Recombinant attenuated salmonella vaccines to reduce Campylobacter colonization in poultry

Campylobacter is an important foodborne pathogen. In Europe, Campylobacteriosis is the foodborne disease most frequently identified and the second in USA, preceded by infections due to Salmonella. The CDC estimates that Campylobacter is not only among the most common causes of acquired foodborne illnesses in humans (some 1.3 million per year with over 500 deaths), but also is a leading cause of hospitalization (over 8,000 annually). Campylobacter is a commensal microorganism of the gastrointestinal tract of many wild animals (birds such as ducks and gulls), farm animals (chickens, cattle and pigs) and companion animals (such as dogs and cats) and it is responsible for zoonoses. The transmission occurs via the fecal-oral route through ingestion of contaminated food and water. The disease varies from a watery diarrhea to a severe inflammatory diarrhea with abdominal pain and fever and can be burdened by complications such as Guillain-Barré Syndrome (GBS), Reactive Arthritis (REA) and irritable bowel syndrome (IBS). Recently, many cases of Campylobacter isolated from human infections, showed resistance to various antibiotics such as tetracyclines and fluoroquinolones. The majority of human Campylobacter infections are associated with poor handling of raw chicken or consumption of undercooked chicken. There is a high prevalence of Campylobacter in both live birds and on carcasses and findings from epidemiological studies, and detection of identical genotypes in both poultry and human infections support the idea that transmission to humans in predominantly the result of the consumption of undercooked contaminated chicken. Reducing the Campylobacter load at the farm level and reducing contamination at slaughter are therefore important to prevent transmission to humans and is a high priority and it has been predicted that decreasingCampylobactercolonization of poultry by 2-log10will reduce human infections by 30-fold. Curtiss Healthcare is developing next generation live synthetic vaccines to address bacterial, viral, fungal and parasitic pathogens. Our technology is centered around the construction of engineered recombinant attenuated Salmonella vaccines (RASVs) strains with high immunogenicity, complete safety and attenuation, and inability to persist or be shed in the environment after administration. These RASVs are engineered to display regulated delayed attenuation in vivo, regulated delayed in vivo synthesis of protective antigens specified by codon-optimized DNA sequences and regulated delayed lysis in vivo. The lysis phenotype permits RASVs to deliver in lymphoid tissues a bolus of protective antigen(s) and confers complete biological containment. These cost-effective vaccines are manufactured under special conditions that enable them to display after oral administration the capabilities of a wild-type strain to survive host defense stresses and efficiently colonize effector lymphoid tissues before manifesting attenuation to preclude disease symptoms and to synthesize protein antigens to induce protective immune responses. In addition, since these vaccines are based on a Salmonella delivery platform they have the added benefit of induction of cross-protective immunity to some Salmonella serotypes which like Campylobacter are transferred via the fecal-oral route and responsible for zoonoses. RASVs stimulate mucosal, systemic and cellular immunities and can be inexpensively manufactured by fermentation with frozen or lyophilized preparation that can be reconstituted at the time and place of use. Our goal is to build on our successful preliminary work and construct new RASVs to deliver by course spray/oral immunization multiple conserved C. jejuni protective antigens to prevent infection and persistence of Campylobacter in chickens. If successful we would look to develop the most promising constructs for licensure by the USDA as part of a Phase 2 SBIR applicationTo achieve our goal of developing a vaccine for use in chickens to prevent/reduce campylobacter colonization we propose the following objectives:Objective 1: Complete cloning of the Cj0427 antigen into our two proprietary plasmid vectors and characterize the stability and inducible protein expression in our proprietary c12341 Salmonella vector (see table 1).Objective 2: Further characterization of the additional nine constructs already cloned into our two proprietary plasmid backbones (see table 1) will be performed to further optimize protein expression and where necessary obtain clones with optimized signal sequences to facilitate secretion of antigen. We expect this will result in approximately 20 plasmid constructs to be evaluated. This includes a prelminary evaluation of the constructs for their ability to be scaled-up in a suitable manufacturing process.Objective 3: We have identified antigens that are conserved among Campylobacter jejuni isolates and can be expressed stably in our RASV system (see table 1). We now aim to evaluate these selected antigens in our c12341 Salmonella vector backbone and assess their ability to generate an immune response in chickens. Following vaccination with the selected vaccine candidates (see table 1) we will evaluate the sera and mucosal tissue from chickens for antibody responses against the vaccinated antigens. Constructs with the best responses will then be evaluated in combination to look for interference.Objective 4: Based on the results from the objectives listed above we will evaluate the two most promising combinations of antigens in a model of Campylobacter colonization in chickens. The results from these studies will be used to select one of the combinations for further development.Table 1. Campylobacter antigens to be used in this proposal.AntigenGeneFunctionSignalPeptide Stable Protein ExpressionConserved*ReferencePYA3764PG8R17Omp18Cj0113peptidoglycan associated lipoproteinYesYesYesYesUS Patent 9328148 B2Peb1Cj0921cbifunctional adhesin/ABC transporter aspartate /glutamate-binding proteinYesYesYesYesUS Patent 9328148 B2DpsCj1534cDNA starvation/stationary phase protection protein, putative bacterioferritinNoYesNoYesUS Patent 9328148 B2Cj0998cCj0998chypothetical proteinYesYesNoYesUS Patent 9328148 B2CjaACj0982csurface antigen/glutamine ABC transporter substrate-binding protein YesYesYesYesUS Patent 9328148 B2Cj0427Cj0427hypothetical proteinYesTo bemadeTo bemadeYesUS Patent 9328148 B2Peb3Cj0289cmajor antigenic peptide PEB3YesYesYesYes(32)TlyACj0588putative hemolysinNoYesNoYesUS Patent 9328148 B2CadFCj1478couter membrane fibronectin-binding proteinYesYesYesYes(33)FlaACj1339cflagellin A (FlaA)YesYesNoYesUS Patent 9328148 B2*Indicates >95% identify with a consensus sequence constructed using amino acid sequences for the indicated protein fromover 10,000 isolates used in our analysis.