HK1216149B - Campylobacter vaccine - Google Patents

Description

Campylobacter vaccine

Technical Field

The present invention relates to a Campylobacter vaccine. More specifically, the present invention relates to a Campylobacter vaccine comprising Escherichia coli cells expressing a Campylobacter jejuni heptasaccharide glycan derived from the N-glycosylation pathway.

Background Art

In North America and many industrialized countries, the Gram-negative bacterium Campylobacter is the most common bacterial cause of gastroenteritis in humans. Campylobacter is also a significant foodborne pathogen in livestock, including poultry, which is considered the main source of campylobacteriosis in humans. Therefore, on-farm control of Campylobacter in poultry will reduce the risk of human exposure to this pathogen and have a significant impact on food safety and public health.

Campylobacter is endemic to many developing countries, primarily due to poor sanitation and close human contact with animals that serve as reservoirs for the pathogen. A report by Katarzyna et al. (Expert Rev. Vaccines 8:625-645, 2009) suggests that Campylobacter infection is responsible for between 1.5 million (World Health Organization data) and 2.4 million (U.S. Centers for Disease Control data) cases of illness annually in the United States. Furthermore, according to the World Health Organization, approximately 1% of the population in Western Europe is infected with Campylobacter annually. Human infection is primarily caused by two species: Campylobacter coli (C. coli) and Campylobacter jejuni, which together account for over 95% of cases of campylobacteriosis. The clinical manifestations of Campylobacter infection can range from asymptomatic cases to severe gastroenteritis, sometimes accompanied by persistent, mucous, bloody, or watery diarrhea.

Jun Lin, "Novel Approaches for Campylobacter Control in Poultry" (FOODBORNE PATHOGENS AND DISEASE, Vol. 6, No. 7, pp. 755-765, 2009), a publication incorporated herein by reference, discusses various strategies for reducing Campylobacter infection in poultry. Lin proposes three general strategies for controlling Campylobacter in poultry at the farm level: (1) reducing environmental exposure (biosecurity measures), (2) improving host resistance in poultry to reduce Campylobacter transport in the digestive tract (e.g., competitive exclusion, vaccination, and host genetic selection), and (3) using antimicrobial alternatives to reduce and even eliminate Campylobacter from colonized chickens (e.g., phage therapy and bacteriocin treatment). Lin further states that, in addition to biosecurity measures, other intervention methods are not commercially available and are still under development.

Eliminating these pathogens from livestock can be used as a means to reduce the incidence of human infection and prevent spread among farm animals. Vaccination on farms can also reduce the risk of contamination from humans who eat or handle animal products and from livestock manure that sheds bacteria through feces. Treating campylobacteriosis with antibiotics is also becoming increasingly challenging as antibiotic resistance of Campylobacter to previously effective antibiotics becomes more common.

Glycosylation was once clearly considered a eukaryotic phenomenon, but was later shown to be widespread in both the archaeal and bacterial domains. Bacterial O- and N-bonds are formed with a wider range of sugars than those observed in eukaryotic glycoproteins. The general glycosylation pathway for proteins in bacteria was first demonstrated in Campylobacter jejuni (Szymanski et al., Molecular Microbiology, 32: 1022-1030, 1999). The glycosylation machinery of Campylobacter jejuni has been characterized and has even been successfully transferred to Escherichia coli (Wacker et al., Science, 298: 1790-1793, 2002) and demonstrated active N-glycosylation of proteins (Young et al., J Biol Chem, 277: 42530-42539, 2002; Wacker et al., Science, 298: 1790-1793, 2002). A gene locus in Campylobacter jejuni, called pgl (for protein glycosylation), is involved in the glycosylation of multiple proteins. Mutational silencing of this locus results in loss of immunogenicity of multiple proteins, among other biological phenotypes.

U.S. Patent Application Publication 2006/0165728A1 (now U.S. Patent No. 7,598,354, incorporated herein by reference) identified a specific and highly immunogenic heptasaccharide present in multiple periplasmic and surface-exposed glycoproteins of Campylobacter jejuni. This heptasaccharide is common to at least several Campylobacter species and many strains that are important as human and veterinary pathogens (Nothaft et al. Mol. Cell. Proteomics 11: 1203-1219, 2012). The heptasaccharide has the following formula (I): GalNAc-α1,4-GalNAc-α1,4-[Glc-β-1,3]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-diNAcBac, where diNAcBac (also known as di-N-acetylbactamido) is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose, GalNAc is N-acetyl-galactosamine, and Glc is glucose. This glycan moiety is a component of various glycoproteins. In Campylobacter jejuni, N-glycans are important for the interaction of Campylobacter jejuni with host cells. Mutations in the glycosylation machinery cause reduced colonization of the intestine in mice and chickens. In C. jejuni, N-glycans are important for attachment and invasion of human epithelial cells (Szymanski et al. Infect Immun 70:2242-2244, 2002), intestinal colonization of mice and chickens (Kelly et al. J Bacteriol 188:2427-2434, 2006; Szymanski et al. Infect Immun 70:2242-2244, 2002; Hendrixson and DiRita, Mol Microbiol. (Microbiol. 52:471-484, 2004; Karlyshev et al. Microbiology 150:1957-1964, 2004), natural competence in strains with a type IV secretion system (Larsen et al. J. Bacteriol. 186:6508-6514, 2004), and binding to the human macrophage C-type lectin MGL (van Sorge et al. Cell Microbiol. 11:1768-1781, 2009). In addition, Campylobacter surface N-glycans have been shown to protect against chicken digestive tract proteases, resulting in increased bacterial fitness (Alemka et al. Infect. Immun. 81:1674-82, 2013).

U.S. Patent Application Publication No. 2012/0100177 describes a Salmonella enterica strain that contains at least one pgl operon of Campylobacter jejuni or a functional derivative thereof and displays at least one N-glycan of Campylobacter jejuni or a glycan derivative thereof on its cell surface. This recombinant Salmonella enterica strain is hypothesized to be suitable for use in vaccines against Campylobacter infection, particularly in livestock (e.g., poultry). Unfortunately, however, subsequent publications demonstrated that, although recombinant Salmonella enterica expressing N-glycans from Campylobacter on its surface was able to colonize chickens without causing disease, no humoral immune response against Campylobacter N-glycans was detectable in vaccinated chickens (Thommen, "Campylobacter N-glycan presenting Salmonella Typhimurium: a new vaccine for broiler chickens?" Zurich Open Repository and Archive, University of Zurich, Dissertation, Vetsuisse Faculty, 2011). Furthermore, C. jejuni colonization was not reduced in vaccinated chickens following challenge infection with C. jejuni.

