Protection against Lethal Influenza with a Viral Mimic

Characterization of pH1N1/E3-sciIV. Plasmid-based reverse genetics techniques were used to rescue a sciIV with genes derived from pH1N1, except the fourth segment which codes for HA(45)GFP(80), as previously described (12). (A) GFP is expressed by cells infected with pH1N1/E3-sciIV. MDCK cells were mock infected or infected with pH1N1 or pH1N1/E3-sciIV at an MOI of 5. Ten hours postinfection, monolayers were fixed, permeabilized, and incubated with a MAb against NP (HT103) and subjected to nuclear counterstaining with DAPI. Representative ×20 images (NP, GFP, DAPI, and merged [bottom row]) are shown. Scale bars, 20 μM. (B and C) Multicycle growth of pH1N1/E3-sciIV. WSN-HA, pH1N1/E3-HA, or parental MDCK cells were infected with pH1N1/E3-sciIV at an MOI of 0.001. At various times postinfection, GFP expression was visualized by fluorescence microscopy (B) (×10 representative images shown; scale bars, 40 μM), and TCSs were collected for titration in MDCK WSN-HA cells (C). Data represent means ± SD of the results determined for triplicates. (D) Multicycle growth of pH1N1/E3-sciIV and pH1N1/E3. Parental or pH1N1/E3-HA-expressing MDCK cells were infected with pH1N1/E3 or pH1N1/E3-sciIV at an MOI of 0.001. At various times postinfection, TCSs were collected for titration on MDCK cells by IFA using the NP MAb HT103. Data represent means ± SD of the results determined for triplicates. (E) Plaque morphology of pH1N1/E3-sciIV. WSN-HA, pH1N1/E3-HA, or parental MDCK cells were infected with pH1N1 or pH1N1/E3-sciIV for plaque assay. Three days postinfection, monolayers were stained with crystal violet. (F) pH1N1/E3-sciIV can infect cells that do not express HA. Parental, WSN-HA, or pH1N1/E3-HA MDCK cells were infected at an MOI of 5 with pH1N1/E3-sciIV. Ten hours postinfection, monolayers were analyzed by fluorescence microscopy for GFP expression. Representative images (×10 magnification) are shown. Scale bars, 40 μM.

Immunogenicity of pH1N1/E3-sciIV in mice. (A to C) Humoral immune response to vaccination. Female 6-to-8-week-old C56BL/6 mice (n = 5) were inoculated at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV once (orange triangles, prime only) or twice (red triangles, prime plus boost) or with PBS (black circles) as a negative control. Two weeks postvaccination, sera were collected and evaluated for IgG Abs against total influenza virus protein (A), recombinant NP from PR8 (B), or recombinant HA from pH1N1 (C) by ELISA. Convalescent-phase sera (green) collected 2 weeks after naive mice were infected with 0.02 MLD₅₀ pH1N1/E3 were used as a positive control (n = 3). Data represent means ± standard errors of the means (SEM) of the results from 4 independent experiments. Ag, antigen; OD, optical density. (D) CD8⁺ T cells present in the lungs after immunization with pH1N1/E3-sciIV. Female 6-to-8-week-old C56BL/6 mice were inoculated twice at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV or PBS (Naive) or were infected with 0.02 MLD₅₀ pH1N1/E3 (n = 3). Ten days after the final vaccination or infection, lungs were extracted and resident cells were pooled and prepared for flow cytometry. Live, CD3⁺ CD4⁻ CD8⁺ cells specific for pH1N1 NP_366-374 tetramer were counted. Data represent means ± SEM of the results from 4 independent experiments. Statistical analysis was performed using Student's one-tailed paired t test. * = P ≤ 0.05; ** = P ≤ 0.01; ns = not significant.

pH1N1/E3-sciIV vaccination protects mice from lethal homologous challenge. (A) Representation of the immunization and challenge schedule. (B) Tolerability of pH1N1/E3-sciIV. Female 6-to-8-week-old C56BL/6 mice were inoculated at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV once or twice or with PBS as a negative control (n = 5). Weight loss was monitored daily for 2 weeks after the PBS or first (or ange triangles, prime only) or second (red triangles, prime plus boost) sciIV inoculation. Plotted data represent means ± SEM. (C and D) Protection from lethal challenge conferred by pH1N1/E3-sciIV vaccination. Immunized mice from the experiment described for panel B were challenged with 10 MLD₅₀ pH1N1/E3, and weight loss (plotted data represent means ± SEM) (C) and survival (D) were monitored daily for 2 weeks postchallenge. (E) Virus lung titers. Mice immunized and challenged as described for panels B and D were sacrificed at 3 and 6 days postchallenge (dpc) to evaluate levels of replicating challenge virus in the lungs by IFA as described for Fig. 2D (n = 3). Symbols represent data from individual mice; bars represent geometric mean lung virus titers, and the dotted line denotes the limit of detection (20 FFU/ml). Statistical analysis was performed using the Mann-Whitney test; * = P ≤ 0.05.

UV-inactivated pH1N1-sciIV does not fully protect mice from lethal homologous challenge. (A) UV light treatment renders sciIV noninfectious. SciIV was exposed to UV light for 20 min on ice, and infectivity was determined by TCID₅₀ assay on MDCK WSN-HA cells. (B) Immunogenicity of UV-inactivated sciIV. Female 6-to-8-week-old C56BL/6 mice were inoculated twice at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV that was (purple triangles; UV-inactivated sciIV) or was not (red triangles; ‘Live’ sciIV) exposed to UV light on ice for 20 min or with PBS (black circles) as a negative control (n = 5). Two weeks postvaccination, sera were collected and evaluated for IgG Abs against total influenza virus protein by ELISA. Convalescent-phase sera (green) were used as a positive control (n = 3); plotted data represent means ± SEM. (C and D) Protection from lethal challenge by pH1N1/E3-sciIV vaccination. Immunized mice from the experiment described for panel B were challenged with 10 MLD₅₀ pH1N1/E3, and weight loss (C) (plotted data represent meana ± SEM) and survival (D) were monitored daily for 2 weeks postchallenge. (E) Virus lung titers. Mice immunized and challenged as described for panels C and D were sacrificed at 3 and 6 days postchallenge to evaluate levels of replicating challenge virus in the lungs by IFA (n = 3). Symbols represent individual mice; bars represent geometric mean lung virus titers, and the dotted line denotes the limit of detection (20 FFU/ml). Statistical analysis was performed using the Mann-Whitney test; * = P ≤ 0.05.

