NZ729153B2 - Vaccine for livestock production systems - Google Patents

Abstract

The invention relates to Salmonella enterica subsp. enterica strains that possess a mutation in the dam and a second mutation in at least one of sifA, spvB and mgtC that leads to loss of gene function. The combined loss of gene function provides an improved Salmonella vaccine.

Description

Vaccine for ock production s Field of the invention The invention relates to a live vaccine for protection against enteric bacterial infection.

Background of the invention Reference to any prior art in the ication 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, ed as relevant, and/or combined with other pieces of prior art by a skilled person in the art. hoidal Salmonella is the t 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 ring, 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 0 deaths annually and has emerged as the leading cause of bacteremia in sub-Saharan Africa, where its fatality rate reaches up to 25%.

The health and economic burden ated with Salmonella is poised to worsen as the prolonged administration of antibiotics has resulted in the emergence of multidrug-resistant strains that have disseminated worldwide; e.g., S. Typhimurium DT104 has caused several food-borne disease outbreaks over the last two decades and is resistant to four of the five most commonly used otics in veterinary medicine (tetracycline, B-lactams, aminoglycosides, and sulfonamides). These multidrug-resistant strains are oftentimes associated with more alizations and bacteremia, and their maintenance in nature can occur at very low antibiotic concentrations that are commonly found in the environment including ground water. Further, a new class of carbapenem-resistant Enterobacteriaceae that are resistant to B-lactams, quinolones, and aminoglycosides was isolated from a patient in 2009, and such resistance has now shown widespread distribution among egative pathogens ing Salmonella. Additionally, ‘hypervirulent’ ella have been recently isolated (2012) from natural ial populations derived from livestock. These hypervirulent strains are 100-times more virulent then most clinical isolates, are more capable of killing vaccinated animals, and are not detectable under standard laboratory test conditions due to rapid switching to a irulent state ex vivo. Together, these findings support the view that the Salmonella disease burden is poised to worsen with the potential emergence of more virulent multidrug-resistant strains that are difficult to control with currently available antibiotics.

Salmonella enterica is acquired via the fecal-oral route and is comprised of six subspecies that are subdivided into more than 2500 serovars (serological variants) based on carbohydrate, lipopolysaccharide (LPS), and flagellar composition, with cies enterica containing more than 99% of human enic isolates. S. enterica infection can result in any of four distinct disease syndromes: enterocolitis/diarrhea, bacteremia, enteric (typhoid) fever and chronic asymptomatic carriage. Many serovars infect both humans and animals, and disease severity is a function of the serovar, strain virulence and host tibility.

Salmonella control efforts in livestock continue to be problematic for the following reasons: 1) most livestock infections are subclinical; 2) disease outbreaks are sporadic and frequently caused by specific serotypes although many serotypes are endemic to livestock tion systems; 3) environmental persistence provides an ongoing reservoir for livestock infection; 4) the recent emergence of strain variants that are more virulent and can kill vaccinated animals; 5) some strains derived from human salmonellosis ts are distinct from those of animal ; and 6) management and environmental events can increase pathogen re and/or compromise host Vaccination represents a sustainable approach to any food safety plan, reducing pathogen exposure at the outset of the food production chain [1]. However, the immunity conferred by conventional vaccines is restricted to a narrow range of closely- related strains, and on-farm control requires the development of vaccines that elicit protection against many pathogenic serotypes [1]. Recent advancements have ed in the development of modified live Salmonella cross-protective vaccines, many of which contain mutations in global regulatory networks that favor antigen production; and that are also le for the expression of heterologous antigens [2-6]. The molecular basis of cross-protective vaccine efficacy is not entirely clear. Relevant ters might include: the expression of multiple antigens shared among pathogenic serotypes; diminished vaccine-induced immunosuppression; ed removal of immunodominant antigens to expose cross-protective epitopes; type III secretion of recombinant antigens; and/or delayed vaccine attenuation for enhanced stimulation of immune responses (reviewed in [1, 3, 7, 8]).

Modified live attenuated S. enterica r Typhimurium that harbor loss of function ons in genes may be useful for providing protection against a diversity of salmonella. The number of loci that might be considered for providing an able loss of function on is large, as is the number of applicable mutations at each locus.

Some examples of loci for providing loss of function include loci involved in adherence, invasion, and intra- and ellular survival of the bacteria ding many genes encoding proteins involved in metabolic processes). Some mutations of the gene encoding the DNA adenine methylase (dam) are capable of eliciting protection against a diversity of salmonellae. These appear to be well tolerated when applied as modified live vaccines in mice [2, 9], poultry [10, 11], sheep [12] and calves [13-15]. Induction of immunity is rapid and the vaccine can be administered with delivery via drinking water for low cost and low-stress immunization of livestock populations [12, 16].

The commercial success of any vaccine is dependent on the therapeutic index, the ratio of safety/toxicity, and safety is of particular concern for modified live vaccines that have the ial to revert to heightened virulence. Generally, a vaccine should y 4 safety categories to be considered as a candidate for commercial use in a livestock production system. The relevant safety phenotypes are as s: reduced i) vaccine shedding; ii) challenge strain shedding; iii) persistence in systemic tissues (liver/spleen); and iv) persistence in the environment.

It is understood that in ing an attenuated strain ning loss of function mutations, it is important that the improved safety profile arising from the relevant mutations does not decrease the efficacy of the vaccine in terms of the protection that it provides. Ultimately what one is looking for is a mutation that does not decrease the persistence of low level infection in the immunized individual and that does not increase the persistence of the immunogen in the environment when the pathogen is shed from the immunized animal and released to the environment.

It is difficult to predict which loss of function mutations are more useful for attenuation than others, ularly given that the data on potency and reversion to pathogenicity relevant to each locus and on arises from different laboratory systems.

A further complication is that it is desirable to select more than one locus for on, so as to prevent reversion to a pathogenic phenotype should at least one loss of function mutation be lost. Such an approach requires one to combine at least 2 loci from a large list of ate loci and yet with limited guidance as to whether a particular combination is likely to increase or se the likelihood of reversion, or likely to increase or decrease the potency of the resulting attenuated vaccine.

There is a need for an attenuated live vaccine for protection t Salmonella infection.

There is also a need for an attenuated live vaccine for protection against Salmonella infection that has an improved safety ype.

There is a need for an attenuated live vaccine that has limited or no propensity for shedding from an animal, that has minimal persistence in the nment when shed from an animal, and that retains an able level of persistence in an animal to invoke immunity.

Summary of the invention The ion seeks to improve or address one or more of the above mentioned problems, limitations or needs and in one embodiment provides an enteric bacterium including: - a first loss of function on in a gene encoding DNA adenine methylase (herein dam) and - a second loss of function mutation in a gene selected from the group consisting of: sifA, spo and mgtC.

In a further embodiment there is provided a vaccine including: - an enteric bacterium as described above; and - a carrier, t, excipient or adjuvant.

