WO2013164334A1

WO2013164334A1 - Escherichia coli vaccine

Info

Publication number: WO2013164334A1
Application number: PCT/EP2013/058969
Authority: WO
Inventors: Eric Cox; Daisy Vanrompay; Michaela LOOS; Bakr AHMED ABDELRAHMAN MOHAMED
Original assignee: Universiteit Gent
Priority date: 2012-05-04
Filing date: 2013-04-30
Publication date: 2013-11-07

Abstract

The present invention relates to a bacterial type III secreted protein or nucleic acid, and uses thereof. Also provided is a method of preventing Escherichia coli infections in a subject by administering a vaccine comprising a bacterial type III secreted protein or nucleic acid to a subject.

Description

Escherichia coli vaccine

Field of the invention The present invention relates to a bacterial type III secreted protein or nucleic acid, and uses thereof. Also provided is a method of preventing Escherichia coli infections in a subject by administering a vaccine comprising a bacterial type III secreted protein or nucleic acid to a subject.

Background of the invention

Enterohemorrhagic Escherichia coli (EHEC) are a group of human enteropathogens that have been responsible for multiple outbreaks of diarrhea and hemorrhagic colitis (HC) worldwide (1 ). HC occasionally progresses to an hemolytic uremic syndrome (HUS), which is the most common cause of acute renal failure in children, and results in fatalities as high as 50% in the elderly. Although many serotypes of EHEC have been identified, E. coli 0157:H7 remains the most commonly (80%) associated £. coli with the disease outbreaks (2). Unlike the case with the majority of bacterial diseases, previous clinical studies did not demonstrate beneficial effects of antibiotic therapy on the outcome of EHEC infection (3,4,5). Moreover, several retrospective studies have linked antibiotic therapy to higher rates of HUS development, a prolonged period of symptomatic disease, and a prolonged bacterial shedding (6,7,8). Therefore, current strategies are rather focusing on vaccination as a preventive measure. However, an effective and well-utilized vaccine against EHEC is not yet available. HC and HUS are particularly caused by shiga-like toxins, which are produced by these EHEC, and become disseminated in the circulation as a consequence of intestinal colonization. Thus, intestinal colonization is considered a key determinant for EHEC pathogenicity (1 ).

The process of intestinal colonization is essentially mediated by type III secretion system (T3SS) proteins, which are employed by EHEC to establish a tight adherence to enterocytes, and to modify their cytoskelatal proteins, giving rise to the characteristic attaching and effacing (A E) lesions. It has been proposed that since cattle are associated with the majority of cases of E. coli 0157:1-17 infection in humans, reducing levels of the organism in cattle would be an attractive strategy to reduce the risk of human infection. Several different approaches have been proposed to achieve this goal, including modification of feed, probiotics, as well as vaccination. With respect to the latter, a number of different antigens have been tested for their ability to induce immune responses which block colonization, including bacterial type III secreted proteins. Vaccines targeting EHEC virulence determinants to prevent colonization of host cells by E. coli 0157:1-17 have been assessed with variable results in cattle (Potter et al., 2004; McNeilly et al., 2008; Smith et al., 2008; McNeilly et al., 2010; Vilte et al., 201 1 ) and other animal models (Agin et al., 2005; Babiuk et al., 2008; Yekta et al., 201 1 ). Systemic vaccination of cattle with bacterial type III secreted proteins, including Tir and EspA, has been shown to result in decreased E. coli 0157:1-17 shedding following both experimental infection as well as under field conditions (Potter et al., 2004; WO2007101337). Additionally, several studies involving type H ISS protein as vaccine candidates have been reported in mice models of human disease (Gu, Ning et al. 201 1 ), (Amani, Mousavi et al. 201 1 ), (Babiuk, Asper et al. 2008) (Fan, Wang et al. 2012) (Zhang, He et al. 201 1 ). EspB is a member T3SS protein that interacts with other T3SS proteins as well as host-proteins to mediate the colonization process (9). It interacts with EspD and EspA to form a complex that links bacterial and enterocytes membranes resulting in intimate adherence (10). Additionally, it binds to various host proteins including o catenin (1 1 ), a1 -antitrypsin (12) and myosin (13) in order to regulate the host-cell actin networks, resulting in the effacement of the microvilli on enterocytes (10). Moreover, the strong immunogenicity of EspB has been reported in infected patients (14, 15, 16) as well as in experimentally infected or vaccinated animals (14,17). Therefore, the role of EspB in EHEC pathogenicity, together with the high immunogenicity of this protein, might indicate the potential of EspB as a candidate for the development of a vaccine against EHEC. However, Cataldi et al., 2008, suggests intimin to be a more potent stimulator of immunity compared to EspB and demonstrates the need of a strong mucosal adjuvant MALP-2 in order to obtain a mucosal immune response after intranasal immunization. Moreover, it was previously demonstrated that a vaccine containing EspB and the C-terminal fragment of 280 AA of γ-intimin (γ- intimin C280) can decrease EHEC 0157:H7 shedding following systemic immunization in cattle but faecal IgG or IgA response to both antigens was not detected (Vilte et al., 201 1 ). Hence the significance of EspB in and its potential for vaccination is not straightforward.

