A replication-incompetent PB2-knockout influenza A virus vaccine vector

Abstract

Vaccination is the primary form of protection from influenza virus infection. We recently developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that possesses a reporter gene (the green fluorescent protein gene) in the coding region of the PB2 segment. This virus replicated to high titers in PB2-expressing, but not unmodified, cells, suggesting its potential safety and feasibility as a vaccine. Here, we tested its efficacy in a murine model. The levels of IgG and IgA antibodies against influenza virus in sera, nasal washes, and bronchoalveolar lavage fluids of mice immunized with the PB2-KO virus were higher than those induced by a conventional inactivated vaccine. All PB2-KO virus-immunized mice survived challenges with lethal doses of influenza virus. Moreover, importantly, mice immunized with the PB2-KO virus produced antibodies against the reporter protein, suggesting that the PB2-KO virus has potential as a multivalent vaccine to combat infection with not only influenza virus but also other pathogens.

Fig 1

Virus-specific antibody responses in immunized mice. Purified PR8 virus was used as an antigen to analyze IgG and IgA antibody titers in the sera, nasal washes, and BAL fluids (top, middle, and bottom, respectively) of mice mock immunized with medium or immunized with the formalin-inactivated virus or with the PB2-KO virus. Sera (top panels) were obtained at different time points, i.e., prevaccination (bars A), before the second vaccination (bars B), before the third vaccination (bars C), and before challenge (bars D). Nasal washes and BAL fluids (middle and bottom panels, respectively) were obtained 1 day before challenge from mice given 1 vaccination (bars 1), 2 vaccinations (bars 2), or 3 vaccinations (bars 3). Values are expressed as the mean absorbance ± standard deviation (SD) (n = 3). Statistical significance between samples obtained from mice immunized with inactivated virus and PB2-KO virus is indicated.

Fig 2

Body weight change after challenge in mice. Mice immunized with the indicated agents once (A), twice (B), or three times (C) were challenged with 0.5 (i) or 5 (ii) MLD₅₀ of PR8 virus. Values are expressed as mean changes in body weight ± SD (n = 3).

Fig 3

Virus titers in the lungs and nasal turbinates (NT) of immunized mice after challenge. The numbers on the x axis indicate the number of vaccinations. Three BALB/c mice per group were intranasally infected with the indicated doses of PR8 virus (50 μl per mouse) and sacrificed on days 3 and 6 postinfection for virus titration. Bars indicate the virus titer in each organ of each mouse. The absence of bars indicates that virus titers were below the detection limit of 5 PFU/ml/organ.

Fig 4

Detection of antibodies against GFP in the sera of mice immunized with the PB2-KO virus. Confluent 293 cells that transiently express GFP were treated with sera (1/20 dilution) obtained from mice inoculated with medium (A), the formalin-inactivated virus (B), or the PB2-KO virus (C) or were treated with a commercial anti-GFP antibody (D). DNA (first column) was stained with Hoechst 33342. GFP (second column) represents cells transfected with a plasmid for the expression of GFP. GFP antibody (third column) represents the presence of the GFP antibody in the samples. These three images were merged (fourth column). Scale bars, 20 μm.