There remains a need for an effective vaccine for preventing and/or treating Campylobacter infections in humans and animals, particularly livestock, and more particularly poultry.

This background information is provided for the purpose of the applicant believing that known information may be relevant to the present invention. It is not necessarily intended to, or should be construed as, an admission that any of the foregoing information constitutes prior art against the present invention.

Summary of the Invention

An object of the present invention is to provide a vaccine against Campylobacter. According to one aspect, a vaccine composition is provided, comprising bacteria engineered to express at least one N-glycan of Campylobacter or a glycan derivative thereof on their cell surface; and one or more of a physiologically acceptable diluent, excipient, adjuvant, or carrier. In certain embodiments, the Campylobacter species is Campylobacter jejuni. The bacteria can be Escherichia coli or Salmonella, and the engineered bacteria express the Campylobacter jejuni heptasaccharide on their surface.

In certain embodiments, the vaccine composition comprises live, engineered E. coli or live, attenuated, inactivated or killed engineered E. coli cells. The composition may comprise a suspension of engineered bacteria in a suitable buffer diluent (such as phosphate buffered saline) and may be formulated for, for example, oral administration, intravenous administration, parenteral administration (such as by injection or infusion) or spraying. The vaccine composition may also be formulated for addition to livestock feed, feed additives or water and for administration to poultry (such as chickens).

According to another aspect, a method of vaccinating an animal against Campylobacter is provided, the method comprising administering to the animal a vaccine composition as described herein, the vaccine composition comprising a bacterium engineered to express at least one N-glycan of Campylobacter (such as Campylobacter jejuni) or a glycan derivative thereof on its cell surface; and one or more of a physiologically acceptable diluent, excipient, adjuvant, or carrier.

It is known that expression of N-glycans on Salmonella does not induce a protective immune response. Surprisingly, the present inventors found that E. coli (which is a very similar bacterium to Salmonella and similar results would be expected) does induce a protective immune response in chickens when expressing N-glycans.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the present invention, as well as other aspects and further features thereof, reference is made to the following description taken in conjunction with the accompanying drawings, in which:

FIG1 shows E. coli proteinase K-treated cell lysates from E. coli polymerase mutants.

Figure 2 shows the structure of lipid A-N-glycan and NMR experiments of purified lipid A-C. jejuni N-glycan fractions.

FIG3 shows a FACS experiment using E. coli polymerase mutants.

Figures 4A, B, C and D depict vaccination and challenge experiments as described in Example 2, and

Figures 5A and B depict chicken IgY (IgG) N-glycan-specific antibody responses (ELISA).

DETAILED DESCRIPTION

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "comprising" will be understood to mean that the following list is non-exhaustive and may or may not include any other additional suitable items, such as one or more other features, components and/or ingredients, as desired.

The terms "C. jejuni glycan," "C. jejuni heptasaccharide," "N-glycan heptasaccharide," "Campylobacter N-glycan," and "heptasaccharide" are used interchangeably herein to refer to a glycan moiety present in a variety of surface-exposed glycoproteins and free oligosaccharides in various strains and species of Campylobacter. In the case of C. jejuni and as exemplified herein, this glycan has the formula: GalNAc-α1,4-GalNAc-α1,4-[Glc-β-1,3]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-diN AcBac, wherein diNAcBac is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose. These terms may refer to glycosylation by N-glycosylation or using sugars derived from N-glycosylation or other pathways. Recent work by the present inventors has shown that C. jejuni N-glycans and oligosaccharides are reasonably conserved among thermophilic species of Campylobacter (Nossoff et al. Mol. Cell. Proteomics 11: 1203-1219, 2012), and that some species that produce hexasaccharide derivatives of the heptasaccharide lack glucose branches. The use of alternative N-glycan structures and oligosaccharides present in non-thermophilic Campylobacter species described by (Nossoff et al. Mol. Cell. Proteomics 11: 1203-1219, 2012) such as those described in PCT Publication WO/2011/097733 is also contemplated and incorporated herein by reference.

As used herein, the term "antigen" refers to a chemical or biological substance that induces an immune response in an animal or human. In the system described in the present invention, the antigen comprises a hepta-saccharide of Campylobacter jejuni or an N-glycan derivative thereof. As used herein, the term "N-glycan derivative" refers to a derivative of a hepta-saccharide that induces an immune response similar to or better than that induced by the hepta-saccharide itself in an animal. N-glycans can be bound to a carrier, such as a protein (as described in PCT application No. WO2012/027850) and a lipid (Nosaf et al. Molecular and Cellular Proteomics 11: 1203-1219, 2012, Van Sorge et al. Cell Microbiology 11: 1768-1781, 2009), such as a hepta-saccharide or a shorter or longer carbohydrate repeat sequence to a polysaccharide.

As used herein, the term "vaccine" refers to a composition used to improve immunity in animals or humans against certain microorganisms. The vaccines described herein can be used in a variety of animals, such as birds, such as poultry, and mammals. The microorganisms targeted by the vaccines described herein are Campylobacter species.

As used herein, the term "Campylobacter" refers to the bacterial genus comprising any and all species of the genus Campylobacter. The various species of Campylobacter in this genus include, but are not limited to, C. jejuni, C. hominis, C. rectus, C. lari, C. fetus, C. coli, C. upsaliensis, C. fetus subsp. venerealis, C. fetus subsp. fetus, C. peloridis, C. lari subsp. concheus, C. sputorum, C. gracilis, C. showae, C. lanienae, C. curvus, C. helveticus, C. hyointestinalis subsp.hyointestinalis), C.hyointestinalis subsp.lawsonii, C.mucosalis, C.sputorum bv.paraureolyticus, C.sputorum bv.fecalis, C.ureolyticus, C.insulaenigrae, C.concisus, C.subantarcticus, C.avium, C.cuniculorum, and C.volucris.