Oral vaccination is the most preferable strategy to trigger immune responses at the intestinal mucosal surfaces. However, these responses have to be directed against antigens, which are crucial for the virulence of the enteropathogen and preferably in the early phase of the infection. In this context, oral immunization to elicit specific mucosal immunity against T3SS proteins of EHEC, which the pathogen uses in the early phase of colonization could be an interesting approach to interfere with the pathogenesis of EHEC and to prevent HC and HUS. However, oral delivery of vaccine antigens at the site of immunisation is a challenge, since antigen can be degraded by the harsh environment in the gastrointestinal tract: gastric acidity and digestive proteases, bile salts, flora. Hence, there has been a growing interest in using vehicles for oral vaccine delivery, with the use of Lactococcus lactis as one example. Lactococcus lactis is a lactic acid bacterium which is non-colonizing, nonpathogenic and generally regarded as safe. Additionally, genetically modified L. lactis has been successfully used for the oral delivery of recombinant proteins (18, 19 ,20). Therefore, the use of L. lactis for the safe delivery of oral vaccine antigens against EHEC could be particular of importance to children and elderly, who are at higher risk of HUS complications and for whom oral vaccination is much more acceptable than a more painful parenteral immunization. A further example of a delivery vector is Salmonella enterica. Khare et al., 2010, showed a reduced shedding and colonization of E. coli 0157:H7 in cattle after oral administration of a recombinant salmonella expressing intimin. Other studies for oral EHEC vaccines were described in mice models (Gu, Ning et al. 201 1 ) (Amani, Mousavi et al. 201 1 ).

However and despite these promising delivery systems, further challenges for oral vaccination are created by the "intestinal barrier", i.e. the protection of the mucosa by the mucus layer of the intestines and the filter formed by the glycocalyx and brush borders. Antigens have to pass the epithelial barrier to reach the underlying mucosal system. Moreover, very few antigens are immunogenic on its own, i.e. without the need of a strong adjuvant via the oral or mucosal route.

Hence, there is a need for novel mucosal vaccines and methods of inducing mucosal immunity. There is also a need for compositions and methods for preventing or reducing attachment, colonization and shedding of microorganisms, especially Escherichia coli, in a subject. Further, there is a need for novel methods and compositions for immunizing a subject such that protective immunity is obtained thus resulting in a reduction of the pathogenic bacterial load.

It was for the first time demonstrated in the present invention that vaccination with EspB, as recombinant protein or as nucleic acid in a delivery vector, results in a systemic and mucosal immune response against the antigen which significantly decreased 0157:1-17 E. coii colonization and faecal excretion.

Summary of the invention

The present invention relates to a bacterial secreted type III protein EspB, a nucleic acid encoding the EspB protein, or a fragment thereof, for use in preventing Escherichia coii (£. coii) infection in a subject, characterized in that the protein or nucleic acid is administered to the subject at the intestinal mucosa. More specific, the EspB protein or nucleic acid is used in a method of preventing or reducing attachment, colonization and/or shedding of Escherichia coii (£. coii) in a subject. Preferably, the subject is a ruminant or monogastric mammal, in particular cattle, sheep, goat, pig, and horse, even more in particular catle.

As provided herein, the EspB protein is characterized by an amino acid sequence which is at least 80% identical to SEQ ID NO: 2. The EspB nucleic acid is characterized by a nucleotide sequence which is at least 80% identical to SEQ ID NO: 1.

In a particular embodiment, the £. coii infection is an enterohemorragic £ coii (EHEC) infection, and more specific EHEC 0157:1-17.

In a further embodiment, the protein EspB is a recombinant protein. Alternatively, a nucleic acid encoding the EspB protein is inserted in a vector and said vector is administered to the subject at the intestinal mucosa.

The invention also relates to a composition for use as described herein comprising a bacterial secreted type III protein EspB, a nucleic acid encoding the EspB protein, or a fragment thereof, and a pharmaceutically acceptable carrier, diluent and/or excipient, further characterized in that the composition is administered to the subject at the intestinal mucosa. Optionally, the composition comprises an adjuvant, in particular a mucosal adjuvant.

In a further embodiment, the protein, nucleic acid, vector or composition as provided herein are administered orally or rectally.

In an even further embodiment, the invention encompasses a method of vaccinating a subject against £ coli infection, said method comprising the step of administering at the intestinal mucosa a bacterial secreted type III protein EspB, nucleic acid encoding the protein, or fragment thereof, to said subject.

Brief description of the figures Figure 1. Lactococcal expression vector pNZ8150 with the inserted EspB fragment. Figure 2. Total EspB-specific antibody titres in serum of mice one week after vaccination with Lactococcus lactis strain expressing EspB, the same amount of L. lactis containing the expression vector (pNZ8150) or with the bacterial culture medium (B9).

