Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation

Abstract

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.

Keywords: H3N2 subtype; protective efficacy; reverse genetics; swine influenza virus.

Fig. 1

Phylogenetic analysis of hemaggulutinin (HA) gene segment of H3N2 SwIV isolates from southern China. The HA donor of the seed virus in this study is indicated by a dark circle.

Fig. 2

Growth of the reassortant GD/PR8 virus in embryonated eggs. 0.1 mL of 100EID50 of GD/PR8 or GD/06 viruses were inoculated into the allantoic cavities of 10-day-old embryonated eggs and HA titers were checked at 12, 24, 36, 48, 60 and 72 h post-inoculation.

Fig. 3

IgG antibody responses induced by GD/PR8 inactivated vaccines in mice. Sixty 6-week-old SPF female BALB/c mice were randomly divided into three groups (n = 20 per group). Groups of mice were inoculated with one dose of previously prepared vaccine (GD/PR8+F) or non-adjuvanted GD/PR8 virus (GD/PR8) using the same amount of concentrated GD/PR8 virus (10 µg). Mock-vaccinated mice received 50 µL Freund's complete adjuvant (FCA) as a placebo. All inoculations were administered by the muti-point subcutaneous route twice with a two week interval. Serum samples (n = 10 each group) were collected weekly after immunization. All serum samples were assayed for GD/06-specific IgG antibody titers. The results are shown as the mean ± standard deviation for each of the groups of 10 serum samples. Asterisks indicate statistically significant differences (p < 0.05) compared with values for mock-vaccinated control mice. The horizontal broken line represents the detection limit.

Fig. 4

Detection of viral RNA in lungs of infected mice using real-time PCR (RT-PCR). Copy numbers in log₁₀ per 1 µL of cDNA obtained by RT-PCR targeting the matrix protein gene are given for individual mice. cDNA (20 µL) was synthesized using Uni12 primer by reverse transcription PCR. Asterisks indicate statistically significant differences (p < 0.05) compared with values for mock-vaccinated control mice. The horizontal broken line represents the detection limit.

Fig. 5

Microscopic lung lesions in lungs of infected mice. (C) Mock-vaccinated mice with enhanced pneumonia compared to mice immunized with (A) GD/PR8 vaccine and (B) non-adjuvanted GD/PR8 virus. (D) NV/NC mice remained untreated as environmental controls. H&E stain, ×200.