In a further embodiment there is ed a method of preventing or treating a bacterial enteric disease or condition including the step of providing a e as described above to an individual in which a bacterial enteric disease or condition is to be prevented or treated.

In a further embodiment there is provided a method for producing a collection of immunogens suitable for use in a vaccine for preventing or treating a bacterial enteric e or condition including: - providing an enteric bacterium having a loss of function mutation in dam, - introducing a loss of function mutation into a gene of the bacterium selected from the group consisting of: sifA, spo and mgtC.

In a further embodiment there is provided a method for ing a collection of immunogens suitable for use in a e for preventing or treating a ial c disease or condition including: - providing an enteric bacterium having a loss of function mutation selected from the group consisting of: sifA, spo and mgtC, - introducing a loss of function mutation into the dam gene of the bacterium.

Brief description of the drawings Figure 1. Evaluation of Salmonella dam double mutant vaccine candidates for colonization and tence in mucosal and systemic tissues. BALB/c mice were orally immunized with S. Typhimurium UK-1 damA232 double mutant vaccine candidates (dam aroA [MT3138], dam htrA 2], dam mgtC [MT3146], dam sifA [MT3150], dam spiC [MT3154], dam spo [MT3158], dam ssaV [MT3162]) or the parental UK-1 damA232 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 ed for colony forming units (CFU) on LB medium. Limits of detection: PP, MLN, spleen < 100 CFU; Liver < 50 CFU.

Figure 2. gous strain efficacy evaluation of Salmonella dam double mutant vaccine candidates. BALB/c mice were orally immunized with S. Typhimurium UK-1 damA232 double mutant vaccine candidates (dam aroA [MT3138], dam htrA [MT3142], dam mgtC [MT3146], dam sifA 0], dam spiC [MT3154], dam spo [MT3158], dam ssaV[MT3162]) or the parental UK-1 damA232 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 (x3761).

Non-vaccinated control mice all died by day 21 nfection. Differences in the tion of mice surviving virulent challenge was analysed using logistic regression (Genstat 15th edition [34]). Vaccination provided significant tion (P< 0.01). Similar tion was afforded by the dam vaccines incorporating the secondary deletions mgtC, sifA and spo as compared to the parent dam vaccine. A significant reduction in the cy of the dam vaccine was observed following introduction of the secondary deletions aroA, htrA, spiC and ssaV(** P < 0.01 *** P ; < 0.001 ).

Figure 3. Heterologous cross-protective efficacy tion of Salmonella dam double mutant vaccine candidates. BALB/c mice (16 to 25 per cohort) were orally immunized with S. urium UK-1 damA232 double mutant vaccine candidates (dam mgtC [MT3146], dam sifA [MT3150], dam spo [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 ry (8. 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 tion 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 spo provided significant WO 33532 protection against the homologous and heterologous challenge strains assessed (*** P < 0.001).

Figure 4. Vaccine safety tion (reversion to 2-AP resistance) among Salmonella dam double mutant vaccine candidates. BALB/c mice were intraperitoneally infected with 105 CFU of S. Typhimurium UK-1 damA232 double mutant vaccine candidates (dam mgtC [MT3146], dam sifA 0], dam spo [MT3158]) or the dam UK-1 parent vaccine strain [MT3134]. The number of 2—AP sensitive (open boxes) or 2—AP resistant (closed boxes) Salmonella organisms in the spleen (A) or liver (B) was enumerated at day 5 post-infection. The symbols below the zero CFU value represent the number of mice in which the ial load in spleen and liver was below the limit of detection (< 25 CFU). Statistical significance for S.

Typhimurium UK-1 dam double mutant vaccine persistence (2—APS) and reversion to heightened virulence (2—APr) in comparison to the parental Salmonella damA232 e strain was ined using analysis of variance (* P < 0.05).

Figure 5. Vaccine fecal shedding evaluation of Salmonella dam double mutant vaccine ates. Kanamycin-resistant derivatives of S. Typhimurium UK-1 damA232 double mutant vaccine candidates, dam mgtC (MT3183), dam sifA (MT3184), dam spo (MT3186), and the dam UK-1 parent strain (MT3180), were used to vaccinate BALB/c mice by the oral route (109 CFU). Feces was collected from dual mice and plated for CFU/g on kanamycin 50 ug/ml LB plates on days 2, 4, 7, 11, 14, and 21 post- immunization. Fecal shedding of the Salmonella dam double deletion vaccine candidates vs. the parental Salmonella damA232 vaccine strain was analysed using REML repeated measures analysis. Both vaccine and time following ation were icant (* P < 0.05). No significant differences in fecal shedding were observed n the different double deletion dam vaccines. Values given are the model predicted mean number of CFU/g in feces of mice following ation. Limit of detection is 60 CFU.

Figure 6. Challenge strain fecal shedding in mice immunized with Salmonella dam double mutant vaccine candidates. Kanamycin-resistant derivatives of S. Typhimurium UK-1 damA232 double mutant vaccine candidates, dam mgtC (MT3183), dam sifA (MT3184), dam spo (MT3186), and the dam UK-1 parent strain (MT3180), were used to vaccinate BALB/c mice by the oral route (109 CFU). Vaccine strain fecal clearance was achieved four weeks post-immunization. Eleven weeks post- immunization, ated mice were challenged with a dose of 100 LD50 of a kanamycin-resistant derivative of wild-type S. Typhimurium UK-1 (MT2315;107 CFU).

Feces was collected from individual mice and plated for CFU/g on kanamycin 50 ug/ml LB plates on days 2, 4, 7, 11, 14, and 21 post-immunization. Fecal ng of the pe challenge strain following challenge of vaccinated mice was analysed using REML repeated measures analysis. Both vaccine and time following vaccination were significant (* P < 0.05) and there was a trend for significant interaction between time 1O and vaccine (P = 0.075). Pairwise comparisons revealed significant differences between groups at different times ing virulent challenge; a = shedding of double deletion vaccines was significantly less than shedding of the parental dam vaccine; b = shedding of dam sifA and dam spo vaccines was less than shedding of the parental dam vaccine and dam sifA shedding was significantly less than shedding of dam mgtC and dam spo vaccines; c = ng of dam sifA and dam spo vaccines was significantly less than shedding of dam mgtC and dam spo vaccines and shedding ofd dam spo vaccine was significantly less than shedding of dam mgtC vaccine; d = shedding of dam sifA, dam spo and dam mgtC vaccines was significantly less than shedding of dam spo vaccine. Values given are model predicted mean CFU of wildtype challenge strain in feces following challenge. Limit of detection is 60 CFU.

Figure 7. Environmental vaccine persistence (deionized water) tion of Salmonella dam double mutant e candidates. Kanamycin-resistant tives of S. Typhimurium UK-1 damA232 double mutant vaccine candidates, dam mgtC (MT3183), dam sifA (MT3184), dam spo (MT3186), and the dam UK-1 parent strain (MT3180) were used to inoculate 20 ml of deionized water (104 CFU/ml).

