Patent Description:
Vaccines or immunogenic compositions comprising glycan antigens can induce the production of specific antibodies to provide protection against a variety of pathogenic bacteria. Subunit vaccines are typically preferred over inactivated or attenuated pathogens as they often exhibit lower side effects; however, the immunogenicity of subunit vaccines is frequently low and typically fails to generate sufficient memory B-cell response. The coupling of a polysaccharide antigen to a protein carrier, so generating a glycoconjugate, is known to increase the immunogenicity significantly. Currently licensed human glycoconjugate vaccines include those against Haemophilus influenzae, Neisserria meningitidis and Streptococcus pneumonia.

The development and chemical synthesis of glycoconjugate vaccines is laborious and costly requiring several steps including the purification of polysaccharide glycan from the native pathogen and the chemical coupling of the sugar to a suitable protein carrier. The use of organic systems represents often a more rapid and economical method for the production of glycoconjugates. Campylobacter jejuni harbours a gene cluster involved in the synthesis of lipo-oligosaccharides and N-linked glycoproteins involved in the glycosylation of over <NUM> proteins. The oligosaccharyltransferase PglB identified in C. jejuni, the enzyme responsible for the transfer of glycans to protein acceptor proteins and part of the gene cluster, was also found to catalyse the transfer of glycans onto a wide range of different non-species related protein acceptors.

Production of glycoconjugate vaccines in a bacterial system such as E. coli utilising PgIB, a carrier polypeptide and an antigenic saccharide is disclosed in <CIT>. A further oligosaccharyltransferase isolated from the related species Campylobacter sputorum with glycan transfer capability is disclosed in <CIT>.

The production of glyconjugates in a bacterial expression system requires the co-expression of three genes ["tri-plasmid"]: an acceptor protein, a polysaccharide biosynthetic locus and an oligosaccharyltransferase enzyme. <CIT> discloses a method providing the stable integration of the genes encoding the acceptor protein, a glycan biosynthetic locus and an oligosaccharyltransferase into a bacterial genome using transposable elements for the production of glycoconjugates at reasonable levels. <CIT> discloses recombinant modified N-oligosaccharyl transferases from Campylobacter jejuni for use in the production of bioconjugates. <NPL>) discloses the N-linked protein glycosylation system from Campylobater jejuni, glycoconjugate synthesis using the glycosyltransferase pglB and discusses its substrate specificity.

This disclosure relates to the identification of an alternative C. sputorum oligosaccharyltransferase closely related in sequence to the oligosaccharyltransferase disclosed in <CIT> which has enhanced glycan transfer activity when compared to closely homologous oligosaccharyltransferases. Moreover, recombinant expression systems with decreased translational efficiency comprising oligosaccharyltransferases, toxic carrier proteins or genes encoding proteins required for glycan biosynthesis are also disclosed. Translational efficiency is decreased by providing a vector with increased distance between the ribosome binding site [RBS] and the translational start codon thus enabling bacterial growth to a high density and avoiding deleterious effects of expressing recombinant proteins at concentrations which are toxic to the bacterial cell. Chen et al describes that the spacing between the Shine-Dalgarno sequence and the initiation code can affect the translation efficiency (<NPL>).

Furthermore, the disclosure relates to the characterisation of a modified oligosaccharyltransferase polypeptide with altered glycan specificity and/or enzyme activity when compared to the unmodified oligosaccharyltransferase polypeptide.

The present invention, in its various aspects, is as set out in the accompanying claims.

According to an aspect of the invention there is provided a transcription cassette comprising:.

wherein said nucleic acid molecule is operably linked to a promoter adapted for expression in a bacterial host cell.

In a preferred embodiment of the invention there is provided a transcription cassette comprising a nucleic acid molecule encoding an oligosaccharyltransferase wherein said oligosaccharyltransferase comprises an amino acid sequence set forth in SEQ ID NO: <NUM>.

In a preferred embodiment of the invention said transcription cassette further comprises a nucleic acid molecule encoding a carrier polypeptide wherein said carrier polypeptide comprises one or more glycosylation motifs for said oligosaccharyltransferase.

