Delivery of recombinant vaccine antigens by live bacterial vectors and subunit vaccination against necrotic enteritis in chickens
Creators: name: Manohar, Megha Mano
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300304
subject: Animal protection (incl. pests and pathogens)
classificationCode: 300903
subject: Veterinary bacteriology

description: The global poultry industry forms an integral part in supplying sustainable protein sources to the growing population. Broiler chickens are susceptible to various pathogens (bacterial, viral, fungal, parasite) which leads to production losses to the industry. To protect the birds from pathogens alternative means other than antibiotics needs to be considered to prevent the development of antibiotic resistance strains. Consumers’ perspectives on the quality and safety of animal products have also increased the pressure on poultry industry to provide alternative means to protect the birds from pathogens and improve their quality of life. Vaccines are very effective in providing protection against many infectious diseases. However, it has proven difficult to develop highly efficacious vaccines against some pathogens and so there is a continuing need to improve vaccine technologies. The first successful and widely used vaccines were based on attenuated pathogens (e.g., laboratory passaged Pasteurella multocida to vaccinate against fowl cholera) or closely related non-pathogenic organisms (e.g., cowpox to vaccinate against smallpox). Subsequently, live vaccines, either attenuated pathogens or non-pathogenic microorganisms modified to deliver heterologous antigens, have been successfully used to induce protective immune responses against many pathogens. Unlike conventional killed and subunit vaccines, live vaccines can deliver antigens to mucosal surfaces in a similar manner and context as the natural infection and hence can often produce a more appropriate and protective immune response. Despite these advantages, there is still a need to improve the immunogenicity of some live vaccines. The efficacy of injectable killed and subunit vaccines is usually enhanced using adjuvants such mineral salts, oils, and saponin, but such adjuvants cannot be used with live vaccines. Instead, live vaccines can be engineered to produce immunomodulatory molecules that can stimulate the immune system to induce more robust and long-lasting adaptive immune responses. One of the infectious diseases encountered by the poultry industry is Necrotic enteritis (NE), caused by Clostridium perfringens, a debilitating disease that leads to poor quality of life in broiler chickens and results in production losses to the poultry industry. Traditionally, antibiotics have been used to control NE in flocks. However, because of concerns about antibiotic resistant strains, antibiotic residues, and restricted use in some regions, alternative methods to control this disease are required. In previous studies, NetB, the key virulence factor, and alpha-toxin have been used individually as subunit vaccines with only partial protection induced. It appears that a single subunit antigen is insufficient to produce high levels of protection. Here, an experimental multi-antigen vaccine incorporating four antigens has been designed and tested. Live vaccination to deliver NetB and immune modulating protein adjuvant chicken interleukin-6 using different live bacterial vectors was a part of this experimental plan. Three strains of live bacterial vaccine candidates (Salmonella enterica serovar Typhimurium STM1, Salmonella enterica subsp. salamae serovar Sofia 76, Escherichia coli Pers1), confirmed to express recombinant NetB (rNetB), were used to orally vaccinate the birds thrice and birds were then challenged in-feed using pathogenic strain of C. perfringens. For the subunit vaccine approach, to simplify the production and delivery of multiple recombinant antigens, a multi-valent (MV) fusion protein was designed. The MV-protein vaccine and each of the individual proteins, were tested as vaccine candidates in a necrotic enteritis challenge model. Birds were vaccinated twice then challenged with a pathogenic strain of C. perfringens. The development of necrotic lesions was scored, antibody responses were assessed, and FACS analysis of gut and spleen samples were performed. There was no significant reduction in NE lesions seen in birds orally vaccinated with live vaccine candidates expressing rNetB and further work to improve the live vaccine such as MV-protein expression and augmenting immune response using immune modulating adjuvant is required.   Birds vaccinated with the MV-protein showed a 95% reduction in lesions compared to unvaccinated birds. Western blot and ELISA analysis showed that vaccination with MV-protein induced antibodies specific for all four target protein fragments within the MV-protein. The MV-protein vaccine induced specific antibody responses and provided significant protection against the formation of necrotic lesions and provides the basis for ongoing vaccine development.