Triplicate assays were med in 50 ml l tubes with loose caps at room temperature. Samples were vortexed and plated for CFU/ml for a two week period at the time points indicated. Values given are the average CFU/ml with error bars indicating : standard error of the mean (SEM). The number of CFU/ml present in water over time was ed using REML repeated measures analysis. A significant ction between vaccine group and time was observed (P < 0.001). PainNise isons revealed significant differences between groups at different times ( P < 0.05). a = all vaccines have lower CFU/ml than the parent UK-1 wild type ; b = the CFU/ml for the dam A232 strain is significantly less than the CFU/ml for the dam mgtC, dam sifA and dam spo strains; c = CFU/ml for the dam A232 and dam spo strains were significantly less than for the dam sifA and dam mgtC strains.

Figure 8. nmental vaccine persistence (sheep feces) evaluation of Salmonella dam double mutant vaccine candidates. Twenty per cent fecal dry matter was ted by adding 20 ml of deionized water to 5 g of dried sheep feces (gift from Barbara Byrne, University of California, Davis; [32,33]). The 20% fecal dry matter was inoculated (104 CFU/ml) with kanamycin-resistant derivatives of S.

Typhimurium UK-1 damA232 double mutant vaccine ates, dam mgtC (MT3183), dam sifA (MT3184), dam spo (MT3186), or the dam UK-1 parent strain 0).

Triplicate assays were performed in 50 ml conical tubes with loose caps at room temperature. Samples were vortexed and plated for CFU/ml for a two week period at the time points indicated. Values given are the average CFU/ml with error bars indicating : standard error of the mean (SEM). The number of CFU/ml present in feces over time was analysed using REML ed measures analysis. A significant interaction between vaccine group and time was observed (P < 0.001). PainNise comparisons revealed significant ences between groups at different times (P < 0.05). CFU/ml for all e strains were less than for the parent UK-1 wild type for all time points except for day 1 and 6. a = dam sifA significantly less than dam, dam mgtC and dam spo; b = dam sifA significantly less than dam and dam mgtC; c = dam sifA significantly less than dam mgtC; d = dam spo significantly less than dam mgtC; e = dam spo significantly less than dam; f = dam mgtC significantly less than dam.

Detailed ption of the embodiments 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 t 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 ing 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 ock 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 vation are known.

The ors 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 hood 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 on 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.

A. Definitions ‘Loss of function on’ generally refers to a mutation of a gene that tely or partially inactivates a relevant function of the gene in a given biological process. Particular loss of function mutations of interest are those that interrupt the cle of enteric bacteria in a host, while not disrupting the immunogenic profile of the bacteria.

‘Enteric bacteria’ generally refers to bacteria of the intestines or gut. Of particular interest are the obacteriaceae’, a large family of Gram-negative enteric bacteria that includes pathogens, such as Salmonella, Escherichia coli, Yersinia , K/ebsie/la and Shige/la. Other disease-causing bacteria in this family include s, Enterobacter, Serratia, and Citrobacter.

‘Salmone/la’ is an enteric bacteria of the Enterobacteriaceae. ‘dam’ refers to the gene encoding DNA adenine methylase, also known as deoxyadenosine ase, DNA adenine methyltransferase or deoxyadenosyl transferase. An example of an accession number for the S. urium dam gene is NCBI accession number: 1255007. The locus tag for this gene is STM3484. ‘sifA’ refers to the gene encoding the secreted effector protein SifA. An example of an accession number for the S. urium sifA gene is NCBI accession number 1252742. The locus tag for this gene is STM 1224. ‘spo’ refers to the gene encoding the Salmonella plasmid virulence protein B (Spo). An example of an ion number for the S. Typhimurium spo gene is NCBI accession number 1256199. The locus tag for this gene is PSLT039. ‘mgtC’ refers to the gene encoding Mg(2+) transport ATPase protein C, MgtC. An 1O example of an accession number for the S. urium mgtC gene is NCBI accession number 1255288. The locus tag for this gene is STM3764. ‘attenuated’ for example, in "attenuated bacteria" generally refers to a modification of a bacterium that reduces the virulence of the bacterium, but still keeps it viable (or "live") so that it can replicate, albeit at a slower rate or under different conditions. Attenuation takes an ious agent and alters it so that it becomes ss or less virulent. Typically, attenuation does not substantially decrease the immunogenicity of the relevant bacteria. ‘vaccine’ generally refers to a composition that contains an immunogen Le. a substance capable of invoking an immune response. Typically a vaccine is useful for sing, preventing or providing protection against ion, or manifestation of a relevant symptom, on exposure to a pathogen, particularly where the exposure is in the form of challenge. A vaccine may be used for prevention or for treatment of a condition.

A e may be used to minimise the likelihood of infection with a pathogen. ‘bacteria/ enteric disease or condition’ generally refers to a condition arising from the ion of an individual with enteric bacteria. Such a condition may include the following symptoms: gastric inflammation, dehydration, diarrhoea, fever. Salmonellosis is one example of a bacterial enteric disease or condition. ‘immunisation’as used herein, generally refers to a process by which a subject’s immune system is fortified against an immunogen. The attenuated Salmonella microorganisms of the invention have utility in immunising a t against Salmonellaw and thereby prevent infection with other, more virulent Salmonella serovars. ‘gene’ as used herein, refers to the coding sequence and its regulatory sequences such as promoter and termination signals. ‘comprise’ and variations of the term, such as ising’, ises’ and ‘comprised’, are not ed to exclude further additives, components, integers or steps.

The present inventors have found that specific combinations of loss of function mutations in Salmonella genes provide a particular advantage in the generation of live attenuated strains of Salmonella which have utility as live vaccines for conferring immunity from infection with virulent or pathogenic serotypes of Salmonella.

Specifically, the inventors have found that the introduction of mutations in the sifA, spo or mgtC genes in a strain of Salmonella also having a loss of function mutation in the dam gene, s in the generation of rganisms which can be safely administered to subjects, are safe in the environment and maintain the capacity to confer protection to heterologous pathogenic serotypes of ella.

The present invention thus es a live attenuated Salmonella rganism, wherein said microorganism comprises a loss of function mutation in the dam gene and at least one r loss of function mutation in a gene selected from the group consisting of: sifA, spo and mgtC.

In a ularly preferred embodiment, the microorganism according to the invention has a loss of function mutation in dam and a further loss of function mutation in sifA. In this embodiment, the microorganism or enteric bacteria may not have a loss of on mutation in spo or mgtC.

The attenuated Salmonella microorganisms of the present invention can be prepared by known techniques, e.g, by deletion mutagenesis, insertional inactivation or substitution of one or more nucleotides in the target genes. The skilled person will appreciate that the target genes do not necessarily need to be mutated, provided that the expression of the native gene product is in some way disrupted. For example, the mutation may be made upstream of the target gene, for e in a er or regulatory region.

In one embodiment, the loss-of-function ons engineered into dam, sifA, mgtC and spo genes are in-frame deletions. The use of in-frame deletions is such that the transcription of downstream genes is maintained.

