Preparation and efficacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles

Abstract

Background: Newcastle disease (ND) is a highly contagious viral disease of poultry caused by pathogenic strains of the Newcastle disease virus (NDV). Live NDV vaccines are administered by drinking water, eyedrops or coarse aerosol spray. To further enhance mucosal immune responses, chitosan nanoparticles were developed for the mucosal delivery of a live NDV vaccine.

Methodology/principal findings: A lentogenic live-virus vaccine (strain LaSota) against NDV encapsulated in chitosan nanoparticles were developed using an ionic crosslinking method. Chitosan nanoparticles containing the lentogenic live-virus vaccine against NDV (NDV-CS-NPs) were produced with good morphology, high stability, a mean diameter of 371.1 nm, an encapsulation rate of 77% and a zeta potential of +2.84 mV. The Western blotting analysis showed that NDV structural proteins were detected in NDV-CS-NPs. The virus release assay results of NDV-CS-NPs indicated that NDV was released from NDV-CS-NPs. Chickens immunized orally or intranasally with NDV-CS-NPs were fully protected whereas one out of five chickens immunized with the LaSota live NDV vaccine and three out of five chickens immunized with the inactivated NDV vaccine were dead after challenge with the highly virulent NDV strain F48E9.

Conclusions/significance: NDV-CS-NPs induced better protection of immunized specific pathogen free chickens compared to the live NDV vaccine strain LaSota and the inactivated NDV vaccine. This study lays a foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.

Figure 1. Transmission electron microscopy photomicrograph of the chitosan-NDV nanoparticles prepared by an ionic cross linking method under the optimized conditions.

The TEM photomicrograph (magnification 25,000×) showed that the prepared nanoparticles had a regular round shape and good dispersion, but did not have adhesion or subsidence damage. The sizes of particles were between 200 nm and 500 nm.

Figure 2. Detection of the NDV structural proteins after encapsulation by Western blot.

M: Protein marker; 1: Original NDV fluid; 2: NDV recovered from the NDV-CS-NPs. Four positive reaction bands were detected at 75 kDa (hemagglutinin-neuraminidase protein, HN), 54 kDa (fusion protein, F), 45 kDa (phosphoprotein, P), and 41 kDa (matrix protein, M).

Figure 3. In vitro release profiles of NDV from NDV-CS-NPs in PBS (pH 7.2).

The experiment was repeated three times and each measured in triplicate. Mean values were analyzed using the Student’s test. Data were presented as mean values ± SD.

Figure 4. IgA antibody content in intestinal mucus of SPF chickens immunized with either NDV-CS-NPs orally, NDV-CS-NPs intranasally, LaSota live vaccine intranasally, blank CS-NPs control or physiological saline by oral route.