Oral vaccination with attenuated Salmonella enterica serovar Typhimurium expressing Cap protein of PCV2 and its immunogenicity in mouse and swine models

Abstract

Attenuated Salmonella enterica serovar Typhimurium (S. typhimurium) was selected as a transgenic vehicle for the development of oral vaccines against Porcine circovirus type 2 (PCV2). The Cap-encoding gene of PCV2 was amplified by PCR and cloned into expression vector pYA3341. The recombinant plasmid pYA3341-Cap was transformed into attenuated S. typhimurium X4550. BALB/c mice were inoculated orally with various doses of attenuated S. typhimurium X4550/pYA3341-Cap. The bacterium was safe to mice at dose of 2 × 10⁹ cfu and eventually eliminated in the spleen and mesenteric lymph nodes at 4 weeks post-immunization. The flow cytometry analysis showed that the percentage of CD4⁺ T cells and CD4⁺/CD8⁺ ratio were increased significantly in mice immunized with attenuated S. typhimurium X4550/pYA3341-Cap. Vaccine tests in swine showed that the oral immunization with attenuated S. typhimurium X4550/pYA3341-Cap could elicit significantly higher Cap antibody titers in the treated swine than the control groups. Virus neutralization test showed that serum from the swine treated with attenuated S. typhimurium X4550/pYA3341-Cap had significant levels of neutralization activities. The swine lymphocyte proliferative responses indicated that attenuated S. typhimurium X4550/pYA3341-Cap could induce obvious cellular immune response. An in vivo challenge study showed the swine treated with attenuated S. typhimurium X4550/pYA3341-Cap had significantly lower PCV2-associated lesions and PCV2 viremia than the control groups. The results indicated that attenuated S. typhimurium X4550/pYA3341-Cap can be a potential vaccine against PCV2 infections.

Introduction

Porcine circovirus type 2 (PCV2), a member of the Circoviridae family, is known to be associated with post-weaning multisystemic wasting syndrome (PMWS) that is an emerging swine disease in 2–3.5-month-old pigs (Allan and Ellis, 2000). Recently, PCV2-associated disease (PCVAD) has spread rapidly across North America (Horlen et al., 2008). The disease affects most swine production regions worldwide, causing devastating production and economic losses within the industry. Development of an effective vaccine against PCV2 infection has been accepted as a strategy for the prophylaxis of PCVAD. Though commercial vaccines have been used widely in controlling PCV2 infections, occasional disease caused by PCV2 still occur in many areas. Therefore, there is an urgent need to develop a more effective vaccine by different bioreactors. PCV2 is a small non-enveloped single-stranded circular DNA virus with a 1.76 kb ambience genome. The genome contains at least three major open reading frames (ORF1, ORF2, and ORF3) (Cheung and Bolin, 2002). The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies, which makes it a leading target in the design of a new generation of vaccines against PCV2 infection (Pogranichnyy et al., 2000). A recent study has demonstrated that baculovirus virions simultaneously displaying GP5 glycoprotein of PRRSV and capsid protein of PCV2 is a potential bivalent subunit vaccine against PRRSV and PCV2 infections (Xu et al., 2012).

Attenuated Salmonella strains carrying heterologous antigens offer an advantageous alternative to conventional vaccines, especially because they induce humoral, cellular, and mucosal immunization responses effectively (Bai et al., 2004). Other aspects compared with traditional vaccines, Oral vaccines are easy to administer, safe, adequate for large-scale immunization, stable without refrigeration (if lyophilized), needle-free delivery and a relatively low cost of production. The use of attenuated Salmonella strains as a vector for the delivery of heterologous antigens had already been shown to induce protective immune responses in a variety of animal models (Pasetti et al., 2003, Kotton and Hohmann, 2004). The main objective of this work was to construct a recombinant attenuated S. typhimurium X4550/pYA3341-Cap carrying PCV2 Cap protein and to study its biological property and immunogenicity. The recombinant attenuated S. typhimurium X4550/pYA3341-Cap used as a vaccine was evaluated in mouse and swine models.