The present application provides a polysaccharide and immunologically active fragments thereof that can be used as a vaccine against Campylobacter infection in humans and animals. The vaccine can be used to prevent or neutralize Campylobacter infection in livestock, thereby preventing this pathogen from entering the human food chain. In certain embodiments, the Campylobacter jejuni heptasaccharide and fragments thereof, optionally linked to amino acids, oligopeptides, lipids, or other suitable conjugates, can be used as a vaccine. For example, the vaccine can be used in any animal infected with Campylobacter, wherein the N-glycan can be expressed on the surface of Escherichia coli as a lipid A core fusion.

Vaccine composition

The present application provides a vaccine composition comprising recombinant Escherichia coli that has been engineered to express at least one Campylobacter N-glycan or a heptasaccharide derivative thereof on its surface. The recombinant Escherichia coli is alive, dead and/or attenuated.

As described above, the Campylobacter heptasaccharide is common to at least several Campylobacter species and many strains, including those that are important as human and veterinary pathogens. It is a component of a variety of glycoproteins, including, for example, Campylobacter jejuni Nos. Cj0114, Cj0200c, Cj0289c, Cj0367c, and others. This glycan moiety is also highly immunogenic, and therefore, this glycan (and its related derivatives and glycopeptides containing N-glycans or their derivatives) have been identified as good candidates for use as antigens in vaccines for immunization against various strains and species of Campylobacter in mammals, including humans and livestock, including chickens (U.S. Patent No. 7,598,354).

Escherichia coli is a Gram-negative bacterium with an outer membrane covered in lipopolysaccharide (LPS), which contributes to the structural integrity of the bacterium and provides a physical barrier to protect the membrane. LPS is composed of three main components: lipid A, core, and O-antigen. Lipid A anchors LPS to the outer membrane, and O-antigen is the outermost part of LPS. The core is a branched oligosaccharide that bridges the lipid A and O-antigen components of LPS.

The E. coli strains suitable for preparing the vaccine compositions of the present invention are any strains that have been or can be sufficiently attenuated to allow them to be administered to humans and/or animals in a non-pathological manner in a live or dead form. Other bacteria, such as Salmonella or other strains of E. coli, can be used that provide adequate expression and an improved immunogenic response.

As used herein, the term "pgl operon" refers to any physiologically active glycosylation cluster of Campylobacter genes capable of glycosylation of homologous or heterologous structures produced by the E. coli strains employed in the vaccine composition. The pgl operon in Campylobacter jejuni encodes all the enzymes necessary for the synthesis of the Campylobacter jejuni N-glycan heptasaccharide, its transport across the inner membrane, and its transfer to proteins. PglD, E, and F encode enzymes involved in the biosynthesis of di-N-acetylbacteramide, PglC transfers UDP-diN-acetylbacteramide to undecadienyl phosphate, and PglA, H, and J add GalNAc residues. The Glc branches are connected by PglI. The complete heptasaccharide is transferred across the inner membrane via the action of PglK, and the oligosaccharyltransferase PglB transfers the N-glycan to proteins and releases the heptasaccharide in its free form into the periplasm.

Functional derivatives of the pgl operon are gene clusters derived from any Campylobacter operon that have deletions, mutations, and/or substitutions of nucleotides or entire genes, yet are still capable of producing oligosaccharides or polysaccharides that can be linked to homologous or heterologous structures produced by the E. coli strain used in the vaccine composition. One or more pgl operons or their derivatives can be integrated into the chromosome of the E. coli strain or can be introduced as part of at least one plasmid. Chromosomal integration is generally preferred because it is more stable than plasmid vectors, which can be lost during propagation. It should be noted that an E. coli strain can contain more than one pgl operon or its derivative that produces one or more N-glycans or their derivatives. In certain embodiments, the vaccine composition comprises an E. coli strain having more than one type of pgl operon that produces more than one glycan structure expressed on the surface of the recombinant E. coli. This can be advantageous for eliciting a more diverse immune response against different Campylobacter species in humans or animals. In an alternative embodiment, the vaccine composition comprises an E. coli strain having a single type of pgl operon that produces a single glycan structure expressed on the surface of the recombinant E. coli. This may be advantageous for eliciting a specific immune response against a single Campylobacter species in humans or animals.

Optionally, the expression level of C. jejuni glycans can be modulated by using different promoters or other regulatory elements upstream of the pgl operon, including but not limited to promoters of ribosomal protein genes and promoters from antibiotic resistance-encoding genes (such as bla) or similar and preferably strong promoters. Such regulation can be used for plasmid-encoded or chromosomally integrated pgl operons. In addition, plasmid stability can optionally be enhanced by including essential genes on the plasmid while deleting these genes in the genome of the E. coli strain used in the vaccine composition.

In an alternative embodiment, the pglB gene of the pgl operon is inactivated, meaning that the corresponding oligosaccharyl transferase B is not expressed or at least enzymatically inactivated. The pglB gene product transfers the N-glycan to a specific polypeptide acceptor site as described further below and releases the heptasaccharide in its free form. Inactivation of the transferase causes the N-glycan or N-glycan derivative to bind only to the lipid A core of the O-antigen acceptor in E. coli and causes the exchange of GlcNAc for diN-acetylbactamidin, since the E. coli O-antigen ligase recognizes glycans containing GlcNAc only at the ligation site (i.e., the reducing end).

In a related embodiment, the pgl derivative is one in which one or more genes for di-N-acetylbacteramide biosynthesis (pglD, E, F) and transfer are inactivated, and the pglB gene is also inactivated. This embodiment results in the exchange of GlcNAc for di-N-acetylbacteramide. Incorporation of such pgl derivatives into Salmonella results in increased cellular expression and transfer of the modified heptasaccharide to the lipid A core rather than the polypeptide acceptor (see U.S. Patent Application Publication No. 2012/0100177).

The at least one N-glycan of Campylobacter jejuni or a heptasaccharide derivative thereof may be any N-glycan produced by any pgl operon of Campylobacter or a functional derivative thereof, provided that the glycan is immunogenic such that it elicits an immune response specific for the Campylobacter species.

