Preparation and efficacy of Newcastle disease virus DNA vaccine encapsulated in chitosan nanoparticles

Figures & data

Table 1 Optimization of pFNDV-CS-NPs preparation conditions by orthogonal experimental design

Experiment number	Chitosan concentration (μg/mL)	Na2SO4 concentration (mmol/mL)	N/P ratio	Entrapment efficiency (%)
1	100	5	1	74.87±0.92
2	100	10	2	83.03±1.91
3	100	15	3	92.46±1.65
4	100	20	4	87.97±1.47
5	150	5	2	84.57±1.04
6	150	10	1	73.43±0.49
7	150	15	4	89.46±1.17
8	150	20	3	93.10±1.40
9	200	5	3	98.37±0.87
10	200	10	4	93.43±1.00
11	200	15	1	76.37±1.10
12	200	20	2	87.47±0.49
13	250	5	4	91.43±0.50
14	250	10	3	83.40±0.67
15	250	15	2	87.70±0.98
16	250	20	1	71.33±1.80
T1	84.58%	87.31%	74%
T2	85.14%	83.32%	85.69%
T3	88.91%	86.50%	91.83%
T4	83.47%	84.97%	90.57%
R	5.44%	3.99%	17.83%

Notes: T1, T2, T3 and T4 represent the mean value of factors at 1, 2, 3, and 4 levels, respectively; R represents range; data are shown as the mean ± standard deviation (n=3); N/P ratio refers to the number of nitrogen residues per deoxyribonucleic acid (DNA) phosphate.

Abbreviation: pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles.

Table 2 Evaluation of entrapment efficiency and loading capacity

Experiment number	Entrapment efficiency (%)	Loading capacity (%)
1	99.5	36.8
2	98.2	35.2
3	97.4	37.1
4	98.7	38.5
5	99.1	36.7
Mean value	98.58±0.73	36.86±1.05

Note: Values are presented as mean ± standard deviation of five experiments in each group.

Figure 1 Transmission electron microscopy micrograph of the pFNDV-CS-NPs (magnification 30,000×). (A) pFNDV-CS-NPs at pH 5.5; (B) pFNDV-CS-NPs at pH 7.4.

Abbreviation: pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles.

Figure 2 Size distribution and Zeta potential of the pFNDV-CS-NPs. (A) Measurement of these particles showed a narrow distribution of the pFNDV-CS-NPs, and the average diameter was 199.5 nm; (B) measurement of these particles showed a Zeta potential of +12.11 mV.

Abbreviation: pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles.

Figure 3 Stability analysis of the plasmid DNA after encapsulation in the chitosan nanoparticles.

Notes: Lane1: untreated naked plasmid pVAX I-optiF; Lane 2: untreated chitosan encapsulated plasmid DNA; Lane 3: naked plasmid pVAX I-optiF treated by DNase I; Lane 4: pFNDV-CS-NPs treated by DNase I; Lane 5: pFNDV-CS-NPs treated by DNase I and chitosanase; M: DNA marker DL 15000.

Abbreviations: DNA, deoxyribonucleic acid; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles; pVAX I-optiF, eukaryotic expression plasmids.

Figure 4 In vitro release profiles of the plasmid DNA pVAX I-optiF from pFNDV-CS-NPs.

Note: Data are presented as the mean ± standard deviation (n=3).

Abbreviations: DNA, deoxyribonucleic acid; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles; pVAX I-optiF, eukaryotic expression plasmids; pFDNA, DNA in pFNDV-CS-NPs.

Figure 5 In vitro expression of the pFNDV-CS-NPs in 293T cells by the indirect immunofluorescence analysis (×40) and Western blot. (A) The naked plasmid DNA pVAX I-optiF group; (B) pFNDV-CS-NPs transfected group; (C) blank CS-NP group; (D) 293T cell group as the negative control; (E) Lane 1: pFNDV-CS-NPs transfected group; Lane 2: naked plasmid DNA groups; M: Protein marker; Lane 3: 293T cells as the negative control; Lane 4: blank CS-NP group.

Abbreviations: CS, chitosan; DNA, deoxyribonucleic acid; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles; pVAX I-optiF, eukaryotic expression plasmids.

Figure 6 IgG antibody titers in serum of SPF chickens immunized with PBS (IM), blank CS-NP (IM), blank CS-NP (IN), and the naked plasmid DNA (IM), pFNDV-CS-NPs (IM), pFNDV-CS-NPs (IN).

