Sequences and schematic diagram of constructs. The M2e sequence shown is the consensus of the human influenza A virus M2e sequence. In 4.M2e, four repetitive M2e regions (underlined) are located in a tandem. Sequences in italics are flexible linkers. A 6-histidine tag-encoding DNA was fused in frame. In the membrane-anchored 4.M2e-tFliC, the central immunogenic region of FliC (domain 3 [D3], aa 177 to 401) was replaced by the 4.M2e sequence. The melittin signal peptide (SP) was fused to the N terminus of 4.M2e-tFliC (N-terminal domains 1 and 2 [ND1-2] in FliC) to enable its ectodomain to reach the exocytic pathway and is removed in the mature protein by insect cell signal peptidase (44). The transmembrane and cytoplasmic domains (TM/CT) of A/PR8 virus HA were attached to the C terminus of the 4.M2e-tFliC (C-terminal domains 2 and 1 [CD2-1] in FliC).

Characterization of 4.M2e-tFliC VLPs. (A) Diagram of influenza virus 4.M2e-tFliC VLPs. The membrane-anchored form of the 4.M2e-tFliC is inserted into the lipid bilayer of the envelope. (B) Negative-stain EM of VLP. (C to E) Characterization of VLPs. VLP samples were applied to SDS-polyacrylamide gels, followed by Western blotting. Lanes 1, 4.M2e-tFliC VLPs; lanes 2, M1-only VLPs. Protein bands were detected by mouse anti-FliC antibody (FliC-1) (C), mouse anti-M2e antibody (14C2) (D), and anti-M1 antibody (GA2B; Abcam) (E). (F) TLR5 agonist activity of flagellin. The mouse macrophage cell line RAW264.7, which expresses TLR2 and TLR4 without TLR5, was used to determine the bioactivity of 4.M2e-tFliC VLPs. Soluble recombinant FliC (sFliC) was used as a positive control; M1-only VLPs and recombinant 4.M2e were used as negative controls. TLR5-expressing RAW264.7 cells were prepared by transfection with the vector pUNO-hTLR5 expressing the human TLR5, as described previously (40). Data represent means ± standard deviations (SDs) from triplicate repeats.

Antibody responses. (A) Serum IgG recognizing M2e in immunized mice. To determine serum M2e-specific IgG titers, ELISA plates were coated with 100 μl/well of synthesized M2e peptide (5 μg/ml; at the Emory University Biochemical Core Facility). (B) Endpoint titers of IgG recognizing native M2 protein. MDCK cells were infected with A/PR8 viruses at an MOI of 1. Uninfected cells were used as a control. At 12 h postinfection, cells were washed with PBS and fixed with 0.5% glutaraldehyde. Samples diluted 80-fold were applied to detect antibody binding, as described in Materials and Methods. Data depict the OD₄₅₀ (mean ± SD) with infected cells, with the background of uninfected cells subtracted. Groups 1 and 2 represent mouse groups immunized with 4.M2e and 4.M2e-tFliC/M1 VLPs, respectively. (C) Lung lavage IgA. IgA endpoint titers were determined as described for the serum IgG endpoint titer in panel A, but the secondary antibody used was HRP-conjugated goat anti-mouse IgA antibody. (D) Lung lavage IgG. Representative data are the geometric mean (GM) ± 95% confidence interval (CI) of six mice in each group at 4 weeks after the last immunization. Asterisks on the top of a bar present the statistical difference of the group to its counterpart in the M2e group. The statistical difference between other groups is labeled with a connecting line. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

A/Philippines (H3N2) live virus challenge. Four weeks after the last immunization, 6 mice of each group were infected i.n. with 5 LD₅₀s of mouse-adapted A/Philippines viruses (250 PFU). Survival of mouse groups immunized i.m. (A) or i.n. (B) and body weight changes of i.m. (C) and i.n. (D) groups were monitored and recorded for 15 days to indicate the protective efficacy of the vaccines. In panels C and D, the data represent means of the body weight change (percentage of prechallenge body weight) of 6 mice in each group.

A/PR8 (H1N1) virus challenge. Four weeks after the last immunization, mice were infected i.n. with 5 LD₅₀s of mouse-adapted A/PR8 virus (125 PFU). Survival of mouse groups immunized i.m. (A) or i.n. (B) and body weight changes of groups immunized i.m. (C) or i.n. (D) were monitored and recorded as described in the legend to Fig. 4.

Lung viral load on day 4 postchallenge. Four weeks after the last immunization, three mice in each group were infected i.n. with 5 LD₅₀s of mouse-adapted A/PR8 (H1N1) or A/Philippines (Phi; H3N2) virus. Mouse lungs were collected on day 4 postchallenge. Each lung was ground and cleared in 1 ml of Dulbecco modified Eagle medium. Virus titers of lung extracts were titrated using a standard plaque assay with MDCK cells. Lung viral titer was expressed as the number of PFU/lung. Data depict means ± SDs of three mice from each group. Groups 1 and 2 represent mouse groups immunized i.m. or i.n. with 4.M2e and 4.M2e-tFliC/M1 VLPs, respectively. Statistically significant differences between immunized groups and the naive group are indicated: *, P < 0.05; **, P < 0.01.