Figure 3. EspB-specific IgA titres in faeces of mice one week after the booster immunization with Lactococcus lactis strain expressing EspB, Lactococcus lactis containing the expression vector (pNZ8150) or with the bacterial culture medium (B9).

Figure 4. Kinetic of the faecal excretion 0157:1-17 £ coli by mice from 3 immunization groups (pNZ-EspB-SEC, pNZ8150 and BM9) which were challenge infected following streptomycine pretreatment.

Figure 5. Kinetic of the faecal excretion of 0157:1-17 £. coli by mice from 3 immunization groups (pNZ-EspB-SEC, pNZ8150 and BM9) after a challenge infection. These mice were not treated with streptomycine prior to the challenge i nfection an d overal l sh ow a shorter excreti on tha n m ice pretreated with streptomycin.

Figure 6. Lactococcal expression vector pTREX with the inserted EspB fragment. Figure 7. Kinetic of the faecal excretion 0157:1-17 £ coli by mice from 2 immunization groups (pT-EspB-SEC and pTREX) which were challenge infected following streptomycin pretreatment. Figure 8. Kinetic of the faecal excretion of 0157:1-17 E. coli by mice from 2 immunization groups (pT-EspB-SEC and pTREX) after a challenge infection. These mice were not treated with streptomycine prior to the challenge infection and overall show a shorter excretion than mice pretreated with streptomycin.

Figure 9. Kinetic of the faecal excretion of 0157:1-17 E. coli by mice from 2 immunization groups (EspB and control) after a challenge infection.

Figure 10. Levels of IL-4, IL-10 and INF-y produced by Payer's patches lymphocytes isolated from mice immunized using the EspB secreting L. lactis, non engineered vectors or inoculation buffer group, BM9. Immunized mice secreted higher levels of all measured cytokines compared to control groups.

Figure 11. Levels of IL-4, IL-10 and INF-y produced by spleenocytes isolated from mice immunized using the EspB secreting L. lactis, non engineered vectors or inoculation buffer group, BM9. Immunized mice secreted higher levels of all measured cytokines compared to control groups.

Figure 12. EspB nucleotide (A) and amino acid sequence (B).

Detailed description The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound. The terms described above and others used in the specification are well understood to those in the art.

The present invention provides a method of preventing or reducing attachment, colonization and/or shedding of enterohemorrhagic Escherichia coli (EHEC) bacteria in a subject by administering a vaccine comprising a bacterial type III secreted protein or nucleic acid, in particular EspB, or fragment thereof, to a subject. More in particular, the invention relates to a method for stimulating a mucosal immune response. Mucosal surfaces are the sites of entry of most infectious agents and hosts. Therefore, mucosal immunity is important as the first line of defense against infectious agents. It prevents attachment of pathogens to the mucosal epithelium, neutralizes viruses and bacterial toxins, and allows other aspects of the immune system to phagocytose and remove pathogens from the mucosal site. The mucosa- associated immune system functions to prevent the penetration of microbes into the internal regions of the body. Direct application of antigens to a mucosal surface is the most effective way to induce a local immune response. However, this is not always possible or practical either because the antigen is not immunogenic via this route (e.g. oral tolerance), because it becomes degraded by the normal physiological and biochemical processes (e.g. low pH, digestive enzymes, bile salts, ...), the mucosa is to distant (e.g. large intestine), because of the handling involved or because of the toxicity of the antigens to the mucosal surface (e.g. LT enterotoxins). The gut- associated lymphoid tissue (GALT) located in the intestines contains functional T and B lymphocytes and antigen-presenting cells. In contrast to the systemic lymphoid tissues of the body, the B lymphocyte population of GALT includes a significant population of cells which are committed to the synthesis of IgA class antibodies. This antibody type is not effectively induced through conventional intramuscular or subcutaneous immunization.

Hence it is an aim of the present invention to provide a method as defined herein wherein the protein, nucleic acid or composition is administered via a mucosal surface, in particular via oral or rectal administration, or any combination thereof. More specific the invention relates to a method for inducing of secretory IgA in the gut of a subject, comprising administering a composition comprising the bacterial type III secreted protein EspB or a nucleic acid encoding said protein, or fragment thereof, to the subject. The protein, nucleic acid or composition of the present invention is preferably administered via at least one mucosal surface as described herein, in particular the digestive tract, even more in particular the stomach and the intestine (small and large intestine). In a preferred embodiment, administration of the composition is via the mouth or anus, i.e. via oral or rectal administration, preferably rectal.

In one embodiment the protein, nucleic acid or composition is used for preventing, reducing and/or shortening of Escherichia coli (E. coli) infections, in particular enterohemorrhagic Escherichia coli (EHEC) infections. In particular, the method results in preventing, reducing or shortening attachment, colonization and/or shedding of E. coli bacteria in a subject. By the term "colonization" it is meant the continued presence and/or growth of bacteria in the intestinal tract of a subject. By the term "shedding" is meant the presence of bacteria in the subjects faeces.

As used herein the term "enterohemorrhagic" relates to a class of intestinally-related organisms which causes colonic hemorrhaging and results in blood loss. These include E. coli strains 0157:1-17, 026:1-11 1 and 01 1 1 :NM.

