Six-week-old female BALB/c mice were purchased from SLAC Laboratory Animal (China) and housed in a specific pathogen-free (SPF) facility following the approved animal care protocols. The mice were acclimated to their new environment for one week prior to immunization. All experimental protocols involving mice were approved by the Chinese Ministry of Agriculture and the Review Board of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (permit No. SYXK 2011–0116).

A/Swine/Guangdong/164/06 (SwGD164; H3N2), A/Swine/Heilongjiang/1/05 (SwHLJ1; H3N2) and A/Swine/Guangdong/96/06 (SwGD96; H1N1) were isolated from pig farms in China [22]. PR8 (H1N1) is a mouse-adapted virus. All viruses were grown for 48 hours at 37℃ in the allantoic cavities of 10-day-old SPF chicken embryo eggs. The novel tetra-branched M2e-MAP was constructed by fusing four copies of a universal sequence, which is an extracellular part of the M2 protein, with one copy of promiscuous Th cell epitope by a lysine tree with two additional Lys residues defining a putative cleavage site for cathepsin D [12]. It was synthesized by GenScript (China). M2e-MAP was prepared at 10 mg/mL in distilled water and stored at −20℃. Its construction and sequence are shown in Fig. 1.

rgH3N2 harboring the HA and NA genes from SwGD164 and six internal genes from PR8 was generated by plasmid-based reverse genetics as previously described [20]. Briefly, one microgram of each plasmid was added into 250 µL Opti-MEM (Invitrogen, USA) and vortexed. Next, 16 µL of the transfection reagent Lipofectamine 2000 (Invitrogen) were added into 250 µL Opti-MEM and mixed gently. Five minutes later, the diluted transfection reagent was mixed with the diluted plasmids. The DNA-transfection reagent mixture was kept at room temperature for 20 min, then added directly to a monolayer of 293T cells in a 6-well plate (Costar; Corning, USA). After 6 h of incubation at 37℃ in 5% CO₂, the medium was replaced with 2 mL fresh Opti-MEM, and the plate was incubated as described above for 48 h. The supernatant was subsequently inoculated into the allantoic cavity of 10-day-old embryonated SPF eggs. The allantoic fluid was harvested after 48 h of incubation at 37℃, after which the virus was identified by hemagglutination assay. Reverse transcription PCR (RT-PCR) and re-sequencing confirmed that the genome of the rescued virus was identical in sequence to the cDNA in the plasmids used for its rescue [20]. The rescued viruses were then confirmed by sequencing. The titer of the rgH3N2 virus was determined in 10-day-old SPF embryonated chicken eggs, and the 50% embryo infective dose (EID₅₀) was calculated by the Reed-Muench method.

The rgH3N2 virus was propagated in the allantoic cavity of 10-day old embryonated SPF chicken eggs and inactivated with 0.1% (v/v) formalin for 48 h at 4℃. The virus was then purified by sucrose density gradient centrifugation [11].

The novel M2e-MAP was synthesized and stored at −20℃. One dose of M2e-MAP combined vaccine containing 10 µg of each inactivated rgH3N2 and M2e-MAP (in 50 µL sterile PBS) was mixed and emulsified with the same volume of Freund's incomplete adjuvant (FIA; sigma, USA) or Freund's complete adjuvant (FCA; sigma).

A total of 120 female BALB/c mice were randomly divided into four groups (n = 30 in each group), immunized subcutaneously with immunogen plus FCA and boosted with the same amount of immunogen in FIA at 2-week intervals. Mice in group 1 (M2e-MAP+rgH3N2+Freund) received one dose of M2e-MAP combined vaccine. Mice in group 2 (rgH3N2+Freund) were immunized with one dose of the vaccine as prepared above. Mice in group 3 (rgH3N2) and group 4 (Freund) were immunized with inactivated virus and Freund alone as controls, respectively. The immunization schedule of mice in the experiment is shown in Table 1. Serum samples from five mice in each group were collected at 0, 1, 2, 3 and 4 weeks after the first immunization. Mice (n = 10 for each virus) were anesthetized and challenged intranasally with 10⁶ EID₅₀ of SwGD96 and SwHLJ1, as well as 10 LD₅₀ of PR8 on day 14 after the booster. The loss of body weight and symptoms were monitored daily and the survival curves were analyzed by the Kaplan-Meier method.

Enzyme-linked immunosorbent assay (ELISA) was applied to determine titers of antibody specific to rgH3N2 and M2e-MAP in serum samples collected on days 7, 14, 21 and 28 post primary immunization as described previously [21]. Hemagglutination inhibition (HI) assay was performed according to the WHO Manual on Animal Influenza Diagnosis and Surveillance (World Health Organization, Geneva, Switzerland). Neutralization tests (NTs) were performed according to Kida et al. [10].