In a further preferred embodiment of the invention said transcription cassette further or alternatively comprises a nucleic acid molecule comprising a nucleotide sequence encoding a biosynthetic locus comprising one or more polypeptides required for the synthesis of a heterologous glycan antigen.

In a preferred embodiment of the invention said oligosaccharyltransferase and/or said carrier polypeptide and/or biosynthetic locus is operably linked to a regulatable promoter to provide regulated expression of each or all nucleic acid molecules encoding said polypeptides.

In a preferred embodiment of the invention said one or more polypeptides required for the synthesis of a heterologous glycan antigen are operably linked to one or more regulatable promoters to provide regulated expression of each or all nucleic acid molecules encoding said polypeptides.

In a preferred embodiment of the invention said promoter includes an inducible nucleotide element conferring regulated expression in response to an inducer.

In an alternative embodiment of the invention said promoter includes a repressible nucleotide element conferring regulated expression in response to a repressor.

In a preferred embodiment of the invention said promoter is further operably linked to a ribosome binding site wherein there is provided a nucleotide spacer sequence between the <NUM>' prime end of said ribosome binding site and the <NUM>' initiating start codon of the nucleic acid molecule encoding said oligosaccharyltransferase wherein translation from the nucleic acid molecule encoding said oligosaccharyltransferase is reduced when compared to a control nucleic acid molecule encoding said recombinant polypeptide that does not comprise said nucleotide spacer sequence.

In a further preferred embodiment said oligosaccharyltransferase comprises a sequence as set forth in SEQ ID NO: <NUM>.

In a preferred embodiment of the invention said promoter is further operably linked to a ribosome binding site wherein there is provided a nucleotide spacer sequence between the <NUM>' prime end of said ribosome binding site and the <NUM>' initiating start codon of the nucleic acid molecule encoding said carrier polypeptide wherein translation from the nucleic acid molecule encoding said carrier polypeptide is reduced when compared to a control nucleic acid molecule encoding said recombinant polypeptide that does not comprise said nucleotide spacer sequence.

In a preferred embodiment of the invention said carrier polypeptide includes the amino acid motif: Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline.

In an alternative embodiment of the invention said carrier polypeptide includes the amino acid motif: D/E-X-N-X-S/T, wherein X is any amino acid except proline.

In an alternative preferred embodiment of the invention said carrier polypeptide includes the amino acid motif D/E-X-N-X-S/T, wherein X is any amino acid except proline and is selected from the group consisting of: DVNVT (SEQ ID NO <NUM>), EVNAT(SEQ ID NO <NUM>), DQNAT(SEQ ID NO <NUM>), DNNNT(SEQ ID NO <NUM>), DNNNS (SEQ ID NO <NUM>), DQNRT (SEQ ID NO <NUM>), ENNFT(SEQ ID NO <NUM>), DSNST(SEQ ID NO <NUM>), DQNIS (SEQ ID NO <NUM>), DQNVS (SEQ ID NO <NUM>), DNNVS (SEQ ID NO <NUM>), DYNVS (SEQ ID NO <NUM>), DFNVS (SEQ ID NO <NUM>), DFNAS (SEQ ID NO <NUM>), DFNSS (SEQ ID NO <NUM>), DVNAT(SEQ ID NO <NUM>), DFNVT (SEQ ID NO <NUM>) or DVNAS (SEQ ID NO <NUM>).

In a further preferred embodiment said carrier polypeptide comprises a nucleic acid encoding said polypeptide comprising a nucleotide sequence as set forth in SEQ ID NO: <NUM> or <NUM> or <NUM>.

In a preferred embodiment of the invention said promoter is further operably linked to a ribosome binding site wherein there is provided a nucleotide spacer sequence between the <NUM>' prime end of said ribosome binding site and the <NUM>' initiating start codon of the nucleic acid molecule encoding said one or more polypeptides required for the synthesis of a heterologous glycan antigen wherein translation from the nucleic acid molecule encoding said biosynthetic locus is reduced when compared to a control nucleic acid molecule encoding said recombinant polypeptide that does not comprise said nucleotide spacer sequence.

In a preferred embodiment of the invention said heterologous glycan antigen is a heptasaccharide.

In a preferred embodiment of the invention said biosynthetic locus is the Pgl locus.

