Vaccine strains and reagents
Influenza A virus NIBRG-121 (NIBSC, A/California/7/2009 H1N1 virus), virus (A/California/07/2009 H1N1; CDC# 2009712112), hemagglutinin (HA) antigen (A/California/7/2009, NIBSC 09/146), and antiserum (A/California/7/2009, NIBSC 09/152) were obtained from the Centers for Disease Control and Prevention (CDC, Taiwan), or the National Institute for Biological Standards and Control (NIBSC, UK). NIBRG-121 was used as vaccine strain, and virus A/California/07/2009 H1N1 was used as challenge virus.
NIBRG-121 virus strain was amplified in embryonic eggs of 10-day special pathogen free (SPF) for 48 h, it was then harvested and inactivated with 0.04% formalin at 4°C for overnight. Harvested virus was then filtered through 0.45 μm filter and concentrated with Lab scale TM TFF System, treatment with benzonase (cat. no. 1.01656.0001, Merck KGaA, Germany) (9 ~ 90 u/ml at 4°C for overnight), and purified with 10-50% sucrose gradient by Alfa Wassermann PKII Pilot-Scale Ultracentrifuge System. Purified virus was dialyzed with PBS, filtered with 0.45 μm filter and stocked in 1.5-ml stock tube at −80°C until use. Hemagglutinin (HA) antigen concentration was determined by single radial immunodiffusion assay (SRD)  for the quantification of vaccine S-OIV H1N1.
Quality control assays of vaccine
To monitor the quality of produced vaccine, several items of assays were evaluated. Vaccine was quantified with SRD method; ovalbumin was evaluated with Chicken Egg Ovalbumin Elisa Kit (Cat. No. 6050) from Alpha Diagnostic International (ADI); endotoxin was measured with Toxin Sensor TM Chromogenic LAL Endotoxin Assay Kit (Cat. No. L00350) obtained from the GenScript Corporation. Above assays were followed the protocols of commercial kits. Formalin content of vaccine was evaluated according to the guideline of version 6th of Pharmacopoeia Chinensis. For abnormal toxicity assay, male BALB/c mice (body weight: 17–22 gm) were inoculated intraperitoneal with 0.5 ml of produced vaccine (at least 5 mice per dose of vaccine) and then observed their changes of body weight. It fit the demand criteria only if there is no death, accident symptoms of mice, and mice should recovery of their body weight to the level of pre-inoculation 7 days after inoculation. The antigenicity of vaccine was evaluated with hemagglutination (HA) assay as described with HAI assay without antibody.
Determination of vaccine HA concentration
The HA concentration of S-OIV candidate vaccine was measured with single-radial immunodiffusion (SRD) according the protocol described previously [22, 23].
All animal experiments were reviewed by the Institutional Animal Care and Use Committee and approved by the regulatory authorities of Taiwan. All animal experiments were conducted in accordance with Taiwan laws on animal experimentation and guidelines set out by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and the Office of Laboratory Animal Welfare (OLAW). The IACUC certificate No. of this study were AN-99-05、 AN-100-07、 and AN-101-06. Animal were housed according to OLAW and AAALAC guidelines, in housing facilities accredited by the Center of Disease Center (CDC) of Taiwan.
Immunization of mice
Female BALB/c mice (8-week-old) were immunized intraperitoneal with different dosage of produced vaccine (PBS control, 0.001 μg, 0.01 μg, and 0.05 μg) in the absence or presence of adjuvant alum (final 5% of Aluminum hydroxide gel, cat no.A8222, Sigma) or MPLA (Monophosphoryl Lipid A) (Sigma Adjuvant System, cat no.S6322, Sigma) using a single-vaccination regimen.
IgG subclass determination
ELISA plates were coated with 10 ng HA per well of purified vaccine at 4°C for overnight. After blocking nonspecific binding (5% skim milk in PBST, 1 h at 37°C) and subsequent washing, PBST diluted mice sera (1:400) of pre-immune or immunized were added to wells and plated were incubated for 1 h at 37°C, washed again, prior to further incubation for 1 h with IgG subclass-specific peroxidase-conjugated goat or rabbit anti-mouse IgG antibodies (IgG: goat anti-mouse IgG (H + L) HRP, cat. no. 115-035-146, Jackson ImmunoResearch, USA; IgG1: rabbit anti-mouse IgG1 HRP, cat. no. 61–0120, Invitrogen, USA; IgG2b: rabbit anti-mouse IgG2a HRP, cat. no.61-0220, Invitrogen, USA). Bound IgG subclass antibodies were detected colorimetrically using TMB substrate by OD450 nm endpoint reading. The cutoff value was set at ODn plus three standard deviations, where ODn is the mean of ODs of six preimmune serum specimens.
