WO2013113865A1 - Eimeria vector vaccine for campylobacter jejuni - Google Patents

Abstract

The present invention applies to the field of veterinary vaccines, namely vaccines for poultry against Campylobacter jejuni, and in particular to a recombinant Eimeria vector. The invention provides a live recombinant Eimeria vector expressing an immunogenic protein from C. jejuni, for use in a vaccine against C. jejuni in poultry. In addition the invention provides a vaccine and a use comprising the recombinant Eimeria vector, as well as methods for the preparation of the vaccine, and for the vaccination of poultry against C. jejuni.

Description

Eimeria vector vaccine for Campylobacter jejuni

The present invention applies to the field of veterinary vaccines, namely vaccines for poultry against Campylobacter jejuni, and in particular to a recombinant Eimeria vector.

Eimeria are protozoan parasites of the phylum Apicomplexa, and the class Coccidia. When infecting poultry, they cause a medium to severe gastrointestinal disease, called: Coccidiosis. Eimeria have complex lifecycles with multiple stages, some developing outside the host. The major species that infect poultry can be identified in a number of ways, routinely by microscopic size and appearance. Also, the different Eimeria species tend to colonise different areas of the avian intestines.

Eimeria infection occurs by ingestion of sporulated oocysts and can occur when the target host is just one day old. In the gut sporozoites are released which then colonize a section of the bird's intestine, by invading gut epithelial cells. Replication leads to release of merozoite stages, and rupture of host cells. The merozoites reinfect further epithelial host cells, which continues for up to four cycles. Finally a sexual stage develops, which produces oocysts which are released with the faeces. After sporulation in the environment the cycle of 3-10 days (typically 4-6 days) starts anew (Shirley et al., 2005, Adv. in Paras., vol. 60, p. 285).

Symptoms of Eimeria infection in poultry vary from loss of appetite to bloody diarrhoea and organ failure due to build up of necrotic tissue in the intestines. Consequences are a drop in feed conversion rate, reduced growth rate, reduced egg production, and susceptibility to secondary infections, all causing major discomfort to affected birds, and serious economic damage to a commercial poultry operation.

Protection against Eimeria infection and Coccidiosis is based on administering anti-coccidial drugs via the feed, but resistance formation, and drug-residues in animal products are a constant concern. Therefore Eimeria infection in poultry is preferably combated by vaccination using live Eimeria strains, either wildtype or attenuated, which can induce strong immunity in the bird, by both humoral and cellular routes of the immune system. Such vaccines are considered to activate and mobilise intraepithelial lymphocytes -which are the primary immune effector cells involved in the innate response to Eimeria- to defined areas of the intestine.

Attenuated Eimeria for use in vaccine strains are obtained by selection of naturally occurring

Eimeria differing in pathogenicity. A special class of attenuated Eimeria are the so-called precocious strains, which are Eimeria that will complete their lifecycle in a bird in fewer than normal rounds of infection. This will cause lower numbers of oocyst output and also less damage to a target's intestine.

Typically an Eimeria vaccine will contain a combination of several Eimeria species, as immunity is type specific. For example some commercial Coccidiosis vaccines for poultry from MSD Animal Health (Boxmeer, The Netherlands) are: Coccivac® D, containing 8 wildtype strains of Eimeria, and Paracox® 8 containing 8 precocious strains of Eimeria; both provide excellent safety and immunity after inoculation by the oral route into chicks from 1 day old onwards. Such vaccination is routinely performed by mass vaccination to inoculate by the oral route, such as by spraying day old chicks in the hatchery, or alternatively the vaccine may be given by drinking water or on the feed. Several species of Eimeria have been studied in more detail, e.g. a genome sequencing project is in progress for E. tenella. The genome is about 60 megabases in size and has a GC content of about 53%. There are 14 chromosomes that range in size from 1 to 6 Mb (see: www.genedb.org/ Homepage/ Etenella, and: Chapman & Shirley (2003, Avian Pathol., vol. 32, p. 1 15)).

Transfection of Eimeria parasites has been described e.g. in Clark et al. (2008, Mol. Biochem.

Paras., vol. 162, p. 77), in order to generate recombinant Eimeria parasites, such as described in: US 5,976,553.

Eimeria has also been proposed as a live vaccine vector, for delivering and expressing of heterologous genes; e.g. Suo et al. (US 2010/183,668) describe the presentation of antigens from Newcastle disease virus, a respiratory virus of poultry; and Huang et al. (201 1 , J. of Immunol., vol. 187, p. 3595) describe the in vivo expression of a marker gene. Yan et al. (Int. J. for Parasitol., vol. 39, p. 109) described stably transfected Eimeria, constitutively expressing a marker gene, and suggested use as a vaccine vehicle for intracellular microbes such as avian influenza virus. Campylobacter is a genus of gram-negative bacteria, which are motile, non-sporeforming, and have a spiral shape. Several Campylobacter species are pathogenic to humans and animals, but the most notorious member is Campylobacter jejuni, which is a zoonotic hazard, responsible for the bulk of cases of bacterial food-poisoning in humans in developed countries. Typically C. jejuni replicates and survives in an animal's intestines (e.g. cows or chickens) as a commensal organism relatively harmless to the host, although C. jejuni has been reported to cause infectious hepatitis (a.k.a. vibrionic hepatitis) in chickens, and campylobacteriosis in calves. Upon slaughter, produce may become contaminated with the C. jejuni- loaded intestinal content of the animal. The consumption of undercooked meat or organs may then cause severe illness in humans, and a gastro-enteritis can manifest itself in the form of diarrhoea with cramps, fever and pain for up to 7 days. This is also part of the syndrome known as 'traveller's disease'.

C. jejuni can be cultured in vitro and prefers 42°C and micro-aerophilic conditions, which explains the preference for the avian intestines. Campylobacter has in the past been mis-classified as Vibrio, now a different class. The first full genome sequence of C. jejuni was published in 2000 (Parkhill et al., Nature, vol. 203, p. 665), in the mean time many more sequences have become available. Next to hygienic- and bio security measures, also vaccines against C. jejuni are in development, but none are commercially available so far. Such vaccines can aim to reduce infection or disease in humans, or alternatively, reduce the C. jejuni load in animals and so reduce the risk of cross-contamination at slaughter. Several types of vaccines have been tested: attenuated live, killed whole cell, subunit and vector type vaccines. For a recent review see Jagusztyn-Krynicka et al. (2009, Expert Rev. Vaccines, vol. 8, p®. 625).

In the veterinary field, the focus is on the vaccination of chickens which form a biological reservoir for zoonotic infection, as they can carry very high loads of C. jejuni in their intestines. The main target is the broiler type chicken which is reared for meat production. It is routinely vaccinated against a number of diseases at 1 day of age, or even before hatching, in so-called in ovo vaccination at 18 days of embryonic development, but not routinely against C. jejuni.

Some experimental vaccines obtained differing levels of success using a Salmonella enteritidis bacterial vector vaccine, expressing C. jejuni genes: Wyszynska et al. (2004, Vaccine, vol. 22, p. 1379) reported a reduction in C. jejuni load of 6 Log 10 in colony forming units (cfu) per gram of caecal content. The chickens were vaccinated with an attenuated Salmonella vector expressing the gene for the C. jejuni amino-acid transporter periplasmic solute-binding protein: CjaA (also known by its gene number: Cj0982), which is an N-glycosylated lipoprotein, localized in the bacteria's inner membrane. However in that study an important control group was lacking, and no other research institution has been able to replicate this level of reduction. A more recent study by Buckley et al. (2010, Vaccine, vol. 28, p. 1094) reported a reduction of 1.4 Log 10 in level of C. jejuni colonisation, using a Salmonella vector expressing a fusion protein of CjaA and part of tetanus toxin (the C-terminal region of the C-fragment). Good results were also obtained by Layton et al. (201 1 , Clin. & Vaccine Immunol., vol. 18, p. 449) with Salmonella vectors expressing a linear epitope from one of three C. jejuni proteins. A reduction of C. jejuni colonisation up to 4 Log 10 cfu/g was observed for the Salmonella vector expressing the 18 kDa outer membrane protein: omp18 (peptidoglycan associated lipoprotein, a.k.a. Cj01 13). However, it is difficult to compare these different outcomes as a result of the complex nature of Campylobacter challenge studies, which have several critical features which complicate reproducibility.