Note: Data are presented as the mean ± standard deviation (n=5).

Abbreviations: CS, chitosan; DNA, deoxyribonucleic acid; IM, intramuscularly; IN, intranasally; OD, optical density; PBS, phosphate buffered saline; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles; SPF, specific pathogen free; IgG, immunoglobulin G.

Figure 7 IgA antibody content in serum (A), tears (B), tracheal fluid (C), and bile (D) of SPF chickens immunized with PBS (IM), blank CS-NP (IM), blank CS-NPs (IN), and the naked plasmid DNA (IM), pFNDV-CS-NPs (IM), pFNDV-CS-NPs (IN).

Note: Data are presented as the mean ± standard deviation (n=5).

Abbreviations: CS, chitosan; DNA, deoxyribonucleic acid; IM, intramuscular; IN, intranasal; PBS, phosphate buffered saline; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles; SPF, specific pathogen free; IgA, immunoglobulin A.

Table 3 The stimulating index of T lymphocyte proliferation in specific pathogen free chickens after immunization

Group	Stimulating index
	14 days post primary immunization	28 days post primary immunization	42 days post primary immunization
PBS	1.0345±0.0115^c	1.0805±0.0065^c	1.1055±0.0035^d
Blank CS-NPs IM	1.0275±0.0155^c	1.2385±0.0075^c	1.3240±0.003^d
Blank CS-NPs IN	1.1660±0.048^c	1.3065±0.0175^c	1.4170±0.004^d
pVAXI-opti F IM	2.0645±0.0785^a	3.3300±0.0090^a	2.8165±0.0305^c
pFNDV-CS-NPs IM	1.8865±0.1005^b	2.9245±0.0515^b	3.3490±0.008^b
pFNDV-CS-NPs IN	2.1850±0.0400^a	3.4895±0.0585^a	4.1755±0.0375^a

Notes: Values are presented as mean ± standard deviation of five experiments in each group; values within the same column with the different lower case letter (^a–d) in the superscript are significantly different (P<0.05; Student’s t-test).

Abbreviations: CS, chitosan; IM, intramuscularly; IN, intranasally; PBS, phosphate buffered saline; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles.

Table 4 Protective efficacy of the immunized specific pathogen free chickens after challenge with the highly virulent NDV strain F₄₈E₉

Group	Mortality/total	Morbidity(%)	Protective efficacy (%)
PBS	5/5	100	0
Blank CS-NPs IM	5/5	100	0
Blank CS-NPs IN	5/5	100	0
pVAXI-opti F IM	2/5	40	60
pFNDV-CS-NPs IM	1/5	20	80
pFNDV-CS-NPs IN	0/5	0	100

Abbreviations: CS, chitosan; IM, intramuscularly; IN, intranasally; NDV, Newcastle disease virus; PBS, phosphate buffered saline; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles.

Figure 8 Histopathology slides of normal glandular stomach, duodenum, and bursa of Fabricius and the same organs challenged with the highly virulent NDV strain F₄₈E₉.

Notes: (A1–A3) normal tissues of the glandular stomach, duodenum, and bursa of Fabricius; (B1, C1, D1, E1, F1 and G1) tissues of the glandular stomach PBS (IM), blank CS-NP (IM), blank CS-NPs (IN), and the naked plasmid DNA (IM), pFNDV-CS-NPs (IM), pFNDV-CS-NPs (IM); (B2, C2, D2, E2, F2 and G2) tissues of the duodenum PBS (IM), blank CS-NP (IM), blank CS-NPs (IN), and the naked plasmid DNA (IM), pFNDV-CS-NPs (IM), pFNDV-CS-NPs (IN); (B3, C3, D3, E3, F3 and G3) tissues of the bursa of Fabricius PBS (IM), blank CS-NP (IM), blank CS-NPs (IN), and the naked plasmid DNA (IM), pFNDV-CS-NPs (IM), pFNDV-CS-NPs (IN).

Abbreviations: CS, chitosan; DNA, deoxyribonucleic acid; IM, intramuscularly; IN, intranasally; NP, nanoparticles; NDV, Newcastle disease virus; PBS, phosphate buffered saline; pFNDV-CS-NPs, Newcastle disease virus F gene encapsulated in chitosan nanoparticles.