Vaccination with recombinant Lactococcus lactis expressing HA1-IgY Fc fusion protein provides protective mucosal immunity against H9N2 avian influenza virus in chickens

Abstract

Background: H9N2 virus is mainly transmitted through the respiratory mucosal pathway, so mucosal immunity is considered to play a good role in controlling avian influenza infection. It is commonly accepted that no adequate mucosal immunity is achieved by inactivated vaccines, which was widely used to prevent and control avian influenza virus infection. Thus, an improved vaccine to induce both mucosal immunity and systemic immunity is urgently required to control H9N2 avian influenza outbreaks in poultry farms.

Methods: In this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the HA1 proteins derived from an endemic H9N2 virus strain and chicken IgY Fc fragment. We evaluated the immunogenicity and protective efficacy of this recombinant L. lactis HA1-Fc strain.

Results: Our data demonstrated that chickens immunized with L. lactis HA1-Fc strain showed significantly increased levels of serum antibodies, mucosal secretory IgA, T cell-mediated immune responses, and lymphocyte proliferation. Furthermore, following challenge with H9N2 avian influenza virus, chickens immunized with L. lactis HA1-Fc strain showed reduced the weight loss, relieved clinical symptoms, and decreased the viral titers and the pathological damage in the lung. Moreover, oropharyngeal and cloacal shedding of the H9N2 influenza virus was detected in chicken immunized with L. lactis HA1-Fc after infection, the results showed the titer was low and reduced quickly to reach undetectable levels at 7 days after infection.

Conclusion: Our data showed that the recombinant L. lactis HA1-Fc strain could induce protective mucosal and systemic immunity, and this study provides a theoretical basis for improving immune responses to prevent and control H9N2 virus infection.

Keywords: H9N2 influenza virus; Immune responses; Lactococcus lactis; Mucosal immune; Oral vaccine.

Fig. 1

Western blot analyses of L. lactis HA1-Fc. Western blotting identification of the recombinant proteins with the goat anti-rabbit HA antibody. Lane M is protein molecular size page ruler; Lane 1 is the strain of L. lactis; Lane 2 is the strain of L. lactis HA1-Fc

Fig. 2

HI Antibody levels and the specific IgG levels induced by L. lactis HA1-Fc. A Antibody titers determined by HI assay after the chickens were vaccinated with PBS, L. lactis, L. lactis HA1, L. lactis HA1-Fc and inactivated H9N2 vaccine. B The antigen specific IgG titers of chickens after vaccination detected by indirect ELISA. The data were showed as means ± SD (n = 5). **P < 0.01 relative to PBS and L. lactis. ^##P < 0.01 relative to L. lactis HA1

Fig. 3

Mucosal antibody levels induced by L. lactis HA1-Fc. SIgA antibodies in the feces (A) and BALF (B) were assessed by ELISA after the chickens were vaccinated with PBS, L. lactis, L. lactis HA1, L. lactis HA1-Fc and inactivated H9N2 vaccine. The data were showed as means ± SD (n = 5). ^▲▲P < 0.01 relative to PBS. **P < 0.01 relative to PBS and L. lactis. ^##P < 0.01 relative to L. lactis HA1. ^$$P < 0.01 relative to L. lactis HA1-Fc

Fig. 4

IL-2, IL-4, and IFN-γ levels in intestinal tissues induced by L. lactis HA1-Fc. Chickens were vaccinated with PBS, L. lactis, L. lactis HA1, L. lactis HA1-Fc and inactivated H9N2 vaccine, respectively, then intestinal tissues were collected. IL-2 (A), IFN-γ (B), and IL-4 (C) were detected via real-time PCR. The data were showed as means ± SD (n = 5). ^ΔΔP < 0.01 relative to PBS. **P < 0.01 relative to PBS and L. lactis. ^##P < 0.01 relative to L. lactis HA1. ^$$P < 0.01 relative to L. lactis HA1-Fc

Fig. 5

T cells proliferation assay. T cells proliferation was examined by Cell Proliferation ELISA BrdU Kit, and results were expressed as a stimulation index (SI) of chickens vaccinated with PBS, L. lactis, L. lactis HA1, L. lactis HA1-Fc and inactivated H9N2 vaccine. The data were showed as means ± SD (n = 5). **P < 0.01 relative to PBS and L. lactis. ^##P < 0.01 relative to L. lactis HA1. ^$$P < 0.01 relative to L. lactis HA1-Fc

Fig. 6

Weight change of the vaccinated chickens after challenge with H9N2 avian influenza virus. Chickens were immunized with PBS, L. lactis, L. lactis HA1, L. lactis HA1-Fc and inactivated H9N2 vaccine. 10 days after the boost immunization, the immunized chickens were challenged with H9N2 influenza virus. The weight loss was recorded daily for 2 weeks

Fig. 7

Viral shedding from cloacal and oropharyngeal swabs. Immunized chickens were infected with H9N2 avian influenza virus, cloacal and oropharyngeal swabs were collected from challenged chickens on days 2, 4, and 7 post-challenges. Viral titers were detected from oropharyngeal swabs collected from challenged chickens on days 2 (A), 4 (B), and 7 (C) post-challenges. Viral titers were detected from cloacal swabs collected from challenged chickens on days 2 (D), 4 (E), and 7 (F) post-challenges

Fig. 8

Protective effect of L. lactis HA1-Fc on lung injury induced by H9N2 influenza virus infection. Seven days after challenge with H9N2 avian influenza virus, lung tissue of PBS group (A), L. lactis group (B), L. lactis HA1 group (C), L. lactis HA1-Fc group (D) and H9N2 inactivated vaccine (E) was evaluated by histopathological analysis. The sections were stained with H&E. Objective magnification, × 200 (A–E). (F) The virus titers of lung samples from vaccinated chickens were obtained at 7 days after challenge and infectivity was measured by EID⁵⁰. The data were showed as means ± SD (n = 5). **P < 0.01 relative to PBS and L. lactis. ^##P < 0.01 relative to L. lactis HA1