In spite of these efforts, no C. jejuni vector vaccine is commercially available so far. One reason being that Salmonella is itself also a human pathogen, so its use in vaccination of chickens against C. jejuni can only be employed with caution. Current regulations on the use of Salmonella vaccines in chickens, require a zero detectability at slaughter, which is at about 6 weeks of age for broilers (EU regulation 2160/2003). However, even attenuated Salmonella strains have been detected to persist and shed at low level, or intermittently, even beyond 4 weeks after inoculation (see datasheet for

GallivacSE®, Merial). Consequently alternative vector vaccines for Campylobacter with improved safety profiles are highly desired.

It is an object of the present invention to generate a safe and effective vector vaccine for C. jejuni; the vector vaccine should be safe to humans and induce in poultry an effective immune protection to reduce the infection, level of colonisation, and spread of C. jejuni.

Surprisingly it was found that this object can be met, and the disadvantages of the prior art be overcome, by the use of Eimeria parasite species as vaccine vector for the delivery and expression of immunogenic proteins from C. jejuni to poultry.

This is unexpected as there is no such use of Eimeria vectors described in the prior art, leading to an actual and effective expression of a bacterial protein by an Eimeria parasite. In addition, this was previously also unlikely to be successful, because bacterial proteins are normally not expressed in a eukaryotic context such as that of an Eimeria parasite replicating inside a poultry's intestinal host cell. In such environment the gene-expression and protein replicative mechanisms are essentially different compared to bacterial systems; e.g. with respect to codon usage, mRNA editing, and the processing of the expressed protein, such as by glycosylation, folding, or transportation. Any of these may cause the protein to end up in a location or in a form that renders it ineffective as an immunogenic protein. Also it could not be expected that this expressed protein would be properly presented to the immune system of the host. Especially the efficacy of such a vector-insert combination in a life host animal could therefore not be foreseen, and was never investigated. Consequently, the inventors did not expect to find that the delivery and expression of proteins from C. jejuni by recombinant Eimeria parasites as vectors could induce an effective immune response in poultry, which was capable of significantly reducing the establishment of C. jejuni load in the poultry's intestine following a challenge inoculation with wildtype C. jejuni. The level of reduction in C. jejuni load in vaccinates was comparable to that seen with prior art Salmonella based vector vaccines. Surprisingly however, the onset of protection induced by a recombinant Eimeria parasite based vector was apparent already one week earlier then observed with a Salmonella vector. Also the inventors realised that the use of Eimeria as a vaccine vector provides many favourable features: its high species-specificity makes it completely safe for humans; in addition, its strong immunity inducing character clears of the vector in a few weeks, making it a self-limiting infection.

The mechanism behind this unexpected effectiveness is not known; without wishing to be bound by theory, the inventors speculate that this is related to the way the expressed immunogenic C. jejuni protein is presented to the poultry's immune system. Apparently, the Eimeria vector by its replication in the poultry's intestines, in combination with its natural highly immunogenic characteristics, disturbs the normal 'immune tolerance' of C. jejuni bacteria in the poultry's intestines, which now makes the bacteria vulnerable to immune-clearance. This has never been described or suggested before.

Therefore in one aspect the invention relates to a recombinant Eimeria vector for use in a vaccine against Campylobacter jejuni in poultry, wherein the recombinant Eimeria vector comprises a heterologous nucleic acid molecule which comprises a nucleotide sequence capable of expressing an immunogenic protein from C. jejuni, or an immunogenic part of said protein.

The recombinant Eimeria vector for use in a vaccine according to the invention, induces a strong immune response in poultry that can significantly reduce the infection and/or the level of colonisation by C. jejuni, and so reduces the spread of C. jejuni to the environment and thus the chances of zoonotic infection.

In addition, the use of the recombinant Eimeria vector in a vaccine according to the invention, provides an effective immunisation against Coccidiosis caused by the Eimeria species from which the vector is derived.

For the invention, a "recombinant" is a nucleic acid molecule or a micro-organism of which the genetic material has been modified, to result in a genetic make-up that it did not originally posses.

"Eimeria" for the invention are members of the Eimeriidae family that can replicate in poultry species, for example comprising, but not limited to, the species: E. acervulina, E. tenella, E. maxima, E. brunetti, E. mitis, E. mivati, E. necatrix, E. praecox, E. hagani, E. meleagrimitis (type 1 and type 2), E. adenoides, E. gallopavonis, E. dispersa, E. innocua, E. subrotunda, and E. meleagridis.

Preferred Eimeria are: E. acervulina, E. maxima, and E. tenella; most preferred is E. tenella.

This includes also Eimeria that are sub-classified therefrom in any way, for instance as a subspecies, strain, isolate, genotype, serotype, serovar, variant or subtype and the like. Such Eimeria share the characterising features of their taxonomic family-members such as the genomic, the physical, electron-microscopic, and biochemical characteristics, as well as biological characteristics such as immunologic, or pathological behaviour. Conveniently determinations can be based on nucleotide sequencing or polymerase chain reaction (PCR) assays and on serotyping assays, as known in the field.

It will be apparent to a skilled person that while the micro-organism that is used as vector for the invention is currently named Eimeria, this is a taxonomic classification which could be subject to change as new insights lead to reclassification into a new or different taxonomic group. However, as this does not change the micro-organism involved or its characterising features, only its name or classification, such reclassified organisms are considered to remain within the scope of the invention.

Eimeria parasites for use in the invention can be obtained from a variety of sources, e.g. as original field isolates from a poultry house, or as reference- or laboratory strain from various laboratories and institutions (Shirley et al., 2005, supra).

A "vector" for the invention is a live recombinant micro-organism, here: an Eimeria parasite, that carries and expresses a heterologous nucleic acid sequence to a target human or animal, and presents the heterologous protein encoded from said nucleic acid the to the host's immune system.

Different Eimeria species colonise different part of a birds intestines, therefore the selection a specific type of Eimeria as the parental strain for the development of the recombinant vector for the invention, allows control over the area in the poultry's intestines where the vector will establish itself, and present the immunogenic C. jejuni protein to the host's immune system, as well as over the activation and attraction of epithelial lymphocytes. For the invention this was not found to be a critical aspect, however there may be conditions where it is favourable to let the recombinant Eimeria vector be based on an Eimeria parental strain that replicates in the same habitat as the C. jejuni that it is intended to combat, or alternatively to deliberately select an Eimeria replicating in another section of the intestines, in order to create a new area for immunological presentation.

An example of selecting the same habitat is where the C. jejuni replicates in the central region of the intestines, to use e.g. E. maxima or E. necatrix, or when the C. jejuni replicates in more caudal parts of the intestines or in the ceaca, then E. brunetti, or E. tenella respectively, can be used as vector.

Alternatively an example of selecting a different habitat for the Eimeria vector and C. jejuni, is to use E. acervulina as vector, which will replicate in the rostral part (the duodenal region directly after the gizzard), where few C. jejuni will occur.

Therefore in a preferred embodiment of the recombinant Eimeria vector for use in a vaccine according to the invention, the recombinant Eimeria vector is based on E. tenella, E. maxima or E. acervulina.

As the Eimeria vector for the invention is also effective as a vaccine against Coccidiosis, it is

advantageous to let the recombinant Eimeria vector be based on an Eimeria parental strain that is an established Coccidiosis vaccine strain; preferably such a parental Eimeria vaccine strain is an attenuated Eimeria strain, such as a precocious Eimeria strain.

Therefore in an embodiment of the recombinant Eimeria vector for use in a vaccine according to the invention, the recombinant Eimeria vector is based on an attenuated Eimeria strain. In a preferred embodiment the attenuated Eimeria is derived from a precocious strain of Eimeria.

