Preparation and immunological effectiveness of a swine influenza DNA vaccine encapsulated in chitosan nanoparticles

Abstract

Preparation conditions of a DNA vaccine against swine influenza encapsulated in chitosan nanoparticles were determined. The nanoparticles were prepared according to a complex coacervation method using chitosan as a biodegradable matrix forming polymer. Under the preparation conditions, chitosan nanoparticles containing the DNA vaccine were produced with good morphology, high encapsulation rate and high stability. Transfection test indicated that the vaccine could be expressed as an antigen in cells, and maintained good bioactivity. In addition, better immune responses of mice immunized with the chitosan nanoparticles containing the DNA vaccine were induced and prolonged release of the plasmid DNA was achieved compared to the DNA vaccine alone. These results laid a foundation for further development of DNA vaccines in nanoparticles before ultimate industrial application.

Introduction

Swine influenza, commonly known as ‘Swine Flu’ or ‘Hog Flu’, caused by swine influenza virus (SIV), is an acute, highly infectious disease. It is one of the most prevalent respiratory infections of the swine worldwide [1], [2]. The virus poses an even greater threat when combined with secondary infections from other pathogens. Influenza virus affecting the pigs is capable of producing flu in humans, animals and birds. The current swine influenza vaccine is an inactivated SIV. This type of vaccines must be used at a high dosage with adjuvants, which increases the cost of vaccination. In addition, vaccine production in chicken embryos may alter the antigenicity of some components of the virus, causing a significant reduction in immune response and safety. Therefore, a new type of swine influenza vaccine is needed [3].

Genetic immunization has emerged as one of the most promising applications of non-viral gene therapy [4]. Immunization with antigen-encoding plasmid DNA can elicit strong and long-lasting humoral and cellular immune responses. It has overcome many problems associated with the traditional vaccine, and thus becomes a focus in the research field [5]. A few recent studies on DNA vaccines against hemagglutinin (HA) of SIV showed some positive effects [6], [7]. The potential advantages of DNA vaccines over conventional vaccines include: (i) the high stability of plasmid DNA, (ii) low manufacturing cost, (iii) lack of infection risk associated with attenuated viral vaccines, (iv) the capacity to target multiple antigens on one plasmid, and (v) the ability to elicit both humoral and cellular immune responses [4], [8]. Until recently, intramuscular (i.m.) injection was the primary route for DNA vaccine administration. However, this method is obviously not feasible for field application. In addition, the low bioavailability of plasmid DNA in the muscle coupled with the redundant nature of antigen transfer by muscle cells are obvious problems for this route of delivery [9], [10]. As an alternative to intramuscular administration, researchers have investigated targeting plasmid DNA to the skin using intradermal needle injection, needle-free jet injection devices, or gene gun. Intradermal needle injection of plasmid DNA into the skin has been shown to be more effective than intramuscular injection in several animal species in eliciting immune responses [4]. Furthermore, several preclinical animal studies have reported the use of needle-free jet injection devices and the gene gun to administer plasmid DNA [11], [12], [13]. However, DNA vaccines are administered as either aqueous solutions or injection-ready frozen powders. DNA vaccines contain plasmid molecules of 2–10 kb that have high hydrophilicity and a low coefficiency of distribution between oil and water phase. After intramuscular injection, it is difficult for the vaccines to move through cell membranes, so only a small amount reaches antigen presenting cells (APC) to induce immune responses [14], [15]. Low levels of DNA vaccine expression and weak immune responses, especially in large animal models, have limited the clinical applications of these novel vaccines [20]. As indicated by previous studies [16], [17], the need for more effective delivery systems that would improve the transfection efficiency in vivo and allow for much lower dose of plasmid DNA is pressing.

To meet the need of simple and cost effective DNA vaccine delivery systems for mass vaccination in farms, a number of new techniques have been recently developed to introduce foreign DNA into cells. One approach is non-viral delivery system. Cationic lipids and cationic polymer have been employed as non-viral gene transfer agents. In mice, DNA vaccines have been delivered orally using a variety of carriers [18]. Previous studies have reported the feasibility of gene transfer into fish by encapsulating the DNA into chitosan and incorporating into fish feeds [19]. Chitosan is a natural biodegradable polysaccharide extracted from crustacean shells [20]. It is proved that the chitosan is non-toxic both in experimental animals [21] and humans [22]. Chitosan has been shown to effectively bind DNA in saline or acetic acid solution and partially protect DNA from nuclease degradation [23], [24]. The present study also reported that chitosan nanoparticles improved delivery of DNA to antigen presenting cells (APC) by efficient trafficking through local lymphoid tissue and uptake by dendritic cells.

In this study, DNA vaccines encapsulated chitosan nanoparticles were prepared by a complex coacervation method to enhance the efficacy of a DNA vaccine against swine influenza. The immune response elicited in BALB/c mice by chitosan nanoparticles containing a combined DNA vaccine against swine influenza was evaluated. In addition, bioactivity and safety of the chitosan nanoparticles were studied by in vitro transfection and cytotoxicity analysis. This work has laid a foundation for future work on a wide range of gene delivery systems including those for DNA vaccines.