WO2024018033A1 - Bacterial vaccine comprising probiotic competitor and inactivated pathogen - Google Patents

Abstract

The invention relates to a pharmaceutical composition capable of protecting a patient from disease caused by a pathogenic strain of a bacterium, that displays a wild type surface antigen. It comprises a probiotic component comprising a live avirulent bacterial strain of said pathogen, which displays a variant of said surface antigen, said variant being capable of escaping binding by immunoglobulins capable of specifically recognizing the wild-type surface antigen. The composition further comprises a vaccine component of an inactivated vaccine strain of said bacterium, which displays said wild type surface antigen.

Description

Bacterial Vaccine comprising Probiotic Competitor and Inactivated Pathogen

This application claims the benefit of priority from EP applications no. 22186078.6 filed 20 July 2022 and EP 22207976.6 filed 17 November 2022, both incorporated by reference herein.

Field

The present invention relates to vaccine compositions useful in the treatment and prevention of diseases caused by Proteobacteria. The compositions of the invention comprise a probiotic component composed of living, engineered avirulent variants of a pathogenic strain, and an immunogenic component comprised of inactivated pathogen.

Background

World-wide, the frequency of drug-resistant infections with E. coli and with non-Typhoidal Salmonella have been steadily increasing, posing a looming challenge for health systems. There is a pressing need to develop control and prevention strategies that are independent of antibiotics. Vaccination of both farm animals and humans is a promising alternative. While high-efficacy vaccine candidates against Typhoid fever are currently well advanced, there are no licensed human vaccines for non-Typhoidal Salmonellosis. Moreover, licensed live-attenuated S.Tm (Salmonella Typhimurium) swine and bovine vaccines show only weak protection from colonization and can cause disease in highly susceptible individuals. An ideal solution would be fully inactivated vaccines that can mimic the immunity and protection generated by live-attenuated vaccines but with a much higher safety profile and robustness.

When designing vaccines targeting intestinal bacteria, the field has either focused on mimicking natural infection, or on generating immune responses targeting specific pathogen antigens. However, it is increasingly realized that infection of the gut involves disruption of a densely populated microbial ecosystem, and the intestinal microbiome is itself part of our defences. Therefore, evidence suggests that naturally arising immunity at our mucosal surfaces does not operate in isolation, but rather interacts with the endogenous microbiota to recover homeostasis and evict colonizing opportunistic pathogens.

The inventors’ population dynamics analysis of S.Tm during the early stages of non-Typhoidal Salmonellosis in mouse models indicates that a more than 1000-fold reduction in invasion rate of S.Tm is required during the first day of infection to prevent tissue invasion, inflammation and systemic spread. The overall invasion rate is a product of the gut luminal S.Tm population size and the ability of each individual bacterium to invade. Antibody-mediated clumping, via enchained growth and/or agglutination achieves around a 100-fold reduction in infectivity, but alone cannot be sufficient to fully prevent invasive disease. Therefore, vaccination strategies are required that not only inhibit pathogenicity of invading bacteria, but which also limit their ability to colonize the gut lumen.

At the simplest level, the population size of S.Tm in the gut lumen is determined by its growth rate, clearance rate and the size of the available metabolic niche. The available metabolic niche can be dramatically shrunk by competitors using the same resources. A classic example of this phenomenon occurs when “cheater” mutations spontaneously arise during chronic NTS. Mutations in the Salmonella Pathogenicity Island 1 (SPI1 ) master-regulator of virulence hilD can outgrow and displace wildtype Salmonella from the gut lumen. This is attributed to a fast net-growth rate and increased stress resistance of rt/ZD-mutant strains due to loss of the fitness costs associated with expression of the HilD regulon.

Niche competition can also be favoured by increasing the clearance rate of the pathogen. High-affinity intestinal IgA, induced by oral vaccination with whole-cell inactivated vaccines, can increase S.Tm clearance rates by generating bacterial clumps (via enchained growth or agglutination) that are more efficiently cleared in the flow of the fecal stream. The inventors have recently demonstrated that this IgA-mediated selective pressure can be used to manipulate the evolutionary trajectory of Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm) in the gut lumen. Moreover, part of this work demonstrated the ability of IgA to generate a more than a million-fold ratio of a targeted S.Tm strain over a non-bound strain. Therefore, IgA protects the intestinal environment not only via immune exclusion (i.e. preventing interaction of pathogenic bacteria with the epithelium) but also by competitively eliminating targeted bacteria from the intestinal ecosystem. This suggested a huge potential for specific high-affinity IgA responses to manipulate the outcome of competition between an invading pathogen and an engineered niche competitor.

Critical components of such a prophylactic system are: 1 ) a vaccine capable of inducing high-affinity specific IgA against the pathogen of interest; 2) a non-pathogenic strain with (ideally) complete metabolic niche overlap with the pathogen of interest, a faster growth-rate than the pathogen of interest and absence of surface antigen cross-reactivity to the pathogen of interest. Additionally, questions arise as to how vaccines and competitors can be combined temporally to give maximum effect, and the extent of protection from invasive disease that can be achieved with such an approach. Here the inventors make use of the well-established and severe, murine model of non-Typhoidal Salmonellosis to establish this concept. In this model, oral antibiotics are applied to acutely generate a large open niche for S.Tm in the mouse cecum and upper large intestine. Subsequently, very low numbers of S.Tm can be orally inoculated and will rapidly grow to fill the available niche. Disease depends on the activity of Salmonella Pathogenicity Islands 1 and 2 and includes acute typhlocolitis, colonization of the intestinal tissue, mesenteric lymph nodes, spleen and liver and weight-loss (Barthel, M. et al. Infect Immun 71 , 2839- 2858, doi:10.1128/iai.71.5.2839-2858.2003 (2003)). In the resistant (Nramp1+/+, also known as Slc11a1 ) 129SJL mouse strain, the disease is slowly controlled, but full recovery takes more than 1 month. The inventors also extend our observations to the murine oral Typhoid fever model, in which a high-dose of S.Tm is delivered orally to mice with an intact intestinal microbiota, resulting in a disease that is more reliant on the tissue-invasive stages of disease.

Vaccination with inactivated pathogenic bacteria, such as Salmonella, can be rendered less effective than expected because evolution of the pathogen generates strains that evade vaccine-induced anti-O- IgG responses, a major determinant of immunity, by mutation of the O antigen. W02020239960A1 proposes the use of inactivated vaccine strains that contain modifications found in escape mutants, to pre-empt this evasion. This strategy however has not entirely solved the needs for better antibacterial vaccines. The objective of the present invention is to provide means and methods to provide cheap and effective vaccine compositions generating immunity and avoiding immune escape, for prevention of bacterial pathogen-associated disease in vertebrate hosts, particularly domestic animals and human patients. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

Summary of the Invention

The data generated by the inventors and shown in the examples, demonstrate that combining a benign niche-competitor and a whole-cell inactivated vaccination regimen can profoundly suppress intestinal colonization with virulent Salmonella even on high-dose exposure to pathogen. This is shown in the examples in a very high-susceptibility model. Protection from gut tissue invasion and inflammation conferred by the composition according to the invention is robust. Interestingly, live-attenuated Salmonella vaccines were superior to inactivated vaccines in protecting from colonization of systemic sites, indicating that mechanisms that protect systemic sites are discrete from intestinal protection.

A first aspect of the invention relates to a pharmaceutical composition capable of protecting a patient from infection by, or disease caused by a pathogenic strain of a bacterium, wherein said pathogenic strain displays a bacterial wild type surface antigen, said composition comprising a. a probiotic component comprising bacteria of a live propagation competent avirulent strain of said pathogen, wherein said avirulent strain displays a modified variant of said surface displayed bacterial antigen, said variant being capable of escaping binding by vaccine-induced immunoglobulins capable of specifically recognizing the wild-type surface antigen; b. a vaccine component comprising an inactivated propagation incompetent vaccine strain of said bacterium, wherein said inactivated strain displays said wild type surface displayed bacterial antigen.

Another aspect of the invention relates to a probiotic pharmaceutical preparation for use in protecting a patient from infection by, or disease caused by a pathogenic strain of a bacterial pathogen, said probiotic pharmaceutical preparation comprising live bacteria of an propagation competent avirulent strain of said bacterial pathogen, wherein said pathogenic strain of said bacterial pathogen displays a bacterial wild type surface antigen, and wherein said avirulent strain displays a modified variant of said surface displayed bacterial antigen, said variant being capable of escaping binding by vaccine-induced immunoglobulins capable of specifically recognizing the wild-type surface antigen; wherein said probiotic pharmaceutical preparation is provided after administration of an antibacterial drug at a dosage sufficient to remove competing bacteria from the niche occupied by the avirulent strain.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

"And/or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term Lipopolysaccharide in the context of the present specification relates to a molecule consisting of a lipid and a polysaccharide composed of O-antigen, outer core and inner core joined by a covalent bond that is located in the outer membrane of Gram-negative bacteria. The term lipooligosaccharide ("LOS") is used to refer to a low-molecular-weight form of bacterial lipopolysaccharides. The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.

Detailed Description of the Invention

A first aspect of the invention relates to a pharmaceutical composition capable of protecting a patient from infection by, or disease caused by, a pathogenic strain of a bacterium. The pathogenic strain is characterized by display on its surface of a bacterial wild type surface antigen. The composition comprises two components, a live probiotic bacterial component and a vaccine component.

The probiotic component comprises bacteria of a live propagation competent, or at least gutcolonization competent, avirulent strain of said pathogen, wherein said avirulent strain displays a modified variant of said surface displayed wild type bacterial antigen. The variant is capable of escaping binding by vaccine-induced immunoglobulins capable of specifically recognizing the wild-type surface antigen and colonizes ecological niches inside the patient that otherwise might be occupied by mutant variants of the pathogen that manage to escape immunity induced by the vaccine component.

The vaccine component comprises an inactivated, propagation incompetent vaccine strain of said bacterium. The inactivated strain displays said wild type surface displayed bacterial antigen; it induces immunity against the pathogen.

In certain embodiments, the patient is a human patient. In certain embodiments, the patient is an animal, particularly a vertebrate, more particularly a vertebrate domestic animal, such as a pig, a chicken, a turkey, a sheep, a goat, a cow (cattle), a dog, a rabbit, or a cat.