The level of attenuation of an Eimeria strain can simply be assessed by testing the behaviour of such a strain in vivo, for example by monitoring its effect in experimental animals for relevant parameters such as (histo-)pathological effects on the intestines, feed conversion, pre-patent period, or oocyst output numbers.

A "vaccine" is a formulation for a medical purpose, which induces in a target human or animal an immune response that aids in preventing, ameliorating, reducing sensitivity for, or treatment of a disease or disorder resulting from infection with a micro-organism. The vaccine-induced protection is achieved as a result of administering at least one antigenic molecule derived from that micro-organism. This will cause the target to show a reduction in the number, or the intensity, of clinical signs caused by the microorganism. This may be the result of a reduced invasion, colonisation, or infection rate by the microorganism, leading to a reduction in the number or the severity of lesions and effects that are caused by the micro-organism, or by the target's response thereto.

"Campylobacter jejuni" refers to the bacterial micro-organisms currently classified as such and having the characterising features as described above. Like for Eimeria, this term includes all C. jejuni that are sub- classified therefrom in any way. And, as for Eimeria, it must be realised that the current taxonomic classification could change in future, but such re-classified organisms remain within the scope of the invention.

The present invention provides an advantageous utility for all species of birds used for human consumption, in whole or in part, for example of muscle-tissue, or organs such as liver, kidneys etc., provided those birds can be colonised by C. jejuni, and are susceptible to inoculation with Eimeria.

Therefore the term "poultry" for the invention relates to: chicken, turkey, duck, goose, peacock, partridge, quail, guinea fowl, pheasant, pigeon, and ostrich. Preferred poultry species are: chicken, turkey, duck and goose; with chickens as most preferred species.

The target birds may be of any type such as layers, breeders, broilers, combination breeds, or parental lines of any of such breeds. Preferred type is broilers.

The term "comprises" (as well as variations such as "comprise", "comprised", and "comprising") as used herein, intends to refer to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and not to the exclusion of any of such element(s) or combinations.

Therefore any such text section, paragraph, claim, etc., can also relate to one or more embodiment(s) wherein the term "comprises" (or its variants) is replaced by terms such as "consist of, "consisting of", or "consist essentially of". A nucleic acid is "heterologous" to the Eimeria vector that carries it, if that nucleic acid was not present in the parental Eimeria that was used to generate the recombinant Eimeria vector according to the invention.

For the invention an Eimeria "comprises a heterologous nucleic acid molecule" when such nucleic acid molecule is contained in the Eimeria, for example as an episomal nuclear element on a plasmid, or as an element integrated into the genome of an Eimeria. The integration can be in a single or in multiple loci of the genome, and can be a random, or a directed insertion. When directed, the insertion can be aimed at a non-transcribed area of the genome, or at a non-essential genome region, so as not to disturb or make unstable the survival and replication of the Eimeria vector itself. Alternatively the insertion can deliberately be directed to target an insertion-site in or near a coding or regulatory region, which is then functionally disabled, for example to generate a knock-out recombinant with an attenuated phenotype.

The resulting effect of the insertion of the nucleotide molecule into the Eimeria genome may thus differ, as this genome may become larger, the same, or smaller in size, depending from whether the net result on the genome is an addition, a replacement or a deletion of genetic material, respectively. These choices determine the ultimate composition of the inserted heterologous nucleic acid molecule, and its effect on the vector and ultimately on the vaccinated target poultry.

The insertion of a heterologous nucleic acid molecule into Eimeria can in principle be made by any suitable technique, provided the resulting recombinant Eimeria vector is able to display a stable and productive replication, both in vivo as in vitro, as well as an effective and sustained expression of the inserted protein encoding sequence.

Methods to insert a nucleic acid molecule into Eimeria are well known, and relate to the physical insertion of a nucleic acid into the Eimeria parasite, followed by several rounds of selection for those recombinant Eimeria vectors that have become stably transformed and express the inserted coding sequence. Typically such techniques employ the transfection of sporozoite stage Eimeria by

electroporation, e.g. as described for transient transfection by Hao et al. (2007, Mol. Biochem. Parasitol., vol. 153, p. 213), and for stable transfection by Clark et al. (2008, supra). Clark et al. describe a technique of restriction enzyme mediated transfection (REMI), which allows the insertion into the Eimeria genome in semi-random locations, guided by the occurrence of specific rare restriction enzyme recognition sites. This technique typically generates between 0.01 and 0.1 % of a recombinant Eimeria vector which is subsequently selected and amplified. This selection can be done in a variety of ways, such as by in vitro selection using an anti-coccidial drug in case its resistance gene was inserted, or by fluorescence activated cell sorting (FACS) in case a fluorescent marker gene was inserted. Subsequent rounds of in vivo selection in a poultry host then significantly increase the number and the relative percentage of a recombinant Eimeria vector. For example a certain amount of recombinant oocysts, or even a single oocyst, can be inoculated into a chicken and the next generation isolated from its droppings some days later. To prevent the acid barrier of the gizzard, inoculation can also be done by cloacal, instead of oral route. Generally, using these methods of selection and amplification, a 100 % pure preparation of a recombinant Eimeria vector can be obtained within 5 rounds of in vivo infection and reinfection.

The construction of the Eimeria vector for the invention can thus be done by well-known molecular biological techniques, involving cloning, transfection, recombination, selection, and amplification. These are extensively described in handbooks such as: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989); Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986); and: Sambrook & Russell, 2001 , in: 'Molecular cloning: a laboratory manual', 3rd edn. New York, USA: Cold Spring Harbour Laboratory Press.

Detailed methods for transfection and selection of Eimeria are also described and exemplified herein. Therefore, a person skilled in the art will readily be able to apply, adapt, modify and improve upon these techniques, using nothing but routine methods and materials.

One additional advantage of the use of Eimeria as a recombinant vector, is that because of the mega- base size of the parasite's genome, the size or the number of the inserted heterologous nucleic acid molecule(s) does not quickly become limiting to the vector's ability to replicate, as compared to vectors of much smaller size, such as a bacterium, or even a virus.

Therefore in an embodiment, the recombinant Eimeria vector for the invention comprises more than one heterologous nucleic acid molecule. These inserts may be integrated or episomal, and may be the same or different. When episomal they may be on the same or on different plasmids; when integrated they may be in the same or in different locations on the genome.

In a further embodiment, the heterologous nucleic acid molecule comprises more than one nucleotide sequence encoding an immunogenic C. jejuni protein. These may be expressed from a single promoter, e.g. connected as a fusion protein, or as individual proteins from separate promoters.

A "nucleotide sequence capable of expressing" a particular protein is a well known concept in molecular biology and refers to the central dogma of molecular biology wherein a DNA sequence is transcribed into mRNA, and the mRNA is translated into the amino acid sequence of (a part of) a protein.

Typically such a nucleotide sequence capable of encoding a protein is called an open reading frame (ORF), indicating that no undesired stop-codons are present that would prematurely terminate the translation into protein. Such a nucleotide sequence may be a gene (i.e. an ORF encoding a complete protein), or be a gene-fragment. It may be of natural or synthetic origin.

To allow the expression of the nucleotide sequence, this needs to be under the control of a promoter sequence, which initiates the transcription process. This is commonly referred to as the promoter being "operatively linked" to the nucleotide sequence, where both are connected on the same

DNA, in effective proximity, and with no signals or sequences between them that would intervene with an effective transcription.

For the invention, the promoter sequence can in principle be any promoter, derived either from Eimeria, or from another source, as long as an effective and sustained expression of the inserted nucleic acid sequence is provided. Commonly promoters also comprise an enhancer area which is a regulatory region involved in the regulation of the time point, the duration, the conditions, and the level of the transcription. This way, the selection of the promoter allows the control over the type of expression. For example when using a promoter derived from an Eimeria microneme protein gene, this will be active in the motile zoite stages of the parasite, while the use of a promoter from an Eimeria housekeeping gene such as the actin protein, will provide for a constitutive expression in all of the developmental stages of the recombinant Eimeria vector. An other example is the Eimeria heat shock protein 90 promoter, primarily promoting expression by intracellular schizont life cycle stages which can influence the nature of the immune responses stimulated.

