A recombinant influenza virus vaccine expressing the F protein of respiratory syncytial virus

Abstract

Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.

Fig. 1. Characterization of the PB2 (120)GFP(336) virus

(A and B) Schematic diagram of wild-type PB2, PB2(120)GFP(120), and PB2(120)GFP(336) vRNAs. PB2(120)GFP(120) and PB2(120)GFP(336) vRNA possess 120 and 336 nucleotides (nt), respectively, of the coding sequence of the PB2 vRNA, along with both the 3′ and 5′ non-coding sequences, 120 nt of the 3′ coding sequence, and the GFP gene. The non-coding and coding sequences of PB2 vRNA are represented by the gray and black bars, respectively. The GFP gene is represented by the green bar. (C) Growth kinetics of PB2(120)GFP(120) and PB2(120)GFP(336) viruses. AX4 (upper panel) and AX4/PB2 (lower panel) cells were infected with the wild-type PR8, PB2(120)GFP(120), or PB2(120)GFP(336) virus at an m.o.i. of 0.001. Supernatants collected at the indicated time points were assayed for infectious virus by use of plaque assays in AX4/PB2 cells.

Fig. 2. Characterization of the PB2-RSVF virus

(A) Schematic diagram of wild-type PB2 and PB2-RSVF vRNAs. PB2(120)FRSV(336) vRNA possesses the 3′ non-coding sequence of the PB2 vRNA, 120 nt of the 3′ coding sequence of the PB2 vRNA with two ATG mutations (red crosses), the full-length RSV F gene, 336 nt of the 5′ coding region of the PB2 vRNA, and the 5′ non-coding sequence of PB2 vRNA. The non-coding and coding sequences of the PB2 vRNA are represented by the gray and black bars, respectively. The RSV F gene is represented by the blue bar. (B) Expression of RSV F protein in PB2-RSVF virus-infected cells. AX4/PB2 cells were infected with wild-type PR8 (top panels) or PB2-RSVF (bottom panels) virus at an m.o.i. of 0.1. At 16 h postinfection, the cells were fixed and stained with anti-influenza virus NP (left panels) and anti-RSV F (right panels) antibodies. (C) Growth kinetics of the PB2-RSVF virus. AX4 and AX4/PB2 cells were infected with wild-type PR8 or PB2-RSVF virus at an m.o.i. of 0.001. Supernatants collected at the indicated time points were assayed for infectious virus by use of plaque assays in AX4/PB2 cells.

Fig. 3. Body weight changes of mice infected with the PB2-RSVF virus

BALB/c mice (three mice per group) were inoculated intranasally with 10⁴, 10⁵, or 10⁶ PFU of the PB2-RSVF virus (50 μl). Body weight was monitored daily for two weeks.

Fig. 4. Influenza virus-specific antibodies in immunized mice

BALB/c mice (three mice per group) were immunized twice intranasally with 10⁴, 10⁵, or 10⁶ PFU of the PB2-RSVF virus. Two weeks after the boost immunization, anti-influenza virus IgG (A) and IgA (B, C, and D) antibodies in 100-fold diluted serum (A and B), nasal wash (C), and BAL (D) samples were detected by using an ELISA. Mice immunized with formalin-inactivated PR8 virus (FI-PR8), infected with live PR8 virus (PR8), or mock immunized with PBS (PBS) served as controls. Values are expressed as the mean absorbance ± standard deviation (SD) (n=3). Statistical significance is indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Fig. 5. RSV-specific antibodies in immunized mice

BALB/c mice (three mice per group) were immunized twice intranasally with 10⁴, 10⁵, or 10⁶ PFU of the PB2-RSVF virus. Two weeks after the boost immunization, IgG (A) and IgA (B, C, and D) antibodies in undiluted serum (A and B), nasal wash (C), and BAL (D) samples were detected by using an ELISA. Mice immunized with heat-inactivated RSV (HI-RSV), infected with live RSV (values for 1:100 dilution), or mock immunized with PBS served as controls. Values are expressed as the mean absorbance ± SD (n=3). Statistical significance is indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Fig. 6. Body weight changes of mice after virus challenge

(A) Body weight changes and survival rates of mice challenged with influenza virus. Mice were immunized twice with 10⁴, 10⁵, or 10⁶ PFU of the PB2-RSVF virus or controls (formalin-inactivated PR8 [FI-PR8] virus and PBS). Two weeks after the boost immunization, mice were challenged with 10 MLD₅₀ of PR8 virus. Body weight (upper panel) and survival (lower panel) were monitored daily for two weeks. (B) Body weight changes and survival rates of mice challenged with RSV. Mice were immunized twice with 10⁴, 10⁵, or 10⁶ PFU of the PB2-RSVF virus or controls (heat-inactivated RSV [HI-RSV] and PBS). Two weeks after the boost immunization, mice were challenged with 10^6.5 TCID₅₀ of RSV. Body weight (upper panel) and survival (lower panel) were monitored daily for two weeks. Values are expressed as mean changes in body weight (n=3). Bars represent SD.