The term “avirulent” means here that the virulence of the strain used as probiotic is attenuated by mutations inactivating virulence factors.

The glycan molecules displayed on the bacterium’s surface (i.e., the O antigen = the O side chain of the lipopolysaccharide) are modified in the probiotic component. In one specific example in Salmonella typhimurium, the acetylation of abequose is disabled by mutating the acetyltransferase responsible for the acetylation. The probiotic is therefore of serotype “04” while the pathogen is “05” (acetylated).

In certain embodiments, virulence factors are disabled in the probiotic by i) mutating hilD, coding for the master regulator of virulence, and/or ii) ssaV, coding for a structural component of the Type three secretion system 2 involved in systemic dissemination. Bacterial pathogens for which the invention is particularly suitable

In certain embodiments, the bacterium belongs to the class Gammaproteobacteria. In certain particular embodiments, the bacterium belongs to the order Enterobacterales. In certain more particular embodiments, the bacterium belongs to the family Enterobacteriaceae.

While the examples presented herein illustrate the proof of the inventive principle with Salmonella typhimurium, the skilled artisan readily understands that the invention can be practiced with minor if any alterations on other strains and serotypes of Salmonella. Furthermore, extension of the principle to other strains and serotypes of E. coli and Klebsiella spp. should be possible. The same applies to the generalization to Enterobacteriaceae as a family.

In certain embodiments, the bacterium belongs to the family Enterobacteriaceae and belongs to a genus selected from the group of Salmonella, Escherichia, Enterobacter, Klebsiella, Shigella, Citrobacter.

In certain particular embodiments, the bacterium belongs to the genus Salmonella.

In certain more particular embodiments, the bacterium belongs to the genus Salmonella and belongs to the species S. enterica.

In certain even more particular embodiments, the bacterium belongs to the subspecies S. enterica subsp. enterica.

In certain other even more particular embodiments, the bacterium belongs to the subspecies S. enterica subsp. typhimurium.

In certain particular embodiments, the bacterium belongs to the genus Escherichia.

In certain more particular embodiments, the bacterium belongs to the genus Escherichia and belongs to the group of Entero-pathogenic Escherichia coli (EPEC).

In certain even more particular embodiments, the bacterium is an EPEC double mutant comprising: a. a deletion of the gene ler encoding Ler (Uniprot ID E9N650), the master regulator of expression of virulence genes in the Locus of Enterocyte Effacement (LEE). Ler would be the equivalent of HilD in Salmonella; b. a deletion of wzy encoding the O antigen polymerase

This protein is also known as enterobacterial common antigen (ECA) polymerase; the ECA is polymerized by wzy, the same enzyme than the O antigen.

The LEE is essential in the pathogenicity of the EPEC. Deleting wzy makes the probiotic escaping recognition by IgA in vaccinated hosts.

The vaccine component can be prepared both for the EPEC as for Salmonella, by killing a preparation of the infectious bacteria with peracetic acid.

Delta HilD mutants show abolished virulence, increased growth, and are generally very competitive in occupying ecological niches in the patient. In certain particular embodiments, the bacterium belongs to the genus Enterobacter.

In certain particular embodiments, the bacterium belongs to the genus Klebsiella.

In certain particular embodiments, the bacterium belongs to the genus Shigella.

In certain particular embodiments, the bacterium belongs to the genus Citrobacter.

The surface displayed antigen and mutations therein

In certain embodiments, the surface displayed antigen is an O antigen.

In certain embodiments, the avirulent strain is characterized, relative to the pathogenic strain, by one or more modifications functional deletions of gene function selected from the following groups of gene functions: c. Virulence Factor group: i. Main regulator of virulence expression HilD (e.g. for Salmonella enterica); and/or Ler (for E. coli EPEC) ii. SsaV a key component of the Type three secretion system 2, not controlled by HilD, are removed by targeted in-frame gene deletion. d. O-antigen Modifier group: i. abequose acetyltransferase OafA ii. wzyB (O antigen polymerase) iii. wbaP (galactosyl transferase); iv. wbaN; wbaU; wbaV; and/or wzx v. wecA, wzm, wzt, waaL, msbA, wbbE and/or wbbF.

Generally, any genes in the pathway of O-antigen I LPS synthesis can be targeted to provide the avirulent strain.

Conserved genes that make good targets to create a probiotic from E. coli, Salmonella or Klebsiella include, but are not limited to: wbaP, wbaN, wbaU, wbaV, wzx, and wzyB (also known as wzy or rfc)

HilD regulation in Salmonella is detailed in Petrone et al., J Bacteriol. (2014) 196: 1094-1101.

LPS biosynthesis involves a large number of enzymes and assembly proteins encoded by more than 40 genes, reviewed in:” Raetz and Whitfield (2002) Annu. Rev. Biochem., 71 , 635-700; Samuel and Reeves (2003) Carbohydr. Res., 338, 2503-2519; Valvano (2003) Front. Biosci., 8, s452-471 , all of which are incorporated herein as reference.

Candidates for deletion other than Ssav include any T3SS-2 structural components: sseC, sseD, sseB, ssaG, ssaC, ssal, ssaJ, ssaD, ssaR, ssaS, ssaT, ssaX, ssaU, ssaQ, ssaV, ssaK, ssaO, SsaL, ssaM, SpiC, and ssaN.

In certain embodiments, more than one virulence factor is removed in the avirulent strain.

In certain embodiments, the avirulent strain is characterized by having a modification selected from each of the Virulence Factor group and the O-antigen Modifier group. A Salmonella typhimurium probiotic is therefore of serotype 04 012 (oafA is knocked out) while the pathogen is 05 012 (acetylated O antigen, oafA present and expressed).

The change in O-antigen composition drives avoidance of the host response in the probiotic strain.

Another aspect of the invention relates to a pharmaceutical composition according to the first aspect of the invention, optionally characterized by any one of the specific embodiments of its separate features alone or in combination, for use as a vaccine for preventing disease caused by the bacterial pathogen.

In certain embodiments, the vaccine targets Salmonella spp. and is for administration in a chicken or turkey.

In certain other embodiments, the vaccine targets E. coli and/or Salmonella spp. and is for administration in a pig.

In certain other embodiments, the vaccine targets E. coli, and is for administration in a human.

In certain other embodiments, the vaccine targets Klebsiella spp and is for administration in a human.

In certain other embodiments, the vaccine targets Citrobacter spp and is for administration in a human.

In certain other embodiments, the vaccine targets Enterobacter spp., and is for administration in a human.

In certain other embodiments, the vaccine targets Salmonella spp. and is for administration in a human.

In certain other embodiments, the vaccine targets more than one, particularly three or more bacterial species selected from the group consisting of E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., and/or Salmonella spp. and is for administration in a human.

Another aspect of the invention relates to an administration form prepared for oral administration, comprising the pharmaceutical composition as specified in the above aspects and embodiments.

Yet another aspect of the invention relates to a probiotic pharmaceutical preparation for use in protecting a patient from infection by, or disease caused by a pathogenic strain of a bacterial pathogen. This probiotic pharmaceutical preparation comprises live bacteria of a propagation competent avirulent strain of said bacterial pathogen. The pathogenic strain of said bacterial pathogen displays a bacterial wild type surface antigen, and the avirulent strain displays a modified variant of said surface displayed bacterial antigen. The variant is capable of escaping binding by vaccine-induced immunoglobulins capable of specifically recognizing the wild-type surface antigen. The probiotic pharmaceutical preparation is provided after administration of an antibacterial drug at a dosage sufficient to remove competing bacteria from the niche occupied by the avirulent strain. “After administration” is understood by the skilled person as meaning “Sufficient time having elapsed after administration of the antibacterial drug to allow the antibacterial effect having had time to run its course and diminish the population of the bacterial pathogens, not so soon for the probiotic component to be significantly affected by the antibacterial drug’s effect, but soon enough to allow for the probiotic to colonize the ecological niche liberated by diminishing the pathogenic strain”. The skilled person will be able to determine the ideal time point depending on species treated, and the pathogenic strain at hand, with straightforward testing with routine procedures.

In yet another aspect of the invention, the probiotic pharmaceutical preparation for use according to the invention is provided after administration of a vaccine comprising an inactivated vaccine strain of said bacterial pathogen, wherein said inactivated strain displays said wild-type surface antigen.

In certain embodiments, the probiotic pharmaceutical preparation provided for use according to the previous paragraph relates to a bacterial pathogen and an associated avirulent strain as specified according to the first aspect of the invention.

Petrone et al. J Bacteriol. 2014 Mar; 196(5): 1094-1101 discusses SPI-1 virulence regulation.

At least 46 O antigens of Salmonella are known (see Liu et al., Structural diversity in Salmonella O antigens and its genetic basis; FEMS Microbiol Rev. 2014 Jan;38(1 ):56-89. doi: 10.1111/1574- 6976.12034). This structural variation corresponds to the serological specificity of the 46 recognized serogroups. O antigens are the main immunogenic surface structures of E.coli and Salmonella (Mostowy and Holt, Diversity-Generating Machines: Genetics of Bacterial Sugar-Coating; Trends in Microbiology 26(12)(2018)). At least 46 O antigens of Salmonella are known (see Liu et al., Structural diversity in Salmonella O antigens and its genetic basis; FEMS Microbiol Rev. 2014 Jan;38(1 ):56-89. doi: 10.1111/1574-6976.12034). This structural variation corresponds to the serological specificity of the 46 recognized serogroups, but there is also structural variation within these serogroups. A similar situation exist for E.coli with 181 recognized O-antigens at present (Liu et al., FEMS Microbiology Reviews, Volume 44, 2020, 655-683). The genetics underlying O-antigen synthesis and modification in both E.coli and Salmonella is quite well characterized (see citations above), with considerable homology in the synthetic pathways. Notably O-antigen variation by acetylation (Knirel et al., J Bacteriol. 2015 Mar; 197(5): 905-912), and by glucosylation (Mann et al., J Biological Chemistry 290, (42) 25561-25570) has also been reported in E.coli and is known to alter surface binding properties. Thus engineering of immunologically distinct avirulent probiotic strains of E.coli is equivalent to that in Salmonella.

