Article Figures & Data
Figures
- FIG 1
Generation and characterization of influenza antigen-expressing VSV replicons. (A) Phylogenetic relationship of influenza A virus NA genes included in this study. Amino acid sequences were aligned using ClustalW and a phylogenetic tree constructed using the neighbor-joining method and 1,000 bootstrapped replications with observed amino acid differences as the distance on the aligned sequences using MEGA7. (B) Scheme of the genomes of the parental VSV and the recombinant VSV*ΔG replicons. VSV*ΔG lacks the gene encoding the VSV G protein and includes the eGFP gene in an additional transcription unit, depicted in gray. VSV*ΔG(X) encodes the respective heterologous influenza virus antigens, depicted in dark gray. (C) Vero E6 cells were surface biotin labeled with EZ-Link sulfo-NHS-biotin (Pierce) 12 h after infection with the respective VSV*ΔG replicons, followed by cell lysis and immunoprecipitation of 50 μg of cell extracts with homologous mouse antiserum. Proteins in the pellet fraction were separated on an SDS-PAGE gel, and surface biotin-labeled proteins were visualized using an avidin-HRP secondary antibody. (D) Protein bands from three independent replicates were quantified and normalized relative to NAPR8 for each blot and are shown as fold changes. Error bars represent the standard deviations of the means. Groups were compared using one-way ANOVA with a Tukey posttest, and statistical significance is indicated (*, P < 0.05; ns, not significant).
- FIG 2
Humoral and cellular immune responses in mice induced by the different VSV*ΔG replicons. (A) Schematic overview of the immunization strategy and blood and splenocyte sampling time points. Six- to eight-week-old naive C57BL/6 mice were immunized intramuscularly twice with 106 FFU of the VSV*ΔG replicons or with 2.5 μg of BPL-inactivated PR8 virus. (B and C) Kinetics of total antibody titers (B) and 50% neuraminidase-inhibiting (NI) antibody titers (C) against PR8 in animals immunized with matched antigens. Symbols represent the means for each group (n = 6), and error bars indicate the standard deviations of the means. (D and E) Total antibody titers (D) and NI titers (E) against all PR8, USSR, and H5N1 viruses. Bars represent the means for each group (n = 6), and error bars indicate the standard deviations of the means. Log2-transformed groups were compared to the NAPR8 group using one-way ANOVA with a Tukey posttest. (F) IFN-γ ELISpot analysis results after restimulation with purified PR8 virus. H1N1 PR8-infected mice were used as positive controls, and noninfected mice were used as negative controls. Symbols indicate spot-forming units (SFU) for individual animals, and black bars represent the respective means. Groups were compared using one-way ANOVA with a Tukey posttest, and statistical significance is indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
- FIG 3
Protective efficacy of the different vaccine candidates. Mice were vaccinated once or twice in 4-week intervals and then challenged with 1 × 104 TCID50 of H1N1 PR8 4 weeks later. Upon challenge, animals were monitored daily for clinical signs and body weight and euthanized as soon as the weight loss reached 25%. (A to D) Percent weight changes after challenge following one (A) or two (B) immunizations and the respective percent survivals (C and D) (n = 6; N1 H5N1, n = 5). (E and F) Correlation between mean NI titer and percent survival 4 weeks after the first (E) or the second (F) immunization.
- FIG 4
Efficacy of passive serum transfer. (A) Schematic overview of the experimental design. Mice were vaccinated on days 0 and 28 with the respective VSV*ΔG replicons, and serum was isolated 7 days after the boost immunization. Sera were subsequently transferred into naive mice. Blood samples were taken 24 h later, and animals were challenged with 1 × 104 TCID50 of H1N1 PR8. (B) Total antibody responses against PR8 and NI titers of donor mice. Bars represent the means for three repeated measurements of pooled serum, and error bars indicate the standard deviations of the means. Log2-transformed groups were compared to the NAPR8 group using one-way ANOVA with a Tukey posttest. Statistical significance is indicated (*, P < 0.05; ****, P < 0.0001). (C and D) Percent weight change (C) and percent survival (D) in recipient mice. Upon challenge, body weight was monitored as a measure of morbidity. Animals were euthanized as soon as the weight loss reached 25%.
- FIG 5
Humoral immune responses in ferrets induced by the different VSV*ΔG replicons and viral load after infection. (A) Schematic overview of the immunization-and-infection strategy and blood sampling time points. Naive ferrets were immunized twice with 108 FFU VSV*ΔG replicons intramuscularly. All animals were challenged intranasally with 105 TCID50 A/Mexico/InDRE4487/2009 and sacrificed for virus titration on day 3 after infection. (B and C) Kinetics of total antibody titers (B) and NI titers (C) against pdmH1N1 in animals immunized with matched or mismatched antigens. Log2-transformed groups were compared using two-way ANOVA with a Tukey posttest. (D and E) Temperature changes (E) and clinical scores (F) measured over 3 days after infection. Symbols represent the means for each group (n = 4; postinfection N1 H5N1, n = 2), and error bars indicate the standard deviations of the means. (F and G) Virus titers in the nasal turbinates (F) and lungs (G) in terms of TCID50 per gram on MDCK cells. Symbols indicate data for individual animals, and black bars represent the respective means.
- FIG 6
Linear comparison of the NA proteins. The cytoplasmic domain, transmembrane region (TM), stalk region, and head domain are depicted in dark, medium and light gray, and white, respectively. Putative glycosylation sites are shown above, active sites are in red, and previously identified critical residues for inhibiting antibody binding are indicated by gray arrowheads and the respective amino acid numbering.