In enterohaemorrhagic Escherichia coli (EHEC), the type III secretion protein EspB is translocated into the host cells and plays an important role in adherence, pore formation and effector translocation during infection. EspB is a member T3SS protein that interacts with other T3SS proteins as well as host-proteins to mediate the colonization process (9). It interacts with EspD and EspA to form a complex that links bacterial and enterocytes membranes resulting in intimate adherence (10).

Additionally, it binds to various host proteins including ocatenin (1 1 ), a1 -antitrypsin (12) and myosin (13) in order to regulate the host-cell actin networks, resulting in the effacement of the microvilli on enterocytes. The EspB protein and nucleic acid sequences included herein are any homolog or artificial sequence that is

substantially identical, i.e. at least 80%, 85%, 87%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding EspB sequence identified by NCBI Accession number BA000007 Gl:471 18301 AP002550- AP002569 (incorporated herein by reference) or as represented herein by SEQ ID NO:1 and 2. EspB as used herein encompasses also natural variants of the aforementioned specific EspB proteins. Such variants have at least the same essential biological and immunological properties as the specific EspB protein. The percentage identity of nucleic acid and polypeptide sequences can be calculated using commercially available algorithms which compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) may be used to determine homologies/identities: BLAST, gapped BLAST, BLASTN and PSI BLAST, which may be used with default parameters.

It is contemplated that the EspB protein may be obtained from the respective bacterium or bacterial strain that produces it in nature, for example, by purifying it therefrom. Alternatively, the proteins may be produced in a suitable recombinant system as would be known to a person of skill in the art. It is also possible that the EspB protein, or fragments thereof, may be synthesized chemically according to well known methods. In a particular embodiment, the EspB protein or fragment thereof, may be attached to a heterologous carrier protein, non-protein carrier or some other amino acid sequence. The term "fragment" as used herein refers to partial amino acid sequences (and nucleic acid sequences coding therefore) having at least one immunologic or immunogenic property in common with the native molecule. Such fragments will include at least one epitope (or antigenic determinant) of the native molecule. Normally, they will have a length of at least 8 amino acids, preferably at least 15 or 20 amino acids. In particular, the fragment is an immunogenic fragment. The present invention also contemplates a DNA based vaccine comprising a nucleotide sequence encoding the EspB protein, or fragments thereof, which are capable of being expressed in the subject. As will be evident to a person of skill in the art, a nucleotide sequence encoding the EspB protein or one or more fragments thereof may form part of a nucleotide construct, for example, but not limited to a vector or the like. Such a construct may comprise a promoter, terminator and/or one or more regulatory sequences, as would be known in the art. Recombinant or attenuated strains of various bacteria such as Salmonella, Escherichia coli, Listeria, Shigella, and Lactobacilli can been used as a vectors to deliver the antigen. Suitable vectors contemplated by the present invention include Lactococcus lactis, as described herein, Lactobacillus spp, Salmonella spp, Escherichia coli spp, Listeria monocytogenies, and Shigella. Also viruses are often used as vectors, e.g. Vaccinia viruses, Herpesviruses, Adenoviruses and Retroviruses, as is well known in the art.

The composition as provided herein may optionally further comprise an adjuvant. In a preferred embodiment, the adjuvant is a mucosal adjuvant, for example, but not limited to a cholera toxin (CT) or thermolabile subunit (LT), a bacterial cell wall extract, a bacterial cell wall extract complexed with bacterial DNA, a mycobacterial cell wall extract (MCW), a mycobacterial cell wall-DNA complex (MCC), an immunostimulatory oligonucleotide including, but not limited to a CpG containing oligonucleotide (ODN), a non-CpG containing oligonucleotide, any other other well- recognized vaccine adjuvant, or a combination thereof. In a particular embodiment, the composition is a pharmaceutical composition or vaccine comprising an effective amount of EspB and a pharmaceutically acceptable diluent, carrier and/or excipient, optionally further comprising an adjuvant, in particular a mucosal adjuvant, even more in particular a thermolabile subunit (LT). As used herein, the term "vaccine" or "pharmaceutical composition" relates to a component or a composition comprising a component that is capable of eliciting an immunological response to the bacterial type III secreted protein EspB, or fragment thereof, in a subject. Usually, an "immunological response" includes, but is not limited to, one or more of the following effects: the production of antibodies, the production and/or activation of B-cells, the production and/or activation of helper T- cells, the production and/or activation of suppressor T- cells, the production and/or activation of cytotoxic T-cells, the production and/or activation of M cells, the production or activation of dendritic cells, or a combination thereof. In a preferred embodiment, administration of the protein, nucleic acid, vaccine or composition results in induction of immunity at one or more mucosal surfaces, especially the intestinal surface. Further, this may involve a local immunological response (slgA and in ruminants also lgG1 ) that includes both humoral and cellular immune responses. Hence the immune response confers some beneficial, protective effect to the subject against a subsequent challenge with the infectious agent. More preferably, the immune response prevents the onset of or ameliorates at least one symptom of a disease associated with the infectious agent, or reduces the severity of at least one symptom of a disease associated with the infectious agent upon subsequent challenge.