O antigen diversity is reviewed in Raetz and Whitfield C (2002), Annu. Rev. Biochem. 71 : 635-700. doi:10.1146/annurev.biochem.71.110601.135414.

Pharmaceutical Compositions, Administration/Dosacie Forms and Salts

According to one aspect of the compound according to the invention, the compound according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form.

Similarly, a dosage form for the prevention or treatment of bacterial infection or bacterial pathogen- associated disease is provided, comprising a composition according to any of the above aspects or embodiments of the invention.

Certain embodiments of the invention relate to a dosage form for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

Method of Manufacture and Method of Treatment according to the invention

The invention further encompasses, as an additional aspect, the use of a composition as identified herein in detail above, for use in a method of manufacture of a vaccine for the prevention of bacterial infection or bacterial pathogen-associated disease.

Similarly, the invention encompasses methods of administration, with the objective to prevent bacterial infection or bacterial pathogen-associated disease, in a patient possibly exposed to such pathogen. This method entails administering to the patient an effective amount of a composition as identified herein.

Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention further encompasses the following items:

1. A pharmaceutical composition capable of protecting a patient from disease caused by a pathogenic strain of a bacterium, wherein said pathogenic strain displays a wild type surface antigen, said composition comprising a. a probiotic component comprising bacteria of a live avirulent strain of said pathogen, wherein said avirulent strain displays a variant of said surface antigen, said variant being capable of escaping binding by immunoglobulins capable of specifically recognizing the wild-type surface antigen; and b. a vaccine component comprising an inactivated vaccine strain of said bacterium, wherein said inactivated strain displays said wild type surface antigen.

2. The pharmaceutical composition according to item 1 , wherein the bacterium belongs to the class Gammaproteobacteria, particularly wherein the bacterium belongs to the order Enterobacterales; more particularly wherein the bacterium belongs to the family Enterobacteriaceae.

3. The pharmaceutical composition according to item 2, wherein the bacterium belongs to a genus selected from the group of Salmonella, Escherichia, Enterobacter, Klebsiella, Shigella, Citrobacter.

4. The pharmaceutical composition according to item 3, wherein the bacterium belongs to the species S. enterica, particularly wherein the bacterium belongs to the subspecies S. enterica subsp. enterica; or wherein the bacterium belongs to the subspecies S. enterica subsp. typhimurium.

5. The pharmaceutical composition according to item 3, wherein the bacterium belongs to the group of Entero-pathogenic Escherichia coli (EPEC) particularly wherein the bacterium is an EPEC double mutant comprising: a. a deletion of the gene ler; b. a deletion of wzy. The pharmaceutical composition according to any one of the preceding items, wherein the surface antigen is an O antigen. The pharmaceutical composition according to any one of the preceding items, wherein the avirulent strain is characterized, relative to the pathogenic strain, by one or more functional deletions of gene function selected from the following groups of gene functions: a. Virulence Factor group: i. Main regulator of virulence expression HilD; and/or Ler (for E. coli EPEC). ii. T3SS-2 structural components of Salmonella spp: sseC, sseD, sseB, ssaG, ssaC, ssal, ssaJ, ssaD, ssaR, ssaS, ssaT, ssaX, ssaU, ssaQ, ssaV, ssaK, ssaO, SsaL, ssaM, SpiC, and ssaN. b. O-antigen Modifier group: i. abequose acetyltransferase OafA ii. wzyB (O antigen polymerase)

Hi. wbaP (galactosyl transferase); iv. wbaN; wbaU; wbaV; and/or wzx v. wecA, wzm, wzt, waaL, msbA, wbbE and/or wbbF. The pharmaceutical composition according to item 7, wherein more than one virulence factor is removed in the avirulent strain. The pharmaceutical composition according to item 7 or 8, wherein the avirulent strain is characterized by having a modification selected from each of the Virulence Factor group and the O-antigen Modifier group. The pharmaceutical composition according to any one of the preceding items, wherein the probiotic component and the vaccine component are provided as part of one administration form. The pharmaceutical composition according to any one of the preceding items 1 to 9, wherein the probiotic component and the vaccine component are provided each as a distinct administration form. The pharmaceutical composition according to any one of the preceding items, for use as a vaccine for preventing disease caused by said bacterium. The pharmaceutical composition for use according to item 12, wherein the vaccine a. targets Salmonella spp. and is for administration in a chicken or turkey b. targets E. coli and/or Salmonella spp. and is for administration in a pig c. targets E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., and/or Salmonella spp. and is for administration in a human. The pharmaceutical composition for use according to any one of the preceding items, as an administration form prepared for oral administration. A probiotic pharmaceutical preparation for use in protecting a patient from disease caused by a pathogenic strain of a bacterial pathogen, said probiotic pharmaceutical preparation comprising live bacteria of an avirulent strain of said bacterial pathogen, wherein said pathogenic strain of said bacterial pathogen displays a wild-type surface antigen, and wherein said avirulent strain displays a variant of said surface antigen, said variant being capable of escaping binding immunoglobulins capable of specifically recognizing the wild-type surface antigen.

16. The probiotic pharmaceutical preparation for use according to item 15, wherein said probiotic pharmaceutical preparation is provided after administration of an antibacterial drug.

17. The probiotic pharmaceutical preparation for use according to item 15 or 16, wherein said probiotic pharmaceutical preparation is provided after administration of a vaccine comprising an inactivated vaccine strain of said bacterial pathogen, wherein said inactivated strain displays said wild-type surface antigen.

18. The probiotic pharmaceutical preparation for use according to any one of claims 15 to 16, wherein the bacterial pathogen and the avirulent strain are specified in any one of items 1 to 9.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows that combining oral vaccination with niche competition protects mice from intestinal inflammation and leads to rapid S.Tm^WT clearance. PBS (blue symbols) or PA-S.Tm- vaccinated (pink symbols) 129S6/SvEv mice were pretreated with streptomycin and infected with a total of 10⁴ of a 1 :1 mixture of two isogenic S.Tm^WT strains (open circles) or S.Tm^WT and S.Trn^Comp (S.Tm^/,'^{/D ssaV oafA}, filled diamonds). (A) Experimental procedure. (B) S.Tm^WT (O:4[5],12-0) specific intestinal IgA and serum IgG titres as determined by flow cytometry. (C) Change in body weight over the course of infection. Solid lines show the mean. (D) Fecal CFUs as determined by selective plating. (E) A 4-parameter logistic curve fit was fitted to the fecal S.Tm^WT CFUs and the area under the curve was statistically compared. Shaded areas depict the 95% Cl of the fits. (F-l) S.Tm CFUs in cecum content (F), MLN (G), liver (H) and spleen (I). (J) Intestinal inflammation was determined by measuring fecal lipocalin-2. (K) Histology of cryo-embedded H&E-stained cecal tissue sections. Arrowheads show exemplary goblet cells. Scale bars: 100 pm. Pooled data from two independent experiments with switched antibiotic resistances (n = 5-8 mice/group). Solid lines depict the median unless stated otherwise, error bars the interquartile range. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. Open triangles show mice that had to be euthanized prematurely due to excessive weight loss (> 15%) or disease symptoms. Statistics were performed by mixed-effects analysis (C) or one-way analysis of variance (ANOVA) (C) on log-normalized data (B, F-l) or area under the curve (AUC) (E, J). Where only two groups were compared, an unpaired two-tailed t-test on log-normalized data was done (F-l). CFU, colony forming unit; DF, dilution factor; LCN2, lipocalin-2; Lu., Lumen; MFI, median fluorescence intensity; MLN, mesenteric lymph node, S.E., submucosal edema.

Fig. 2 shows modelling of S.Tm^WT extinction for vaccination and/or niche competition. (A)

Adjustment of the model to the data shown in Figure 1. The thick lines correspond to the numerical simulation and the thin lines with dots to the experimental data. Initial S.Tm^WT and S.Trn^Comp CFUs: 5-10³. (B) Extinction probability of the WT Salmonella over time for vaccinated mice +.Trn^Comp. The red line shows the prediction of the model when S.Tm^WT and S.Trn^Comp are given at the same time, the blue line for the case that S.Trn^Comp is given 3 days prior to S.Tm^WT. The simulations were performed with the deterministic model for large population size and with the Gillespie algorithm (1000 realizations) for small size of WT population. (C) Extinction time probability distribution for S.Tm^WT and S.Trn^Comp given at the same time. The y-axis corresponds to (bin height*bin width). Simulations were performed with the deterministic model for large population sizes. For a small S.Tm^WT population size, simulations were performed with the Gillespie algorithm (2500 realizations). The gray area shows trajectories of the 2.5^th percentile extinction time and the 95^th percentile extinction time. Mean of the distribution: 9.2 days, standard deviation: 0.9 days. Initial S.Tm^WT CFUs: 5-10³. Initial S.Trn^Comp CFUs: 5'10³. (D) Extinction time probability distribution for a priori colonization with S.Trn^Comp. The y-axis corresponds to (bin height*bin width). Simulations were performed with the deterministic model for large population sizes. For a small S.Tm^WT population size, simulations were performed with the Gillespie algorithm (5000 realizations). The gray area shows trajectories of the 2.5^th percentile extinction time and the 95^th percentile extinction time. Mean of the distribution: 6.25 days, standard deviation: 0.4 days. Initial S.Tm^WT CFUs: T10⁶. Initial S.Trn^Comp CFUs: 5- 10³. (A, C, D) Parameter values: r_w = 33.7 day^-1, Cw = 6.4 day^-1, r_c = 38.7 da ¹, Cc = 5.9 day¹, r_m = 18.7 day¹, c_m = 1.7 day¹, Ki = 5.9- 10⁹, K₂ = 2.3- 10⁴, K₃ = 10⁷ and mo = 10⁵. r_w, r_c and r_m: growth rate of S.Tm^WT, S.Trn^Comp and the microbiota, respectively. Cw, c_c and c_m: clearance rates of S.Tm^WT, S.Trn^Comp and the microbiota. Ki, K₂, K3: carrying capacities of S.Tm and the microbiota, S.Tm^WT and S.Trn^Comp and the microbiota alone, mo: size of microbiota after antibiotic clearance.