Because a promoter is by definition located upstream of the gene of which it controls the transcription in the native context, knowing the location of a gene, or the transcription start of its mRNA, inherently discloses the position of its accompanying promoter. Therefore, a suitable promoter from an Eimeria genome can simply be selected by subcloning the region upstream of a particular gene. Because (regions of) the genomes of several Eimeria species have been sequenced, the skilled person can readily identify and obtain suitable Eimeria promoters by routine techniques.

Alternatively sequences of suitable promoters from Eimeria can be derived from GenBank®, such as promoters from: the E. tenella apical membrane antigen-1 gene (JN032081 ), and the microneme protein 4 gene (AJ306453) or the E. maxima immune mapped protein 1 gene (FN813228).

For the invention, a "protein" is a molecular chain of amino acids. A protein can be a native or a mature protein, a pre- or pro-protein, or a part of a protein. Inter alia: peptides, oligopeptides and polypeptides are included within the definition of protein.

The "immunogenic protein from C. jejuni" for use in the invention, can in principle be any protein from C. jejuni as long as it is immunogenic, that is: it should be able to induce an effective immune- response when expressed in vivo via a recombinant Eimeria vector.

An 'effective immune response' for the invention is an immune response that is capable of significantly reducing the level of colonisation by C. jejuni in a poultry's intestines.

The selection of such immunogenic proteins, as well as the assessement of their immunogenic efficacy is well within the routine capabilities of the skilled artisan, and can for example employ a vaccination-challenge experiment in poultry, followed by an assessment of any reduction in level of C. jejuni colonisation, e.g. by the instructions as described and exemplified herein.

The inventors have identified several immunogenic C. jejuni proteins that can advantageously be used for the invention as they have been found to be highly effective immunogens, providing reduction in the level of C. jejuni colonisation in different assays. Therefore preferred immunogenic C. jejuni proteins for expression by a recombinant Eimeria vector according to the invention are: CjaA (Cj0982); omp18

(Cj01 13); jlpA (surface-exposed lipoprotein adhesin, a.k.a. Cj0983); mapA (outer membrane lipoprotein, a.k.a. Cj1029); wlaK (UDP-4-keto-6-deoxy-GlcNAc C4 aminotransferase, a.k.a. Cj1 121 ); or cadF (Campylobacter adhesion to Fibronectin, a.k.a. Cj1478). The system of Campylobacter gene numbering is described by Gundogdu et al. (2007, BMC Genomics, vol. 8, p. 162).

Therefore, in an embodiment of the recombinant Eimeria vector for use in a vaccine according to the invention, the immunogenic protein from C. jejuni, or the immunogenic part thereof, is selected from the group consisting of the C. jejuni proteins: CjaA, omp18, jlpA, mapA, wlaK, and cadF. In addition, as is well known in the art, variants or homologs of such proteins can equally be used, provided they can induce an effective immune-response against C. jejuni colonisation when expressed in vivo via a recombinant Eimeria vector. Such a variant or homologous protein is at least 90% identical in amino acid sequence, and is therefore considered to be within the scope of the invention, providing a similar immuno-protective result, in a similar way.

Such variants or homologs can be of natural or synthetic origin, and can for example be derived by employing as the encoding nucleic acid sequence, a sequence that differs in nucleotide sequence, but that encodes essentially the same amino acid sequence compared to a natural C. jejuni gene. This is a result of the "degeneracy of the genetic code", wherein a heterology of up to 30% may exist between two nucleic acid sequences, while both still encode essentially the same protein.

For the invention, the immunogenic C. jejuni protein expressed and presented by the recombinant Eimeria vector preferably is a complete C. jejuni protein. However a part of such a protein may also be employed, as it can be advantageous for the stability and replication speed of the vector-insert construct to use an inserted coding sequence that is relatively short. The protein part can for example represent only the mature form of an immunogenic C. jejuni protein, i.e. without a 'leader', 'anchor', or 'signal sequence'. The expressed C. jejuni protein part may even be a specific section of a protein, comprising a particular immunoprotective epitope, as long as the expressed protein part induces an immune response in vivo that is capable of reducing the level of C. jejuni colonisation of a poultry's intestine to a significant degree. Therefore, for the invention, the expressed immunogenic C. jejuni protein is at least an immunogenic part of a C. jejuni protein.

What constitutes "an immunogenic part" for the invention can conveniently be determined using well known techniques; for example by generating tryptic digests of C. jejuni proteins and testing the immunogenicity of the fragments obtained. Also the fragments can be synthesized and tested as in the well known PEPSCAN method (WO 84/003564, WO 86/006487, and Geysen et al. (1984, PNAS USA, vol. 81 , p. 3998)). Alternatively, immunogenically relevant areas can be predicted by using well known computer programs. An illustration of the effectiveness of using these methods was published by Margalit et al. (1987, J. of Immunol., vol. 138, p. 2213) who described success rates of 75 % in the prediction of T- cell epitopes. Also, in general antigenicity frequently correlates with hydrophilicity as such regions will be found on the surface of a protein where it can interact with the host immune system. Therefore a highly hydrophilic region of a C. jejuni protein is an excellent candidate antigen. As is well known, protein parts in order to be immunogenic need to be of a minimal length; typically 8-1 1 aa for MHC I receptor binding, and 1 1-15 aa for MHC II receptor binding (reviewed e.g. by Germain & Margulies, 1993, Annu. Rev. Immunol., vol. 1 1 , p. 403). Therefore, for the invention, an immunogenic fragment of the polypeptide according to the invention is at least 8 amino acids in length.

Polypeptide fragments that still do not generate an effective immune response may be presented to a target's immune system attached to, or in the context of, an immunogenic carrier molecule. For the invention this relates to the expression by the recombinant Eimeria vector of the immunogenic C. jejuni protein as part of a fusion-protein with a carrier protein. Well known carriers are bacterial toxoids, such as Tetanus toxoid, e.g. as employed by Buckley et al. (2010, supra), or Diphteria toxoid, but also green fluorescent protein (GFP) has been used in a fusion construct to enhance the immunogenicity of recombinant expressed parasitic proteins (Kaba et al., 2002, Parasitology, vol. 125, p. 497). Like Eimeria, the C. jejuni for use in the invention may be obtained from animal- or field samples, or from a laboratory or institution, for example from ATCC or ECACC. Such a bacterial isolate can then be used to obtain the nucleotide sequence encoding an antigenic protein of C. jejuni using routine molecular biological techniques. However, more conveniently, the encoding nucleotide sequence information can also be taken from C. jejuni protein-gene sequences published e.g. in Genbank®, and the DNA can be synthesized in vitro. Many C. jejuni complete genomic sequences are published, for example: (EMBL according to the invention, nr.: AL1 1 1 168). But also a great many individual gene-sequences are published in GenBank or GeneDB under their genomic annotation, e.g.: CjaA is Cj0982; omp18 is Cj01 13; jlpA is Cj0983; mapA is Cj1029; wlaK is C j 1 121 ; and cadF is Cj1478.

In a preferred embodiment, the expression of the immunogenic C. jejuni protein by the recombinant Eimeria vector is targetted to arrive at a specific intra- or extracellular domain. For example, the expressed protein is targeted to the surface of the parasite, or to be secreted out of the parasite (ending up either in the cytosol of the host cell, or in the gut lumen. This can be achieved by employing suitable signal- or leader sequences, and different anchoring sequences, etc. For example, expression within the cytosol, the parasitophorous vacuole, or the micronemes are described by Shi et al. (2009, Paras. Res., vol. 104, p. 315), and Huang et al., 201 1 (supra).

The immunogenic C. jejuni protein-encoding nucleotide sequence can for example be incorporated into a standard DNA cloning plasmid, for example from pUC or pBR series; these are commercially available. This allows convenient subcloning, adaptation, and amplification, using routine molecular biological techniques. The resulting plasmid is then commonly referred to as a 'transfervector', and is suitable for use in transfection protocols. Exemplary embodiments of heterologous nucleic acid molecules for use in the invention, comprising a nucleotide sequence capable of expressing an immunogenic C. jejuni protein, are disclosed herein.