Lung tissues from euthanized mice were removed at 3 days post infection (dpi) and homogenized in 500 µL of ice cold PBS. Virus titers in mice lungs were determined by EID₅₀ titration and quantitative real-time PCR as previously described [20]. The specific primers and labeled fluorescent-probe were as follows: InfA-forward: 5-GACCGATCCTGTCACCTCTGAC-3; InfA-rerverse: 5-AGGGCATTCTGGACAAAGCGTCTA-3; TaqMan probe: 5-FAM-TGCAGTCCTCGCTCACTGGGCACGBHQ-3.

The lung tissues of challenged mice were collected on day 4 and fixed in 10% neutral buffered formalin, and histopathological changes of the mouse lungs were examined by hematoxylineosin stain and observed under a light microscope.

Data were analyzed and charted with Excel and Graphpad Prism 5. The survival curves were analyzed by the Kaplan-Meier methods, and the significance of differences in overall survival between groups was determined by the log-rank test. Statistical significance was determined by two-way ANOVA.

The rgH3N2 virus harboring the HA and NA genes from SwGD164 and six internal genes from PR8 was generated by plasmid-based reverse genetics and confirmed by sequencing analysis. The HA titer of rgH3N2 reached 512 HAU/50 µL, which was 2 fold higher than that of SwGD164. The rgH3N2 virus (10^6.7EID₅₀/mL) grows to a higher titer than SwGD164 in embryonated chicken eggs.

M2e-MAP combined vaccine induced strong M2e-specific and rgH3N2-specific antibody responses in mice (Fig. 2). The titer of M2e-sepcific antibody reached 1 : 10³ at 7 days post primary immunization and increased to the highest titer of over 1 : 10⁵ at 14 days post boost. In contrast, only background levels of antibody were detected in mice vaccinated with Freund's alone (Fig. 2). Both the antibody titers to rgH3N2 and M2e-MAP increased after the boost. The antibody titer of rgH3N2 in group 3 was lower than the one emulsified with Freund (groups 1 and 2) at each sampling point.

The HI and NT tests were performed on serum collected 2 and 4 weeks post primary immunization. As shown in Table 2, HI antibodies and neutralizing antibodies were detected in all groups except for the control. The HI and NT titers were almost equal in groups 1 and 2, where the average titers of HI and NT were found to be 256 and 10³ after the first immunization, respectively. The mean titers of HI and neutralizing antibodies were up to 1,152 and 10⁴ until viral infection, respectively. In group 3, the average titer of HI antibodies was 160 and that of the neutralizing antibody was 10² after the first immunization. Until viral infection, the mean titers of HI and neutralizing antibody were up to 512 and 10³, respectively. The above results indicated that immunization with M2e-MAP combined with H3N2 inactivated vaccine induced high levels of humoral responses in mice.

Mouse lungs were collected on 3 dpi and the virus titers were determined by EID₅₀ titration and quantitative real-time PCR (Fig. 3). No detectable influenza virus was present in the lungs of mice challenged with SwHLJ1 (H3N2) or SwGD96 (H1N1) (< 1.0 log10 EID₅₀/mL), and the titer decreased to 10^{3.25 ± 0.35} EID₅₀/mL in PR8 challenged mice (Table 2). The addition of M2e-MAP significantly increased the ability of the vaccine to reduce heterologous virus replication in group 1. Real-time quantitative PCR indicated M2e-MAP combined vaccine provides strong protection against PR8 (H1N1), SwGD96 (H1N1) and SwHLJ1 (H3N2) with significant differences in viral titers among four groups (p < 0.001).

Mouse lungs were collected for histopathologic examination at 3 dpi. In groups 2, 3 and 4, moderate to serve broncho-interstitial pneumonia was observed (Fig. 4). In groups of mice challenged with PR8, lung tissues were damaged and inflammatory cell infiltration was observed in large areas in the control group (panel J in Fig. 4). However, the lung tissues of mice that were challenged with PR8 in group 1 appeared relatively normal aside from slight alveolar dilatation (panel A in Fig. 4). No histopathologic changes were observed in the group 1 mice challenged with SwGD96 (panel B in Fig. 4) or SwHLJ1 (panel C in Fig. 4).

The body weight and survival rate of the mice that received lethal challenge (10LD₅₀) of PR8 were monitored for 14 days post challenge (Fig. 5). Survival rates of mice in groups 1, 2 and 3 were 100%, 40% and 30%, respectively (p = 0.0002) (Fig. 5A). All mice in group 4 died on day 9. As shown panel B in Fig. 5, the body weight of mice in group 1 decreased slightly at first, then increased on day 10. Lethargy, loss of appetite, fever and shiver were observed in groups 2, 3 and 4 at 2 dpi.