Other suitable techniques include the use of a suicide vector comprising a d gene and a selective marker. The suicide vector is introduced into the Salmonella rganism carrying the wild-type gene sequence (although, as the skilled person will appreciate, may comprise one or more mutations at alternative loci) by conjugation. The wild-type gene is ed with the mutated gene via homologous recombination, and the mutated microorganism is identified using the selective marker.

Other suitable techniques are bed, for example, in The skilled person will also be able to readily determine whether the introduced mutation has resulted in a loss of function or if gene function is impaired. For example, the mgtC gene is required for survival of Salmonella in environments having low magnesium concentration.

The loss of function mutations introduced into the dam gene and any one of the sifA, spo or mgtC genes are effective for resulting in attenuation of the microorganism.

Preferably the microorganism is an enteric bacterium, and in particular a pathogenic enteric bacterium, such as a member of Enterobacteriacea.

Most preferably the microorganism is Salmonella. It will be appreciated by the skilled person, that any number of Salmonella serotypes which are normally virulent or enic, can be treated using the above techniques to generate live attenuated strains. For example, the Salmonella microorganisms may be from a wide variety of Salmonella enterica subsp. Enterica serovars, including, but not limited to serovars S.

Typhimurium, S. Enteritidis, S. , S. t, S. Choleraesuis, or S.

Bovismorbificans. In a particularly red embodiment, the loss of function mutations are introduced into an S. urium microorganism.

In yet a r red embodiment, the attenuated live rganism is an S.

Typhimurium having loss of function mutations in both dam and sifA genes.

The inventors have found that the microorganisms of the present invention are particularly suitable for use as vaccines for sing ts against virulent serotypes of Salmonella and minimising the likelihood of infection with virulent serotypes. In particular, the inventors have found that compared with ella having loss of function mutations in other combinations of genes, Salmonella having mutations in the dam gene as well as in any of the sifA, spo or mgtC genes, exhibited improved vaccine safety in the subject to be immunised and in the environment.

Thus, in a further aspect, the present invention provides a vaccine composition for inducing an immune se in a subject to an enteric bacteria, preferably a pathogenic bacteria such as Salmonella. The vaccine composition comprises a live attenuated Salmonella microorganism in an amount sufficient to elicit an immune response in the subject and a le carrier or diluent, wherein said live attenuated microorganism comprises a loss of on mutation in the dam gene and at least one further loss of function mutation in a gene selected from the group consisting of: sifA, spo and mgtC.

In a ularly preferred embodiment, the vaccine composition comprises an amount of a live attenuated ella comprising loss of function mutations in both the dam and sifA genes.

To formulate the vaccine compositions, the attenuated microorganisms may be present in the composition together with any suitable excipient. For e, the compositions may comprise any suitable adjuvant. Furthermore, the compositions may be d for a variety of means of administration. Preferred administration routes include the oral, mucosal (e.g., nasal) or systemic routes (e.g. parenteral injection) and the vaccines are live attenuated Salmonella microorganisms. In one particular embodiment, the vaccine compositions can be provided for inclusion in the drinking water or food or feedlot of the subject to which it is to be delivered.

The number of attenuated microorganisms present in the vaccine compositions can readily be determined by the skilled person, depending on the intended route of administration of the vaccine composition and the subject to which it will ultimately be delivered.

The particular suitable carriers or diluents employed in the vaccine compositions are not critical to the present invention and are conventional in the art. Examples of diluents include: buffer for buffering against gastric acid in the stomach, such as e buffer (pH 7.0) containing e, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose and optionally aspartame. Examples of carriers e: proteins, e.g., as found in skimmed milk; sugars, e.g., sucrose; or polyvinylpyrrolidone.

The present inventors have found that administration of the attenuated microorganisms of the present invention or vaccine compositions sing the same to a t, confers ance in that subject to subsequent infection with a wild-type or pathogenic serovar.

Accordingly, in yet a further aspect, the present invention provides a method of preventing infection with a virulent strain of Salmonella, said method comprising: - stering to a subject in need thereof: - an amount of a live attenuated Salmonella microorganism, wherein said microorganism comprises a loss of function mutation in the dam gene and at least one further loss of function mutation in a gene selected from the group consisting of: sifA, spo and mgtC, or - a vaccine composition comprising a live attenuated ella microorganism and a suitable carrier or diluent, wherein said live attenuated microorganism comprises a loss of function mutation in the dam gene and at least one further loss of on mutation in a gene ed from the group consisting of: sifA, spo and mgtC. - wherein the amount of microorganism or e administered is sufficient to elicit an immune response in the subject.

WO 33532 It will be appreciated that in preventing infection with a virulent strain of Salmonella, the present invention also es a method of immunising a t against infection with a virulent serovar of Salmonella.

The attenuated microorganisms of the invention and vaccine compositions comprising the same are suitable for immunising subjects against infection with virulent and pathogenic serovars of Salmonella which normally result in salmonellosis. The attenuated microorganisms of the invention and vaccine compositions sing the same are particularly suitable for immunising any animal which is susceptible to infection with Salmonella rganisms. For example, in some embodiments, the subjects which can be immunised may be humans. Alternatively, the subjects to be immunised may be veterinary species and livestock. Examples of subjects to be immunised in accordance with the t invention include pigs, sheep, calves, cattle, deer, goats, , horses, n, , ducks, quails etc.

The amount or number of attenuated ella microorganisms or vaccine can readily be determined by the skilled person. In general, about 10‘2 cfu to about 101° cfu, ably about 105 to about 101° cfu of microorganism is administered. An immunising dose varies according to the route of administration. The skilled person will appreciate that the effective dose for a vaccine administered parenterally (for example, by intravenous, intraperitoneal or subcutaneous injection) is likely to be smaller than a similar vaccine which is administered orally, for example in drinking water or in food.

By an ‘immunising amount’ as used herein, is meant an amount that is able to induce a protective immune response in the subject that receives the attenuated microorganism or vaccine comprising the same. The immune response may be a humoral, mucosal, local and/or cellular immune response. Further, as the skilled person will appreciate, the amount of attenuated microorganism or e required will also depend on age, weight and other factors relating to the subject being immunised.

The d person will appreciate that in order to produce sufficient numbers of the live attenuated microorganism described herein, it may be necessary to culture the microorganism in suitable conditions. For example, depending on the intended route of stration of the microorganism, it may be necessary to culture the microorganism under aerobic or anaerobic conditions. The skilled person will be readily be able to determine the relevant culturing conditions. Furthermore, it may be desirable, once ient numbers of the microorganism have been produced in culture (for example, once the microorganism has reach log-phase growth), to purify the culture to remove any elements of the growth medium which are not intended for inclusion in downstream use of the microorganism.

Accordingly, in one embodiment, the present invention provides a purified culture of a live ated Salmonella microorganism as described above.

The culture comprising the live attenuated Salmonella microorganism may be purified so that it may be used in downstream applications including for use as a vaccine or in the manufacture of a vaccine composition to induce an immune response in a subject to a Salmonella microorganism.