This way a series of plasmid constructs were made, which have been used to transfect Eimeria sporozoites and to generate recombinant Eimeria vectors according to the invention. For some plasmids tested, the expression of the immunogenic C. jejuni protein gene was driven by the E. tenella actin gene promoter, and with the termination region from the E. tenella MIC3 gene. Some of these plasmids also expressed a marker gene encoding Citrine (an enhanced version of yellow fluorescent protein) to allow FACS sorting and selection of a recombinant Eimeria vector. The Citrine gene was expressed using the E. tenella Mid promoter and -terminator sequences. Examples of plasmids tested were: pCIT_cjaA, pCIT_cj01 13, and pCIT_cj1029, each expressing a different immunogenic C. jejuni protein. See Figure 1 for a graphical representation of the pCIT_cjaA plasmid; the other two are similar, only comprising a different C. jejuni gene. The nt sequence of all three plasmids is presented in SEQ ID NO's: 1-3 respectively.

The recombinant Eimeria vector for use in a vaccine according to the invention, is advantageously employed in a vaccine composition for poultry as described herein. Therefore in a further aspect the invention relates to a vaccine against C. jejuni in poultry, comprising the recombinant Eimeria vector as described for the invention, and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" aids in the effective administration of an active vaccine compound, without causing (severe) adverse effects to the health of the target human or animal to which it is administered. Such a carrier can for instance be sterile water or a sterile physiological salt solution. In a more complex form the carrier can e.g. be a buffer, which can comprise further additives, such as stabilisers or preservatives. Details and examples are for instance described in well-known handbooks such as: "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472), and: "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681 ).

The vaccine according to the invention is prepared from live recombinant Eimeria vector parasites as described for the invention, by methods as described herein that are readily applicable by a person skilled in the art. For example, the recombinant Eimeria vector as described for the invention is constructed in vitro, transfected and the desired recombinant Eimeria vector is selected as described herein. Next the recombinant Eimeria vector parasites are produced industrially in smaller or larger volumes in donor poultry animals, and isolated from their droppings by well known techniques such as salt flotation, followed by sporulation and sterilisation, and finally counting by light microscopy. Sporulation can be performed e.g. using potassium-dichromate, and sterilisation can be done using sodium-hypochlorite or beta-propiolactone. The sporulated Eimeria are formulated into a vaccine and the final product is packaged. After extensive testing for quality, quantity and sterility such vaccine products are released for sale.

General techniques and considerations that apply to vaccinology are well known in the art and are described for instance in governmental regulations (Pharmacopoeia) and in handbooks such as: "Veterinary vaccinology" and: "Remington" (both supra).

The recombinant Eimeria vector vaccine according to the present invention in principle can be given to target poultry by different routes of application, and at different points in their lifetime, provided the administered recombinant Eimeria vector can establish an effective infection.

A C. jejuni infection can originate from day 1 , and be established by 2 - 3 weeks of age; also as

Eimeria are ubiquitous, therefore it is advantageous to apply the vaccine of the invention as early as possible, to provide the earliest possible protection.

Therefore, the vaccine according to the invention is preferably applied at the day of hatch or shortly thereafter, i.e. on day 1-3 of age after hatch, or even in ovo, e.g. at 18 days ED.

Next to in ovo inoculation the vaccine according to the invention may also be applied by other methods of mass vaccination. This minimises both the discomfort to the target animals as well as the labour costs. Suitable methods for mass application that are applicable at early age for a recombinant Eimeria vector vaccine according to the invention, should of course be compatible with a live Eimeria micro-organism such as the present vector vaccine, and aim for inoculation via the oral route. Preferred methods are: by coarse spray, by feed or drinking water, or by automated injection into the egg. Suitable equipment for industrial scale application is available commercially.

When applied by spray vaccination, the selection of droplet size used is important; generally a coarse spray applies a droplet size of over 50 μιτι. Different in ovo inoculation routes are known, such as into the yolk sac, the embryo, the amniotic fluid, or the allantoic fluid cavity; these can be optimised as required.

Therefore, in a further preferred embodiment, the vaccine according to the invention is applied in ovo.

Depending on the circumstances of the application of the vaccine according to the invention, e.g. the route and the target poultry species, it may be necessary to adapt the vaccine composition. This is well within the capabilities of a skilled person, and generally involves the fine-tuning of the efficacy or the safety of the vaccine. This can be done by adapting the vaccine dose, quantity, frequency, or the route, by using the vaccine in another form or formulation, or by adapting the other constituents of the vaccine (e.g. a stabiliser or an adjuvant). Typically when applied as coarse spray, an Eimeria vaccine may be mixed with a suspending agent, and with a red or green colorant to induce the birds to active pick-up the vaccine droplets; when applied on the feed, the vaccine may be formulated as a gel.

Similarly, to be suitable for application in ovo, the vaccine composition is required to be very mild, in order not to reduce the hatchability of the eggs. Some reduction of hatchability can be acceptable, e.g. by 10 %, more preferably by 5% or even less.

As will be apparent to the skilled practitioner, for a use in ovo, the nature of the parental Eimeria strain to be used as the vector needs to be carefully selected. In that regard, an attenuated, or even a precocious Eimeria strain is advantageously used. These are generally available and known to be suitable for in ovo inoculation. The incorporation of a heterologous nucleic acid molecule is not likely to increase their pathogenicity (on the contrary), and no return to a wildtype pathogenicity is applicable.

The exact amount of the recombinant Eimeria vector as described for the invention in a vaccine dose is not as critical as it would be for an inactivated type vaccine, because the recombinant Eimeria vector will readily replicate itself and thus colonise the host. The vaccine dose only needs to be sufficient to initiate such a productive infection. A higher inoculum dose hardly shortens the time it takes to reach the optimal colonisation in the host; and very high doses are not attractive for economic reasons. Evidently, too low a dose, although capable of establishing an Eimeria infection, may take too much time for a proper onset of immunity.

A preferred inoculum dose is therefore between 1 x10^Λ1 and 1χ10^Λ5 sporulated oocysts of the recombinant Eimeria vector per animal-dose, more preferably between 1x10^Λ2 and 1x10^Λ4 oocysts per dose, even more preferably between 100 and 5000 oocysts / dose. As will be apparent to the skilled person, the optimal vaccine dose will depend e.g. on the species and the virulence of the parental Eimeria strain used for the recombinant vector, as well as on the target animal species, the level of C. jejuni colonisation to be combated, etc..

Similarly, the determination of the immunologically effective amount of the vaccine according to the invention is well within reach of the skilled person, for instance by monitoring the reduction in the level of C. jejuni colonisation, and comparing this to responses seen in unvaccinated animals. The dosing schedule for applying the vaccine according to the invention to a target poultry can be in single or multiple doses, which may be given at the same time or sequentially, in a manner compatible with the formulation of the vaccine, and in such an amount as will be immunologically effective. The vaccine according to the invention can be used both for prophylactic and for therapeutic treatment, and so interferes either with the establishment and/or with the progression of a C. jejuni colonisation.

The vaccine according to the invention may effectively serve as a priming vaccination, which can later be followed and amplified by one or more further doses, as booster vaccination(s).

Preferably the vaccine according to the invention is applied only once, and ideally the protocol for the administration of the vaccine according to the invention is integrated into existing vaccination schedules of other vaccines. Typically vaccination via the feed or the drinking water will only be applicable from several days of age when the chicks will start feeding and drinking.

Preferably the vaccine according to the invention is applied at the day of hatch, or in ovo at day

18 ED.

The volume per animal dose of the recombinant Eimeria vector vaccine according to the invention can be optimised according to the intended route of application, e.g.: in ovo inoculation is commonly applied with a volume between 0,05 and 0,5 ml/egg. The determination of the optimal dosage volume is well within the capabilities of the skilled artisan.

The age, weight, sex, immunological status, and other parameters of the poultry to be vaccinated are not critical, although it is evidently favourable to vaccinate healthy targets, and to vaccinate as early as possible to prevent (the consequences of) an early colonisation by C. jejuni.

