Construction of the L. lactis vectored vaccine
The Spax (411 bp) gene was used as an anchor domain and amplified by PCR from the Staphylococcus aureus (S. aureus) genome as described previously . The HA gene fragment (1650 bp) of A/Vietnam/1203/2004 (H5N1) (GenBank accession No. EU122404) without the signal and transmembrane region was amplified by PCR from pcDNA3.1-HA (kindly provided by Institute of Virology, Chinese Academy of Science, Wuhan, China) using the following primers (the GS linker sequence and Hind III are underlined): H-F, 5' GGCGGCGGCGGCGCCGATCAGATTTGCATTGGTTAC 3'; and H-R, 5' CCGAAGCTTTTAAATGCAAATTCAGCATT 3'. The Spax and HA fragments were fused into Spax-HA using the primers S-F (5' CTAGCTAGCAGTCTTCTAACCGAG 3') and H-R via a GS linker. The resulting Spax-HA containing Nhe I/Hind III was subcloned into an L. lactis expression vector, pNZ8148 (Fig. 1A), and then electroporated into competent L. lactis NZ9000. The positive clone of L. lactis/pNZ8148-Spax-HA was screened and expressed as described previously . L. lactis/pNZ8148-Spax was used as a negative control for the subsequent analyses.
Nisin-controlled induction of the L. lactis vectored vaccine
L. lactis/pNZ8148-Spax and L. lactis/pNZ8148-Spax-HA cells were individually cultured in M17 broth medium with 10 μg/ml chloramphenicol and 0.5% (wt/vol) glucose (GM17) at 30°C overnight without agitation. 1:25 diluted overnight cultures of L. lactis/pNZ8148-Spax or L. lactis/pNZ8148-Spax-HA cells were transferred into the fresh GM17 medium, respectively. When an optical density (OD) at 600 nm (OD600nm ) reached 0.3 to 0.4, nisin A was added at a final concentration of 10 ng/ml, and then continued to culture for 3 h at 30°C without agitation.
Western bolt analysis
The HA protein expression level in recombinant L. lactis was determined by Western blot analysis as described previously . Briefly, 108 cells of L. lactis/pNZ8148-Spax-HA pellets were washed three times with 500 µL of sterile phosphate-buffered saline (PBS), resuspended in 50 µL of 6× loading buffer and boiled for 10 min. The treated samples were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% non-fat milk at room temperature for 2 h, the membrane was incubated with a monoclonal mouse anti-HA antibody overnight at 4 ℃ and then with affinity-purified horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma-Aldrich Corporation, St. Louis, MO, USA). The membrane was subsequently reacted with the West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA) and imaged using the Molecular Imager ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Meanwhile, Precision Plus Protein™ WesternC™ (Bio-Rad, USA) was served ed as a protein marker.
Immunofluorescence assay and flow cytometry analysis
The display of HA protein on the surface of L. lactis was confirmed by immunofluorescence assay (Olympus IX70, Japan) and flow cytometry (FACS) analysis (BD FACS Calibur, BD Bioscience, San Jose, CA, USA). Briefly, 108 cells of L. lactis/pNZ8148-Spax-HA were washed three times with sterile PBS containing 0.5% bovine serum albumin (BSA), incubated with monoclonal mouse anti-HA antibody for 1 h at 4 ℃ and then with FITC-conjugated goat anti-mouse IgG for 30 min at 4 ℃, and resuspended in 500 µL of sterile PBS. The resulting L. lactis/pNZ8148-Spax-HA cells were subjected to immunofluorescence assay and FACS analysis, respectively.
Quantification of HA protein expressed by L. lactis/pNZ8148-Spax-HA
The expression of HA protein by L. lactis/pNZ8148-Spax-HA was determined by indirect ELISA . Briefly, 1012 colony forming units (CFUs) of L. lactis/pNZ8148-Spax-HA pellets were resuspended in 100 μL of a monoclonal mouse anti-HA antibody (0, 10, 25, 50, 75, 100, 125, 150, 175, and 200 μg/mL) in PBS containing 2% BSA, incubated at room temperature for 2 h, and incubated with goat anti-mouse IgG antibody conjugated to horseradish peroxidase (1 mg/mL) (Sigma-Aldrich Corporation, St. Louis, MO, USA) at room temperature for 1 h. The cells were then washed with sterile PBS and resuspended in 100 μL of HRP substrate 3,3’,5,5’-tetramethylbenzidine (TMB) (Sigma-Aldrich Corporation, St. Louis, MO, USA) in the dark for 25 min, and 100 μL of 2 mol/L H2SO4 was then added to stop the reaction. The OD450nm value of the supernatant was measured using a microplate reader. L. lactis/pNZ8148-Spax was used as a negative control.