Fig. 3 shows that a priori colonization with S.Trn^Comp can generate sterilizing immunity in the gut when combined with vaccination. PBS (blue symbols) or PA-S.Tm-vaccinated (pink symbols) 129S6/SvEv mice were pretreated with streptomycin and infected with T10⁶ S.Tm^WT. Two groups were pre-colonized with 5- 10³ S.Trn^Comp 3 days before infection. (A) Experimental procedure. (B) S.Tm^WT-specific intestinal IgA and serum IgG titres as determined by flow cytometry. (C) Change in body weight over the course of infection. Solid lines depict the mean. Fecal (D) and cecum content (E) S.Tm CFUs as determined by selective plating. (F) Comparison of the predictions of the model to the experimental data. The thick solid lines correspond to the numerical simulation and the thin lines with dots to the experimental data. Initial S.Tm^WT CFUs: T10⁶. Initial S.Trn^Comp CFUs: 5- 10³. Parameter values: r_w = 33.7 day^-1, Cw = 6.4 day¹, r_c = 38.7 day¹, c_c = 5.9 day¹, r_m = 18.7 day¹, c_m = 1.7 day¹, Ki = 5.9- 10⁹, K₂ = 2.3' 10⁴, K₃ = 10⁷ and m₀ = 10⁵. r_w, r_c and r_m: growth rate of S.Tm^WT, S.Trn^Comp and the microbiota, respectively. Cw, c_c and c_m: clearance rates of S.Tm^WT, S.Trn^Comp and the microbiota. Ki, K2, K₃: carrying capacities of S.Tm and the microbiota, S.Tm^WT and S.Trn^Comp and the microbiota alone, mo: size of microbiota after antibiotic clearance. (G-l) Intestinal inflammation as determined by fecal lipocalin-2 (G) and histopathological scoring of cecal tissue sections (H) Solid lines in (H) show the mean. (I) Representative images of H&E-stained cecal tissue sections. Arrowheads show exemplary goblet cells. Scale bars: 100 pm. (J-L) S.Tm CFUs in MLN (J), liver (K) and spleen (L). Pooled data from two independent experiments with switched antibiotic resistances (n = 5-8 mice/group). Solid lines depict the median unless stated otherwise, error bars the interquartile range. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. Open triangles show mice that had to be euthanized prematurely due to excessive weight loss (> 15%) or disease symptoms. Statistics were performed by mixed-effects analysis (C) or one-way ANOVA (C, G) on log-normalized data (B, E, l-K) or area under the curve (AUC) (D, F). Where only two groups were compared, an unpaired two- tailed t-test on log-normalized data was done (E, l-K). CFU, colony forming unit; DF, dilution factor; FMT, fecal microbial transplant; LCN2, lipocalin-2; Lu., lumen; MFI, median fluorescence intensity; MLN, mesenteric lymph node; S.E., submucosal edema.

Fig. 4 shows that vaccination together with the commensal competitor E. coli 8178 can prevent intestinal pathology albeit S.Tm^WT cannot be cleared from the gut. PBS (blue symbols) or EvoT rap- vaccinated (pink symbols) 129S6/SvEv mice were pretreated with ampicillin and infected with 1 -10⁶ S.Tm^WT. Two groups were pre-colonized with 5-10³ E. coli 8178 3 days before infection. (A) Experimental procedure. (B) S.Tm^WT-specific intestinal IgA and serum IgG titres as determined by flow cytometry. (C) Change in body weight over the course of infection. Solid lines depict the mean. (D-H) CFUs in feces (D) cecum content (E), MLN (F), liver (G) and spleen (H). Intestinal inflammation was determined by fecal lipocalin-2 (I) and histopathological scoring of cecal tissue sections (J). Solid lines in (J) show the mean. (K) Representative images of H&E-stained cecal tissue sections. Arrowheads show exemplary goblet cells. Scale bars: 100 pm. Pooled data from two independent experiments (n = 6-8 mice/group). Solid lines depict the median unless stated otherwise, error bars the interquartile range. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. Open triangles show mice that had to be euthanized prematurely due to excessive weight loss (> 15%) or disease symptoms. Statistics were performed by mixed-effects analysis (C) or one-way ANOVA (C, J) on log-normalized data (B, E-H) or area under the curve (AUC) (D, I). Where only two groups were compared, an unpaired two-tailed t-test on log-normalized data was done (E-H). CFU, colony forming unit; DF, dilution factor; LCN2, lipocalin-2; Lu., lumen; MFI, median fluorescence intensity; MLN, mesenteric lymph node; S.E., submucosal edema.

Fig. 5 shows EvoTrap vaccination together with niche competition provides better intestinal protection than a licensed animal vaccine in the murine NTS model. PBS (blue circles), EvoT rap- vaccinated (pink diamonds) or S.Tm^aroA vaccinated (green triangles) 129S6/SvEv mice were pretreated with ampicillin and infected with T10⁶ S.Tm^WT. EvoTrpa-vaccinated mice were pre-colonized with T10⁹ E. coli 8178 3 days before infection. (A) Experimental procedure. (B) S.Tm^WT-specific intestinal IgA and serum IgG titres as determined by flow cytometry. (C) Change in body weight over the course of infection. Solid lines show the mean. Fecal (D) and cecum content (E) CFUs as determined by selective plating. Intestinal inflammation as determined by fecal lipocalin-2 (F) and histopathological scoring of cecal tissue sections (G). Solid lines in (G) show the mean. (H) Representative images of H&E-stained cecal tissue sections. Arrowheads show exemplary goblet cells. Scale bars: 100 pm. (I-K) CFUs in MLN (I), liver (J) and spleen (K). N = 5 mice/group. Solid lines depict the median unless stated otherwise, error bars the interquartile range. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. Open triangles show mice that had to be euthanized prematurely due to excessive weight loss (> 15%) or disease symptoms. Statistics were performed by mixed-effects analysis (C) or one-way ANOVA (C, G) on log-normalized data (B, E, l-K) or area under the curve (AUC) (D, F). CFU, colony forming unit; DF, dilution factor; LCN2, lipocalin-2; Lu., lumen; MFI, median fluorescence intensity; MLN, mesenteric lymph node; S.E., submucosal edema.

Fig. 6 shows S.Tm^aroA vaccination protects better against systemic invasion of S.Tm^WT in the murine Typhi model. PBS (blue circles), EvoT rap-vaccinated (pink diamonds) or S.Tm^aroA vaccinated (green triangles) 129S6/SvEv mice were infected with 1 ■ 10⁶ S.Tm^WT without antibiotic pretreatment. EvoTrpa-vaccinated mice were pre-colonized with 1 ■ 10⁹ E. coli 8178 3 days before infection. (A) Experimental procedure. Fecal (B) and cecum content (C) CFUs as determined by selective plating. (D) Intestinal inflammation as determined by fecal lipocalin-2. (E-G) CFUs in MLN (E), liver (F) and spleen (G). N = 5 mice/group. Solid lines depict the median, error bars the interquartile range. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. Open triangles show mice that had to be euthanized prematurely due to excessive weight loss (> 15%) or disease symptoms. Statistics were performed by ANOVA on log- normalized data (C, E-G) or area under the curve (AUC) (B, D). CFU, colony forming unit; LCN2, lipocalin-2; MLN, mesenteric lymph node.

Fig. 7 shows that S.Trn^Comp is not pathogenic in 129S6/SvEv and C57BL/6J mice. (A-F)

129S6/SvEv mice were pretreated with streptomycin and infected with a total of 10⁵ of a 1 :1 mixture of two isogenic S.Trn^Comp strains and colonization was followed for 10 days. S.Trn^Comp CFUs were determined by selective plating in feces (A) cecum content (B), MLN (C), liver (D) and spleen (E). (F) Intestinal inflammation was determined by measuring fecal lipocalin-2. (G- L) C57BL/6 mice were pretreated with streptomycin and infected with a total of 10⁴ of a 1 :1 mixture of two isogenic S.Trn^Comp strains and colonization was followed for 7 weeks. S.Trn^Comp CFUs were determined by selective plating in feces (G) cecum content (H), MLN (I), liver (J) and spleen (K). (L) Intestinal inflammation was determined by measuring fecal lipocalin-2. N = 5-7 mice. Solid lines depict the median, error bars the interquartile range. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. Statistics were performed by an unpaired two-tailed t-test on log-normalized area under the curve (AUC) (A, G). CFU, colony forming unit; LCN2, lipocalin-2; MLN, mesenteric lymph node.

Fig. 8 shows bacterial generations in vivo at 12 hours post infection. (A) PBS (blue symbols) or PA-S.Tm-vaccinated (pink symbols) 129S6/SvEv mice were pretreated with streptomycin and infected with a total of 10⁴ of a 1 :1 mixture of two isogenic S.Tm^WT strains (circles) or S.Tm^WT and S.Tm^Comp (S^m^ossavoaM^ fj||_ec| diamonds). Number of generations 12 hours post infection was estimated based on the loss of pAM34. Open and filled circles show the two isogenic S.Tm^WT strains. (B) PBS (blue symbols) or PA-S.Tm-vaccinated (pink symbols) 129S6/SvEv mice were pretreated with streptomycin and infected with 1 ■ 10⁶ S.Tm^WT. Two groups were pre-colonized with 5- 10³ S.Trn^Comp 3 days before infection. Number of generations 12 hours post infection was calculated based on the loss of pAM34. Solid lines depict the mean. Statistics were performed by one-way ANOVA.

Fig. 9 shows that vaccination together with S.Trn^Comp a priori colonization prevents S.Tm^WT transmission. Naive 129S6/SvEv mice were pretreated with streptomycin and a FMT was performed with feces collected from 9 days post infection of untreated (blue circles) or vaccinated and S.Trn^Comp pre-colonized mice (pink diamonds; see Fig. 3). S.Tm^WT and S.Trn^Comp CFUs were determined by selective plating in feces (A) cecum content (B), MLN (C), liver (D) and spleen (E). Pooled data from two independent experiments with switched antibiotic resistances (n = 2 or 8 mice/group). Solid black lines depict the median. Dotted lines show the detection limit and the shaded area the range for cases in which the detection limit is dependent on sample weight. CFU, colony forming unit; FMT, fecal microbial transplant; MLN, mesenteric lymph node.