In a specific embodiment, the glycan is a heptasaccharide of formula (I) as described above, i.e., GalNAc-α1,4-GalNAc-α1,4-[Glc-β-1,3]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-diNAcBac, wherein diNAcBac (also known as di-N-acetylbacillus) is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose.

An alternative embodiment in which the genes for di-N-acetylbacteramide biosynthesis in the pgl operon are inactivated or largely or completely deleted leads to the synthesis of the derivative heptasaccharide of formula (II) (GalNAc-α1,4-GalNAc-α1,4-[Glc-β-1,3]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-GlcNAc).

In a specific embodiment, the N-glycans or derivatives produced by at least one pgl operon or derivative thereof can be linked to at least one homologous or heterologous E. coli polypeptide that will ultimately be translocated to and displayed on the cell surface. The polypeptide linked to the N-glycan (derivative) can be any type of polypeptide, such as a pure polypeptide (amino acids only) or a post-translationally modified polypeptide (e.g., a lipid-linked polypeptide).

In another embodiment, the glycan or its derivative is purified from the native host in its free oligosaccharide form and then chemically conjugated to a polypeptide or lipid carrier.

In a specific embodiment, at least one glycan or derivative thereof produced by at least one pgl operon or derivative thereof is linked to the E. coli lipid A core or a functionally equivalent derivative thereof. The E. coli lipid A core is an oligosaccharide structure composed of, but not limited to, hexoses, heptoses, and KDO (3-deoxy-D-mannose-octulose acid), which is linked to an acyl chain via two glucosamines, anchoring the structure in the outer membrane of the bacterium. A functionally equivalent derivative of the lipid A core is one that is capable of accepting one or more glycans or derivatives thereof and presenting them on the cell surface. It should be noted that in this case, the heptasaccharide or derivative thereof is not N-linked. This is because the E. coli structural lipid A core is not a polypeptide.

Optionally, at least one heptasaccharide or its derivative replaces the O-antigen side chain in LPS (lipopolysaccharide). The inner and outer lipid A cores of E. coli remain unchanged, while O-antigen biosynthesis is abolished, for example, by mutation of wzy and/or other mutations. In certain embodiments, at least one heptasaccharide, its derivative, or a mixture of both is expressed simultaneously with the O-antigen side chain in LPS, producing a heterogeneous LPS containing both the host O-antigen and the heptasaccharide.

Preferably, and highly important for medical use, the E. coli strains of the invention do not induce pathogenic effects when administered to animals or humans in live and/or inactivated form. The skilled person is aware of many ways to attenuate virulent E. coli species by mutation. For example, mutations that attenuate pathogenic E. coli (1) CarAB mutants of the avian pathogenic E. coli O2 are attenuated and effective as live oral vaccines against colibacillosis in turkeys (Kwaga et al. Infect Immun. 62:3766-3772, 1994); (2) mutations in the RNA chaperone protein Hfq significantly reduce the pathogenicity of VTEC, EAEC, and UPEC in a nematode model (Bojer et al. Microbes Infect. 14:1034-1039, 2012); (3) mutations in genes within the phosphate-specific transport system (Pst) attenuate E. coli strains (Buckles et al. Microbiology 152:153-160, 2006; Daigle et al. Infect Immun. 63:4924-4927, 1995).

In a specific embodiment, the E. coli strain employed in the vaccine composition is attenuated by partial or complete inactivation of O-antigen expression, for example, by mutations in the wzy gene (generating an O-antigen polymerase mutant) (Baba et al. Mol. Syst. Biol. 2:2006).

The above-mentioned E. coli strains are highly immunogenic and generate immune responses against infection with Campylobacter (e.g., Campylobacter jejuni). In addition, once prepared, they can be easily propagated and produced in large quantities. They can be administered as killed or live vaccines, with live vaccines allowing long-term propagation in the host and sustained immune stimulation and a full immune response with or without adjuvants.

Therefore, the present application also relates to the medical use of live or dead E. coli strains engineered to present one or more Campylobacter N-glycans or derivatives thereof on their surface, in particular for the preparation of a medicament, preferably a vaccine.

Preferably, the medicament is suitable for preventing and/or treating Campylobacter jejuni infection and/or colonization, preferably in livestock, more preferably in cattle and poultry, most preferably in poultry like chickens, turkeys, geese and ducks.

According to one aspect, the present application provides a vaccine composition, which is a pharmaceutical composition, food or feed (additive), comprising dead or live Escherichia coli engineered to present one or more Campylobacter N-glycans or derivatives thereof on its surface and a physiologically acceptable excipient, diluent or carrier. Optionally, the vaccine composition includes other components (such as adjuvants) or is administered together with other components. In another alternative, the vaccine composition described herein is formulated for administration together with another vaccine composition.

Adjuvant generally comprises the material that strengthens host immune response in a non-specific manner.A plurality of different adjuvants are known in the art.The example of adjuvant is Freund's complete and incomplete adjuvant (Freunds Complete and Incomplete adjuvant), vitamin E, nonionic blocking polymer and polyamine, such as dextran sulfate, carbopol (carbopol) and pyran.It is equally suitable that surface active substance, such as Span, Tween, hexadecylamine, lysolecithin, methoxy hexadecylglycerol and saponin (such as Quil).In addition, peptide is usually used, such as muramyl dipeptide, dimethylglycine and tuftsin (tuftsin).Being immediately following these adjuvants, immune stimulating complex (ISCOMS), mineral oil (such as or), vegetable oil or its emulsion and Forte can be advantageously used.

Optionally, the vaccine is mixed with one or more stabilizers, for example to protect components susceptible to degradation from degradation, to increase the shelf life of the vaccine, or to improve lyophilization efficiency. Suitable stabilizers are, for example, SPGA (Bovarnik et al. J. Bacteriology 59:509, 1950), skim milk, gelatin, bovine serum albumin, carbohydrates (e.g., sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (e.g., albumin or casein or their degradation products), and buffers (e.g., alkali metal phosphates).

The vaccine composition may be in the form of, for example, a solution, a suspension, or a lyophilized composition suitable for reconstitution prior to administration.