Preferably said Pgl locus comprises genes encoding said one or more polypeptides selected from the group consisting of: PgIG, PgIF, PgIE, optionally Cj1122c; PgID, PgIC, PgIA, PgIJ, Pgll, PglH, PgIK, Gne
In a further preferred embodiment of the invention said nucleic acid molecule encoding one or more polypeptides required for the synthesis of a heterologous glycan antigen comprises a nucleotide sequence as set forth in SEQ ID NO: <NUM>, wherein said SEQ ID NO <NUM> does not include a functional version of PgIB (SEQ ID NO: <NUM>).

Ribosome Binding Sites [RBS] in prokaryotic nucleic acid molecules are referred as a Shine Dalgarno [SD] sequence and is a consensus sequence that is typically positioned <NUM>-<NUM> nucleotides upstream of an initiating codon of the nucleic acid molecule. The consensus RBS sequence consists of a purine rich region followed by an A and T-rich translational spacer region, for example the consensus AGGAGG or AGGAGGU. Initiating codons are commonly AUG but translation can also be initiated at codons such as GUG, UUG, AUU or CUG.

In a preferred embodiment of the invention said nucleotide spacer sequence is at least <NUM> nucleotides in length.

In a preferred embodiment of the invention said nucleotide spacer sequence is <NUM> and <NUM> nucleotides in length; preferably the nucleotide spacer sequence is between <NUM> and <NUM> nucleotides in length.

In a preferred embodiment of the invention said nucleotide spacer sequence is <NUM> nucleotides in length.

In a preferred embodiment of the invention said nucleotide spacer sequence is <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> nucleotides in length.

In a preferred embodiment of the invention said nucleotide spacer sequence is between <NUM> and <NUM> nucleotides in length.

In a preferred embodiment of the invention said nucleotide spacer sequence is <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> nucleotides in length.

The reduction in nucleic acid molecule translation of said oligosaccharyltransferase and/or said carrier polypeptide and/or biosynthetic locus is reduced by at least <NUM>% when compared to a control nucleic acid molecule that encodes said oligosaccharyltransferase and/or said carrier polypeptide and/or biosynthetic locus but does not comprise said spacer nucleotide sequence.

The reduction in nucleic acid translation is <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% when compared to a control nucleic acid that encodes said recombinant polypeptide but does not comprise said spacer nucleotide sequence.

Bacterial expression systems that utilize inducers and repressors of gene expression are well known in the art and include modifications that are well established which enhance induction or repression of gene expression. For example is laclq carries a mutation in the promoter region of the lacl gene that results in increased transcription and higher levels of Lac repressor within the cells. Moreover, the Ptac, a strong hybrid promoter composed of the -<NUM> region of the trp promoter and the -<NUM> region of the lacUV5 promoter/operator and is strongly inducible.

Alternative heterologous glycan antigens include O-antigen. O-antigens comprising repetitive glycan polymers are the polysaccharide component of lipopolysaccharides (LPS) found associated with the outer membrane of gram negative bacteria. O-antigens typically elicit a strong immune response in animals. The composition of the O chain varies from bacterial strain to bacterial strain. For example, there are over <NUM> different O-antigen structures known produced by different E. coli strains. O-antigens are exposed on the outer surface of the bacterial cell and serve a target for recognition by host antibodies. Examples of polysaccharide synthesis loci are well known in the art and can be found in: "<NPL>; "<NPL>; "<NPL>; and "<NPL>.

We disclose but do not claim an oligosaccharyltransferase polypeptide selected from the group:.

wherein said oligosaccharyltransferase is for use in the transfer of one or more heterogeneous glycans to at least one carrier polypeptide.

Preferably said nucleic acid molecule comprises a nucleotide sequence that is <NUM>% or <NUM>% identical to the nucleotide sequence set forth in SEQ ID NO: <NUM>.

More preferably said nucleic acid molecule comprises a nucleotide sequence that is at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% identical to the nucleotide sequence set forth in SEQ ID NO: <NUM>.

We disclose but not claim a transcription cassette comprising a nucleic acid molecule encoding an oligosaccharyltransferase wherein said oligosaccharyltransferase comprises an amino acid sequence that is at least <NUM>% identical to the amino acid sequence set forth in SEQ ID NO: <NUM>.