The term "pharmaceutically acceptable" refers to a material which can be administered to a subject along with the composition without causing significant undesirable biological effects and without interacting in a deleterious manner with any other component of the pharmaceutical composition. Pharmaceutical compositions suitable for administration illustratively include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions e.g. IgA-coated liposomes, phosphate buffered saline with LT, mutantLT or CT as adjuvant

As used herein, the "subject" is a human or animal, especially a mammal, more specific a ruminant or monogastric mammal, even more specific cattle, sheep, goat, pig, horse, etc. The term "cattle" refers to bovine animals including but not limited to steer, bulls, cows, and calves. The term "effective amount" refers to an amount sufficient to elicit an immune response in the subject to which it is administered. The amount of a protein that is therapeutically effective may vary depending on the condition of the subject and/or the degree of infection, and can be determined by a (veterinary) physician. The extent and nature of the immune responses can be assessed by using a variety of techniques. For example, sera can be collected from the inoculated subjects and tested for the presence of antibodies, e.g. specific for EspB. I n a particular embodiment, an effective amount or suitable (single) dose of EspB is at least 25μg, preferably at least 5C^g, even more particular at least 75μg, e.g. 8C^g, 90 μg, 95μg, 10C^g, or more.

The vaccine or composition may be administered in a single dose or in multiple doses, for example over a period of time. The timing and total dose(s) administered may be determined based on characteristics of the subject including species, sex, age, weight, health, any livestock management practice, and the like, the particular vaccine or composition used, and the route of administration. In a particular embodiment, at least two or three doses are administered to the subject with intervals of two to ten weeks. In a more preferred embodiment, the composition is administered in a prime boost regimen. For example, the administration regimen of the present invention encompasses two immunizations with a 3-week- interval.

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Particular embodiments and examples are not in any way intended to li mit the scope of the invention as claimed . Add itionally, th roughout th is application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

Examples

Example 1 : immunization of mice with EspB in inducible L. lactis Material and Methods:

1. Expression of EspB in inducible L. lactis vector: The lactococcal expression vector pNZ8150 was used for the extracellular expression of EspB under nisin-inducible control. The EspB coding sequence (SEQ ID NO:1 ) was inserted downstream of the nisin-inducible (PnisA) promoter and upstream of a secretion signal (UspS), forming the EspB expression plasmid, pNZ- EspB-SEC (Fig 1 ). The L. lactis MG1363 strain transformed with EspB expression plasmid was designated the same plasmid name. The expression of EspB by the recombinant L. lactis was confirmed and quantified by a specific sandwich ELISA.

2. Immunizing BALB/c mice with EspB-expressing strains:

Groups (n=7) of BALB/c mice were immunized orally either with the EspB- expressing strain, pNZ-EspB-SEC, the strain containing the corresponding empty expression vector, pNZ8150, with recombinant EspB in the medium for the oral inoculation of the bacteria (BM9-salts medium) or with the medium alone. Mice received a primary immunization, which was followed by a booster immunization 21 days later. Both primary and booster immunizations consisted of oral administration of 2 x 10⁹ CFU of the bacteria, 5 μg rEspB in the medium or the medium, on three consecutive days. Fourteen days after the primary immunization or seven days after the boost, mice were screened for specific serum total Ig or faecal IgA using a specific direct ELISA.

3. Challenge infection:

In order to evaluate the protective capacity of the induced immune responses, mice of each group were randomly divided into two subgroups which were challenged using a different protocol. One subgroup of each group was treated with streptomycin (5g/L) in drinking water for 24 hours prior to the challenge. Streptomycin treatment has been reported to enhance the colonization of EHEC in mice by clearing the commensal intestinal flora (21 ) (high colonization subgroup n= 4). The other subgroups did not receive streptomycin treatment and thus the intestinal flora was physiologically maintained (low colonization group n=3). Mice were challenged with 10¹⁰ CFU of a non-Stx expressing 0157:1-17 E. coli, NCTC12900. Faecal shedding of the challenge strain was monitored in the faeces of all infected mice at a 2-day interval until negative for two consecutive samplings, which indicated cessation of shedding. Medians of the shedding durations were compared among the experimental groups. Results

1. EspB expression by the inducible L. lactis strain:

The expression of EspB in the supernatant of an induced culture of pNZ- EspB-SEC was confirmed and quantified by specific sandwich ELISA using a standard curve of purified EspB as a reference. The strain produced an antigen yield of 3.8± 1 .1 μg ml culture medium. In addition the bacteria contained 826 ng EspB/ml culture intracellular.

2. Total EspB-specific antibody titres in serum and IgA titres in faeces of vaccinated mice

Serum and faeces of immunized mice were tested by ELISA's at day seven after each immunization for total EspB-specific immunoglobulin (Ig) and EspB- specific IgA titres, respectively. After the primary or the booster immunization, the group that received the vaccine strain showed significantly higher titres (P = < 0.001 ) of total serum EspB-specific I g (Fig . 2) and faecal EspB-specific IgA (Fig. 3) compared to the groups that received the empty pNZ8150 vector, the recombinant EspB or the inoculation medium.