Animals, immunization, sample collection and virus challenge
Specific pathogen-free (SPF) white Leghorn chickens (aged 7 days) were purchased from the Veterinary Research Academy of Agricultural Sciences of Jiangxi Province (Jiangxi, China) and housed in ventilated cages (five chickens per cage). The chickens were administered pelleted feed and sterile water and maintained in an SPF environment.
Three groups of 30 chickens each vaccinated orally with 2 mL of sterile PBS, 1012 CFU of L. lactis/pNZ8148-Spax or 1012 CFU of L. lactis/pNZ8148-Spax-HA, respectively. Prime immunization was performed at day 0, 1, and 2 and boosted at day 17, 18, and 19. PBS and L. lactis/pNZ8148-Spax cells were used as controls.
At days 15 and 34 after the initial vaccination, blood samples were collected from the wing vein. Sera were separated by centrifugation of the blood at 2,000 × g for 10 min and stored at -20 °C until use. Meanwhile, the intestines were isolated from the vaccinated chickens (n=3 per group at day 15, n=3 per group at day 34) and washed with 500 µL of sterile PBS. Feces were also collected, resuspended in 500 µL of PBS and stored at -20 °C until use.
Two weeks after the final vaccination, all the vaccinated chickens (n=24 per group) were transferred into an animal biosafety level-3 (BSL-3) containment facility. Slight ether narcosis-anaesthetized chickens were intranasally infected with 20 µL of 5 × 50% lethal dose (5 × LD50) of HPAI H5N1 virus strains belonging to clade 1 (A/Vietnam/1203/2004, VN1203), clade 2.3 (A/Anhui/1/2005, Anhui) or clade 8 (A/chicken/Henan/12/2004, Henan).
Three chickens in each group were sacrificed at day 3 post challenge to check the virus titres in the lungs, as described previously . Briefly, 10-fold dilutions of lung homogenate supernatants were mixed with MEM containing trypsin, and reach up to 100 µL. Dilutions were added with 100 µL of Madin-Darby Canine Kidney (MDCK) cells at 2.5 × 106 cells/mL, and incubated overnight at 37°C. After 24 h incubation, 50 µL of 0.5% chicken red blood cells (CRBCs) was added, and then incubated for 1 h at room temperature and recorded hemagglutination afterwards to determine 50% tissue culture infective dose (TCID50). The other five chickens remaining in each group were used for survival records. The chickens were monitored every alternate day at a fixed time point to record their weight loss and survival. The humane endpoint of the challenge studies was a body weight loss exceeding 25% relative to the weight at the time of challenge inoculation. After final monitoring, all the surviving chickens were euthanized by CO2 inhalation for 5 min.
All animal immunizations were performed at a BSL-2 facility, and the virus challenge experiments were strictly performed in BSL-3 containment facilities, complied with the Guidelines for the Use and Care of Experimental Animals and were approved by the Institute Animal Care and Use Committee of Nanchang University.
Determination of antibody responses by ELISA
Total serum antibody (IgY) and secretory IgA (sIgA) in the intestinal washes and feces were determined by ELISA using recombinant HA protein (2 µg/mL) from A/Vietnam/1203/2004 as a coating antigen as described previously . In a brief, pNPP phosphatase substrate (MP Biomedicals, USA) was used for determining IgY titres. The OD value was measured at 405 nm using using an ELISA plate reader (Bio-Tek, USA). Furthermore, the intestinal washes andfeceswere used for determining sIgA using indirect ELISA. 3,3’,5,5’-Tetramethylbenzidine (TMB) was used as a substrate, and OD values were measured at 450 nm using an ELISA plate reader (Bio-Tek, USA). The IgY or sIgA titre was determined as the lowest dilution with an OD greater than the mean OD of the naïve controls plus two standard deviations.
The neutralization activity of serum against different H5N1 viruses was assessed as described previously . Briefly, receptor-destroying enzyme (RDE)-treated sera (n=24 chickens per group) were serially diluted (2-fold) and incubated with 100 TCID50 of viruses belonging to clade 1 (A/Vietnam/1203/2004, VN1203), clade 2.3 (A/Anhui/1/2005, Anhui) or clade 8 (A/chicken/Henan/12/2004, Henan) for 1 h at room temperature and plated in duplicate in a 96-well plate with MDCK cells. The neutralizing titre was assessed as the highest antibody dilution for which no cytopathic effect was observed by light microscopy.
The statistical significance of the differences was assessed by Student’s t test and one-way ANOVA with multiple comparisons. Significant differences in the survival curves were determined by log-rank analysis. A p value less than 0.05 was considered to indicate statistical significance.