Lyophilization is an effective method for preserving vaccine compositions. Lyophilized materials can be stored stably for many years. Lyophilized materials can be stored at temperatures above freezing without adversely affecting the material. Lyophilization can be performed according to all well-known standard lyophilization procedures.

Immunization method

The present application provides a method for immunizing an animal against infection with Campylobacter spp. The method comprises administering to the animal a vaccine composition comprising a recombinant Escherichia coli engineered to display at least one Campylobacter N-glycan or a heptasaccharide derivative thereof on its surface as described above.

Campylobacterosis is a disease caused by infection with Campylobacter. The most common symptoms are diarrhea, abdominal pain, fever, headache, nausea and/or vomiting. These symptoms usually only last for about three to six days. However, rarely, Campylobacter infection can cause persistent complications, such as Guillain-Barré Syndrome (GBS), arthritis and bacteremia. Therefore, the vaccine described herein is suitable for immunizing animals (including humans and livestock, such as chickens, which are the main causes of human foodborne diseases). Therefore, the present application further provides a method for preventing the effects of the disease or illness caused by Campylobacter infection or minimizing the effects. In a specific embodiment, the disease or illness caused by Campylobacter infection is Campylobacterosis, Guillain-Barré Syndrome (GBS) and/or arthritis and/or bacteremia, but other Campylobacter species are relevant to other diseases (such as periostitis and abortion).

In embodiments where the vaccine composition is used to vaccinate livestock, there are multiple routes for administering the composition that can facilitate large-scale vaccination. Administration can be performed via drinking water, food or feed, spray/atomization (e.g., to day-old chickens in transport boxes, or to animals in confinement environments such as poultry houses), eye drops, puncture and scarification (skin routes in the flippers or feet), injection (e.g., intramuscular or subcutaneous), or in ovo administration.

Optionally, the animal is treated with an initial dose of the vaccine composition, followed by one or more booster doses at appropriate intervals. Those skilled in the art will readily identify dosages and dosing schedules suitable for specific applications. In the present example, we perform an initial vaccination in one-week-old birds using 1×10 ⁸ live or formalin-fixed E. coli cells (or with any other saccharide conjugate vaccine, i.e., Campylobacter jejuni N-glycan linked to ToxC). We perform a booster two weeks later with the same amount of bacterial cells (or protein). Campylobacter is typically challenged one week later, and the birds are euthanized one week after challenge (as described below).

In order to better understand the present invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of the present invention in any way.

Examples

Example 1: Preparation of vaccines

Campylobacter jejuni N-glycan fused to protein

Expression and Purification of ToxC-GT Protein Glycosylated with N-Glycans from C. jejuni: ToxC-GT protein glycosylated with N-glycans from C. jejuni was expressed in E. coli BL21 expressing the C. jejuni pgl operon and purified by Ni-NTA chromatography as described in International Published PCT Application No. WO 2012/027850. The protein was further purified by ion exchange chromatography using an AEKTA FPLC system equipped with a 2.5 ml MonoQ anion exchange column. The mobile phase was 50 mM Tris-HCl buffer (pH 8.0), and a NaCl gradient was set from 0 to 500 mM NaCl over 30 column volumes. Fractions containing glycoconjugates were analyzed by 12.5% SDS-PAGE, passed twice through 1 g of lipid removal absorbent (LRA, Supelco), dialyzed against sterile PBS, and brought to a protein concentration of 0.5 mg/ml before use. Protein concentration was determined using a standard method (Bradford test) using PBS containing increasing concentrations of BSA to generate a standard curve.

Campylobacter jejuni N-glycans fused to the lipid A core structure of Escherichia coli: preparation of E. coli vaccines

E. coli cells expressing the Campylobacter jejuni heptasaccharide were previously described (Nosaf et al. Mol. Cell. Proteomics 11: 1203-1219). Cells were grown in liquid broth (2×YT broth (yeast extract and tryptone broth)) at 37° C. with vigorous shaking (220 rpm) until growth lag phase was reached. Cells were collected by centrifugation and washed twice with sterile PBS. Cell counts were determined by inoculating serial dilutions of the cell suspension (set to an OD ₆₀₀ of 2.0 and used directly or fixed with formalin).

The cells of the Escherichia coli overnight culture expressing the Campylobacter jejuni pgl locus were collected by centrifugation and washed twice with sterile PBS buffer, as described in Nosaf et al. Molecular and Cellular Proteomics 11: 1203-1219, 2012. Then, 1 ml of cells with an OD ₆₀₀ adjusted to 1.0 by sterile PBS were centrifuged and suspended in 100 μl of 1 times Laemmli sample buffer and heated at 95°C for 10 minutes. Proteinase K was added to a final concentration of 200 μg/ml, and the sample was incubated at 60°C for 1 hour, then incubated on ice for 5 minutes and centrifuged for 15 minutes. Aliquots of the supernatant were separated by standard 12.5% SDS-PAGE. Using Campylobacter jejuni N-glycan specific antiserum hR6 as primary antibody and anti-rabbit conjugated to alkaline phosphatase as secondary antibody, the heptasaccharide fused to the lipid A core was observed by Western blotting as described (Nosaf et al. Molecular and Cellular Proteomics 11: 1203-1219) (Figure 1). Lane 1 shows the cell lysate of proteinase K treatment of an E. coli O-antigen polymerase _mutant (pACYC184pglBmut) expressing the protein glycosylation operon of Campylobacter jejuni with an inactive pglB gene. The formation of lipid A-glycan fusions is marked by arrows. Lane 2 shows the cell lysate of proteinase K treatment of an E. coli O-antigen polymerase mutant blank vector control (pACYC184). Molecular weight markers (MW, kilodaltons, kDA) are indicated on the left. The higher molecular weight bands are components of the E. coli that cross-react in the two preparations.