3. Protection against EHEC colonization

The orally immunized mice with L. Lactis expressing EspB showed a marked decrease in duration of bacterial faecal shedding following challenge with the non- toxinogenic 0157:1-17 E. coli compared to mice of all control groups. This was the case in both challenge models. In the high colonization model, mice immunized with the EspB expressing bacteria stopped shedding 15 days post challenge, while the control groups continued to shed the bacteria till 32 days post challenge (Fig. 4). Similarly, in the low colonization model, mice immunized with the EspB expressing L. lactis stopped shedding significantly more rapid (9 days post challenge), than mice of all control groups (remained shedding for 22 days following infection) (Fig. 5).

In conclusion

The oral immunization of BALB/c mice using the T3SS protein EspB has resulted in systemic and mucosal immune responses that significantly decreased colonization of mice with 0157:1-17 E. coli in two different colonisation models. Therefore, we report for the first time the potential of EspB given via the oral route to protect against an EHEC infection. Example 2:

1. Expression of EspB in constitutive L. lactis vector:

The lactococcal expression vector pTREX was used for the constitutive expression of EspB in the extracellular medium. Constitutive expression systems could deliver higher doses of EspB, since they are capable of the constant release of the antigen in vivo. This is in contrast to inducible systems, where antigen release is dependent to induction prior immunization. Since inducers are ideally removed from the culture medium prior immunization, a more limited capability of in vivo antigen release could be expected. The EspB coding sequence was inserted downstream of the constitutive (P1 ) promoter and upstream of a secretion signal (UspS), forming the EspB expression plasmid, pT-EspB-SEC (Fig 6). The L. lactis MG1363 strain transformed with EspB expression plasmid was designated the same plasmid name. The expression of EspB by the recombinant L. lactis was confirmed and quantified by a specific sandwich ELISA.

2. Immunizing BALB/c mice with EspB-expressing strains:

The immunization protocol, including the number of animals per group was performed as described in example 1 .

3. Challenge infection:

Mice groups were challenged using the same protocol described in example

1 . Results

1. EspB expression by the constitutive L. lactis strain:

The expression of EspB in the supernatant of culture supernatant of pT-EspB- SEC was confirmed and quantified by specific sandwich ELISA using a standard curve of purified EspB as a reference. The strain produced an antigen yield of 1 .9± 0.4 μg ml culture medium. In addition the bacteria contained 147± 53 ng EspB/ml culture intracellular. These levels are less than those obtained by the previously constructed inducible strain, pNZ-EspB-SEC.

2. Total EspB-specific antibody titres in serum and IgA titres in faeces of vaccinated mice Serum and faeces of immunized mice were tested by ELISA's at day seven after each immunization for total EspB-specific immunoglobulin (Ig) and EspB- specific IgA titres, respectively. The responses induced by pT-EspB-SEC were qualitatively similar to these obtained by pNZ-EspB-SEC. However, the responses are characterized by lower titres of both serum Ig and fecal IgA (data not shown).

3. Protection against EHEC colonization

The orally immunized mice with L. Lactis expressing EspB showed a marked decrease in duration of bacterial faecal shedding following challenge with the non- toxinogenic 0157:H7 E. coli compared to mice of all control groups. This was the case in both challenge models. In the high colonization model, mice immunized with the EspB expressing bacteria stopped shedding 29 days post challenge, while the control groups continued to shed the bacteria till 32 days post challenge (Fig. 7). Similarly, in the low colonization model, mice immunized with the EspB expressing L. lactis stopped shedding significantly shorter (15 days post challenge), than mice of all control groups (remained shedding for 18 days following infection) (Fig. 8). Comparative analysis of the duration of shedding observed for pNZ-EspB-SEC and pT-EspB-SEC revealed a shorter duration of shedding being achieved by the inducible strain, pNZ-EspB-SEC in both models (High colonization model; 15 versus 9 days, low colonization model; 29 versus 15 days, respectively).

In conclusion

In this example the constitutive of EspB did not resulted in higher immune responses compared to the inducible one. Nevertheless, immunized mice were significantly less colonized with 0157:H7 E. coli upon the challenge in both colonization models, indicating protection.

Example 3: immunization of mice with rEspB 1. Immunizing BALB/c mice with purified EspB:

In the present example a group of BALB/c mice (n= 5) were orally immunized with purified EspB. The immunization protocol described in example 1 was followed, using a dose of 25 μg of EspB per immunization day. A control group (n=5) received only the inoculation medium. 2. Challenge infection:

Mice groups were challenged using the non-streptomycin treated model described in example 1 . 3. Total EspB-specific antibody titres in serum and IgA titres in faeces of vaccinated mice

Serum of immunized mice were tested by ELISA's at day seven after each immunization for total EspB-specific immunoglobulin (Ig). Mice immunized with purified EspB showed higher titres of serum total-lg compared to the control group. (data not shown).