Preferably, said oligosaccharyltransferase comprises an amino acid sequence that is <NUM>% or <NUM>% identical to the amino acid sequence set forth in SEQ ID NO: <NUM>.

Preferably said use is in a microbial host cell and said oligosaccharyltransferase polypeptide is transformed into said microbial host cell.

According to a further aspect of the invention there is provided a vector comprising a transcription cassette according to the invention.

In a preferred embodiment of the invention said vector is a plasmid.

In an alternative preferred embodiment of the invention said vector is a transposon.

In a preferred embodiment of the invention said transposon is selected from the group consisting of: Tn5, Tn10, Himar1 and other mariner elements, Tn7, Tn917 and Tn916.

In a preferred embodiment of the invention said transposon is Tn5.

According to a further aspect of the invention there is provided a bacterial cell genetically modified with a transcription cassette or vector according to the invention.

In a preferred embodiment of the invention said nucleic acid molecule encoding said oligosaccharyltransferase is stably integrated into the genome of said bacterial cell.

In a further preferred embodiment of the invention said nucleic acid molecule encoding said carrier polypeptide is stably integrated into the genome of said bacterial cell.

In a yet further preferred embodiment of the invention said nucleic acid molecule encoding said biosynthetic locus is stably integrated into the genome of said bacterial cell.

Genetic transformation of an attenuated pathogenic bacterial cell according to the invention using a transcription cassette as herein disclosed can be via transformation using episomal vectors that are replicated separately from the genome of the attenuated pathogenic bacterial cell to provide multiple copies of a gene or genes. Alternatively, integrating vectors that recombine with the genome of the attenuated pathogenic bacterial cell and which is replicated with the genome of said attenuated pathogenic bacterial cell.

In a preferred embodiment of the invention said nucleic acid molecule encoding a oligosaccharyltransferase polypeptide, a carrier polypeptide and a biosynthetic locus comprising one or more polypeptides required for the synthesis of a heterologous glycan antigen are each integrated into the genome of said bacterial cell.

In a preferred embodiment of the invention said bacterial cell is a pathogenic Gram-positive bacterial cell.

In a preferred embodiment of the invention said bacterial cell is a pathogenic Gram-negative bacterial cell.

In a preferred embodiment of the invention said bacterial cell is a human pathogen.

In a preferred embodiment of the invention said human pathogen is selected from the group: Neisseria, Moraxella, Escherichia, Salmonella, Shigella, Pseudomonas, Helicobacter, Legionella, Haemophilus, Klebsiella, Enterobacter, Cronobacter and Serratia.

In a preferred embodiment of the invention said bacterial cell is a non-human pathogen.

In a preferred embodiment of the invention said non-human pathogen is selected from group: Mannheimia spp. , Actinobacillus spp. g Actinobacillus pleuropneumoniae, Pasteurella spp. , Haemophilus spp. or Edwardsiella spp.

In a preferred embodiment of the invention said bacterial cell is a zoonotic bacterial species. In a preferred embodiment of the invention said zoonotic bacterial species is selected from the group: Brucella spp. , Campylobacter spp. , Vibrio spp. , Yersinia spp. and Salmonella spp. According to a further aspect of the invention there is providing a bacterial cell culture comprising a genetically modified bacterial cell according to the invention.

We disclose but not claim a transcription cassette or vector according to the invention for use in the production of one or more glycoconjugates.

According to an aspect of the invention there is provided the use of an oligosaccharyltransferase polypeptide encoded by a nucleic acid molecule selected from the group:.

in the transfer of one or more heterologous glycans to at least one carrier polypeptide in a microbial cell.

According to a further aspect of the invention there is provided a process for the production of one or more glycoconjugates comprising:.

We disclose but not claim a cell culture vessel comprising a bacterial cell culture according to the invention.

Preferably said cell culture vessel is a fermenter.

Bacterial cultures used in the process according to the invention are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, bacteria are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between <NUM> and <NUM>, preferably between <NUM> and <NUM>, while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The products produced can be isolated from the bacteria as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography. In this process, the pH value is advantageously kept between pH <NUM> and <NUM>, preferably between pH <NUM> and <NUM>, especially preferably between pH <NUM> and <NUM>.

