Oral immunization with an attenuated <i>Salmonella</i> Gallinarum encoding the H9N2 haemagglutinin and M2 ectodomain induces protective immune responses against H9N2 infection in chickens

H9N2, a low pathogenic avian influenza virus, causes significant economic losses in the poultry industry worldwide. Herein, we describe the construction of an attenuated <i>Salmonella</i> Gallinarum (SG) strain for expression and delivery of H9N2 haemagglutinin (HA) 1 (SG-HA1), HA2 (SG-HA2) and/or the conserved matrix protein 2 ectodomain (SG-M2e). We demonstrated that recombinant SG strains expressing HA1, HA2 and M2e antigens were immunogenic and safe in a chicken model. Chickens (<i>n </i>= 8) were vaccinated once orally with SG alone, SG-HA1, SG-HA2, SG-M2e, or mixture of SG-HA1, SG-HA2 and SG-M2e, or vaccinated once intramuscularly with an oil-adjuvant inactivated H9N2 vaccine. Our results demonstrated that vaccination with SG mutants encoding influenza antigens, administered individually or as a mixture, elicited significantly (<i>P </i>P P P <b>RESEARCH HIGHLIGHTS</b><i>S. gallinarum</i> expressing HA1, HA2 and M2e antigens are immunogenic and safe.<i>Salmonella</i> has dual function of acting as a delivery system and as a natural adjuvant.Vaccine constructs elicit specific humoral and cell-mediated immune responses. <i>S. gallinarum</i> expressing HA1, HA2 and M2e antigens are immunogenic and safe. <i>Salmonella</i> has dual function of acting as a delivery system and as a natural adjuvant. Vaccine constructs elicit specific humoral and cell-mediated immune responses.