Nuclear magnetic resonance (NMR) spectroscopy of purified lipid N-glycan fractions. Glycolipids were prepared from eight liters of an Escherichia coli O-antigen polymerase mutant culture (pACYC184pglBmut) expressing the protein glycosylation operon of Campylobacter jejuni with an inactive pglB _{gene ( OD600} = 1.0). LPS was extracted with phenol and water, dialyzed, treated with AcOH to precipitate nucleic acids, dialyzed, dried, hydrolyzed with 2% AcOH, and separated on Biogel P6. Fractions were analyzed by NMR. Fractions containing C. jejuni N-glycan signals were combined and separated using a gradient on an anion exchange Hitrap column. Fractions were analyzed by NMR. Fractions containing C. jejuni N-glycan signals were desalted by Sephadex G-15 chromatography. Association was confirmed by nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multiple bond correlation (HMBC) spectroscopy. Specific C. jejuni N-glycan chemical shifts were observed, with all 1-4 bonds of C. jejuni N-glycan components receiving transglycosidic NOEs 1:4 and 1:6, and the assignments were in good agreement with previously published data ( FIG. 2 and Table 1 , and Nosaf et al., Mol. Cell. Proteomics 11:1203-1219, 2012). Derivatives of C. jejuni N-glycans with GlcNAc rather than diNAcBac as reducing end sugars ( FIG. 2 ) were linked to the O-7 of the L-glycerol-D-mannose-heptose of the lipid A core. All signals for Hep(L) were found by analyzing the relevant primary stacks, and the assignments for the E. coli lipid A core portion were consistent with published data (Muller-Loennies et al., Journal of Biological Chemistry 278:34090-34101, 2003 and Table 1).

Table 1: Chemical shifts

1 2 3 4 5 6 7 α-GalNAc A 5.47 4.27 3.23 4.07 3.92 3.70; 3.75 98.2 51.0 68.0 77.6 72.7 60.8 α-GalNAc C 5.13 4.29 4.19 4.13 4.49 3.66; 3.78 98.2 51.5 67.8 77.4 71.6 59.9 α-GalNAc D 5.07 4.23 4.04 4.06 4.39 3.70; 3.73 99.3 51.4 68.4 69.6 71.9 61.8 α-GalNAc E 5.04 4.30 4.15 4.13 4.43 3.65; 3.68 99.3 51.5 67.8 77.4 72.3 60.5 α-GalNAc F 5.02 4.53 4.17 4.36 4.45 3.56; 3.64 99.5 50.5 67.8 75.6 72.3 60.3 β-Glc G 4.60 3.32 3.48 3.38 3.43 3.71; 3.92 74.1 76.8 70.9 76.9 61.8 α-GlcNAc N 4.54 3.82 3.74 3.68 3.45 3.75; 3.92 55.3 79.6 72.3 76.9 61.9 α-Hep L 4.89 3.97 3.80 3.86 3.57 4.16 3.80; 4.01 100.4 71.1 72.0 67.3 72.8 68.4 73.2 α-Glc K 5.18 3.61 3.75 3.51 4.15 3.67; 3.92 97.1 72.4 74.6 70.4 71.2 65.9 α-Glc I 5.47 3.68 3.85 3.54 4.08 3.82; 3.90 98.2 76.8 72.1 70.3 72.6 61.2

Fluorescence-activated cell sorting (FACS) analysis. First, 1 ml of E. coli cells with an OD ₆₀₀ = 1.0 were pelleted by centrifugation and resuspended in 1 ml of blocking solution (PBS, 5% skim milk). Cells were probed with C. jejuni N-glycan-specific antiserum hR6 and Alexa Flour-546-conjugated anti-rabbit antiserum and analyzed by FACS. FACS data were processed using FACS Diva software. DAPI counter staining was used to identify and gate intact cells. Analysis of a 2×10 ⁴ cell population demonstrated a significant increase in fluorescence in E. coli cells expressing C. jejuni N-glycans compared to E. coli empty vector control (pACYC184) cells, confirming that C. jejuni N-glycans are displayed on the cell surface ( FIG. 3 ). Peak appearance and peak geometry demonstrated that each E. coli cell displayed comparable amounts of C. jejuni N-glycans on its surface.

Example 2: Vaccination and challenge

Exposure of chickens to injections of the ToxC-GT glycoconjugate ( FIG. 4D ) or oral doses of the dead ( FIG. 4A ) and live ( FIG. 4B and C ) strains of the modified E. coli described in Example 1 resulted in a significant reduction in the cecal content of Campylobacter in the challenged chickens. Three chicken vaccination experiments were conducted using E. coli expressing the Campylobacter jejuni heptasaccharide, demonstrating increased immunity of chickens to Campylobacter. The results of these experiments are shown in FIG. 4A , B and C.

In the first chicken vaccination experiment, three groups of chickens were challenged. The control PBS group contained four chickens, while Groups 2 and 3 each contained eight chickens. The conditions for Groups 1 and 2 are shown in Table 2. Group 3 was orally gavaged with dead E. coli cells expressing C. jejuni N-glycan heptasaccharide on the surface on days 7 and 21. The birds were then challenged as follows: Group 1 (negative control) was orally gavaged with 300 μl PBS; Groups 2 and 3 were orally gavaged with 300 μl PBS containing ¹⁰² C. jejuni 81-176 cells. On day 35, the chickens were euthanized, and colonization levels were determined by inoculating serial dilutions of the cecal contents of each bird on selective Karmalia agar. After the plates were incubated under microaerobic conditions for 48 hours, colony forming units (CFU) were determined. The results are graphically shown in Figure 4A, and colonization levels are shown as CFU/gram of cecal contents. The horizontal bars represent the median for each group. Specifically, the results demonstrate that the N-glycan-based vaccine reduced Campylobacter colonization in chickens. No colony-forming units were detected on the plates of Group 1 (PBS control), while birds in Group 2 were colonized with an average of approximately ¹⁰¹⁰ Campylobacter cells per gram of cecal contents. Colonization was reduced by approximately 4 logs in Group 3.

In the second chicken vaccination experiment, three groups of chickens were challenged. Groups 1 and 2 contained 6 chickens, and Group 3 contained 8 chickens. The conditions for Groups 1 and 2 are shown in Table 2. Group 3 was orally gavaged with live E. coli cells expressing the Campylobacter jejuni N-glycan heptasaccharide on days 7 and 21. The challenge concentration and colonization levels were determined as described in the first experiment. The results are shown graphically in Figure 4B, and the colonization levels are shown as cfu/gram of cecal contents. The horizontal bars represent the median for each group. The results show that the N-glycan-based vaccine reduced Campylobacter colonization in chickens. No colony forming units were detected on the culture plates of Group 1 (PBS control), while the birds in Group 2 were colonized with an average of approximately ¹⁰¹⁰ Campylobacter cells/gram of cecal contents. Colonization was not detectable in any of the birds in Group 3.