Fig. 10 shows that vaccination with the EvoTrap vaccine generates an intestinal IgA and serum IgG response against all S.Tm O-antigen variants. 129S6/SvEv mice were mock-vaccinated with PBS (blue symbols) or EvoT rap-vaccinated (pink symbols) and later infected with T10⁶ S.Tm^WT with or without pre-colonization with 5 10³ E. 00118 78. 10 days after S.Tm^WT infection S.Tm specific antibody responses were determined in intestinal lavage (A-C) and serum (D- E) by flow cytometry. (A+D) S.Tm 0:4,12-0. (B+E) S.Tm O:4[5],12-2. (C+F) S.Tm 0:4,12-2. Pooled data from two independent experiments (n = 6-8 mice/group). Solid lines depict the. Dotted lines show the detection limit. Open triangles show mice that had to be euthanized prematurely due to excessive weight loss (> 15%) or disease symptoms. Statistics were performed one-way ANOVA on log-normalized data (A-C). Where only two groups were compared, an unpaired two-tailed t-test on log-normalized data was done (D-F). DF, dilution factor; MFI, median fluorescence intensity.

Fig. 11 shows that vaccination with the EvoT rap vaccine leads to emergence of S.T m^WT clones with short O-antigen. 129S6/SvEv mice were EvoT rap-vaccinated and later infected with T10⁶ S.Tm^WT with or without pre-colonization with 5 10³ E. coli 8178. S.Tm^WT clones were isolated from feces from 4 days post infection onwards. (A) S.Tm^WT clones were monitored for decreased 0:5 and 0:12-0 staining intesity by flow cytometry. (B) Silver-stained gel of LPS from selected S.Tm^WT clones from 4 different EvoTrap vaccinated mice.

Fig. 12 shows different regimes used for mathematical modelling. PA-S.Tm-vaccinated 129S6/SvEv mice were pretreated with streptomycin and infected with a total of 10⁴ of a 1 :1 mixture of S.Tm^WT (blue symbols) and S.Trn^Comp (purple symbols). Ri defines the time window where S.Tm^WT, S.Trn^Comp and the microbiota are all below their carrying capacity. R2 is defined by S.Trn^Comp being more abundant than S.Tm^WT and the microbiota, and being in the order of magnitude of Ki. R3 is defined by the microbiota being more abundant than S.Tm^WT and S.Trn^Comp but S.Tm^Comp is still present in higher numbers than its carrying capacity in equilibrium K2. R4 is defined by the microbiota being much more abundant than S.Tm^WT and S.Trn^Comp and S.Trn^Comp being in the order of magnitude of K2.

Examples

The inventors hypothesized that the critical components of a prophylactic system against intestinal bacterial disease are: 1 ) a vaccine capable of inducing high-affinity specific IgA against the pathogen of interest; 2) a non-pathogenic strain with (ideally) complete metabolic niche overlap with the pathogen of interest, a faster growth-rate than the pathogen of interest and absence of surface antigen crossreactivity to the pathogen of interest. Additionally, questions arise as to how vaccines and competitors can be combined temporally to give maximum effect, and the extent of protection from invasive disease that can be achieved with such an approach. They made use of the well-established and severe, murine model of non-Typhoidal Salmonellosis (NTS) to establish this concept. In this model, oral antibiotics are applied to acutely generate a large open niche for S.Tm in the mouse cecum and upper large intestine (Barthel, M. et al., Infect Immun 71 , 2839-2858, (2003)). Subsequently, very low numbers of S.Tm can be orally inoculated and will rapidly grow to fill the available niche. Disease depends on the activity of Salmonella Pathogenicity Islands 1 and 2 and includes acute typhlocolitis, colonization of the intestinal tissue, mesenteric lymph nodes, spleen and liver and weight-loss. In the resistant (Nramp1+/+, also known as Slc11 a1 ) 129SJL mouse strain, the disease is slowly controlled, but full recovery takes more than 1 month. They also extend their observations to the murine oral Typhoid fever model, in which a high-dose of S.Tm is delivered orally to mice with an intact intestinal microbiota, resulting in a disease that is more reliant on the tissue-invasive stages of disease.

Example 1 Combining oral inactivated bacterial vaccine immunization with a bacterial niche competitor protects mice from intestinal inflammation and leads to rapid S. Tm^WT clearance in a model of NTS.

As a first proof-of-principle the inventors made use of S.Tm carrying a mutation in the “Salmonella Pathogenicity Island 1 ” (SPI-1 ) master regulator hilD, that has previously been demonstrated to outgrow wild-type S.Tm in the gut lumen during long-term infections. This was combined with inactivating mutations in ssaV to disrupt the function of “Salmonella Pathogenicity Island 2” (SPI-2) and deletion of oafA to prevent acetylation of the S.Tm O-antigen, i.e. preventing generation of the 0:5 epitope. The resulting strain is fully avirulent (Fig. 7), fast-growing’ and less bound by IgA induced by a wild-type 0:5,12-0 Salmonella vaccine. The resulting mutant S.Tm ^{,D ssaV oaM} was used as niche competitor hereafter named S.Trn^Comp.

To demonstrate the effect of pathogen-targeting IgA on competition, SPF 129S6/SvEv wildtype mice received an inactivated whole-cell oral S.Tm vaccine once weekly for 4 weeks or a mock PBS treatment. Subsequently, mice were antibiotic treated to eliminate a large part of the microbiota and infected with either virulent wildtype S.Tm (S.Tm^WT) alone, or S.Tm^WT combined 1 :1 with S.Trn^Comp. Intestinal colonization and inflammation were monitored for 10 days post infection and tissue invasion and histopathology was monitored at endpoint (Fig 1A). In line with published data, the inventors could detect high levels of S.Tm O:5,12-0-specific IgA in the intestine as well as IgG in serum of vaccinated mice at endpoint, regardless of the subsequent infection (Fig. 1B). This S.Tm specific antibody response was able to protect the vaccinated mice from weight loss upon infection with WT S.Tm (Fig. 1C). In contrast, all mice that were not vaccinated experienced severe weight loss until 5 days post infection. One unvaccinated mouse of each of two independent experiments had to be euthanized at 5 days post infection due to excessive weight loss.

As antibiotic pre-treatment in this model generates a huge empty niche for S.Tm colonization in the gut lumen, in vivo binding of IgA to S.Tm^WT could not prevent initial expansion of S.Tm, and total S.Tm counts were similar in all groups. However, over time the differences between groups become clear: S.Tm^WT CFU/g feces remains constant over 10 days in unvaccinated mice without S.Trn^Comp. The presence of either vaccination alone, or S.Trn^Comp results in a drop in S.Tm^WT CFU from day 2-3 post infection, with CFU 10⁵ CFU/g feces at day 10. In contrast, the combination of vaccination and S.Trn^Comp generates an exponential drop in fecal CFU of S.Tm^WT over the first 5 days of infection, with some mice completely clearing the pathogenic strains by day 5 (Fig. 1D and E). The cecum content counts reflected the picture found in feces with a much lower burden of S.Tm^WT in all treated groups and absence of S.Tm^WT in 50% of the mice that received vaccination and the niche competitor (Fig. 1F). Interestingly, despite a very dramatic clearance of S.Tm^WT from the gut lumen, colonization of systemic sites was apparently unaltered by either vaccination or by introduction of the niche competitor (Fig. 1G-I). This is consistent with quantitative modelling of tissue invasion in murine NTS, which predicted that a more than 1000-fold reduction in Salmonella intestinal load is required to completely inhibit systemic spread. As it takes several days for the S.Tm^WT levels to be reduced below this level in our treatment groups, there remains a considerable time-window for systemic spread to occur. While the inventors cannot exclude that differences may have been more apparent at earlier time-points, this clearly indicates that encounter of a niche competitor simultaneous to infection, even in orally vaccinated mice that display both intestinal IgG and serum IgA responses, is not sufficient to prevent colonization of the spleen and liver of mice.

The partial-protective capacity of IgA with and without niche competition was also apparent when looking at intestinal inflammation as measured by lipocalin-2 levels in feces. In both vaccinated groups, inflammation not only started later but also the maximum fecal lipocalin-2 was significantly lower throughout the whole course of infection (Fig 1 J). This was underpinned by histological stainings at day 10 post challenge that showed no pathological changes in mice that were both vaccinated and colonized with S.Trn^Comp, and only mild changes in the vaccine-alone group (Fig. 1K). Intestinal inflammation could not be significantly prevented by the presence of the niche-competitor alone when it is introduced simultaneously with the infectious challenge (Fig. 1J and K).

These experiments indicated the feasibility of combining vaccination and niche competition, while revealing the need to optimize the procedure for protection of both the gut and systemic sites.

Example 2: Modelling the interaction of niche competition and vaccination

As intestinal colonization is necessarily a highly dynamic process, the inventors built a simple mathematical model to generate predictions on the requirements for extinction of S.Tm^WT and the time- to-exti notion. The inventors predict that minimizing the time-to-exti notion in the gut lumen will best inhibit systemic spread of S.Tm and can minimize the risk of immune escape.

In this model the inventors assumed that the microbiota (of size M(t)), S.Trn^Comp (of size C(t)) and S.Tm^WT (of size W(t)) compete for undefined shared nutrient resources, which leads to a carrying capacity Ki. Additionally, the inventors assumed a second independent nutrient source for S.Trn^Comp and S.Tm^WT on the one hand, and for the microbiota on the other hand leading to carrying capacities K2 and K3. In a deterministic view, the size of the populations evolves as

Eq.

where r_w, r_c and r_m correspond to the growth rate of S.Tm^WT, S.Trn^Comp and the microbiota, respectively. The parameters Cw, c_c and c_m correspond to the clearance rates of these populations.

In order to predict “extinction probability” and “extinction time”, the inventors required realistic estimates of the population dynamics of the two S.Tm strains and the “average microbiome”. S.Tm population dynamics parameters in the presence or absence of IgA were estimated by fitting the competition data shown in Figure 1 and Figure 8A (see materials and methods) (Fig 2A). This confirmed the predicted higher net growth rate for S.Trn^Comp as compared to S.Tm^WT and the elevated clearance rate of S.Tm^WT in vaccinated mice (see Table 1 ).