An overview of known cultivation methods can be found in the textbook <NPL>)) or in the textbook by <NPL>)).

The culture medium to be used must suitably meet the requirements of the bacterial strains in question. Descriptions of culture media for various bacteria can be found in the textbook "<NPL>).

As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogenphosphate or dipotassiumhydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

The fermentation media used according to the invention for culturing bacteria usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "<NPL>). Growth media can also be obtained from commercial suppliers, for example Standard <NUM> (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (<NUM> at <NUM> bar and <NUM>) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

The culture temperature is normally between <NUM> and <NUM>, preferably at from <NUM> to <NUM> and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from <NUM> to <NUM>, preferably around <NUM>. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally <NUM> to <NUM> and preferably <NUM> to <NUM>. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within <NUM> to <NUM> hours.

The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.

However, the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the fatty acids present therein.

We disclose but not claim a process for the identification of novel glycoconjugates comprising:.

We disclose but not claim a glycoconjugate formed by the process according to the invention.

We disclose but not claim an isolated oligosaccharyltransferase polypeptide wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: <NUM>, or polymorphic sequence variant thereof, wherein the amino acid sequence is modified by deletion or substitution of at least one amino acid residue and said modified polypeptide has altered substrate specificity and/or increased oligosaccharyltransferase activity when compared to an unmodified oligosaccharyltransferase polypeptide comprising the amino acid sequence set forth in SEQ ID NO: <NUM>.

Preferably said modification is a deletion or substitution of one or more of the amino acid residues selected from the group consisting of amino acid residue <NUM> and/or amino acid residue <NUM> and/or amino acid residue <NUM> as set forth in SEQ ID NO: <NUM>.

Preferably said modification is amino acid substitution wherein the substitution is amino acid residue <NUM> as set forth in SEQ ID NO: <NUM> wherein amino acid residue serine is substituted with amino acid residue arginine.

Preferably said modification is amino acid substitution wherein the substitution is amino acid residue <NUM> as set forth in SEQ ID NO: <NUM> wherein amino acid residue asparagine is substituted with amino acid residue proline.

Preferably said modification is amino acid substitution wherein the substitution is amino acid residue <NUM> as set forth in SEQ ID NO: <NUM> wherein amino acid residue asparagine is substituted with amino acid residue valine.

Preferably said modified polypeptide or polymorphic sequence variant comprises the amino acid sequence set forth in SEQ ID NO: <NUM>.

Preferably said modified nucleic acid sequence encodes a polypeptide comprising a sequence set forth in SEQ ID NO <NUM>.

We disclose but not claim an isolated nucleic acid molecule that encodes a polypeptide according to the invention.

Preferablysaid isolated nucleic acid molecule is selected from the group consisting of:.

Preferably said isolated nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: <NUM>.

Preferably said nucleic acid molecule is part of a transcription cassette.

We disclose but not claim a vector comprising a nucleic acid molecule according to the invention.

Preferably said vector is an expression vector.

We disclose but not claim a cell transformed or transfected with a nucleic acid molecule or expression vector according to the invention.

Preferably said cell is a microbial cell, for example a bacterial cell.

"Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.

An embodiment of the invention will now be described by example only and with reference to the following figures;.

A codon optimised version of C. sputorum pgIB2 was generated by DNA synthesis in the cloning vector pUC57km and designed to have EcoRI (GAATTC) restriction enzyme sites at the <NUM>' and <NUM>' end of the construct. The plasmid pEXT21 was grown in E. coli DH5α cells and purified by plasmid extraction (QIAGEN Ltd UK). <NUM>µg of pUC57Km containing CsPgIB2 and <NUM>µg of pEXT21 were digested with EcoRIHF (New England Biolabs U. ) cloned into the EcoRI site of the IPTG inducible expression vector pEXT21 to generate the vector pELLA1.

