A novel bivalent vaccine based on a PB2-knockout influenza virus protects mice from pandemic H1N1 and highly pathogenic H5N1 virus challenges

Abstract

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.

Fig 1

Characterization of PR8/PB2-Ca04HA and PR8/PB2-VN1203HA virus. (A) Schematic diagrams of wild-type PB2, PB2(120)Ca04HA(336), and PB2(120)VN1203HA(336) vRNAs. PB2(120)Ca04HA(336) and PB2(120)VN1203HA(336) vRNAs possess the 3′ noncoding region, 120 nucleotides (nt) of the coding sequence of PB2 vRNA, the Ca04HA or the VN1203HA gene, and 336 nt of the 3′ and 5′ noncoding regions of PB2 vRNA. The noncoding region and coding regions of PB2 vRNA are represented by shaded and filled bars, respectively. (B) Growth kinetics of PR8/PB2–Ca04HA or PR8/PB2-VN1203HA virus. AX4 and AX4/PB2 cells were infected with PR8, PR8/PB2–Ca04HA, or PR8/PB2–VN1203HA virus at an MOI of 0.001. Supernatant were collected at 12 h, 24 h, 36 h, 48 h, and 72 h postinfection for virus titration by plaque assays in AX4/PB2 cells. (C and D) HA expression in AX4/PB2 cells infected with PR8/PB2-Ca04HA or PR8/PB2-VN1203HA virus. (C) AX4 and AX4/PB2 cells were mock infected or infected with PR8, PR8/PB2-Ca04HA (left panels), or PR8/PB2-VN1203HA (right panels) virus at an MOI of 5. At 24 h after infection, the cells were collected and Ca04HA (left), VN1203 HA (right), M1, and beta-actin were detected by Western blotting with specific antibodies. (D) AX4 and AX4/PB2 cells were mock infected or infected with PR8, PR8/PB2-Ca04HA (left panels), or PR8/PB2-VN1203HA (right panels) virus. At 48 h after infection, the cells were fixed and stained with anti-influenza virus polyclonal (R309), -H1N1pdm09 HA monoclonal (9C4C11), and -H5 HA monoclonal (9B2) antibodies.

Fig 2

Virus-specific antibody responses in immunized mice. Virus-specific antibodies were detected by means of an ELISA with purified PR8 (A and B), Ca04 (C), and VN1203 (D) viruses as antigens. IgG (top) and IgA (bottom) antibody titers in the serum (left), BALF (middle), and nasal washes (right) from mice intranasally mock immunized with PBS or immunized with the formalin-inactivated (FI) virus, PR8/PB2-Ca04HA (A and C), or PR8/PB2-VN1203HA virus (B and D) were measured. Values are expressed as the mean absorbance ± standard deviation (SD) (n = 3) of samples. The neutralizing antibody titers in the serum of mice intranasally mock-immunized with PBS or immunized with the formalin-inactivated (FI) virus, PR8/PB2-Ca04HA virus (E, left), or PR8/PB2-VN1203HA virus (E, right) were measured by using microneutralization assays.

Fig 3

Body weight changes and survival of mice after challenge with PR8. Four mice per group were mock immunized with PBS or immunized with the formalin-inactivated (FI) virus, PR8/PB2-Ca04HA, or PR8/PB2-VN1203HA twice or three times at 2-week intervals. Three weeks after the final vaccination, mice were intranasally challenged with 3 or 10 MLD₅₀ of PR8 (A, vaccinated with FI-Ca04 or PR8/PB2-Ca04HA virus; B, vaccinated with FI-VN1203 or PR8/PB2-VN1203HA virus). Body weight (top) and survival (bottom) were monitored for 14 days after challenge. Values are expressed as mean changes in body weight ± SD (n = 4).

Fig 4

Virus titers in the respiratory tract of PR8/PB2-Ca04HA or PR8/PB2-VN1023HA-immunized mice challenged with PR8 virus. Four mice per group were mock immunized with PBS or immunized with the formalin-inactivated (FI) virus, PR8/PB2-Ca04HA, or PR8/PB2-VN1203HA twice with a 2-week interval between the vaccinations. Three weeks after the final vaccination, mice were intranasally challenged with 3 or 10 MLD₅₀ of PR8 (A, vaccinated with FI-Ca04 or PR8/PB2-Ca04HA virus; B, vaccinated with FI-VN1203 or PR8/PB2-VN1203HA virus). Nasal turbinates and lungs were collected from mice (n = 3) on days 3 and 6 after challenge and subjected to virus titration by plaque assays in AX4/PB2 cells. The results are expressed as the mean titers ± SD. The asterisk indicates that virus titers in the respiratory tracts of mice immunized with FI, PR8/PB2-Ca04HA virus, or PR8/PB2-VN1203HA virus were significantly lower than those in the respiratory tracts of mock-immunized mice (P < 0.05).

Fig 5

Body weight changes and survival of mice after challenge with mouse-adapted Ca04 or VN1203 virus. Four mice per group were mock immunized with PBS, the formalin-inactivated (FI) virus, or PR8/PB2-Ca04HA or PR8/PB2-VN1203HA virus twice or three times at 2-week intervals. Three weeks after the final vaccination, mice were intranasally challenged with 3 or 10 MLD₅₀ of mouse-adapted Ca04 (MACa04) or VN1203 virus (A, vaccinated twice [left] or three times [right] with FI-CA04 or PR8/PB2-Ca04HA virus; B, vaccinated twice [left] or three times [right] with FI-VN1203 or PR8/PB2-VN1203HA virus). Body weight (top) and survival (bottom) were monitored for 14 days after challenge. Values are expressed as mean changes in body weight ± SD (n = 4).

Fig 6

Virus titers in multiple organs of immunized mice challenged with mouse-adapted Ca04 virus or VN1203 virus. Four mice per group were mock immunized with PBS or immunized with the formalin-inactivated (FI), PR8/PB2-Ca04HA, or PR8/PB2-VN1203HA virus twice or three times at 2-week intervals. Three weeks after the final vaccination, mice were intranasally challenged with 3 or 10 MLD₅₀ of mouse-adapted Ca04 virus or VN1203 virus (A, vaccinated with FI-Ca04 or PR8/PB2-Ca04HA virus; B, vaccinated with FI-VN1203 or PR8/PB2-VN1203HA virus). Multiple organs were collected from mice (n = 3) on days 3 and 6 after challenge and subjected to virus titration by plaque assays in AX4/PB2 cells. The results are expressed as the mean titers ± SD. The asterisk indicates that virus titers in the organs of mice immunized with FI virus, PR8/PB2-Ca04HA virus, or PR8/PB2-VN1203HA virus were significantly lower than those in the organs of mock-immunized mice (P < 0.05).