Dose-dependent immunogenicity and protective efficacy conferred by sciIV vaccination. Female 6-to-8-week-old C56BL/6 mice were inoculated once (A to D, dotted lines) or twice (E to H, solid lines) at 2-week intervals i.n. with 10-fold dilutions of pH1N1/E3-sciIV (red, 10⁵; orange, 10⁴; yellow, 10³; pink, 10²) or with PBS (black) as a negative control (n = 5). Two weeks postvaccination, sera were collected and evaluated for IgG Abs against total influenza virus protein by ELISA (A and E; plotted data represent means ± SEM). Convalescent-phase sera (green) were used as a positive control (n = 3). Immunized mice from the experiment described for panels A and E were challenged with 10 MLD₅₀ pH1N1/E3, and weight loss (B and F; plotted data represent means ± SEM) and survival (C and G) were monitored daily for 2 weeks postchallenge. (D and H) Virus lung titers. Mice immunized and challenged as described above were sacrificed at 3 and 6 days postchallenge to evaluate levels of replicating challenge virus in the lungs by IFA (n = 3). The experiments represented in panels D and H were performed simultaneously; thus, the same PBS-vaccinated controls were plotted in both graphs. Symbols represent individual mice; bars represent geometric mean lung virus titers, and the dotted line denotes the limit of detection (20 FFU/ml). Statistical analysis was performed using the Mann-Whitney test; * = P ≤ 0.05.

Homosubtypic and heterosubtypic immunity conferred by sciIV vaccination. (i and ii) Female 6-to-8-week-old C56BL/6 mice were inoculated (vax) twice at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV (filled symbols) or with PBS (open symbols) as a negative control (n = 5). Two weeks postvaccination, mice were challenged with 10 MLD₅₀ pH1N1/E3 (A, red circles) or PR8 (B, green squares) or with 10⁴ PFU X31 (C, blue triangles) and weight loss (panels i; plotted data represent means ± SEM) and survival (panels ii) were monitored daily for 2 weeks postchallenge. (iii) Virus lung titers. Mice immunized and challenged as described above were sacrificed at 3 and 6 days postchallenge to evaluate levels of replicating challenge virus in the lungs by IFA (n = 3). Symbols represent individual mice; bars represent geometric mean lung virus titers, and the dotted line denotes the limit of detection (20 FFU/ml). Statistical analysis was performed using the Mann-Whitney test; * = P ≤ 0.05. §, no mice surviving at time of lung extraction.

Passive immune transfer does not protect from lethal heterosubtypic challenge. (i and ii) Female 6-to-8-week-old C56BL/6 mice were inoculated twice at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV or with PBS as a negative control (n = 5). Two weeks after the final vaccination, sera were collected over the course of 1 week, checked for seroconversion by ELISA against total influenza virus protein (data not shown), and pooled. Naive mice received 100 μl of immune (sciIV sera, filled symbols) or nonimmune (PBS sera, open symbols) sera (n = 11) i.p. 1 day prior to challenge with 10 MLD₅₀ pH1N1/E3 (A, red circles) or PR8 (B, green squares). Weight loss (panels i; plotted data represent means ± SEM) and survival (panels ii) were monitored daily for 2 weeks postchallenge. (iii) Virus lung titers. Mice passively immunized and challenged as described above were sacrificed at 3 and 6 days postchallenge to evaluate levels of replicating challenge virus in the lungs by IFA (n = 3). Symbols represent individual mice; bars represent geometric mean lung virus titers, and the dotted line denotes the limit of detection (20 FFU/ml). Statistical analysis was performed using the Mann-Whitney test; * = P ≤ 0.05. §, no mice surviving at time of lung extraction.

T cells in sciIV-vaccinated mice enhance clearance of heterologous challenge. (A and B) Female 6-to-8-week-old C56BL/6 mice were inoculated twice at 2-week intervals i.n. with 10⁵ FFU of pH1N1/E3-sciIV or with PBS (naïve; black circles) as a negative control (n = 11). Two weeks postvaccination, immunized mice were depleted of CD4 (yellow open triangles) or CD8 (orange open triangles) or both CD4 and CD8 (pink open diamonds) T cells by i.p. injection with 3 doses of blocking Abs (−2, 0, and 2 days postchallenge) or were given an IgG2b isotype control (green filled squares). All mice were challenged with 10 MLD₅₀ PR8, and weight loss (A; plotted data represent means ± SEM) and survival (B) were monitored daily for 2 weeks postchallenge. (C) Virus lung titers. Mice immunized, depleted of T cells, and challenged as described above were sacrificed at 3 and 6 days postchallenge to evaluate levels of replicating challenge virus in the lungs by IFA (n = 3). Symbols represent individual mice; bars represent geometric mean lung virus titers, and the dotted line denotes the limit of detection (20 FFU/ml). Statistical analysis was performed using the Mann-Whitney test; * = P ≤ 0.05. Statistical significance (P ≤ 0.05) was also achieved in comparisons of each treatment group with the naive group (not shown).