunization experiments were repeated three times under the same conditions. Data are

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presented as mean±standard deviation. One-way analysis of variance (ANOVA) statistical test with Tukey’s

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post test was performed using Origin 7.5 software (OriginLab Corporation, USA) to determine the

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significance of the differences between various groups. P0.05). In vivo cytotoxicity analysis demonstrated that the drinking, feeding, weight and

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other behavior of chickens immunized with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. or i.m. were

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normal compared with those of the control group, and the morbidity and mortality of chickens were 0 % in

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both groups, indicating that immunization of the chickens with a high dose of

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N-2-HACC-CMC/pFDNA-C3d6 NPs is safe. Histopathological analyses of glandular stomach and

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duodenum were shown in Fig. 3A-F. These results indicated that the N-2-HACC-CMC NPs as delivery

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carrier had little cytotoxicity but had higher safety level by the intranasal administration route.

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In vitro expression of the N-2-HACC-CMC/pFDNA-C3d6 NPs

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As shown in Fig. 4, intensive fluorescence was observed in 293T cells transfected with the 13



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N-2-HACC-CMC/pFDNA-C3d6 NPs (Fig. 4A) and pVAX I-F(o)-C3d6 (Fig. 4B), and the level of

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expression was greater for nanoparticle-mediated delivery than the pVAX I -F(o)-C3d6, probably due to the

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protection from DNase degradation. In contrast, no fluorescence was detected in the 293T cells transfected

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with N-2-HACC-CMC nanoparticles (Fig. 4C) and the negative cells control group (Fig. 4D). These results

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indicate that the pVAX I -F(o)-C3d6 can be effectively encapsulated by the N-2-HACC-CMC nanoparticles

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and expressed in vitro.

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Immune efficacy in SPF chickens

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Serum HI antibody

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As shown in Fig. 5A, N-2-HACC-CMC/pFDNA-C3d6 NPs i.n., N-2-HACC-CMC/pFDNA-C3d6 NPs

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i.m. and pVAX I -F(o)-C3d6 i.m. induced significant antibody responses in chickens when they were

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immunized, and the antibody titers of chickens were quickly increased and peaked at the sixth week post

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immunization. After the sixth week, chickens immunized with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n.

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or N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. produced higher titers of anti-NDV than those immunized with

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the pVAX I -F(o)-C3d6 i.m. until the tenth week (P0.05), however, there was significant difference at the tenth week (P

Enhancing Mucosal Immune Response of Newcastle Disease Virus

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