Table 1 : Kinetic parameter values inferred from the competition data shown in Fig. 1 (for vaccinated + S.Trn^Comp group). r_w, r_c and r_m: growth rate of S.Tm^WT, S.Trn^Comp and the microbiota, respectively. Cw, c_c and c_m: clearance rates of S.Tm^WT, S.Trn^Comp and the microbiota. Ki, K2, K3: carrying capacities of S.Tm and the microbiota, S.Tm^WT and S.Trn^Comp and the microbiota alone, mo: size of microbiota after antibiotic clearance at time point of infection with S.Tm^WT.

Using 2500 iterations of a stochastic version of this model, the inventors could derive extinction-related parameters. Based on continuous growth and clearance of the gut luminal Salmonella population, these models indicate clearance of virulent Salmonella within 10 days of infection in the mice treated with vaccination and S.Trn^comp, compared to continuous colonization in mice untreated or receiving vaccine only (Fig 2B and C). Interestingly, the model predicted that the time-point for introduction of the niche competitor was not a major variable, as long as this occurred prior to infection. Major determinants of success are the relative growth rates and loss rates of the competitor versus the virulent bacterium as well as the carrying capacity, which is in turn determined by the regrowth of the microbiota. Therefore, a major conclusion of the model is that the niche competitor should be already present in the gut for effective protection (Fig. 2B and D): this is also a more realistic situation for disease prophylaxis.

As our model allowed scaling to larger animals and transmission processes, the inventors additionally modelled the potential benefit of such a vaccination/competitor regimen in the realistic situation of transmission between domestic pigs under typical farm conditions. A very simple link exists between the duration and intensity of Salmonella shedding and the probability of transmission. As the duration and intensity of shedding can be dramatically reduced by vaccination/competition, the model reveals a major potential for this intervention to reduce the Salmonella burden in farm animals.

Example 3: Introduction of the niche competitor a priori allows complete pathogen clearance when combined with oral vaccination

Based on the modelling data, the inventors carried out a vaccination and challenge experiment, this time introducing the competitor strain 3 days prior to infection (Fig. 3A). The inventors additionally increased our S.Tm^WT infection dose to T10⁶ CFU, in order to increase the stringency of the challenge. Again, the inventors could confirm that vaccination leads to a robust induction of S.Tm-specific intestinal IgA as well as serum IgG (Fig. 3B), and this protected animals from weight loss upon S.Tm^WT infection, regardless of the higher dose. In contrast to simultaneous infection, introducing S.Trn^Comp a priori into naive mice also prevented weight loss (Fig. 3C). In animals infected without S.Trn^Comp, the inventors observe very similar infection kinetics to those induced by challenging with 5 10³ CFU. By contrast, S.Tm^WT struggled to expand at all in mice already colonized with the competitor(Fig 3D) and was eliminated as soon as 4 days post infection in mice protected by both vaccination and pre-colonization with S.Trn^Comp. S.Tm^WT was undetectable in the cecum content in 5 of 8 “vaccinated, S.Trn^Comp colonized” mice on the final day of infection (Fig. 3E). This fitted well to predictions from the model derived above (Fig. 3F) Intestinal inflammation could also be completely prevented by the combined prophylaxis, remaining within the range of healthy animals for the full duration of the infection (Fig. 3G- I). Moreover, systemic spread of S.Tm^WT could be effectively prevented by the combination of vaccination and S.Tm^Comp in around 50% of treated animals (Fig. 3J-L), and correlated well with early loss of S.Tm^WT from the gut, rendering some mice completely pathogen free at 10 days post infection. Therefore, combining an inactivated oral vaccine with a live niche competitor can generate sterilizing immunity against virulent Salmonella in a very severe model of non-Typhoidal Salmonellosis.

To further corroborate our findings of sterilizing immunity, the inventors performed fecal microbial transplants from vaccinated/S.Trn^Comp mice into naive Streptomycin pre-treated 129S6/SvEv mice. All but one mouse from this group, did not efficiently transmit S.Tm^WT by fecal microbial transplantation (FMT) (Fig. 9). In contrast, all untreated control mice used for this experiment efficiently transferred S.Tm^WT and disease to the naive mice (Fig. 9).

In summary, the inventors could show that our niche competitor when given a priori is able to establish a partial colonization resistance thereby limiting intestinal S.Tm^WT expansion. When combined with vaccination, a major fraction of the animals could completely clear S.Tm^WTfrom all examined sites i.e. generating sterilizing immunity. Importantly, the inventors could not only greatly ameliorate disease in animals treated with this method, but the inventors could also prevent transmission in almost 90% of the cases.

Example 4: Commensal competitors and vaccination

While the combination of inactivated oral vaccines and an engineered Salmonella-ci erived competitor is highly effective, there remain potential safety/legislative issues associated with the application of live GMOs and a very low, but non-zero, risk of reversion of the competitor strain via horizontal gene transfer. An attractive alternative would be to develop a non-pathogenic commensal bacterial strain that can nevertheless compete efficiently with S.Tm^WT. For this purpose, the inventors chose the mouse commensal ECOR B2 strain E. coli 8178 (Ec⁸¹⁷⁸). This has been demonstrated to grow well in S.Tm- permissive conditions and could limit S.Tm infection after dietary perturbation. Moreover, this E. coli produces an unrelated O-antigen structure, allowing the inventors to use an “evolutionary trap” version of our S.Tm vaccine covering all possible variations of the S.Tm O-antigen. This adds robustness to the technique as selective pressure exerted by vaccine-induced IgA can provide a selective advantage both to the competitor strain, and to naturally emerging S.Tm variants with a short O-antigen. As mutants with short LPS are susceptible to complement, bile acids and other membrane-targeting stresses, this is associated with decreased virulence of S.Tm^WT in vaccinated mice.

The inventors therefore repeated our previous experiment using the evolutionary trap (EvoTrap) vaccine together with Ec⁸¹⁷⁸ as a competitor given 3 days prior to challenge (Fig. 4A). Vaccination with the EvoTrap vaccine led to high levels of S.Tm 0:5,12-0 specific antibodies in small intestine and blood (Fig. 4B) as well as to the other S.Tm O-anitgen variants (Fig. 10). Similarly, to what the inventors have observed with giving S.Trn^Comp prior to S.Tm^WT infection, also Ec⁸¹⁷⁸ alone could reduce weight loss after challenge and weight loss was prevented in both vaccinated groups (Fig. 4C). Ec⁸¹⁷⁸ colonized the mouse gut to high levels and was able to decrease initial S.Tm^WT expansion. However, Ec⁸¹⁷⁸ was clearly less effective as a competitor in the intestine when compared with S.Trn^Comp, as S.Tm^WT levels remained high (>10⁶) in feces and cecum of all groups at day 10 post infection (Fig. 4D and E). Despite the high S.Tm^WT numbers in the gut, the inventors observed a significant reduction in systemic counts (mesenteric lymph nodes, spleen and liver) in the vaccinated group that received Ec⁸¹⁷⁸ (Fig. 4F-H). When analysing intestinal inflammation, the inventors again found that the intestinal inflammation marker lipocalin-2 was almost completely absent from the “vaccinated plus Ec⁸¹⁷⁸ pre-colonized” group, indicating robust protection from tissue invasion and disease, despite incomplete clearance of S.Tm^WT from the gut lumen (Fig 4I-K). As predicted with EvoTrap vaccination, a major fraction of luminal S.Tm^WT re-isolated from vaccinated mice carried a spontaneous deletion resulting in loss of wzyB and therefore short O-antigen production, which may explain this discrepancy (Fig. 11).

In summary, the inventors could show that a more distantly related competitor does not compete as well in the gut but nonetheless is able to abolish systemic invasion and intestinal inflammation, and this may represent a more easily translatable model. Moreover, future analysis of the metabolism of S.Tm and commensal E. coli strains may allow for the identification of strain combinations with a more complete metabolic niche overlap.

Taken together, the inventors here demonstrate that the combination of an inactivated oral vaccine and a competitor strain in the gut lumen can protect from S.Tm colonization and disease in murine models. The extent of gut lumen clearance was well-correlated with fecal lipocalin-2 and intestinal inflammation.

Example 5: Comparing vaccination/niche competition to licensed animal vaccines reveals mechanistic differences in protection of the intestine and systemic sites.

Live-attenuated non-Typhoidal Salmonella vaccines, carrying a mutation in aroA, which renders the strain auxotrophic for aromatic amino acids, are already used for broilers and very similar auxotrophic strains are used in pig-farming. In order to benchmark our approach to a known S.Tm vaccine the inventors therefore compared protective efficacy and safety of the combined inactivated oral vaccine+Ec⁸¹⁷⁸ treatment to that of S.Tm^aroA vaccination. To gain mechanistic insight into protection at the tissue level versus the gut lumen, the inventors also compared protection in the NTS model to protection in the murine oral typhoid model, in which no major gut luminal niche is generated.

In the NTS model (Fig. 5A), both the classical S.Tm^aroA vaccination as well as EvoTrap vaccination+Ec⁸¹⁷⁸ led to the induction of S.Tm^WT specific antibodies (Fig. 5B) and prevented weight loss (Fig. 5C). EvoTrap vaccination+Ec⁸¹⁷⁸ resulted in significantly lower levels of S.Tm^WT in feces and cecum (Fig. 5D and E) and completely prevented intestinal inflammation (Fig. 5F-H). S.Tm^aroA vaccination, in contrast, showed no effect on fecal or cecal S.Tm loads (Fig. 5D and E) and only a very small suppression of intestinal inflammation (Fig. 5F-H). Interestingly, although EvoTrap vaccination+Ec⁸¹⁷⁸ showed better protection at the level of the gut and mesenteric lymph nodes (Fig. 5D, E and I), the classical S.Tm^aroA vaccination provided better protection from systemic S.Tm^WT infection. Liver and spleen CFU of S.Tm^WT were not dramatically decreased in this experiment by EvoTrap vaccination+Ec⁸¹⁷⁸ but were strongly reduced by vaccination with live S.Tm^aroA (Fig. 5J and K). Interestingly, although S.Tm^aroA was absent from cecum and feces it could still be found in MLNs and liver at day 46 post-vaccination (Fig 5I-K), consistent with known safety challenges associated with these live-attenuated vaccines.