4. Protection against EHEC colonization

Mice immunized orally with purified EspB showed a marked decrease in duration of bacterial faecal shedding following challenge with the non-toxinogenic 0157:1-17 E. coli compared to mice of the control group ( 12 versus 24 days) (Fig. 9).

Example 4: Cell mediated immunity induced by EspB secreting L. lactis

In this example we evaluated the T-lymphocyte helper function which mediates the immune responses induced upon immunizing BALB/c mice using either pT-EspB-SEC or pNZ-EspB-SEC. Groups of mice were immunized as described in example 1 . Ten days after the booster immunization, mice were sacrificed and cells were isolated from Peyer's patches and spleen. Peyer's patches are the main immune-inductive lymphoid tissue associated with the gut, while spleen is a systemic secondary lymphoid organ. The cells were cultured in vitro in the presence of purified recombinant EspB. Murine Interferon IFN-Y(reflective for T-helper type 1 ) and IL-4 and I L-10 (reflective for T-helper type 2) levels were determined in cell culture supernatants using a quantitative ELISA. Results:

Mice immunized using either the strains have showed a mixed profile of Th-1 and Th-2 functions as indicated by the significantly higher levels of cytokines INF- γ, IL-4 and I L-10 in cell cultures of immunized mice compared to the negative controls. This pattern was observed in cells isolated from both Peyer's patches (Fig 10) and spleen (Fig. 1 1 ). Cytokine profile of lymphocytes driven from mesenteric lymph nodes showed a similar pattern to those of the Peyer's patches (data not shown). Conclusion:

Cell mediated immune response represent an important attribute of the vaccine associated immune response, since they define the mechanism by which the vaccine overcomes infection. Mice immunized using either L. lactis strains secreting EspB elicits a mixed Th1/Th2 phenotype in both mucosal and systemic lymphoid tissues. Eliciting a Th2 response is particularly important, since it is the T-helper type associated with augmenting humoral immunity, especially IgA, which has been described to play a role in clearing EHEC in mice models.

Example 5: rectal immunization of calves

Bacterial inoculum

NCTC12900 is a well-characterized Shiga-toxin negative E. coli 0157:1-17

strain of human origin with spontaneous nalidixic acid (Nal) resistance (Dibb-Fuller et al., 2001 ; Wales et al., 2002; Woodward et al., 2003). We use a Shiga-toxin negative strain for biosafety reasons. Bacteria are grown overnight in Luria Bertani broth (LB) with aeration (200 rpm) at 37°C, harvested by centrifugation and re-suspended in sterile phosphate-buffered saline (PBS) to a concentration of 10¹⁰ CFU.

Preparation of recombinant proteins

Plasmids pCVD468 and pCVD469 are used for recombinant expression of EspA and EspB, respectively (Karpman et al., 2002), and plasmid pMW103 to express the C- terminal 380 amino acids of intimin (referred to as intimin) (Sinclair and O'Brien, 2004). Briefly, transformed bacteria are induced with 1 mM isopropyl^-d- thiogalactopyranoside and recombinant His-tagged proteins are purified by nickel- affinity chromatography. For rectal vaccination EspB is formulated at 100 μg protein in a total volume of 1 ml PBS. Expression of EspB in inducible L. lactis vector:

The lactococcal expression vector pNZ8150 is used for the extracellular expression of EspB under nisin-inducible control. The EspB coding sequence (mentioned below) is inserted downstream of the nisin-inducible (PnisA) promoter and upstream of a secretion signal (UspS), forming the EspB expression plasmid, pNZ-EspB-SEC (Fig 1 ). The L. lactis MG1363 strain transformed with EspB expression plasmid is designated the same plasmid name. The expression of EspB by the recombinant L. lactis is confirmed and quantified by a specific sandwich ELISA. For rectal vaccination, 10¹⁰ bacteria in suspension are locally inserted in the rectal.

Animals and experimental procedures

Calves (Holstein-Friesian race), culture negative for £ coli 0157:1-17 in faeces and seronegative for antibodies against intimin, EspA, EspB, and Tir, are allowed to acclimatize for one week after arrival in our facility. Calves are fed grain-based pellets, with free access to hay and water. During the acclimatisation period and vaccination, calves are given colistin (Promycine 100 000 I.U./kg), twice daily during 5 days, to prevent £ coli infections due to handling and transport of the animals or accidental contact.

Calves are immunized rectally (at the recto-anal junction) with either L. lactis expressing EspB (group 1 ) or rEspB (group 2). These immunizations are performed adding the mucosal adjuvant LT (or mutant LT). A third group of calves is not vaccinated and serves as a negative control. A boost immunization is performed 3 weeks later, and one week later, animals are inoculated orally with E.coli strain (NCTC12900). Prior to the inoculation (2 days after the last colistin dose), calves receive a 10% NaHC0₃ solution via a nursing bottle, to close the oesophageal groove in order to direct the inoculum into the abomasum. Each animal is inoculated orally with 10¹⁰ CFU E.coli strain (NCTC12900) for 2 consecutive days and receive a second similar dose seven and eight days later, needed to obtain a chronic infection in non-vaccinated animals. In order to monitor antibody response, serum is collected weekly from the vena jugularis. Faecal excretion of E. coli 0157:1-17 is monitored 2 times per week following infection until 5 weeks post infection or otherwise when all vaccinated animals are culture negative for £. coli 0157:1-17 in their faeces. All calves are euthanized after stopping excretion or otherwise 36 days after the second inoculation, to determine presence of NCTC12900 in the intestinal tract.

