Fig 1.
Generation and characterization of M2e-displaying f88 phages.
(A) Construct of M2e-displaying or control peptide-displaying f88 plasmid. (B) Schematic diagram of f88 and f88M2e2-16 bacteriophages. (C, D) Characterization of CsCl-gradient-centrifuge purified f88M2e2-16 and f88ctr phages using coomassie blue staining and western blot. Two μg of f88M2e2-16 and f88ctr bacteriophages were loaded on SDS-PAGE and the corresponding Western blot was visualized using anti-pVIII mAb, anti-pIII mAb, anti-M2e mAb 37 and anti-M2e mAb 148, as indicated. MAb 37 is an anti-M2e IgG1 specifically recognizing amino acids 4–14 of human consensus M2e; mAb 148 is an anti-M2e IgG1 that specifically binds the conserved first nine amino acids of M2e. (E) Recombinant pVIII-M2e2-16 and f88 phages were separated using Tricine-SDS-PAGE with 15% acrylamide gel. Mouse anti-pVIII antibody was then used to probe protein samples in Western Blot. The upper faint protein band in the left lane corresponds to pVIII-M2e2-16 and is absent in f88 phages. The faster migrating and more intense band corresponds to wild type pVIII. (F) phage preparation titration using TG1 Escherichia coli strain containing F pilus. 100 μl bacteriophage containing 1010 tetracycline transducing units were diluted with 5x106, 5x107 and 5x108 times and were plated on tetracycline-containing LB agar plates. Each colony forming unit represents one viable bacteriophage particle.
Table 1.
List of different M2e sequences used in this study.
Fig 2.
Immunization of mice with f88M2e2-16 phages results in robust anti-M2e specific serum IgG responses.
(A-D) Groups of female BALB/c mice (n = 10 per group) were intraperitoneally vaccinated with antigens in the presence of incomplete Freund’s adjuvant via the intraperitoneal route. Purified phages were administered at 1010 tetracycline transducing units (f88M2e2-16 and f88ctr groups). As a positive control, one group was immunized with 10 μg M2eHBc per vaccination. A PBS plus adjuvant was included as a negative control. Blood was collected 10 days after priming and each boost injection and serum was prepared and tested in ELISA. (A) Anti-f88 total IgG titration following priming and boosting. (B) Anti-human consensus M2e total IgG following priming and boosting. (C) Anti-human consensus M2e IgG1 and (D) IgG2a endpoint titration following priming and boosting. Endpoint serum IgG titers after the third boost were determined from BALB/c mice that had been immunized with 1010 tetracycline transducing units of f88ctr, 1010 tetracycline transducing units f88M2e2-16 or 10 μg of avian M2e-HBc using ELISA plates coated with M2e peptides from different influenza A virus strains: (E) M2e from A/swine/Ontario/42729A/01 (sOnM2e), (F) M2e from A/chicken/HongKong/258/1997 (cHKM2e), (G) M2e from A/chicken/Vietnam/36/2004 (cVNM2e). In A-D error bars represent standard deviations.
Fig 3.
Immunization of mice with f88M2e2-16 phages induces anti-M2e specific serum IgG that binds to human and avian influenza A virus infected mammalian cells.
Preimmune and immune sera collected after the third immunization with avian M2e-HBc, f88M2e2-16 or f88ctr were used to immuno-stain Madin-Darby canine kidney (MDCK) cells that had been infected with A/Memphis/106/76 (H3N2), A/Belgium/145/2009 (pandemic H1N1), A/chicken/Nanchang/3-120/2001 (H3N2), PR8 (H1N1) or mock infected. Alexa Fluor 555 Donkey anti-Mouse secondary antibody (red fluorescence) was used to reveal mouse IgG binding. An Alexa Fluor 488 labeled polyclonal goat anti-RNP staining (green fluorescence) was used to trace influenza A virus infected cells. All cells were stained with DAPI (blue fluorescence).
Fig 4.
Vaccination with f88M2e2-16 protects against influenza A virus challenge.
Six—eight weeks old female BALB/c mice (n = 10 per group) were immunized with 1010 f88M2e2-16 bacteriophage particles, avian M2eHBc or f88ctr via the intraperitoneal route for 3 times at 3 weeks interval in the presence of incomplete Freund’s adjuvant. Two weeks after the last boost, groups of mice were challenged with 4 LD50 mouse adapted PR8 H1N1. (A) Morbidity and (B) mortality following challenge. (C) Lung virus titers in lung homogenates isolated on day 10 after challenge of mice vaccinated as in (A) with 1 LD50 of mouse adapted PR8 virus. (D) Mice vaccinated as in A were sacrificed on day 10 after challenge with 1 LD50 of mouse adapted PR8. Ten μm-thick lung section were prepared and stained with Hematoxylin—eosin. Representative slides magnified 10 times are shown. Black arrows indicate bronchial infiltration of immune cells.
Fig 5.
Vaccination with f88M2e2-16 protects against challenge with an influenza A virus that carries a Pro10 to Leu change in M2e.
Six—eight weeks old female BALB/c mice (n = 6 per group) were immunized with 1010 f88M2e2-16 bacteriophage particles, 1010 f88ctr bacteriophage particles, 10 μg human M2eHBc or PBS. Immunization was done via the intraperitoneal route, in the presence of incomplete Freund’s adjuvant and 3 times with 3 weeks interval. Two weeks after the last boost, mice were challenged with 4 LD50 of mouse adapted A/Memphis/106/76 H3N2. (A) Morbidity and (B) mortality following challenge. (C) Lung virus titers determined on day 6 after challenge with 1 LD50 of mouse adapted A/Memphis/106/76 H3N2 virus.
Fig 6.
Challenge of f88M2e2-16 immunized mice does not boost the anti-M2e serum IgG response.
BALB/c mice (n = 10) were vaccinated with f88M2e2-16, f88-ctr, human M2eHBc or PBS three times in the presence of incomplete Freund’s adjuvant. Three weeks after the last boost, mice were challenged with 0.2 LD50 of mouse-adapted pandemic H1N1 2009 virus. Serum was collected 10 day after the last booster immunization (-) and 15 days after infection (+). The anti-M2e serum IgG response was determined by ELISA coated with (A) human consensus M2e (M2e2-23), M2e from (B) A/swine/Ontario/42729A/01 (sOnM2e), (C) M2e from A/chicken/Hong Kong/258/1997 (cHKM2e), (D) M2e from A/swine/Belgium/1/1998 (sBelM2e) and (E) M2e from A/duck/Vietnam/NCVD-9/2007 (dVNM2e). (F) Hemagglutination inhibition titers against pandemic H1N1 2009 virus before and after infection of mice. Error bars represent standard deviations.