It is highly efficient to formulate the vaccine according to the invention as a combination-vaccine, because in this way multiple immunologic agents can be administered at once, providing a further reduction of discomfort to the vaccinated target animals, as well as of the time and labour costs. A combination vaccine comprises in addition to the vaccine according to the invention, another immunologically active compound. In principle this can be any live or killed micro-organism or subunit product, provided this does not reduce the stability in replication, or the expression from the recombinant Eimeria vector construct. Also, the additional immunoactive component(s) must be compatible with the application route (in ovo or oral) of the Eimeria vaccine. The additional immunologically active compound may be an antigen, an immune enhancing substance, a cytokine, and/or a vaccine

Alternatively, the vaccine according to the invention, may itself be added to a vaccine.

Therefore, in a further preferred embodiment, the vaccine according to the invention is characterised in that the vaccine comprises one or more additional immunoactive component(s).

In a more preferred embodiment the vaccine according to the invention is a combination vaccine, comprising at least one additional antigen derived from a micro-organism that is pathogenic to poultry. The additional antigen may be a live, live attenuated, or killed micro-organism, or a subunit antigen. Preferably the additional antigen from a micro-organism that is pathogenic to poultry is selected from the groups consisting of: viruses: infectious bronchitis virus, Newcastle disease virus, Adenovirus, Egg drop syndrome virus, Infectious bursal disease virus (i.e. Gumborovirus), chicken anaemia virus (CAV), avian encephalomyelitis virus, fowl pox virus, turkey rhinotracheitis virus, duck plague virus (duck viral enteritis), pigeon pox virus, Marek's disease virus, avian leucosis virus, ILTV, avian pneumovirus, avian influenza, and reovirus;

bacteria: Escherichia coli, Salmonella spec, Ornitobacterium rhinotracheale, Haemophilis paragallinarum, Pasteurella multocida, Erysipelothrix rhusiopathiae, Erysipelas spec, Mycoplasma spec, and Clostridium spec;

endo- or ecto-parasites: Eimeria spec, Histomonas spec, and Dermanyssus spec; and

- fungi: e.g. Aspergillus spec.

Most preferred additional antigen is: IBV, NDV, IBDV, ILT, TRT, AIV, MDV, Mycoplasma, Salmonella or Eimeria. Because of its highly immunogenic nature, the recombinant Eimeria vector vaccine according to the invention would not normally be formulated to comprise an adjuvant. However, when formulated as a combination vaccine, or to accommodate specific requirements of the target poultry or the severity of the level of C. jejuni colonisation, a suitable adjuvant may be added.

An "adjuvant" is a well known vaccine ingredient, which in general is a substance that stimulates the immune response of the target in a non-specific manner. Many different adjuvants are known in the art. Examples of adjuvants are Freund's Complete and -Incomplete adjuvant, vitamin E, non-ionic block polymers and polyamines such as dextransulphate, carbopol, pyran, and Saponin, such as Quil A®.

Furthermore, peptides such as muramyldipeptides, dimethylglycine, tuftsin, are often used as adjuvant, and mineral oil e.g. Bayol® or Markol®, vegetable oils or emulsions thereof and DiluvacForte® can advantageously be used.

Evidently the adjuvant should be mild enough not to damage the live recombinant Eimeria vector. An alternative therefore is to add an immunostimulatory molecule, for example a cytokine, e.g. gamma- interferon; an immune-stimulatory oligonucleotide, e.g. carrying a so-called CpG motif; or any suitable kind of TLR agonist.

The vaccine according to the invention can advantageously be combined with a pharmaceutical component such as an antibiotic, a hormone, or an anti-inflammatory drug.

The use of an anticoccidial compound is also possible, provided that the recombinant Eimeria vector is not sensitive to that particular drug.

As described, there are various ways the vaccine according to the invention can be composed and formulated, depending on the desired target, route of application, combination of immunologically active compounds, etc.. Therefore, in a further aspect the invention relates to the use of the recombinant Eimeria vector as described for the invention, for the manufacture of a vaccine against C. jejuni in poultry. And, in still a further aspect the invention relates to a method for the preparation of the vaccine according to the invention, said method comprising the admixing of the recombinant Eimeria vector as described for the invention, with a pharmaceutically acceptable carrier. The vaccine manufactured according to the use or the method according to the invention may contain one or more components that aid the viability and quality of the recombinant Eimeria vector according to the invention, thereby promoting the productive replication and establishment of a protective colonisation in target poultry.

The additive may be a stabiliser, to stabilise the quantity and the quality of the recombinant Eimeria vector according to the invention during storage, handling, and inoculation, such as by injection or ingestion. Generally stabilisers are large molecules of high molecular weight, such as lipids, carbohydrates, or proteins; for instance milk-powder, gelatine, serum albumin, sorbitol, trehalose, spermidine, dextrane or polyvinyl pyrrolidone.

Also suitable preservatives may be added, such as thimerosal, merthiolate, phenolic compounds, or gentamicin.

In a preferred embodiment, the compounds used for the manufacture of the (combination) vaccine composition according to the invention are serum free (i.e. without animal serum); protein free (without animal protein, but may contain other animal derived components); animal compound free (ACF; not containing any component derived from an animal); or even 'chemically defined', in that order of preference.

It goes without saying that admixing other compounds, such as carriers, diluents, emulsions, and the like to vaccines according to the invention are also within the scope of the invention. Such additives are described in well-known handbooks such as: "Remington", and "Veterinary Vaccinology" (both supra).

For reasons of stability or economy a vaccine according to the invention may be manufactured in freeze- dried form. In general this will enable prolonged storage at temperatures above zero °C, e.g. at 4°C. Procedures for freeze-drying are known to persons skilled in the art, and equipment for freeze-drying at different scales is available commercially. Evidently, the recombinant Eimeria vector should be in a relatively robust form, in order to survive the freezing, drying, storage, and thawing periods. Therefore a favourable embodiment is to freeze-dry the sporozoite form of the recombinant Eimeria vector according to the invention.

Therefore, in a further preferred embodiment, the vaccine manufactured according to the use or to the method of the invention is in a freeze-dried form.

To reconstitute a freeze-dried vaccine composition, it is commonly suspended in a physiologically acceptable diluent. Such a diluent can e.g. be as simple as sterile water, or a physiological salt solution, e.g. phosphate buffered saline (PBS); alternatively the diluent may contain an adjuvating compound, such as a tocopherol, as described in EP 382.271. In a more complex form the freeze-dried vaccine may be suspended in an emulsion e.g. as described in EP 1.140.152. As described, the vaccine according to the invention can advantageously be applied to poultry by a method of mass-vaccination such as by spray, via the feed or drinking water, or by in ovo application.

Therefore, in a further aspect the invention relates to a method for the vaccination of poultry against C. jejuni, comprising the step of inoculating said poultry with the vaccine according to the invention.

As the recombinant Eimeria vector will also induce a (type-specific) immune-response against Eimeria, therefore a preferred embodiment of the method for the vaccination of poultry according to the invention, comprises a method for the vaccination of poultry against both C. jejuni and Eimeria.

The invention will now be further described with reference to the following, non-limiting, examples.

Examples

1. Materials and Methods 1.1. Parasites and animals

E. tenella oocysts of the Wisconsin (Wis) laboratory strain (McDougald et al., 1976, Science, vol. 192, p. 258) were propagated in vivo in three to seven week old Light Sussex chickens under specific pathogen free (SPF) conditions using established methods (Long et al., 1976, Folia Veterin. Lat., vol. 6, p. 201 ). Oocysts were cracked and sporozoites hatched and purified through columns of DE-52 supported by nylon wool using standard procedures (Shirley et al., 1995, in: 'Guidelines on techniques in coccidiosis research', Ed. Eckert et al., European Commission, p. 1-24).