In the murine typhoid model (Fig. 6A), both vaccine regimens could prevent intestinal colonization (Fig. 6B and C) and could supress any detectable increase in intestinal lipocalin-2 levels (Fig. 6D). MLN colonization (Fig. 6E) correlated well with intestinal colonization levels at day 10 post infection. As in the murine NTS model, vaccination with S.Tm^aroA resulted in superior protection against infection of liver and spleen (Fig. 6F and G). However, persistent S.Tm^aroA was again found in systemic sites a full 17 days after the last vaccination.

These data imply that protection of the gut tissue and of systemic sites require different mechanisms and can be uncoupled: vaccination/niche competition provides superior protection of all examined gut sites and gut-draining lymphoid tissues, while live-attenuated vaccines provide better protection of deep systemic sites. This therefore reveals both a promising approach for improved safe prophylaxis of intestinal bacterial infections and a system to investigate missing stimuli from whole-cell inactivated oral vaccines to improve their protection of systemic sites.

Example 6: Estimation of in vivo growth rates in the gut by plasmid dilution

Absolute S.Tm growth rates in the gut were assessed using replication-incompetent plasmid pAM34, which has been described previously. Briefly, pAM34 is a ColE1 -like vector in which the replication of the plasmid is under the control of the Lacl repressor, whereby plasmid replication only occurs in the presence of isopropyl p-d-1 -thiogalactopyranoside (IPTG). S.Tm carrying the pAM34 plasmid was therefore cultured overnight in the presence of 1 mM IPTG in LB containing streptomycin. Cultures were diluted 1 :20 into fresh LB broth without IPTG or antibiotics and sub-cultured for 3 h at 37 °C. Inocula for infection were prepared as described above. Concurrently, the inoculum was serially diluted into fresh LB broth without IPTG and cultured for 20 h at 37 °C to generate a standard curve relating plasmid loss to generations undergone for each experiment. pAM34-carrying bacteria within the overnight cultures and the feces were determined by selective plating on agar plates containing 50 pg/ml ampicillin and 1 mM IPTG. To quantify the total population size, samples were further plated on agar plates containing 100 pg/ml streptomycin. The fraction of pAM34-carrying bacteria was calculated using the ratio of pAM34-carrying CFU to the total population CFU and generations estimated by interpolation from the matched standard curve.

Example 7: Non-typhoidal Salmonella transmission

Donor mice were vaccinated with PA-S.Tm as described above, orally pretreated with 25 mg streptomycin, and colonized 24 h later with 5- 10³ S. Trn^Comp. 2 days later, mice were treated again with 25 mg Streptomycin per os, and 24 h later infected with T10⁶ S. Tm^WT. On day 9 post infection, one fecal pellet was collected from each mouse, weighed and homogenized in 200 pl PBS. Large debris was removed by centrifugation at 500x g for 1 minute and 50 pl of the supernatant were immediately given by oral gavge to streptomycin pretreated naive recipient mice. As a control, the same procedure was done using naive mice without competitor colonization as donor mice. Recipient mice were euthanized, and organs were collected on day 3 post transmission. In both donor and recipient mice, fecal pellets were collected daily and selective plating was used to enumerate Salmonella and determine the relative proportions of both competing bacterial strains.

Example 8: Flow cytometry for analysis of 0:5 and 0:12-0 intensity on Salmonella clonal cultures Overnight cultures (1 pl) made in 0.2-pm-filtered lysogeny broth was stained with 0.2-pm-filtered solutions of STA5 (human recombinant monoclonal lgG2 anti-O:12-0; 3.2 pg/ml)¹⁶ or rabbit anti- Salmonella 0:5 (Difco; 1 :200). After incubation at 4 °C for 30 min, the bacteria were washed twice by centrifugation at 7000x g and resuspension in PBS/2% BSA. The bacteria were then resuspended in 0.2-pm-filtered solutions of appropriate secondary reagents (Alexa 647-anti-human IgG (Jackson ImmunoResearch; 1 :100) and Brilliant Violet 421 -anti-rabbit IgG (BioLegend; 1 :100)). This was incubated for 30 min at 4°C before the cells were washed as above and resuspended for acquisition on a Beckman Coulter Cytoflex S.

Example 9: LPS purification and silver staining

LPS was isolated by applying the hot phenol-water method (Luderitz, O. T. T. O., et al. "Isolation and chemical and immunological characterization of bacterial lipopolysaccharides." Microbial toxins 4 (2016): 145-233), followed by buffer exchange against 15 ml PBS and concentration in 500 pl PBS. LPS samples were separated on a 13% Tricin gel by gel electrophoresis and silver staining was performed.

Example 10: Material and Methods

Strains and plasmids

Bacteria were cultivated in lysogeny broth (LB) containing appropriate antibiotics (100 pg/ml streptomycin (AppliChem); 15 pg/ml chloramphenicol (AppliChem); 50 pg/ml kanamycin (AppliChem); 50 pg/ml ampicillin (AppliChem)). Dilutions were prepared in Phosphate Buffered Saline (PBS, Difco).

Gene-deletion mutants were created by generalized transduction with bacteriophage P22 HT105/1 int- 201 as described in. When needed, antibiotic resistance cassettes were removed using the temperatureinducible FLP recombinase encoded on pCP20. Deletions originated from in-frame deletions made in S.Tm 14028S, kind gifts from Prof. Michael McClelland (University of California, Irvine). Primers used for verifications of gene deletions or genetic background are listed Table 2.

Table 2: Listing of primers used for verification of gene deletions or genetic background.

Plasmids were transferred by electro-transformation into competent cells (Wotzka, S. Y. et al. Nat Microbiol 4, 2164-2174, doi:10.1038/s41564-019-0568-5 (2019); Gil, D. & Bouche, J. P. Gene 105, 17- 22, doi:Doi 10.1016/0378-1119(91 )90508-9 (1991 ).). Mice

All animal experiments were performed in accordance with Swiss Federal regulations approved by the Commission for Animal Experimentation of the Kanton Zurich (licenses 193/2016, 158/2019 and 120/2019; Kantonales Veterinaramt Zurich, Switzerland). Specific opportunistic pathogen-free (SPF, containing a complete microbiota free of an extended list of opportunistic pathogens) 129S6/SvEvTac mice were used in all experiments. Mice were bred and housed in individually ventilated cages with a 12 h light/dark cycle in the ETH Phenomics Center (EPIC, RCHCI), ETH Zurich and were fed a standard chow diet. Wherever possible an equal number of males and females was used in each group. As strong phenotypes were expected, the inventors adhered to standard practice of analysing at least 5 mice per group. Researchers were not blinded to group allocation to decrease the risk of contamination.

Vaccinations

Mice were either vaccinated with peracetic acid (PA) killed vaccines or live-attenuated S.Tm^aroA.

Peracetic acid killed vaccines were produced as previously described (Moor, K. et al., Front Immunol 7, 34, doi:10.3389/fimmu.2016.00034 (2016)). Briefly, bacteria were grown overnight to late stationary phase, harvested by centrifugation and re-suspended to a density of 1O⁹-1O¹⁰ per ml in sterile PBS. Peracetic acid (Sigma-Aldrich) was added to a final concentration of 0.4% v/v. The suspension was mixed thoroughly and incubated for 60 min at room temperature. Bacteria were washed three times in 50-100 ml sterile PBS. The final pellet was resuspended to yield a density of 10¹¹-10¹² particles per ml in sterile PBS. The exact number was determined by flow cytometry with counting beads (Fluoresbrite® Multifluorescent Microspheres). Vaccines were stored at 4 °C for up to three weeks. Each batch of vaccine was tested for sterility before use. Vaccine lots were released for use only when a negative enrichment culture had been confirmed. Mice were vaccinated with 10¹°-10¹¹ PA-killed bacteria by oral gavage, once weekly for 4 weeks. Where multiple strains were used, equal numbers of each strain were given.

Live-attenuated S.Tm^aroAwas grown overnight in LB containing chloramphenicol. The cells were washed in PBD and resuspended at a density of 10¹° bacteria per ml. Mice were orally vaccinated with 10⁹ S.Tm^aroA in 100 pl three time in bi-weekly intervals without antibiotic treatment. Vaccinations were started in mice at an age of 4-6 weeks.

Colonization with the bacterial niche competitor and Salmonella challenge infections

The competitor strain was grown overnight in LB containing the appropriate antibiotics. In the morning, the bacteria were washed with sterile PBS and diluted. The competitor was introduced by oral gavage into the respective groups either at 5-10³ CFUs after antibiotic pre-treatment or at T10⁹ CFUs without antibiotic pr-treatment of the animals.

Non-typhoidal Salmonella infections were carried out as previously described (Barthel, M. et al. ibid). In brief, mice were orally pretreated 24 h before infection with 25 mg streptomycin or 20 mg ampicillin. Strains were cultivated overnight separately in LB containing the appropriate antibiotics. Subcultures were prepared before infections by diluting overnight cultures 1 :20 in fresh LB without antibiotics and incubation for 3 h at 37 °C. The cells were washed in PBS, diluted, and 100 pl of bacteria were used to infect mice per os with either 5-10³ or 1 ■ 10⁶ S.Tm CFUs, as indicated in the respective figure legends/text. Competitions were performed by inoculating 1 :1 mixtures of each competitor strain. For mouse typhoid-like infection, the animals were infected with 1 -10⁶ S.Tm CFUs without prior antibiotic treatment. A detailed layout of the vaccination and infection schedule is shown in the figures.

Feces were sampled daily, homogenized in 500 pl PBS by bead beating (3 mm steel ball, 25 Hz for 2.5 min in a TissueLyser (Qiagen)), and large particles were sedimented by centrigugation at 500x g for 1 minute. Bacteria were enumerated by selective plating on MacConkey agar supplemented with the appropriate antibiotics. Fecal samples for lipocalin-2 measurements were kept homogenized in PBS at -20 °C. At endpoint, blood was collected from the heart into 1.1 ml serum gel tubes (Sarstedt). Intestinal lavages were harvested by flushing the small intestinal content with 2 ml of PBS using a cannula. The middle part of the cecum was placed into OCT Compound (Tissue-Tek), snap-frozen and stored at - 80 °C until analysis. Spleen, liver, mesenteric lymph nodes were collected and homogenized in 1 ml PBS at 30 Hz for 3 min. Cecum content was collected and homogenized in 500 pl PBS at 25 Hz for 2.5 min. After centrifugation at 500x g for 1 minute, bacteria were plated on selective MacConkey agar.