It will be appreciated that the purified e may be freeze dried, frozen or reconstituted, depending on the intended downstream application of the culture. r aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual es mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. All patents, patent applications, and publications cited in this ication are herein incorporated by reference in their entirety to the same extent as if each ndent patent application, or publication was specifically and individually indicated to be incorporated by reference.

EXAMPLES 1. als and Methods 1.1. Bacterial s and growth conditions Salmonella animal isolates were derived from ent outbreaks, individual cases, or surveillance submissions to stic laboratories [31]. Virulent S.

Typhimurium UK-1 was used in all studies for comparison [17]. Unless otherwise specified, bacteria were derived from stationary phase cultures aerated at 37°C containing Luria-Bertani (LB) medium [18]. Antibiotics were used at the following trations: kanamycin (Kn), 50 ug/ml, ampicillin (Ap), 50 ug/ml. 1.2. Construction of S. Typhimurium dam vaccine candidates comprising an additional attenuating mutation S. Typhimurium UK-1 Adam was constructed by introducing an in-frame 300 bp deletion of defined dam sequence, termed 2 [19], using standard genetic protocols [20]. The resultant S. Typhimurium UK-1 damA232 strain (MT3134) was 1O shown to be ive to the purine analog, 2-aminopurine , which is toxic to strains lacking a non-functional DNA adenine methylase [21, 22], and was used as the parental Salmonella dam vaccine strain for all studies. Secondary virulence-attenuating on mutations were introduced into the parental S. urium UK-1 damA232 strain utilizing suicide vector pCVD442 as described [20], resulting in the construction of in-frame deletions of defined coding sequence in the following targeted genes: dam aroA (MT3138; 1056 bp deletion); dam htrA (MT3142; 1341 bp deletion); dam mgtC (MT3146; 606 bp deletion); dam sifA (MT3150; 807 bp deletion); dam spiC 4; 306 bp on); dam spo (MT3158; 1563 bp on); and dam ssaV(MT3162; 1959 bp deletion). The resultant genetic ucts were confirmed by PCR using primers that flank the deleted sequences. 1.3. Virulence and protection assays Oral and lntraperitoneal Lethal Doseso (LD50): The dose required to kill 50% of infected animals was determined via the oral (via gastrointubation) and intraperitoneal (i.p.) routes by infecting at least 10 mice [30, 19]. Salmonella test strains and wild-type S. Typhimurium reference strain 14028 were grown overnight in LB medium. Bacterial cells ended in 0.2 ml of 0.2M 4 pH 8.1 or 0.1 ml of 0.15M NaCl (for oral and i.p. administration, respectively) were used to infect mice, which were examined daily for morbidity and mortality up to 3 weeks post infection. The oral and i.p. LD50 for S. Typhimurium UK-1 is 105 and < 10 organisms, respectively [30]. Six- to- eight week old BALB/c mice were used in all nce studies. Protection assays. Mice were orally immunized with S. Typhimurium dam vaccine strains at a dose of 109 CFU [30, 19]. To WO 33532 avoid transient, non-specific cross-protective immune ses attributed to the persistence of the vaccine strain within host s [23-25], immunized mice were not challenged with virulent Salmonella until 4 to 5 weeks after the vaccine strain was cleared from mucosal (Peyer’s s; mesenteric lymph nodes) and systemic tissues (liver; spleen) of immunized animals. Eleven weeks post-immunization, mice were orally challenged with nt Salmonella enterica serotypes at an infection dose equivalent to 100- to 200- fold LD50. Mice were examined daily following challenge for morbidity and mortality for up to 3 weeks hallenge. 1.4. Construction of antibiotic resistant derivatives of Salmonella vaccine candidates to assess e and nge strain fecal shedding, and persistence within deionized water and sheep feces Kanamycin resistant (Knr) derivatives of S. Typhimurium UK-1 damA232 double mutant vaccine candidates were ucted to assess vaccine fecal shedding. S.

Typhimurium strain MT2057 is a Knr derivative of wild-type reference strain 14028, containing a Lac+ MudJ transcriptional fusion encoding Knr which is used to discern it from other ella that are inherently Lac' [19, 26]. Phage P22 grown on donor strain MT2057 was used to transduce recipient Salmonella dam vaccine ates to kanamycin resistance [18], generating Knr S. Typhimurium UK-1 damzi232 double mutant vaccine candidates, dam mgtC (MT3183), dam sifA (MT3184), dam spo (MT3186), and the dam UK-1 parent strain (MT3180). Vaccine strain ng. BALB/c mice were vaccinated with Knr S. Typhimurium UK-1 dam double mutant vaccine candidates by the oral route (109 CFU). Feces was collected from individual mice and plated for CFU /g on kanamycin 50 ug/ml LB plates on Days 2, 4, 7, 11, 14, and 21 post-immunization. Challenge strain shedding. BALB/c mice were vaccinated with Knr S. Typhimurium UK-1 dam double mutant vaccine candidates by the oral route (109 CFU). Vaccine strain fecal clearance occurred by four weeks post immunization. Eleven weeks post- immunization, vaccinated mice were challenged with a dose of 100 LD50 of Knr derivative of S. Typhimurium UK-1 (MT2315; 1O7 CFU). Feces was collected from individual mice and plated for CFU/g on kanamycin 50 ug/ml LB plates on Days 2, 4, 7, 11, 14, and 21 post-immunization. Persistence within de-ionized water and sheep feces.

Twenty per cent fecal dry matter was prepared by adding 20 ml of deionized water to 5g of dried sheep feces (gift from Barbara Byrne, University of California, Davis; [27, 28]).

De-ionized water (20 ml) and 20% sheep feces was inoculated with Knr derivatives of S.

Typhimurium UK-1 2 double mutant vaccine candidates, dam mgtC (MT3183), dam sifA (MT3184), dam spo (MT3186), or the dam UK-1 parent strain (MT3180) (2 x105 CFU). Triplicate assays were med in 50 ml conical tubes with loose caps at room temperature. Samples were vortexed and plated for CFUs over a two week penod. 1.5 Statistical analysis Continuous repeated measures data were analyzed using residual (or restricted) maximum likelihood (REML) analysis (Genstat, 15th n, VSN International, UK, id="p-34" id="p-34" id="p-34" id="p-34"

[34]). A single e, repeated measures model was fitted for the factors time and treatment for the variable CFU. The Wald chi-square test was used to determine significant dual effects and or significant ctions between factors. Any non- significant terms were dropped from the model and analysis repeated. Following analysis data are presented as predicted model based means. Predicted means are those obtained from the fitted model rather than the raw sample means. This is important as predicted means represent means adjusted to a common set of variables, thus ng valid comparison between means. A P value less than 0.05 was considered to be tically significant. The number of CFU present in tissues at necropsy was ed using analysis of variance (ANOVA, Genstat, 15th n, VSN International, UK). Differences between the individual means calculated using REML and ANOVA were determined by calculating an imate least icant difference (LSD). A difference of means that exceeded the calculated LSD was considered significant.