1.2. PCR amplification

PCR amplification was completed using BIO-X-ACT® Short DNA Polymerase (Bioline Ltd.). Each PCR reaction contained 5 ng template DNA, 20 pmol of relevant forward and reverse primers, 0.5 U Taq polymerase, 10 mM Tris-HCI, 1 .5 mM MgCI₂, 50 mM KCI and 0.2 mM dNTPs. Standard cycling parameters were 1 x (5 min. at 95 °C), 30 x (30 sec. at 95 °C, 30 sec. at 50 °C and 1 min. at 68 °C) and 1 x (10 min. at 70 °C). After amplification the PCR products were resolved by standard agarose gel electrophoresis. PCR fragments of interest were gel excised and purified (minelute® gel purification kit, Qiagen), cloned using pGEM®-T Easy (Promega) in XL1-Blue Escherichia coli (Stratagene), and DNA was miniprepped (Qiagen) and sequenced (Beckman CEQ 8000 genetic analysis system), all according to the manufacturer's instructions. Sequence assembly, annotation and interrogation were undertaken using VectorNTI® v1 1.0 (Invitrogen) or Staden version 1.7.0.

1.3. Transfection construct production The C. jejuni CjaA gene coding sequence was amplified from plasmid pTech-CjaA-M 1 (Buckley et al., 2010, supra), incorporating Xbal and Pad restriction sites using the primers F-CjaA-Xbal (SEQ ID NO: 4) and R-CjaA-Pacl (SEQ ID NO: 5) to amplify nucleotides 4-831 of the CjaA coding sequence. Following Xba l/Pac I restriction enzyme digestion the amplified CjaA coding sequence was cloned into an Xba l/Pac I digested Eimeria core transfervector (Clark et al., 2008, supra), replacing the dihydrofolate reductase-thymidylate synthase coding sequence. The resulting plasmid, named pCIT_cjaA, was sequenced to confirm integrity as described above (Figure 1 , SEQ ID NO: 1 ). Plasmid DNA was purified using the Qiagen EndoFree® Plasmid Maxi Kit as described by the manufacturer.

1.4. Parasite transfection

Parasite transfection was carried out essentially as described previously using a restriction enzyme mediated integration (REMI) strategy (Clark et al., 2008, supra), with some modifications, briefly: 12.5 μg purified plasmid DNA was digested with Seal overnight prior to electroporation using Basic Parasite Nucleofector Kit 2® buffers and reagents (Lonza) supplemented with the restriction enzyme Seal and using program U-033 in an AMAXA® Nucleofector II (Lonza) electroporation device. Negative controls included no plasmid and no electroporation transfections (mock-transfected and untransfected respectively).

1.5. Experimental design

Post-electroporation sporozoites were cultured in vitro and/or in vivo. For in vivo culture 1.0 x 10⁵ electroporated sporozoites were administered to each of ten chickens via the cloacal route. No in vivo drug selection was used. Seven days post infection birds were euthanized for caecal oocyst harvest. The oocysts were sporulated and examined microscopically for expression of the Citrine fluorescent protein. Subsequent selective passage of the recombinant (i.e. reporter-expressing) parasite sub-population was initiated using oral administration of 9,500 FACS sorted fluorescent sporocysts purified from the first generation recombinant population. Two further serial selective in vivo passages were undertaken using doses of 12,500-15,000 FACS sorted sporocysts administered orally to each of two chickens per generation. Oocysts were harvested from the caecae on each occasion with the exception of the fourth generation, when a faecal harvest was used from days seven to nine post-infection. Recombinant Eimeria vector parasites were examined using an inverted fluorescent microscope (Leica DM IRB) equipped with a GFP S filter.

1.6. FACS selection of transfected Eimeria

Released and purified E. tenella sporocysts were sorted using an excitation wavelength of 488 nm and emission filter of 330/30 on a FACSCalibur® flow cytometer (Becton Dickinson). FCS Express® (De Novo Software, Ontario, Canada) was used for data analysis.

1.7. RT-PCR

Transcription of the heterologous gene in the recombinant, was determined using total RNA purified from electroporated sporozoites after 24 hours in vitro culture (processed using all cells, both parasite and MDBK, recovered from each culture plate well) and fourth generation oocysts using an RNeasy® kit (Qiagen) according to the manufacturer's instructions, with an oocyst smashing step included where appropriate (Blake et al., 2003, Paras. Res., vol. 90, p. 473). A DNase digestion step was included to remove any residual plasmid used in the electroporation. Recovered RNA was reverse transcribed to produce cDNA using Invitrogen Oligo dT with Superscript® II reverse transcriptase. PCRs were performed as described above using total RNA (without reverse transcription) in addition to no template, mock transfected and transfected parasite cDNA samples. Primers targeting the CjaA recombinant were: F-CjaA_RT1 (SEQ ID NO: 6), R-CjaA_RT1 (SEQ ID NO: 7), F-CjaA_RT2 (SEQ ID NO: 8), and R- CjaA_RT2 (SEQ ID NO: 9). These were supplemented with primers targeting the Citrine reporter-gene to provide positive controls: mCit_RTf (SEQ ID NO: 10), and mCit_RTr (SEQ ID NO: 1 1 ).

1.8. Vaccination trial design

A total of 64 White Leghorn chickens were split into four groups of 16 in independent wire-floored isolators (Groups A-D). Birds in Group A were vaccinated using a single oral infection with 300 fourth generation CjaA-transfected parasites (study day 1 ; two days post-hatch). Birds in Group B were vaccinated by serial oral infection with 100, 500, 3,000 and 5,000 fourth generation CjaA-transfected parasites (days 1 , 3, 7 and 20 respectively). Birds in Group C were vaccinated by serial oral infection with wild-type E. tenella Wis strain oocysts following the same schedule as Group B. Birds in Group D were left unvaccinated.

All birds were cloacally swabbed on day 23 to pre-screen for Campylobacter infection prior to challenge, by direct plating onto cefoperzone charcoal desoxycholate agar (CCDA; Oxoid) followed by microaerophilic incubation at 42°C for 48 h (Oxoid CampyGen® system). On day 28 the C. jejuni challenge inoculum was prepared using C. jejuni strain 02M6380 (obtained from Public Health service, Australia) grown overnight at 42°C in tryptone soy broth + 1 % Yeast extract (growth medium), diluted to about 10⁶ colony forming units per ml in growth medium based upon optical density. In total 0.1 ml, representing about 10⁵ cfu C. jejuni, was dosed orally to each challenged bird. The actual challenge dose was calculated retrospectively by serial dilution in growth medium and plating onto CCDA followed by microaerophilic incubation at 42°C for 48 h. Caecal swabs were collected at post-mortem for quantitative culture of C. jejuni on day 42 using a tenfold dilution series in growth medium and CCDA agar as described. 1.9. Statistical analysis

Statistical analyses including calculation of arithmetic means, associated standard error of the mean, ANOVA and associated post-hoc Tukey's tests were performed using PASW® Statistics 18 (IBM, 2009). Bacterial counts were logarithmically transformed. Differences were deemed significant with a p value < 0.05.

2. Results

2.1. E. tenella CjaA transfection and expression in vitro

Microscopic examination of transiently transfected sporozoites in cell culture 24 hours post- electroporation revealed that 3.3% ± 0.6% expressed the fluorescent Citrine reporter (the average of four replicate wells). No fluorescence was detected in mock-transfected or untransfected negative controls. RT-PCR for the CjaA transcript using RNA purified from whole cell culture lysates with the primer sets CjaA_RT1 and 2, yielded single products of the expected sizes (both about 500 bp ) from pCIT_CjaA transfected E. tenella infected cells (Figure 2). 2.2. CjaA-E. tenella passage and expression in vivo

Following in vivo passage 0.8% (± 0.3%) of the first generation sporulated oocysts expressed the fluorescent Citrine reporter. Subsequent serial passage used FACS selection to sort reporter-expressing sporocysts for each inoculum, resulting in gradually higher proportions of reporter-expressing oocysts in each generation up to the fourth generation (Table 1 ). RT-PCR for the CjaA transcript using primer sets CjaA_RT1 and -2 with RNA purified from fourth generation recombinant oocysts yielded a product of the expected size (Figure 3).

Table 1 : Summary statistics defining the creation and selection of the CjaA-expressing recombinant E. tenella population.