The gene coding for C. sputorum PgIB2 was amplified by PCR with the pTac promoter and Lacl repressor from plasmid pEXT21 as a template using accuprime Taq hifi with (SEQ ID <NUM>: <NUM>'-TTTTGCGGCCGCTTCTACGTGTTCCGCTTCC-<NUM>') as forward primer and (SEQ ID <NUM>: <NUM>'-TTTTGCGGCCGCATTGCGTTGCGCTCACTGC-<NUM>') reverse primer using the following cycling conditions, <NUM>/<NUM> minutes followed by <NUM> cycles of <NUM> for <NUM> seconds, <NUM> for <NUM> seconds and <NUM> for <NUM> minutes. and ligated into the unique Notl site in pJCUSA1 a Zeocin® resistant transposon where the antibiotic marker is flanked by loxP sites allowing for downstream removal of antibiotic marker from the final target strain via the introduction of the CRE enzyme. It has a pMB1 origin of replication and thus can be maintained in any E. coli strain prior to being cut out and transferred along with the Zeocin® resistance cassette using Sfil restriction enzyme digestion and transfer into the pUT delivery vector thus generating a functional transposon. The sequence of the transposon is shown below (SEQ ID <NUM>):
<IMG>.

The insertion of CspglB2 into this transposon and transfer into the pUT delivery vector resulted in plasmid pELLA2 and maintained in Transformax E. coli strain EC100D pir+ (Cambio U.

To enable transfer of the CspgIB2 transposon cargo into the chromosome of a recipient E. coli strain or any other bacterium the plasmids pELLA2 was transferred into E. coli MFD a diaminopimelic acid (DAP) auxotroph. Growth medium was supplemented with Zeocin® <NUM>µg/ml and ampicillin <NUM>µg/ml. Both donor and recipient bacteria were growth until late exponential phase. Bacterial cells were pelleted by centrifugation, washed <NUM> times with PBS and mixed together in a ratio of <NUM>:<NUM> recipient to donor and spotted on a dry LB agar plate with no antibiotics for <NUM> -<NUM> hrs. The cells were scraped and suspended in PBS and dilutions plated on LB agar with appropriate selection antibiotics to select for transconjugants. Individual colonies were picked up and screened for loss of the pUT backbone and for the presence of the transposon.

The transposon carrying CspgIB2 and loxP recombination sites around a Zeocin® resistance cassette was introduced into PoulVAc E. Following selection for Zeocin® resistant colonies, the antibiotic selection marker was removed by introduction via electroporation, the temperature sensitive vector pCRE5 (Reference: <NPL>).

coli was cultured at <NUM> in the presence of kanamycin <NUM>µg/ml, rhamnose was added to induce expression at <NUM> % final concentration and the organism subcultured several times to select for colonies that had lost resistance to Zeocin® but maintained resistance to kanmaycin indicating that the bleomycin resistance gene had been flipped out of the chromosome.

coli mutant was then sub-cultured at <NUM> to cure out the pCRE5 plasmid. Screening for colonies that had once again become sensitive to kanamycin confirmed loss of pCRE5 and completed generation of an unmarked inducible copy of pglB on the chromosome of E.

Attenuated bacterial strains are transformed with the plasmid pGVXN150:GT-ExoA encoding a modified carrier polypeptide [GT-ExoA]. The GT-ExoA construct was engineered to express a modified version of P. aeruginosa Exotoxin A in the vector pGH and closed into a vector derived from pEC415 using the restriction enzymes Nhel and EcoRI (NEB). The synthesized protein contains two internal modifications that allow glycosylation of the protein by Pgl, as well as containing four N-glycosylation sequons at the N terminal and an additional <NUM> at the C terminals glycotags. In addition, a hexa-histidine tag was added to the C-terminus of the protein to facilitate putification and an and an E. coli DsbA signal peptide was added to the N-terminal sequences enabling Sec-dependent secretion to the periplasm. pGVXN150: GT-ExoA is ampicillin resistant and L- (+) - Arabinose inducible. The construct sequence was then confirmed using Sanger sequences with the primers GTExoA NF (SEQ ID NO <NUM>; GCGCTGGCTGGTTTAGTTT), GTExoA NR (SEQ ID NO <NUM>; CGCATTCGTTCCAGAGGT), GTExoA CF (SEQ ID NO <NUM>; GACAAGGAACAGGCGATCAG) and GTExoA CR (SEQ ID NO <NUM>; TGGTGATGATGGTGATGGTC).