Binomial data (shedding [yes/no] and outcome [live/dead]) were analyzed using a logistic regression model (Genstat, 15th Edition, VSN International, UK, [34]). Vaccine was fitted to the model. Overall significance was assessed using the Wald statistic (P < 0.05). Significance of fixed effects (vaccine) was assessed according to the t parameter estimates relative to the reference group. P values less than 0.05 were considered statistically significant. 1.6. Ethics statement All animal mentation was conducted following the National Institutes of Health guidelines for housing and care of laboratory animals and performed in accordance with Institutional regulations after pertinent review and approval by the Institutional Animal Care and Use Committee at the sity of California, Santa Barbara. 2. Results 2.1. Construction of Salmonella dam vaccine candidates containing a secondary virulence-attenuating mutation The commercial success of modified live vaccines is dependent upon the therapeutic index, the ratio of safety/efficacy and, thus, secondary virulence-attenuating mutations were introduced into the S. enterica serovar Typhimurium dam e to improve vaccine safety. An otic ive, dam-deletion derivative of parental strain UK-1 was constructed to eliminate the potential transmission of antibiotic resistance to other microbial s (Materials and Methods). The resultant S. Typhimurium UK-1 damA232 (MT3134) was used as the parental vaccine background for all studies.

Secondary virulence-attenuating mutations were subsequently introduced into S.

Typhimurium UK-1 damA232 to improve vaccine safety (Materials and Methods). These mutations were targeted to genes involved in intracellular and/or systemic survival, including aroA (amino acid biosynthesis); htrA (stress response); mgtC (magnesium ort); sifA, spiC, ssaV (Salmonella Pathogenicity Island- 2 (SPI-2); and spo (cytotoxin production). The resultant Salmonella dam double mutant vaccine ates, dam aroA, dam htrA, dam mgtC, dam sifA, dam spiC, dam spo, dam ssaV, were subsequently ted for improved safety / efficacy in comparison to the parental Salmonella dam vaccine strain. 2.2. Evaluation of Salmonella dam double mutant vaccine ates for colonization and persistence in mucosal and systemic s A principal concern of introducing secondary virulence-attenuating mutations into modified live vaccines is the potential of loss of efficacy due to reduced antigen exposure as a uence of accelerated vaccine nce. Thus, the S.

Typhimurium UK-1 damA232 double mutant vaccine candidates, dam aroA, dam htrA, dam mgtC, dam sifA, dam spiC, dam spo, dam ssaV, were ed for those that ined colonization and persistence parameters similar to those found in the parental S. Typhimurium UK-1 damzi232 vaccine strain. BALB/c mice were orally ed with the Salmonella dam double mutant vaccine candidates (109 CFU), and zation / persistence of the vaccine strains was assessed in mucosal (Peyer’s patches; mesenteric lymph nodes) and systemic tissues (liver and spleen) at 2 and 4 weeks post infection (Figure 1). The Salmonella dam double mutant candidates were classified into two groups, Class I: those that showed similar colonization / persistence relative to that of the parental S. Typhimurium UK-1 damzl232 single mutant vaccine strain (dam mgtC; dam sifA; dam spo); and Class II: those that exhibited colonization / persistence relative to that exhibited by the parental Salmonella dam vaccine (dam aroA, dam htrA, dam spiC, dam ssaV). These data indicate that Class | vaccine candidates vaccines sustained a low grade persistence in host tissues, s Class II vaccines showed rapid clearance in vaccinated animals. 2.3. Efficacy evaluation of ella dam double mutant vaccine candidates The Salmonella dam double mutant vaccine ates (Class | and Class II) were examined to discern whether a low-grade persistence is necessary to confer protective immune responses similar to that elicited by the parental Salmonella dam vaccine. BALB/c mice were orally immunized with each of the seven Salmonella dam 2O double mutant vaccine candidates (109 CFU). To avoid transient, non-specific cross- protective immune responses attributed to the persistence of the vaccine strain within host tissues [23-25], immunized mice were not challenged with virulent ella until 4 to 5 weeks after the vaccine strain was d from mucosal (Peyer’s patches; mesenteric lymph nodes) and systemic tissues (liver; ) of immunized animals.

Eleven weeks post-immunization, mice were orally challenged with a 200-fold LD50 infection dose with the virulent parental strain, S. Typhimurium UK-1. Mice immunized with all (3 of 3) Class | vaccine candidates (dam mgtC, dam sifA, dam spo) exhibited robust protection against virulent homologous nge, r to that exhibited by the parental S. urium UK-1 damzi232 strain (Figure 2). Conversely, none (0 of 4) of the Class II vaccine ates (dam aroA, dam htrA, dam spiC, dam ssaV) that exhibited accelerated clearance conferred significant protection to virulent homologous challenge in comparison to the parental S. urium UK-1 damA232 strain (**P < 0.01, *** P < 0.001).

Class | vaccine ates were assessed for the capacity to elicit cross- protection to heterologous strains as has been shown for Salmonella dam vaccine s in murine [2, 9], avian [10, 11], ovine [12] and bovine [13-15] models of salmonellosis. BALB/c mice were orally immunized with Class | vaccine candidates (dam mgtC, dam sifA, or dam spo; 109 CFU). Eleven weeks post-immunization, mice were challenged with livestock-industry nt pathogenic Salmonella strains derived from sheep (S. Bovismorbificans 174, S. urium 131) and cattle (S. Dublin 8895), sing serogroups C2-C3, B, and D, respectively. All 3 Class | vaccine candidates conferred robust cross-protection to the three heterologous virulent strains tested (Figure 3; *** P< 0.001), similar to the levels of protection exhibited previously against these 3 heterologous nge strains in mice vaccinated with a S.

Typhimurium 14028 2 vaccine strain [2]. These data are tent with the hypothesis that the low grade persistence of Class | vaccine candidates in host tissues (dam mgtC, dam sifA, and dam spo) may provide a stable source of antigens over the time needed to transition to the development of strong adaptive immune responses [2,9,19]. 2.4. Vaccine safety evaluation via assessment of reversion to 2—AP resistance.

Reversion to ened virulence is a concern for all modified live vaccines.

