Figure 1.
Schematic diagram of the 10B-M2e chimeric capsid protein displayed on the T7-M2e phage nanoparticles.
M2e peptide with a GGGS linker is fused to C-terminus of 10B capsid protein after amino acid residue 348 of 10B coat protein. TAA stop codon (#) was inserted downstream of the M2e sequence to avoid any C-terminus extension of the M2e peptide.
Figure 2.
Plaque lift assay on T7-M2e and T7-wt plaques.
Plaques developed after in vitro packaging reaction were transferred onto nitrocellulose membranes. After incubation with 14C2 Ab followed by anti-mouse polyclonal conjugate and development by BCIP/NBT chromogen, sharp spots were developed immediately (2–3 min) indicating accessibility and immunoreactivity of M2e peptide when displayed on the surface of T7 nanoparticles (A). No spots are observed on T7-wt phage plaque lift (B). Following amplification and purification of T7 phage nanoparticles, capsid proteins were separated by SDS-PAGE on 12% polyacrylamide gel (C) and 10B-M2e fusion protein (40 kDa MW) was detected by Western blotting on nitrocellulose membrane using 14C2 anti-M2e monoclonal antibody (D). As shown, no bands were detected on the T7-wt lane indicating no reactivity of 14C2 mAb with capsid proteins. M lane: protein marker. (E) Assessment of T7-M2e stability against various harsh conditions. There was no significant change in the T7-M2e phage titer after incubation for 60 min at 37°C. Results are reported as PFU/ml.
Figure 3.
Antibody responses elicited by T7-M2e nanoparticles.
(A) Total IgG, IgG1 and IgG2a antibody titers against the influenza virus M2e peptide. Results are expressed as an antibody endpoint titer, where the O.D. value is 3-fold higher than the background value obtained with a 1∶50 dilution of pre-immune serum from BALB/c mice. Data represent the mean ±SD of five mice. Statistically significant differences are represented by *p<0.05, **p<0.01, and ***p<0.001. (B) Measurement of anti-T7 nanoparticle IgG antibodies by ELISA using T7-wt-coated microplates. Each value represents mean ± SD in each group of mice. (C) Binding capacity of antibodies in vaccinated mice sera to native M2 expressed on PR8 virus-infected MDCK cells. Results from PBS and CFA/IFA groups are not shown as there was no significant difference compared to T7-wt group. Details are described in Results.
Figure 4.
M2e-specific T cell responses were measured using IFN-γ ELISPOT.
Splenocytes were isolated from immunized mice, pooled in each group and assayed for IFN-γ-producing cells by ELISPOT as described in Materials and Methods. (A) Data are represented as number of IFN-positive spot forming cells (SFCs) per 106 pooled splenocytes in each group ± SD. All samples were tested in triplicate. (B) Frequency of IFN-γ spots in each group of mice.
Figure 5.
Antiviral effects of T7-M2e nanoparticles.
The immunized mice were challenged by 4 LD50 of mouse-adapted PR8 (H1N1) or X47 (H3N2) virus 21 days after the second booster dose and monitored for survival and clinical symptoms (body weight loss) for 14 days. T7-M2e nanoparticles without adjuvant significantly protected mice against a lethal infection with both PR8 (A) and X47 (B) viruses as analyzed by Log-rank test. However, addition of CFA/IFA adjuvant resulted in 100% protection of mice. (C) Body weight was monitored for all mice for 14 days post-challenge with PR8 virus. (D) Five days post-challenge, six mice in each group were sacrificed and residual virus titers were measured in lungs as TCID50 per ml of each lung homogenate.