Protein glycan coupling technology requires the use of Campylobacter jejuni PgIB. This enzyme has <NUM> transmembrane domain and is toxic when overexpressed in E. The pgIB gene was originally amplified by PCR with oligonucleotides PglBEcoRI (EcoRI in bold) using the primers (SEQ ID NO <NUM>: AAGAATTCATGTTGAAAAAAGAGTATTTAAAAAACCC) and PgIBNcol-HA (SEQ ID NO <NUM>: AACCATGGTTAAGCGTAATCTGGAACATCGTATGGGTAAATTTTAAGTTTAAAAACCTTA GC), using Pfu polymerase with pACYC(pgl) as template. Oligonucleotide PgIBNcol-HA encodes an HA-tag to follow PgIB expression by Western blot. The PCR product was digested with EcoRI and Ncol and cloned in the same sites of vector pMLBAD. The plasmid obtained was named pMAF10. Arabinose-dependent expression of PgIB was confirmed by Western blot (Feldman et al. This construct has been subcloned into the EcoRI site of the vector pEXT21 allowing for IPTG dependant inducible expression of CjpglB. This plasmid and ORF combination has been used for several years in order to produce several glycoconjugate vaccines. In a recent modification using PgIB from Campylobacter sputorum we have carried out tests and found that the ribosome binding site is encoded within the pEXT21 vector itself. This means that translational efficiency is partly controlled by the distance between the RBS and the ATG start codon of pgIB. We noticed that inserting the PgIB coding gene into the vector pEXT21 with an extended <NUM> base pairs of DNA sequence resulted in reduced toxicity of the enzyme and subsequently increased growth in the carrier E. coli strain as measured by optical density. Therefore it may be possible to reduce the toxicity of C. jejuni PgIB by the simple modification of insertion of additional nucleotides before the ATG start codon or alternatively clone the gene further away from the RBS carried within the expression plasmid.

The pgIB gene from C. sputorum was amplified using the primers CsPglB1fwd: TTTT GAATTCGATTATCGCCATGGCGTCAAATTTTAATTTCGCTAAA (SEQ ID NO <NUM>) and the reverse primer CsPglB1rev: TTTT GAATTC TTATTTTTTGAGTTTATAAATTTTAGTTGAT (SEQ ID NO <NUM>) using Accuprime Taq Hifi and the following cycling conditions <NUM>/<NUM>, followed by <NUM> cycles of the following conditions <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>. The PCR product was cut with the restriction enzyme EcoRI HF for <NUM> hr at <NUM>. The plasmid pEXT21 was also cut with the restriction enzyme EcoRI HF for <NUM> hr at <NUM>. Both plasmid and PCR product were purified with a PCR purification kit (QIAGEN UK) and the plasmid pEXT21 was dephosphorylated by treating with Antarctic phosphatase (NEB UK Ltd) at <NUM> for <NUM> hr. The enzyme was heat inactivated by heating at <NUM> for <NUM> before the plasmid and the insert were ligated together using T4 DNA ligase (Promega UK) and the reaction was incubated overnight at <NUM>. The ligation reaction was transformed into E. coli Dh10β cells (NEB UK Ltd) and recovered on LB Spectinomycin plates (<NUM>µg/ml). Constructs were then sequenced to confirm that the cloned C. sputorum PgIB had not gained any mutations during the cloning process. This new construct was named pELLA3.

Mutagenesis of <NUM> genes in the C. jejuni <NUM> glycosylation locus cloned in pACYC184 (pACYCpgl) was performed in vitro using a customised EZ::TN transposon system (Epicentre, Madison, WI, USA). Briefly, a kanamycin resistance cassette (Trieu-Cuot et al. , <NUM>) lacking a transcriptional terminator and therefore unable to exert downstream polar effects was amplified by PCR and cloned into the multiple cloning site of the vector pMOD™<MCS> (Epicentre). This construct was linearized by Scal digestion and the kanamycin resistance cassette along with flanking mosaic ends was amplified by PCR using primers FP-<NUM> and RP-<NUM> (Epicentre). The PCR product was combined with plasmid pACYCpgl (Wacker et al. , <NUM>) in an in vitro transposition reaction performed according to manufacturer's instructions (Epicentre). The resultant pool of mutated pACYCpgl plasmids was electroporated into E. coli XL1-Blue MRF' (Stratagene) and putative mutants were screened by PCR to identify the location and orientation of the kanamycin cassette. We only used those mutants having the kanamycin resistance cassette inserted with the same transcriptional orientation as the genes of the glycosylation locus, which were also confirmed by sequence analysis.