Stage dosed Dose/bird Oocysts Generation Oocysts % fluorescent in/bird ^a) recovered out/bird

Transfected sporozoites 1.00χ10^Λ05 1.25χ10^Λ04 1 1.50χ10^Λ07 0.8

FACS sporocysts 9.50x10^Λ03 2.38x10^Λ03 2 2.20x10^Λ07 18

FACS sporocysts 1.50χ10^Λ04 3.75x10^Λ03 3 6.50x10^Λ07 25

FACS sporocysts 1.25χ10^Λ04 3.13χ10^Λ03 4 2.20x10^Λ07 56 ^aDose per bird presented as oocyst equivalents.

SDS-PAGE resolution of recombinant E. coli-produced CjaA protein including 100 mM DTT in the loading buffer resulted in five bands of about 7.5, 29, 30, 36 and 60 KDa, potentially representing different protein isoforms and/or processing (not shown). Addition of 100 mM β-mercaptoethanol to the loading buffer reduced the appearance of all bands with the exception of the about 30 KDa band (Figure 4, lane 1 ).

2.3. Protective capacity of E. tenella delivered CjaA against level of C. jejuni colonisation

Pre-challenge caecal swabbing revealed the absence of C. jejuni from all test birds. Comparison of caecal C. jejuni load 14 days after bacterial challenge revealed a significantly lower colonisation of about one order of magnitude in both groups previously vaccinated using the recombinant CjaA-expressing E. tenella population (p<0.001 ; Table 2). No significant difference was noted between single and multiple vaccination strategies.

Cloacal swabbing 14 days after C. jejuni challenge revealed that delivery by a recombinant Eimeria vector provided effective immunity already at two weeks post vaccination. Table 2: The influence of immunisation using E. tenella-delivered CjaA on the level of caecal C. jejuni colonisation.

Group Treatment Avg. Log-io CFU (SEM)

E. tenella-CjaA, single immunisation Test 1 7.28^a (0.15)

E. tenella-CjaA, multiple immunisations Test 2 7.50^a (0.15)

E. tenella-WT, multiple immunisations Control 1 8.53^b (0.07)

Unimmunised Control 2 8.34^b (0.14)

NB: Figures in the last column that are labelled with a different superscript letter are significantly different (p < 0.05, oneway ANOVA, followed by post-hoc Tukey's test).

3. Discussion

An E. tenella population was produced which constitutively transcribed the C. jejuni vaccine candidate gene CjaA under the control of an actin promoter. Inclusion of the fluorescent Citrine reporter gene on the transfection construct provided a means of selectively isolating transfected parasites and supported the validation of successful transfection. Serial selective passage utilising FACS sorting of purified sporocysts has yielded a recombinant parasite population defined by 56% fluorescent reporter expression by the fourth generation. Despite the unfixed nature of this population, experimental vaccination trials using such fourth generation oocysts clearly displayed the capacity of the E. tenel la-vectored CjaA to induce significant levels of immune responses, protective against C. jejuni challenge inoculation.

Comparison with studies using Salmonella typhimurium to express and deliver CjaA to SPF birds

(Buckley et al., 2010, supra), revealed a comparable level of immune protection even in the absence of any drug selection in the Eimeria system, however delivery by Eimeria induced significant protection already at two weeks post vaccination rather than at three weeks.

Because reduction of level of C. jejuni colonisation by a factor 10 dramatically reduces the bacterial contamination of poultry meat products, this is an impressive result.

The transfection construct used in these studies included a constitutive promoter (from the actin gene) which supported continuous expression of the inserted heterologous genes throughout the multiple rounds of invasion and intracellular development that characterise the eimerian life cycles. Probably this accounts for the lack of significant difference between single and multiple vaccine inoculation strategies tested here. Since each ingested Eimeria species oocyst is expected to be capable of producing 0.5-1.0 x 10^Λ6 progeny oocysts under optimal conditions (in chickens), each vaccine recipient will be exposed to a massive number of overlapping invasion (or vaccination) events starting within a few hours of inoculation and lasting for up to ten days. Although the recombinant Eimeria population used as the vaccine inocula here was not 100% pure, there was clearly no lack of stability, and this did not prevent the development of significant and strong immune protection. Complete purity will be obtained by further serial selective passage or direct selection-cloning by passage of a single recombinant sporocyst per chicken.

Using quantitative PCR between 1 and 15 genomic insertions in stable recombinant E. tenella lines have been identified following a REMI strategy. This had no significant impact on the viability of the recombinant Eimeria vector. Similarly, two different heterologous genes have been successfully expressed, following their introduction on a single construct.

CjaA gene expression, and effective immune-delivery by the recombinant E. tenella population was therefore conclusively demonstrated by evidence of transcription as well as the observed immune protection.

4. List of sequences described: Transfer vector plasmids:

SEQ ID NO: 1 : pCIT_cjaA

SEQ ID NO: 2: pCIT_cj01 13

SEQ ID NO: 3: pCIT_cj1029 PCR-cloninq primers:

SEQ ID NO: 4: F-CjaA-Xbal

SEQ ID NO: 5: R-CjaA-Pacl

SEQ ID NO: 6: F-CjaA_RT1

SEQ ID NO: 7: R-CjaA_RT1

SEQ ID NO: 8: F-CjaA_RT2

SEQ ID NO: 9: R-CjaA_RT2

SEQ ID NO: 10 : mCit_RTf

SEQ ID NO: 1 1 : mCit RTr

Legend to the figures

Figure 1 :

Graphical representation of the assembled transfervector: pCIT_cjaA (SEQ ID NO: 1 ). Light grey block arrows denote coding sequences pointing in the direction of transcription. Black block arrows indicate promoter- and terminator regions. CIT = Citrine gene, cjaA = C. jejuni antigen A gene, MIC = microneme protein genes derived regions.

Figure 2:

RT-PCR confirmation of CjaA transcription in reporter-expressing recombinant sporozoites. Lanes 1-4: no template, mock-transfected, no reverse transcription and transfected parasites.

Figure 3:

RT-PCR confirmation of CjaA transcription in reporter-expressing fourth generation recombinant oocysts. Lanes 1-4: no template, mock-transfected, no reverse transcription and transfected parasites.

Figure 4:

Gel-electrophoretic analysis of recombinant Eimeria vector-expressed CjaA protein. M= marker lane; Lane 1 = Coomassie-stained recombinant CjaA, electrophoresed using SDS-PAGE in buffer including 100 mM DTT and 100 mM β-mercaptoethanol.

Claims

Claims:

1 . A recombinant Eimeria vector for use in a vaccine against Campylobacter jejuni in poultry, wherein the recombinant Eimeria vector comprises a heterologous nucleic acid molecule which comprises a nucleotide sequence capable of expressing an immunogenic protein from C. jejuni, or an immunogenic part of said protein.

2. The recombinant Eimeria vector according to claim 1 , wherein the recombinant Eimeria vector is based on Eimeria tenella, Eimeria maxima or Eimeria acervulina.

3. The recombinant Eimeria vector according to any one of claims 1 or 2, wherein the

recombinant Eimeria vector is based on an attenuated Eimeria strain.

4. The recombinant Eimeria vector according to any one of claims 1 - 3, wherein the

immunogenic protein from C. jejuni, or the immunogenic part thereof, is selected from the group consisting of the C. jejuni proteins: CjaA, omp18, jlpA, mapA, wlaK, and cadF.

5. Vaccine against C. jejuni in poultry, comprising the recombinant Eimeria vector according to any one of claims 1 - 4, and a pharmaceutically acceptable carrier.

6. Use of the recombinant Eimeria vector according to any one of claims 1 - 4, for the

manufacture of a vaccine against C. jejuni in poultry.

7. Method for the preparation of the vaccine according to claim 5, said method comprising the admixing of the recombinant Eimeria vector according to any one of claims 1 - 4, with a pharmaceutically acceptable carrier.

8. Method for the vaccination of poultry against C. jejuni, comprising the step of inoculating said poultry with the vaccine according to claim 5.