Vaccine for livestock production systems

#The invention relates to a live vaccine for protection against enteric bacterial infection.

#Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

#Nontyphoidal Salmonella is the largest foodborne-disease burden in the United States, causing the most infections, hospitalizations and deaths, with 1.03 million illnesses reported annually. The economic burden associated with the disease is staggering, with the medical costs alone reaching more than $11 billion per year and substantial additional costs incurred by the food industry (recalls, litigation, reduced consumer confidence) and by state, local and federal public health agencies in response to NTS outbreaks. Globally, nontyphoidal Salmonella is estimated at 93.8 million cases and 155,000 deaths annually and has emerged as the leading cause of bacteremia in sub-Saharan Africa, where its fatality rate reaches up to 25%.

#FIG. 1. Evaluation of Salmonella dam double mutant vaccine candidates for colonization and persistence in mucosal and systemic tissues. BALB/c mice were orally immunized with S. Typhimurium UK-1 damΔ232 double mutant vaccine candidates (dam aroA [MT3138], dam htrA [MT3142], dam mgtC [MT3146], dam sifA [MT3150], dam spiC [MT3154], dam spvB [MT3158], dam ssaV [MT3162]) or the parental UK-1 damΔ232 vaccine strain (MT3134) (109 CFU). At 2 weeks (A) and 4 weeks (B) post oral immunisation, bacteria recovered from Peyer's patches (PP), mesenteric lymph nodes (MLN), liver (L) and spleen (S) were assessed for colony forming units (CFU) on LB medium. Limits of detection: PP, MLN, spleen <100 CFU; Liver <50 CFU.

#FIG. 2. Homologous strain efficacy evaluation of Salmonella dam double mutant vaccine candidates. BALB/c mice were orally immunized with S. Typhimurium UK-1 damΔ232 double mutant vaccine candidates (dam aroA [MT3138], dam htrA [MT3142], dam mgtC [MT3146], dam sifA [MT3150], dam spiC [MT3154], dam spvB [MT3158], dam ssaV [MT3162]) or the parental UK-1 damΔ232 vaccine strain (MT3134) (109 CFU). Eleven weeks post-immunization, vaccinated mice were challenged with an oral dose of 200 LD50 of homologous strain, wild-type S. Typhimurium UK-1 (χ3761). Non-vaccinated control mice all died by day 21 post-infection. Differences in the proportion of mice surviving virulent challenge was analysed using logistic regression (Genstat 15th edition [34]). Vaccination provided significant protection (P<0.01). Similar protection was afforded by the dam vaccines incorporating the secondary deletions mgtC, sifA and spvB as compared to the parent dam vaccine. A significant reduction in the efficacy of the dam vaccine was observed following introduction of the secondary deletions aroA, htrA, spiC and ssaV (** P<0.01; *** P<0.001).

#FIG. 3. Heterologous cross-protective efficacy evaluation of Salmonella dam double mutant vaccine candidates. BALB/c mice (16 to 25 per cohort) were orally immunized with S. Typhimurium UK-1 damΔ232 double mutant vaccine candidates (dam mgtC [MT3146], dam sifA [MT3150], dam spvB [MT3158]; 109 CFU). Eleven weeks post-immunization, vaccinated mice were challenged with an oral dose of 100 LD50 of heterologous Salmonella serotypes of clinical relevance to the livestock industry (S. Dublin 8895 [cattle], S. Bovismorbificans 225 [sheep], S. Typhimurium 131 [sheep]). Non-vaccinated control mice all died by day 21 post-infection. Differences in the proportion of mice surviving virulent challenge were analysed using logistic regression (GenStat 15th Edition, [34]). Vaccination with each of the dam vaccines incorporating the secondary deletions mgtC, sifA and spvB provided significant protection against the homologous and heterologous challenge strains assessed (*** P<0.001).

#As discussed above, enteric bacterial disease, for example gastroenteritis and other conditions characterised by diarrhoea, fever, and dehydration remain as major problems in livestock production. Salmonella infection, and salmonellosis are of key concern. To date all of the attempts to prevent or treat these conditions in livestock animals have met with limited success, either because of comprised or limited potency of the vaccine or comprised safety profile. Of particular concern has been vaccines that are shed from the animal and that persist in the environment.

#The challenge has been to provide an attenuated bacterium that is sufficiently robust so as to be able to persist in a livestock animal, thereby providing immunity, and that has limited potential for shedding and persistence in feces and more generally the environment, such as a feedlot or other part of the livestock production chain. The need to provide double mutation to prevent reversion to a pathogenic phenotype is an additional level of complexity, particularly where a very large number of candidate genes for inactivation are known.

#The inventors have been interested to provide enteric bacteria useful as live immunogens for example in an attenuated live vaccine, that have an improved safety profile or phenotype, insofar as having a lesser likelihood of reversion to a pathogenic phenotype, a lesser likelihood of shedding, and lesser likelihood of persistence in the environment. From an extensive list of potential candidate loci, the inventors have identified 3 loci that can be used to introduce a loss of function mutation in a dam inactivated strain to provide an attenuated microorganism that has a desirable safety profile, while retaining potency to protect against or treat a broad range of enteric bacteria, and in particular a broad range of Salmonella.