Construction of plasmid expressing NA and expression on L.lactis
The NA gene (1459 bp) of A/Vietnam/1203/2004 (H5N1) was PCR-amplified from pCDNA3.1-HA (kindly provided by St. Jude Children’s Research Hospital, Memphis, TN, USA) using the following primers: NA-F: CTAGCTAGCGGTACCGCCGCCACCATGAA (Nhe I); NA-R: CCGAAGCTTACAGGAAGTATTCAATC (Hind III) and cloned into L.lactis based constitutive expression plasmid pNZ2103 (purchased from MoBiTec, Goettingen, Germany), the resulting plasmid was transformed into competent L.lactis NZ3000, the positive clone was named as L.lactis/pNZ2103-NA.
Western blot analysis was described previously [14] and L.lactis/pNZ2103 was used as a negative control.
Animal experiments and sample collection
For oral administration of chickens, 7-day-old specific-pathogen-free (SPF) single comb white leghorn chickens from an in-house flock (Institute of Jiangxi Agriculture, China) were used in this study. The concentration of recombinant L.lactis/pNZ2103-NA was adjusted to 1012 colony forming unit (CFU)/ml with sterile saline.
Three groups of 16 chickens each were immunized with oral administration of 1 ml of sterile saline, 1012 CFU of L.lactis/pNZ2103 or 1012 CFU of L.lactis/pNZ2103-NA, respectively. Prime immunization was performed at day 0, 1, 2, 3 and boosted at day 17, 18, 19, 20.
At day 15 and day 34 after the first immunization, blood samples were collected from the retro-orbital plexus. Sera were separated by centrifugation of blood at 2,000 × g for 10 min and stored at -20°C until use. Intestine and upper respiratory were isolated from the vaccinated chickens and washed with 500 μL sterile saline, respectively.
At two weeks after the last immunization, chickens were lightly anesthetized with CO2 and inoculated intranasally with 25 μl of 104 EID50 of VN/1203/04 virus through the choanal slit to determine protection efficacy. Chickens were observed for illness, weight loss, and death for 14 days after H5N1 virus infection. H5N1 virus challenge experiments must be strictly performed under the enhanced bio-safety level-3 laboratory (BSL-3).
The chickens were managed with pelleted feed and sterile water, maintained in a SPF environment and all efforts were made to minimize suffering following approval from the Institute Animal Care and Use Committee of the Nanchang University (Approval No. 726-14).
Enzyme-linked immunosorbent assay (ELISA)
Immune sera from the vaccinated chickens were collected by bleeding from the wing vein and treated with receptor-destroying enzyme from Vibrio cholerae (Denka-Seiken, San Francisco, CA) before being tested for the presence of H5-specific antibodies as described previously [16].
NA-specific immunoglobulin G (IgG) and secretory immunoglobulin A (IgA) antibodies were detected by enzyme-linked immunosorbent assay (ELISA) using recombinant NA protein as a coating antigen as described previously [14]. ELISA end point titers were expressed as the highest dilution that yielded an optical density greater than twice the mean plus one standard deviation of that of similarly diluted negative control samples.
Neuraminidase inhibition (NI) assay
The anti-NA immune response was evaluated by Bioluminescence-based neuraminidase inhibition kit. To perform this, 50 μl of chickens sera from each group was taken at 1/2 dilutions which were half diluted further till 1/1024 in a 96-well micro-titer plate. 50 μl of purified rNA (0.25 mg/ml) was added to each well and incubated at 37°C for 2 h. The neuraminidase inhibition titer was represented as the highest dilution until there was no neuraminidase activity observed.
Data analysis
Data are presented as the means ± standard deviations (S.D.) and are representative of at least three independent experiments. All analysis for statistically significant differences was performed by the Student t test and one-way ANOVA. A p value less than 0.05 was considered to be significant.