In the third experiment, shown in Figure 4C, four groups of chickens were challenged. Group 1 contained six birds, and Groups 2, 3, and 4 contained eight birds. The conditions for Groups 1 and 2 are shown in Table 2. Groups 3 and 4 were gavaged orally on days 7 and 21. Birds in Group 3 received live E. coli cells that did not express N-glycans on their surface, while birds in Group 4 received live E. coli cells that expressed the Campylobacter jejuni N-glycan heptasaccharide on their surface. The results are shown graphically in Figure 4C, and colonization levels are shown as cfu/gram of cecal contents. The horizontal bars represent the median for each group. The results show that the N-glycan-based vaccine can reproducibly reduce Campylobacter colonization in chickens, and that the E. coli cells that do not express N-glycans do not have a probiotic effect, as Campylobacter colonization levels were similar to those in Group 2 birds after challenge.

In the fourth experiment shown in Figure 4D, six groups of chickens were challenged, each containing eight chickens. The conditions of each group are shown in Table 2.

Table 2: Attack Groups

Group condition 1 Negative control: no exposure to antigen and no challenge 2 Positive control: no exposure to antigen 3 Single IM dose of glycoprotein antigen on day 21 4 Single IM dose of glycoprotein antigen on day 7 5 Glycoprotein antigen IM doses on days 7 and 21 6 Live E. coli cells expressing C. jejuni N-glycan heptasaccharide on their surface were orally gavaged on days 7 and 21

Similar to the previous experiment, cloacal swabs were performed on 10% of the birds (5 randomly selected) on day 1 and plated onto selective Kamari agar to confirm that the birds were not colonized with C. jejuni. No Campylobacter colonies were observed after 48 hours of incubation at 37°C under microaerobic conditions.

On day 7, similar to the previous experiment, a maximum of 50 μl of blood was collected from each bird (pre-bleeding). Serum was prepared as follows: after keeping the blood sample at 37°C for 1 hour followed by centrifugation (5 minutes, 18.000 × g, 4°C), the supernatant (serum) was transferred to a fresh tube and glycerol was added to a final concentration of 10%. The serum was stored at -20°C until further use. Subsequent antimicrobial treatment was performed as follows: Group 1 (PBS control) and Group 2 (colonization control) received 300 μl PBS with Freund's complete adjuvant on day 7 and the same amount of PBS with Freund's incomplete adjuvant on day 21 (150 μl PBS + 150 μl adjuvant), injected at two sites in the chest with 150 μl of vaccine formulation (without glycoconjugate) at each site. Group 3 received no antigen on day 7 but received one dose of ToxC-GT glycosylated with C. jejuni N-glycans (100 μg protein in 150 μl PBS + 150 μl Freund's complete adjuvant) on day 21; Group 4 received one dose of ToxC-GT glycosylated with C. jejuni N-glycans in Freund's complete adjuvant on day 7 and no antigen on day 21. Group 5 received two doses (with Freund's complete on day 7 and Freund's incomplete as adjuvants) of 100 μg of ToxC-GT with C. jejuni N-glycans injected in the legs (150 μl vaccine formulation per leg), and Group 6 received two doses (on days 7 and 21) of 300 μl PBS containing 10 ⁸ live E. coli cells expressing C. jejuni N-glycans on their surface by oral gavage.

Similar to the previous experiment, on day 28, 100 μl of blood was drawn from each bird from Groups 1 to 6 (test bleed) and serum was prepared and stored as described above. The birds were then challenged as follows: Group 1 (negative control) was gavaged orally with 300 μl PBS; Groups 2 to 6 were gavaged orally with 300 μl PBS containing 10 ² C. jejuni 81-176 cells. On day 34, the birds were euthanized, blood was obtained via cardiac puncture (terminal bleed), and serum was prepared and stored as described above. The colonization level was determined by inoculating serial dilutions of the cecal contents of each bird on selective Kamari agar. After incubating the culture plates under microaerobic conditions for 48 hours, colony forming units (cfu) were determined.

The results are graphically presented in Figure 4D, with colonization levels shown as cfu/gram of cecal contents. The horizontal bars represent the median for each group. Specifically, the results again demonstrate that N-glycan-based vaccines reduce Campylobacter colonization in chickens, and that vaccines containing live E. coli cells expressing C. jejuni N-glycans on their surface perform better in chickens than glycoprotein vaccines (see below).

No colony-forming units were detected on the plates of Group 1 (PBS control), while birds in Group 2 were colonized with an average of approximately ¹⁰¹⁰ Campylobacter cells/gram of cecal contents. Colonization decreased in Groups 3, 4, and 5, with averages of 2.2× ¹⁰⁴ , 6.8× ¹⁰⁵ , and 5.5× ¹⁰⁴ cfu/gram of cecal contents, respectively.

Colonization in Group 6 was nearly eliminated, with an average of 100 cfu/gram of cecal contents. Furthermore, 5 of the 8 birds showed no signs of C. jejuni colonization at all. This clearly indicates that treatment with the protein-based C. jejuni-N-glycan vaccine leads to reduced colonization following challenge with Campylobacter, regardless of injection time point and vaccine administration site, and that oral vaccination with live E. coli cells expressing the heptasaccharide on their surface almost completely eliminates Campylobacter colonization. Furthermore, the self-limitation of the E. coli vaccine strain was demonstrated, as no E. coli was observed in the cecal contents of this group when inoculated on selective LB Kan-Cm. Elimination of the live E. coli vaccine strain from the chickens was observed in all experiments.