Quantification of fecal lipocalin-2

Fecal pellets were processed as described above. Homogenized feces was centrifuged at 16000x g for 5 minutes and the resulting supernatant was analysed in duplicates using the mouse lipocalin-2 ELISA duoset (R&D) according to the manufacturer's instructions.

Analysis of specific antibody titres by bacterial flow cytometry

Specific antibody titres in mouse intestinal washes and serum were measured by flow cytometry as described in Moor, K. et al., Nat Protoc 11 , 1531-1553, 2016. Briefly, intestinal washes and blood were collected as described above. Blood was centrifuged at 10000x g for 5 minutes to obtain serum, heat- inactivated at 56 °C for 30 min and stored at -20 °C until further analysis. Intestinal lavage was centrifuged at 16000x g for 5 min to clear all bacterial-sized particles and stored at -20 °C until analysis. Bacterial targets (antigen against which antibodies are to be titred) were grown overnight in LB, then gently pelleted for 2 min at 7000 g. The pellet was washed with 0.2 pm-filtered PBS before resuspending at a density of approximately 10⁷ bacteria per ml. After thawing, intestinal washes were centrifuged again at 16000x g for 5 min. Supernatants were used to perform serial dilutions. 50 pl of the dilutions were incubated with 50 pl bacterial suspension for 15 min at room temperature. Bacteria were washed twice with 150 pl PBS by centrifugation at 7000x g for 5 min, before resuspending in 25 pl of 0.2 pm- filtered PBS containing polyclonal Alexa Fluor 647 Rabbit Anti-Mouse IgG (Jackson ImmunoResearch, 15 pg/ml) or monoclonal Brilliant Violet 421 Rat Anti-Mouse IgA (BD Bioscience, 2 pg/ml). After 5 min of incubation at RT, bacteria were washed twice with PBS as above and resuspended in 100 pl PBS for acquisition on a Beckman Coulter Cytoflex S using FSC and SSC parameters to threshold acquisition in logarithmic mode. Data were analysed using FlowJo (Treestar). After gating on bacterial particles, log-median fluorescence intensities (MFI) were plotted against lavage dilution factor for each sample and 4-parameter logistic curves were fitted using Prism (Graphpad, USA). Titers were calculated from these curves as the dilution factor giving an above-background signal (typically MFI=300). Histological procedures

Tissue embedded in OCT Compound was cut into 5 pm cryosections and mounted on glass slides. Cryosections were air dried overnight at room temperature and stained with hematoxylin and eosin (H&E). Scoring of cecal inflammation was done in a blinded manner assessing the following four criteria as previously described.

(i) Submucosal edema. Submucosal edema was scored as follows: 0 = no pathological changes;

1 = mild edema (the submucosa accounts for <50% of the diameter of the entire intestinal wall - tunica muscularis to epithelium); 2 = moderate edema (the submucosa accounts for 50 to 80% of the diameter of the entire intestinal wall); and 3 = profound edema (the submucosa accounts for >80% of the diameter of the entire intestinal wall).

(ii) PMN infiltration into the lamina propria. Polymorphonuclear granulocytes (PMN) in the lamina propria were enumerated in 10 high-power fields (x400 magnification; field diameter of 420 pm), and the average number of PMN/high-power field was calculated. The scores were defined as follows: 0 = <5 PMN/high-power field; 1 = 5 to 20 PMN/high-power field; 2 = 21 to 60/high- power field; 3 = 61 to 100/high-power field; and 4 = >100/high-power field. Transmigration of PMN into the intestinal lumen was consistently observed when the number of PMN was >60 PMN/high-power field.

(Hi) Goblet cells. The average number of goblet cells per high-power field (magnification, x400) was determined from 10 different regions of the cecal epithelium. Scoring was as follows: 0 = >28 goblet cells/high-power field; 1 = 11 to 28 goblet cells/high-power field; 2 = 1 to 10 goblet cells/high-power field; and 3 = <1 goblet cell/high-power field.

(iv) Epithelial integrity. Epithelial integrity was scored as follows: 0 = no pathological changes detectable in 10 high-power fields (x400 magnification); 1 = epithelial desquamation;

2 = erosion of the epithelial surface (gaps of 1 to 10 epithelial cells/lesion); and 3 = epithelial ulceration (gaps of > 10 epithelial cells/lesion; at this stage, there is generally granulation tissue below the epithelium). The combined pathological score for each tissue sample was determined as the sum of these scores. It ranges between 0 and 13 arbitrary units and covers the following levels of inflammation: 0 = intestine intact without any signs of inflammation; 1 to 2 = minimal signs of inflammation (this was frequently found in the ceca of SPF mice; this level of inflammation is generally not considered as a sign of disease); 3 to 4 = slight inflammation; 5 to 8 = moderate inflammation; and 9 to13 = profound inflammation.

Statistical analysis

Sample size was determined from the data of previous studies. Researchers were not blinded for the assignment of the experiments and the data analysis except for histopathological scoring. Where two groups of data were compared, analysis was carried out using unpaired two-tailed t-test on log normalized data. One-way ANOVA followed by Tukey’s Test was used for comparison of three or more groups. Statistical analysis on time course data was either done by mixed-effects analysis or by calculating the area undeunder the curve (AUC) of the log normalized data and then assessing differences in AUC with one-way ANOVA and Tukey’s test. Statistical analysis was performed with Graphpad Prism Version 9.2.0 for Windows (GraphPad Software, La Jolla, California USA). P values of less than 0.05 were reported.

Non-patent literature cited:

Petrone et al. J Bacteriol. 2014 Mar; 196(5): 1094-1101. Liu et al., Structural diversity in Salmonella O antigens and its genetic basis; FEMS Microbiol Rev. 2014 Jan;38(1):56-89. doi: 10.1111/1574-6976.12034

Raetz and Whitfield C (2002), Annu. Rev. Biochem. 71 : 635-700. doi:10.1146/annurev.biochem.71.110601.135414

Claims

Claims

1. A pharmaceutical composition capable of protecting a patient from disease caused by a pathogenic strain of a bacterium, wherein said pathogenic strain displays a wild type surface antigen, said composition comprising a. a probiotic component comprising bacteria of a live avirulent strain of said pathogen, wherein said avirulent strain displays a variant of said surface antigen, said variant being capable of escaping binding by immunoglobulins capable of specifically recognizing the wild-type surface antigen; and b. a vaccine component comprising an inactivated vaccine strain of said bacterium, wherein said inactivated strain displays said wild type surface antigen.

2. The pharmaceutical composition according to claim 1 , wherein the bacterium belongs to the class Gammaproteobacteria, particularly wherein the bacterium belongs to the order Enterobacterales; more particularly wherein the bacterium belongs to the family Enterobacteriaceae.

3. The pharmaceutical composition according to claim 2, wherein the bacterium belongs to a genus selected from the group of Salmonella, Escherichia, Enterobacter, Klebsiella, Shigella, Citrobacter.

4. The pharmaceutical composition according to claim 3, wherein the bacterium belongs to the species S. enterica, particularly wherein the bacterium belongs to the subspecies S. enterica subsp. enterica; or wherein the bacterium belongs to the subspecies S. enterica subsp. typhimurium.

5. The pharmaceutical composition according to claim 3, wherein the bacterium belongs to the group of Entero-pathogenic Escherichia coli (EPEC) particularly wherein the bacterium is an EPEC double mutant comprising: a. a deletion of the gene ler; b. a deletion of wzy.

6. The pharmaceutical composition according to any one of the preceding claims, wherein the surface antigen is an O antigen.

7. The pharmaceutical composition according to any one of the preceding claims, wherein the avirulent strain is characterized, relative to the pathogenic strain, by one or more functional deletions of gene function selected from the following groups of gene functions: a. Virulence Factor group: i. Main regulator of virulence expression HilD; and/or Ler (for E. coli EPEC). ii. T3SS-2 structural components of Salmonella spp: sseC, sseD, sseB, ssaG, ssaC, ssal, ssaJ, ssaD, ssaR, ssaS, ssaT, ssaX, ssaU, ssaQ, ssaV, ssaK, ssaO, SsaL, ssaM, SpiC, and ssaN. b. O-antigen Modifier group: i. abequose acetyltransferase OafA ii. wzyB (O antigen polymerase)

Hi. wbaP (galactosyl transferase); iv. wbaN; wbaU; wbaV; and/or wzx v. wecA, wzm, wzt, waaL, msbA, wbbE and/or wbbF.

8. The pharmaceutical composition according to claim 7, wherein more than one virulence factor is removed in the avirulent strain.

9. The pharmaceutical composition according to claim 7, wherein the avirulent strain is characterized by having a modification selected from each of the Virulence Factor group and the O-antigen Modifier group. The pharmaceutical composition according to any one of the preceding claims, for use as a vaccine for preventing disease caused by said bacterium. The pharmaceutical composition for use according to claim 10, wherein the vaccine a. targets Salmonella spp. and is for administration in a chicken or turkey b. targets E. coli and/or Salmonella spp. and is for administration in a pig c. targets E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., and/or Salmonella spp. and is for administration in a human. The pharmaceutical composition for use according to any one of the preceding claims, as an administration form prepared for oral administration. A probiotic pharmaceutical preparation for use in protecting a patient from disease caused by a pathogenic strain of a bacterial pathogen, said probiotic pharmaceutical preparation comprising live bacteria of an avirulent strain of said bacterial pathogen, wherein said pathogenic strain of said bacterial pathogen displays a wild-type surface antigen, and wherein said avirulent strain displays a variant of said surface antigen, said variant being capable of escaping binding immunoglobulins capable of specifically recognizing the wild-type surface antigen. The probiotic pharmaceutical preparation for use according to claim 13, wherein said probiotic pharmaceutical preparation is provided after administration of an antibacterial drug. The probiotic pharmaceutical preparation for use according to claim 13 or 14, wherein said probiotic pharmaceutical preparation is provided after administration of a vaccine comprising an inactivated vaccine strain of said bacterial pathogen, wherein said inactivated strain displays said wild-type surface antigen. The probiotic pharmaceutical preparation for use according to any one of claims 13 to 15, wherein the bacterial pathogen and the avirulent strain are specified in any one of claims 1 to 9.