Salmonella dam mutant vaccines have the ty to undergo reversion to a more virulent state after i.p. (but not oral) infection via acquisition of a mutation(s) in methyl- directed mismatch repair genes [29]. Such reversion can be evaluated using the purine analog 2-amino purine (2-AP), which is toxic to bacteria lacking Dam function [21]. That is, the parental dam strain (2-APS) can be assessed for reversion to 2-APr (as a potential indicator of heightened virulence) in systemic tissues [29]. BALB/c mice were i.p. infected with Salmonella dam double mutant vaccine candidates, dam mgtC, dam sifA, dam spo. or parental S. Typhimurium UK-1 damA232 strain (103 CFU). Five days post infection, bacteria recovered from the liver and spleen were assessed for 2-APs stence) and reversion to the 2-APr phenotype (Figure 4). All 3 vaccine candidates (dam mgtC, dam sifA, dam spo) showed significantly reduced colonization / persistence ) and reduced ion to 2-AP resistance relative in the spleen / liver relative to that of the parental S. Typhimurium UK-1 damzi232 strain (* P < 0.05). 2-Apr derivatives of Salmonella dam double mutant e candidates and the parental dam UK-1 e isolated from the spleens of infected mice were evaluated via oral and i.p. lethal dose (LD50) virulence assays. The oral and i.p. LD50 for wild-type UK-1 are 105 and <10 CFU, respectively. The oral LD50 of all .2-APr isolates derived from all Salmonella dam double mutant vaccine candidates (11 of 11) or parental Salmonella dam vaccine (5 of 5) were avirulent by oral administration (Table 2). In contrast, all (11 of 11) 2-APr es derived from Salmonella dam double mutant vaccine candidates were highly ated via i.p. infection, whereas those derived from the parental dam vaccine (5 of 5) were associated with reversion to a more virulent state, as demonstrated previously [29]. These data indicate that Salmonella dam mgtC, dam sifA, and dam spo vaccine strains exhibited significantly improved vaccine safety as evidenced by the failure to give rise to virulent revertants during the infective process, contrary to the Salmonella dam vaccine. 2.5. Vaccine and challenge strain shedding evaluation of Salmonella dam double mutant vaccine strains Reduced vaccine and challenge strain shedding in vaccinated s are desired traits for vaccine safety. Kanamycin-resistant derivatives of S. Typhimurium UK- 1 damA232 double mutant vaccine candidates were constructed used to assess vaccine strain and challenge strain shedding in the feces of immunized animals. BALB/c mice were immunized with either Salmonella dam double mutant vaccine candidates (dam mgtC [MT3183]; dam sifA [MT3184]; dam spo 6]) or the dam UK-1 parent strain (MT3180) by the oral route (109 CFU). Vaccine strain shedding. Fecal pellets were obtained and assessed for Knr bacteria at Days 2, 4, 7, 11, 14, and 21 post- infection. All ella dam double deletion e candidates exhibited significantly reduced vaccine strain fecal shedding in comparison to that of the parental S.

Typhimurium UK-1 damzl232 strain (Figure 5; P<0.05). Challenge strain shedding.

Eleven weeks post- immunization, vaccinated mice were challenged with a dose of 100 LD50 of Knr derivative of S. Typhimurium UK-1 (MT2315; 107 CFU). Fecal pellets were obtained and assessed for Knr ia at Days 2, 4, 7, 11, 14, and 21 post-infection.

Salmonella dam mgtC, dam sifA and dam spo strains exhibited a significantly reduction in challenge strain shedding relative to that of the parental S. Typhimurium UK-1 damA232 vaccine over a 3 week period, with dam sifA and dam spo strains showing d shedding from day 4 to 21 (Figure 6; P<0.05). These data indicate that vaccination with Salmonella dam double mutant vaccines results in less vaccine fecal shedding relative to that of the parental Salmonella dam vaccine and double deletion vaccination provides more robust attenuation of wildtype salmonella shedding following virulent challenge than the dam parent vaccine. 2.6. Environmental persistence (de-ionized water and sheep feces) evaluation of ella dam double mutant vaccine strains Salmonella dam double mutant vaccine candidates were evaluated for environmental persistence in de-ionized water and in sheep feces. De-ionized water.

Deionized water was inoculated with Knr derivatives of either Salmonella dam double mutant vaccine candidates (dam mgtC [MT3183]; dam sifA [MT3184]; dam spo [MT3186]) or the dam UK-1 parent strain (MT3180) (104 CFU/ml) (Figure 7). Water samples were plated for CFU/g over a two week period. All (3 of 3) Salmonella dam double mutant e candidates and the parental dam strain showed significantly reduced viability in de-ionized water over the 2 week tion in comparison to that of the wildtypeUK-1 strain (Figure 7; P<0.05). Further, the low-level vaccine persistence in water may be compatible with trough water e administration. Sheep feces.

Twenty per cent dry matter sheep feces was inoculated with Knr derivatives of either ella dam double mutant vaccine candidates (dam mgtC [MT3183]; dam sifA 4]; dam spo [MT3186]) or the dam UK-1 parent strain (MT3180) (104 ).

Fecal samples were plated for CFU/g over a two week period. All (3 of 3) Salmonella dam double mutant vaccine ates showed significantly reduced viability in sheep feces over the 2 week tion in comparison to that of the wildtype UK-1 strain over the 2 week incubation period (Figure 8; ). r, Salmonella dam sifA showed significantly reduced viability in sheep feces relative to that of the other 3 vaccine strains tests (P <0.05). These data indicate that Salmonella vaccine candidates show reduced environmental tence in both de-ionized water and sheep feces in comparison to that of the wild type UK-1 strain. 3. Discussion Despite good husbandry practices, salmonellosis continues to be a significant problem in intensive production s that favor fecal-oral transmission. e is principally caused by increased en exposure and disease susceptibility.

Fluctuations in environmental conditions cause shifts in the environmental pathogen load and subsequently host challenge. Physiological changes associated with ncy and parturition increase susceptibility to disease as does the naive immune status of neonates. Management practices may also negatively impact on host immunity with cumulative stressors experienced by stock on farm (mustering, yarding, food and water deprivation prior to transport), during transport (food and water deprivation, environmental stress), and in sale yards (co-mingling, pathogen exposure).

Livestock vaccination against salmonellosis is a viable approach to prevent e since it prevents contamination of food and water supplies at the outset, resulting in diminished pathogen exposure, transmission, animal disease, and the direct ination of livestock-derived food products and indirect contamination of fruit and ble food products by contaminated water.

Optimally, ock should be vaccinated on farms of origin to elicit immunity before livestock experience the stressors and pathogen exposure associated with sale, ort, and the high-risk period following entry into the feedlot. The challenge is convincing producers, who supply stock to feedlots, to vaccinate the animals prior to sale since the cost of disease is not incurred on the property of origin, resulting in the current practice of livestock immunization during the high risk period immediately following entry into the feedlot.

If an affordable and effective product is made available to the commercial , the vaccine could be applied broadly across animal production industries as ation is simple, tood by producers, and likely to be adopted and, thus, may play a critical role in the success of any hensive food safety plan.

As a potential means to address this issue, modified live Salmonella dam vaccines have been shown to be effective and well-tolerated in immunized stock [10- ], and can be administered via drinking water [12, 16]. However, the principal concerns of live vaccines are safety, shedding, and environmental persistence.

Herein, secondary virulence-attenuating mutations were introduced into a Salmonella dam strain to screen for e candidates that were safe in the animal and the environment, and maintained the capacity to confer cross-protective efficacy. 8.

Typhimurium dam sifA exhibited improved vaccine safety, reduced vaccine and challenge strain shedding, d environmental persistence, and conferred a low grade persistence in host tissues that was sufficient to confer cross-protection to heterologous pathogenic ellae serotypes d from infected ock [31].