Cell and virus
MDCK (Madin-Darby canine kidney) cells were obtained from the American Type Culture Collection (ATCC), and maintained in Dulbecco’s minimum essential medium (DMEM; GIBCO, Invitrogen, USA) supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin and 10% fetal bovine serum (GIBCO, Invitrogen) at 37°C in a humidified atmosphere with 5% CO2. Influenza A virus NIBRG-121 (NIBSC, A/California/7/2009 H1N1 virus); obtained from the CDC of Taiwan) was amplified in 10-day-old embryonic eggs at 35°C for 48 h. Virus was harvested from allantoic fluid. To determine the LD50 of each batch of virus, female BALB/c mice (6–7 weeks) (n = 10/group) were anesthetized subcutaneously with Zoletil 50 (Virbac Laboratories, France) (0.375 mg/mice) and inoculated intranasally with serial dilutions of the virus. The LD50 was the dilution of the virus that produced lethality in 50% of the mice and LD50 titers were calculated by the method of Reed and Muench . The LD50 was over 105 TCID50 for NIBRG-121.
To measure virus titer, MDCK cells (5×106 /well) were inoculated into 6-well microplates and were incubated at 37°C in a humidified atmosphere with 5% CO2 for overnight. In the second day, a serial of 10-fold dilutions of virus were prepared in PBS; MDCK cells were washed two times with PBS and 100 μl of viral dilutions were inoculated into 6-well microplates for adsorption. After one hour of adsorption, virus suspensions were removed and cells were washed two times with PBS; then 1% Oxoid agars in DMEM/BSA medium were inoculated into microwells, microplates were incubated at 37°C in a humidified atmosphere with 5% CO2 for three days. MDCK cells were fixed with 10% formalin for 1 h, then agars were removed and stained with crystal violet solution (0.5%) for 1 hour, stained cells were washed with tap water and were observed. Microplates were air dried at room temperature for several hours, and plaques were calculated for virus concentration.
To calculate virus titer, MDCK cells (1.5 × 104 /well) in DMEM medium with 10% FBS were inoculated into 96-well microplates and were incubated at 37°C in a humidified atmosphere with 5% CO2 for overnight. In the second day, a serial of 10-fold dilutions of virus were prepared in PBS; MDCK cells were washed two times with PBS and 100 μl of viral dilutions were inoculated into 96-well microplates for adsorption. After one hour of adsorption, virus suspensions were removed and cells were washed two times with PBS; then DMEM/BSA medium were inoculated into microwells, and microplates were incubated at 37°C in a humidified atmosphere with 5% CO2 for additional five days. The TCID50 titers of virus were calculated by the method of Reed and Muench .
Hemagglutination inhibition (HI) assay
Functional H1N1 HA-specific antibody titers were determined by HI assay using chicken erythrocytes. Prior to serological analysis, sera were treated with receptor-destroying enzyme (RDEII, Denka Seiken, Tokyo, Japan). Serum (0.1 ml) was mixed with 0.3 ml receptor-destroying enzyme, incubated at 37°C for 18 h, and adjusted to a final 1:10 dilution by adding PBS, and inactivated the enzyme activity by incubation at 56°C for 30 min. Sera giving a negative signal in the first dilution (1:10) were assigned a nominal HI score of 1:5. HI titers are expressed as reciprocal value of the highest serum dilution that inhibited hemagglutination. Animals with a serum HI titer of 1:40 were considered seroprotected.
Intranasal influenza challenge
Four weeks after immunization with S-OIV H1N1 or NIBRG-14 H5N1 vaccine; immunized mice were blood-letting for antibody assay and were then lightly anaesthetized with Zoletil 50 (VIRBAC Laboratories, France) and challenged intranasal with 1 × 106 TCID50 A/California/7/2009 H1N1 virus. Over the following 14 days, body weights and survival rates of each group of mice were monitored daily.
Neutralization antibodies of mice post vaccinated with S-OIV H1N1 or NIBRG-14 H5N1 vaccines were evaluated using microneutralization assay. Mice sera (pre-immune as negative; A/California/7/2009 NIBSC 09/152 and antiserum to NIBRG-14 H5N1 HA as positive control) were mixed with viruses (100 TCID50 of NIBRG-121 H1N1 or NIBRG-14 H5N1 virus) at room temperature for 1 hour, and were inoculate into 96-well MDCK cells (1.5 × 104/ml). Experiment was performed following WHO protocol of microneutralization assay [21, 25].
In all figures, vertical error bars denote the standard deviation (SD). Significances of differences in antibody responses and cellular responses were evaluated by one-way analysis of variance (ANOVA). T test was used for the comparison of two specific groups in one-way ANOVA. To test the significance of survival rates between each group of immunized mice, a Z’ (alternative critical ratio) test was used [16, 26]. For the comparison of HAI antibody titer, Mann–Whitney U test was used. A P value of < 0.05 was considered significant.