Excretion and intestinal presence of £ coli 0157:1-17

Faecal samples are analysed immediately after being taken, as described by Vande Walle et al., (2000). Briefly, ten grams of feces are homogenized with a stomacher blender in 100 ml sterile modified tryptone soy broth (Oxoid Ltd, Hanst, United Kingdom) supplemented with 20 mg/l novobiocin (Sigma, Aldrich, St.Louis, MO, USA). Enumeration of £ coli is undertaken by ten-fold serial dilutions plated onto Mac-Conkey agar plates supplemented with sorbitol, cefixime, tellurite and Nal (NalCT-SMAC) (MERCK, Darmstadt, Germany) and incu bated at 37°C for 18h. Remaining broth is enriched for 6h at 42°C and subjected to immune-magnetic separation (IMS) with Dynabeads® (Invitrogen, Merelbeke, Belgium) according to the manufacturer's instructions.

Finally, 10ΟμΙ is plated onto NalCT-SMAC agar and incubated 18h at 37°C. Selected sorbitol -negative colonies are confirmed by 0157-specific latex agglutination assay (Oxoid Ltd, Basingstoke, UK). Colony counts are Iog10 transformed for data analysis. If E. coli is not detected by direct plating , but only detected by enrichment, a concentration of 10 CFU/g is assigned (Vande Walle et al., 2000). To determine presence of NCTC12900 in the intestinal tract, intestinal contents and tissues (jejunum, ileum, caecum, colon and rectum) are tested by direct plate counts as described for faecal samples. Before testing, tissues are rinsed with sterile PBS to remove all non-adherent bacteria.

Serum antibody response against virulence factors of E. coli 0157:1-17

Blood samples are collected one time weekly from the vena jugularis and processed directly after collection. Briefly, sera are heat-inactivated (30 min . at 56°C ) and kaolin-treated. Polysorb 96-well plates (NUNC, Polysorb Immuno Plates, Roskilde, Denmark) are coated with 200 ng/well of recombinant intimin, EspA or EspB in PBS and incubated overnight at 4°C. Subsequently, non-specific binding sites are blocked during 1 h at 37°C by adding PBS + 0.2% Tween®80. After washing with PBS + 0.2% Tween®20, plates are incubated with two-fold dilution series of serum in PBS + 0.05% Tween®20, followed by incubation with HRP -conjugated anti-cattle Ig-specific sheep antibodies (AbD Serotec, UK).

Each incubation step of 1 h at 37°C is followed by 3 washes. After addition of 2,2'- azino-di-[3-ethylbenzthiazoli n e su lfon ate] d i a m m on i u m sa lt (ABTS ) ( Roch e Diagnostics Vilvoorde, Belgium), the optical density is measured at 405 nm (OD405). The results are positive if the absorbance exceeds the cut off value of the mean of the negative control sera plus three times the standard deviation.

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Claims

A bacterial secreted type III protein EspB, or nucleic acid encoding the EspB protein, or a fragment thereof, for use in preventing or reducing Escherichia coli (£. coli) infection in a subject, characterized in that the protein or nucleic acid is administered to the subject at the intestinal mucosa.

The protein for use according to claim 1 , comprising an amino acid sequence which is at least 80% identical to the sequence characterized by SEQ ID NO: 2.

The nucleic acid for use according to claim 1 , comprising a nucleotide sequence which is at least 80% identical to the sequence characterized by SEQ ID NO:1

The nucleic acid for use according to claims 1 or 3, wherein the nucleic acid is administered by use of a vector.

The protein or nucleic acid for use according to any one of claims 1 to 4, wherein the £ coli infection is an enterohemorragic £ coli (EHEC) infection.

The protein or nucleic acid for use according to claim 5, wherein the EHEC is EHEC 0157:H7.

The protein for use according to any one of claims 1 to 5, wherein EspB is a recombinant protein.

A composition comprising a bacterial secreted type III protein EspB, a nucleic acid encoding the EspB protein, or a fragment thereof, and a pharmaceutically acceptable carrier, diluent and/or excipient, for use in a method of preventing or reducing Escherichia coli (£. coli) infection in a subject, characterized in that the composition is administered to the subject at the intestinal mucosa.

The protein, nucleic acid or composition for use according to any one of claims 1 -8, wherein the protein, nucleic acid or composition is administered orally or rectally.

10. The protein, nucleic acid or composition for use according to any one claims 1 -9, wherein the subject is a ruminant or monogastric mammal, particular cattle, sheep, goat, pig, and horse.