These data indicate that Salmonella dam sifA exhibits a favorable therapeutic index (safety / efficacy) for commercial ations, supporting improved safety in both vaccinates and the environment, along with the capacity to elicit cross-protective immunity t pathogenic serotypes.

Herein, the safety of the vaccine was ted in vaccinated animals and in conditions mimicking the environment. Salmonella dam mgtC, dam sifA, and dam spo vaccine strains sustained a low grade persistence in host s that was associated with the maintenance of cross-protective immunity against heterologous pathogenic serotypes derived from infected stock. Further, the Salmonella dam sifA vaccines ted improved vaccine safety (vaccine shedding; challenge strain shedding; persistence in systemic tissues; persistence in the environment), while maintaining robust efficacy against nce challenge with homologous and heterologous pathogenic serotypes. Thus, the Salmonella dam sifA vaccine candidate exhibits considerable increased safety without compromising cross-protective efficacy and may prove to be a safe, effective, and low cost means of oral dosing of livestock t significant environmental persistence.

Table 1. Bacterial strains used in this study Strain Genotype Source/Reference S. Typhimurium UK-1 x3761 Wild type en) [17] MT2315 zjf7504::MudJ (Knr) [10] MT3134 Adam232 This work MT3138 Adam232 AaroA This work MT3142 Adam232 AhtrA This work MT3146 Adam232 AmgtC This work MT3150 Adam232 AsifA This work MT3154 Adam232 AspiC This work MT3158 Adam232 Aspo This work MT3162 Adam232 AssaV This work MT3180 Adam232 4::MudJ (Knr) This work MT3183 Adam232 AmgtC zjf7504::MudJ (Knr) This work MT3184 Adam232 AsifA zjf7504::MudJ (Knr) This work MT3186 2 Aspo zjf7504::MudJ (Knr) This work Animal isolates 131 S. Typhimurium (sheep) [31] 225 S. Bovismorbificans (sheep) [31] 8895 8. Dublin cattle isolate (cattle) |31| Table 2. In vivo-selected 2—APr derivatives of dam double mutant Salmonella are avirulent via the intraperitoneal or oral routes of infection |P Virulence Oral Challenge strain Relevant genotype (LD50)a Virulence (LDso) s. Typhimurium UK-1 Wild type < 10 105 MT3134 Adam232 > 104 2 1010 MT3243 Adam232 2—APr isolate #1 3 103 109 -1010 MT3244 Adam232 2—APr isolate #2 <102 109 MT3245 Adam232 2—APr e #3 <102 109 MT3246 Adam232 2—APr isolate #4 <102 109 MT3247 Adam232 2—APr isolate #5 <102 2 1010 MT3146 Adam232 AmgtC > 104 2 1010 MT3248 Adam232 AmgtC 2—APr isolate #1 103 Z 1010 MT3249 Adam232 AmgtC 2—APr isolate #2 3 104 3 1010 MT3250 Adam232 AmgtC 2—APr isolate #3 3 104 109 -1010 MT3150 Adam232 AsifA > 104 2 1010 MT3251 2 AsifA 2—APr isolate #1 3 104 3 1010 MT3252 Adam232 AsifA 2—APr e #2 3 104 109 -1o10 MT3253 Adam232 AsifA 2—APr isolate #3 3 104 3 1010 MT3254 2 AsifA 2—APr isolate #4 3 104 109 -1o10 MT3255 Adam232 AsifA 2—APr isolate #5 3 104 3 1010 MT3158 Adam232 Aspo > 104 2 1010 MT3256 Adam232 Aspo 2—APr isolate #1 2 104 3 1010 MT3257 Adam232 Aspo 2—APr isolate #2 3 104 109 -1o10 MT3258 Adam232 Aspo 2—APr isolate #3 3 104 109 -1010 a Independently isolated, in vivo selected, 2—amino purine resistant ) derivatives of Salmonella dam mutant es strains were isolated from the spleens of infected mice, and evaluated for oral and intraperitoneal (IP) nce in naive mice [29]. The LD50 assay for each of these strains was compared to that of the wild type (UK-1). The IP LD50 was determined by infecting five mice per challenge dose; the peroral LD50 via gastrointubation was determined by infecting ten mice per challenge dose. The oral and i.p. LD50s for wild-type UK-1 are 105 and <10 CFU, tively [30]. Surviving mice were scored > 2 weeks post-infection.

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International V. GenStat for Windows 15th Edition VSN International, Hemel Hempstead, UK. 2012 1003810589

Claims (16)

1. A Salmonella microorganism, n said microorganism comprises a loss of function mutation in the dam gene and at least one further loss of function mutation in a gene selected from the group consisting of: sifA, spvB and mgtC.

2. The Salmonella microorganism according to claim 1, wherein the loss of function mutation is: an insertion, a deletion and/or substitution of one or more nucleotides in said genes. 10

3. The Salmonella microorganism of claim 1 or 2 wherein the microorganism is a Salmonella ca subsp. Enterica serovar ed from the group consisting of S. Typhimurium, S. Enteritidis, S. Dublin, S. t, S. Choleraesuis, and S. Bovismorbificans. 15

4. The Salmonella rganism of any one of the preceding claims, wherein the microorganism is S. Typhimurium.

5. The Salmonella microorganism of any one of the preceding claims, wherein the at least one further loss of function mutation is in the sifA gene.

6. A composition for inducing an immune response in a subject to a Salmonella microorganism, said composition comprising the microorganism of any one of claims 1 to 5, in an amount sufficient to elicit an immune se in the t and an adjuvant, diluent, carrier or excipient.

7. A purified culture of the Salmonella microorganism of any one of claims 1 to 5.

8. The purified culture of claim 7 n the culture is freeze dried, frozen, or reconstituted.

9. A method of preventing or reducing the likelihood of infection of a non-human subject with a virulent Salmonella microorganism, said method comprising: 1003810589 - administering to a non-human subject in need thereof, an amount of the microorganism of any one of claims 1 to 5, the composition of claim 6 or the purified culture of claim 7 or 8; - wherein the amount of microorganism, composition or purified culture 5 administered is sufficient to elicit an immune response in the subject.

10. The method according to claim 9 wherein the microorganism, composition and/or purified culture is administered via the oral, nasal or parenteral routes.

11. The method according to claim 9 or 10 wherein the t is a nary species.

12. The method ing to claim 12 wherein the subject is selected from the group 15 consisting of cow, horse, goat, sheep, pig and poultry.

13. Use of a Salmonella microorganism ing to any one of claims 1 to 5 in the cture of a medicament for preventing or reducing the likelihood of infection of a subject with a nt Salmonella microorganism.

14. Use of the purified culture of claim 7 or 8 in the manufacture of a medicament for preventing or reducing the likelihood of infection of a subject with a virulent Salmonella microorganism. 25

15. The use according to claim 14 or 15 wherein the medicament is a vaccine.

16. A Salmonella microorganism ing to any one of claims 1 to 5 for use in preventing or reducing the likelihood of infection of a non-human subject with a virulent Salmonella microorganism.