ELISA tests were performed to analyze N-glycan-specific immune responses, specifically chicken IgY (IgG) N-glycan-specific antibody responses. Free oligosaccharides (fOS) from Campylobacter jejuni were prepared as described (Dwivedi et al. Biopolymers, 99:772-7830, 2013) and conjugated to BSA via reductive amination as described (Nosaf et al. Molecular and Cellular Proteomics 11:1203-1219, 2012). The formation of BSA-Cj-N-glycan conjugates was confirmed by Western blotting using R1-4 antiserum. After adjusting the concentration to 1 mg/ml with PBS, the glycoconjugates were stored at 4°C until further use. Subsequently, 96-well Maxisorb culture plates were coated with 500 ng of BSA-Cj-N-glycan conjugates at 4°C overnight (18 hours). After removing unbound antigen, the culture plate was blocked with 100 μl PBS-T, 5% skim milk at room temperature for 1 hour under shaking. After discarding the blocking solution, 100 μl of antibody solution was added and incubated for 1 hour as described above. The antibody solution contained N-glycan-specific antiserum diluted 1:3000 in PBS-T, 1% skim milk or chicken serum (prepared by the second bleeding (i.e., day 28) of the vaccination experiment) and diluted 1:50 in PBS-T, 1% skim milk. The culture plate was incubated at room temperature for 1 hour as described, and each well was washed 3 times with 100 μl PBS-T for 5 minutes. After adding 100 μl of secondary antibody solution (anti-rabbit-AP (1:500) for R1-4 controls or anti-chicken IgY (1:500) for experimental samples) and incubating at room temperature for 1 hour, the secondary antibody solution was discarded and each well was washed 4 times for 5 minutes with 100 μl PBS-T. After the last washing step, the remaining washing solution was completely removed from each well and the plates were developed using PNPP as substrate. After scanning the plates in a plate reader at OD ₄₀₅ , the immunoreactivity in each serum was determined.

jejuni N-glycan-specific antibodies were present ( FIG5A ) in serum prepared from blood drawn from birds on day 28 prior to challenge with Campylobacter that had been vaccinated with: one dose of ToxC-GT (breast, IM) with glycans on day 21 (sample group 3), two doses of ToxC-GT (breast, IM) with glycans on days 7 and 21 (sample group 4), two doses of ToxC-GT (leg) with glycans on days 7 and 21 (sample group 5), and two doses of killed E. coli (sample group 6). The antibody response (expressed as OD ₄₀₅ ) was highest in sample group 6, and the majority of birds in this sample group did not exhibit colonization. The antibody response in the positive and negative colonization controls (sample groups 1 and 2) was below the limit of detection. The horizontal bars represent the median for each group.

jejuni N-glycan-specific antibodies were present ( FIG5B ) in serum prepared from blood drawn from birds vaccinated with live E. coli vaccines displaying N-glycans on the surface 28 days before challenge with Campylobacter (sample group 4). This corresponds to vaccine experiment 3 presented in FIG4C . Antibody responses (expressed as OD 405 ) were below the limit of detection in birds vaccinated with live E. coli vaccines without glycans (sample group 3) and in positive and negative colonization controls (sample groups 1 and ₂ ). Horizontal bars represent the median for each group.

The following items were observed:

1) Feeding chickens with killed E. coli cells expressing the C. jejuni heptasaccharide followed by C. jejuni challenge resulted in approximately a 4-log reduction in C. jejuni colonization of the chicken digestive tract ( FIG. 4A ); and

2) Feeding chickens with live E. coli cells expressing the C. jejuni heptasaccharide followed by challenge with C. jejuni consistently resulted in greater than 7 log reductions in C. jejuni colonization of the chicken digestive tract (Fig. 4B, C, and D).

Significantly, vaccines comprising killed or live E. coli cells expressing the C. jejuni heptasaccharide were able to significantly enhance the immunity of chickens to subsequent C. jejuni challenge.

Control experiment

To demonstrate that the reduction in C. jejuni colonization was a result of vaccination with live E. coli expressing the C. jejuni heptasaccharide and not due to a probiotic effect resulting from exposure to live E. coli cells, cecal contents from birds vaccinated with the live E. coli strain were also plated onto E. coli selective media as described above. No E. coli colonies were detected in the live E. coli vaccinated birds, indicating that E. coli had been eliminated prior to termination of the experiment.

All publications, patents, and patent applications mentioned in this specification are indicative of the levels of skill of those skilled in the art to which this invention pertains and are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

The invention having thus been described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as will be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims (15)

1. A poultry vaccine composition comprising: Escherichia coli engineered with the pglB gene of Campylobacter jejuni pgl operon to express at least one N-glycan of Campylobacter jejuni or a derivative thereof on its cell surface; and one or more physiologically acceptable diluents, excipients, adjuvants or carriers, wherein the Escherichia coli is engineered to partially or completely inactivate O-antigen expression, and wherein the N-glycan is expressed on the surface of the Escherichia coli in the form of a lipid A core fusion complex. 2. The poultry vaccine composition according to claim 1, wherein the composition comprises live engineered Escherichia coli. 3. The poultry vaccine composition according to claim 1, wherein the composition comprises inactivated engineered Escherichia coli cells. 4. The poultry vaccine composition of claim 1, wherein the composition comprises a suspension of engineered bacteria in a suitable buffer diluent. 5. The poultry vaccine composition according to claim 4, wherein the buffer diluent is a phosphate-buffered saline solution. 6. The poultry vaccine composition according to claim 1, further comprising an adjuvant, a stabilizer, or a preservative. 7. The poultry vaccine composition according to claim 1, wherein the composition is formulated during the preparation process for oral administration, intraovarian administration, or parenteral administration. 8. The poultry vaccine composition according to claim 7, wherein the composition is formulated during the preparation process for administration by injection or infusion. 9. The poultry vaccine composition according to claim 7, wherein the vaccine composition is formulated during the preparation process for spraying or adding to livestock feed, feed additives or water. 10. The poultry vaccine composition according to claim 1, wherein the poultry is a chicken. 11. Use of the vaccine composition of claim 1 in the preparation of a medicament for vaccinating poultry against Campylobacter jejuni. 12. The use according to claim 11, wherein the poultry is a chicken. 13. The use according to claim 11 or 12, wherein the vaccine composition is suitable for administration by spraying. 14. The use according to claim 11 or 12, wherein the vaccine composition is suitable for administration in pharmaceutical compositions, food, feed additives or drinking water. 15. The use according to claim 14, wherein the pharmaceutical composition is formulated during the preparation process for oral administration, intraovarian administration, injection, or infusion administration.