Protective effect of a polyvalent influenza DNA vaccine in pigs

Abstract

Background: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity.

Objectives: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study.

Methods: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500μg and 800μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus.

Results: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) - as well as cross-reactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dose-dependent manner.

Conclusion: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination.

Keywords: Challenge; DNA vaccine; H1N1pdm09; Needle-free immunization; Protection; Swine influenza.

Fig. 1

HA-specific antibody responses following DNA vaccination and challenge. Pigs were vaccinated twice (arrows) i.d. with needle-free delivery with 800 μg (n = 5) or 500 μg (n = 5), or not DNA vaccinated at all (n = 5). Levels of IgG in the pre- and post-challenge sera were measured by ELISA against recombinant HA (H1N1pdm09) (A). Hemagglutination inhibition antibody responses in the pre- and post-challenge sera against H1N1pdm09 were measured (B). A Hi-titer cut-off of 20 IE/ml is indicated. Error bars indicate the mean ± SEM, and significant differences from the no-vaccine control group are indicated by **: p < 0.01; *: p < 0.05.

Fig. 2

Cross-reactive anti-H1N1 neutralizing antibody responses in vaccinated pig sera. Pigs were vaccinated twice (arrows) i.d. with needle-free delivery with 800 μg (n = 5) or 500 μg (n = 5), or not DNA vaccinated at all (n = 5). The pre- and post-challenge pig sera were tested in a micro-neutralization (MN) assay. Neutralizing antibody titers, MN titers, were evaluated by the capacity of the sera to prevent the infection of MDCK cells by (A) H1N1pdm09 and (B) swine 2008 H1N1 virus isolates. The MN titer was defined as the reciprocal dilution providing 50% infection inhibition, calculated with a linear interpolation method (Reed and Muench, 1938). Serum samples with a titer below the detectable limit of the assay (lowest serum dilution tested was 1:20) were assigned a value of 10 for graphical representation and statistical analyses. Error bars indicate the mean ± SEM, and significant differences from the no-vaccine control group are indicated by **: p < 0.01; *: p < 0.05.

Fig. 3

Cross-reactive anti-H1N1 neuraminidase inhibition (NAI) in vaccinated pig sera. Pigs were vaccinated twice (day −36 and day −14) i.d. with needle-free delivery with 800 μg (n = 5) or 500 μg (n = 5), or not DNA vaccinated at all (n = 5). The pre- and post-challenge pig sera were tested in an NAI assay. The NAI titers, or the serum dilutions that would inhibit 50% of neuraminidase activity (IC₅₀), were tested against (A) H1N1pdm09 and (B) swine 2008 H1N1 isolates. Error bars indicate the mean ± SEM.

Fig. 4

Protective efficacy of the influenza DNA vaccine. Vaccinated and control animals were challenged with pandemic H1N1 A/California/07/2009. Post-challenge viral loads were assessed for up to 13 days in nasal swabs. Data are expressed as the mean log10 virus titer ± SEM.