The construct pELLA1 was transformed into E. coli CLM24 cells alongside a pEC415vector coding for Pseudomonas aeruginosa exotoxin A with a single internal glycosylation site and the plasmid pACYCpgIB::km coding for the entire C. jejuni heptasaccharide with a disruption in the pgIB gene by insertion of a miniTn5km2 element. As a comparison the exotoxin A and C. jejuni heptasaccharide coding constructs were transformed into an E. coli CLM24 cell carrying pEXT21pgIB from C. <NUM> LB containing <NUM>µg/ml-<NUM> cm, <NUM>µg/ml-<NUM> amp, <NUM>µg/ml-<NUM> spectinomycin were inoculated with <NUM> of an O/N culture of either CLM24 construct combination and incubated with shaking at <NUM>. Optical density <NUM> reading were taken at hourly intervals and protein expression induced at an OD<NUM> of <NUM> by the addition of IPTG <NUM> and L-arabinose <NUM>% final concentration. <NUM> hr post initial induction, <NUM>% L-arabinose was added and OD<NUM> continued to be measured (<FIG>).

The growth of E. coli CLM24 cells without any induction of protein expression was also measured. This was carried out in the same way as described above for the E. coli CLM24 cells carrying pELLA1 except that no IPTG or L-arabinose was added (<FIG>).

coli CLM24 cultures carrying plasmids coding for singly glycosylatable exotoxinA, C. sputorum PgIB2 or C. jejuni PgIB were used to inoculate <NUM> of LB broth. Protein expression was induced as described in example <NUM> with the modification that the cultures were incubated for a further <NUM> hr after the second <NUM>% L-arabinose addition. At this point cells were pelleted by centrifugation at 4000xg for <NUM> and lysed using a high pressure cell homogeniser (Stansted Fluid power) HIS tagged exotoxinA was purified from CLM24 cells using NiNTA binding. Protein was separated on a <NUM>% Bis-tris gel (Invitrogen) before transferring onto a nitrocellulose membrane. This was probed with primary rabbit hr6 anti-campy glycan antibody and mouse anti-HIS. Goat anti-rabbit and anti-mouse infrared dye labelled secondary antibodies were used to enable visualisation of glycoprotein using an Odyssey LI-COR scanner (LI-COR Biosciences UK Ltd) (<FIG>).

pACYCpgIB::kan was introduced into PoulVAC E. coli by electroporation alongside the plasmid pWA2 coding for a HIS tagged diglycosylatable CmeA and pELLA1. After induction with <NUM> IPTG and a total of <NUM> hr incubation at <NUM> with shaking. <NUM> of culture was obtained and centrifuged at <NUM>,<NUM>×g for <NUM>. Cells were lysed by high pressure and purification carried out using NiNTA. The protein was then purified according to manufacturer's instructions (QIAExpressioninst, Qiagen UK) and eluted in 4x <NUM> before concentrating the sample to <NUM>µl. An equal volume of <NUM>×SDS PAGE loading dye was added <NUM>µl was loaded into a <NUM>% Bis-Tris gel and stained by coomassie (<FIG>).

Salmonella Typhimurium strain SL3749 was transformed with pUA31 (coding for the acceptor protein CjaA), pACYCpgIB::km (coding for C. jejuni heptasaccharide coding locus but with pgIB knocked out) and pELLA1 (coding for IPTG inducible C. jejuni pgIB2). A <NUM> O/N <NUM> shaking culture was prepared and used to inoculate <NUM> of LB broth. This continue to be shaken <NUM> until an OD600nm of <NUM> was reached. At this point <NUM> IPTG was added to induce CsPgIB2 expression. The culture was incubated for a further <NUM> hr at <NUM> with shaking. Bacterial cultures were pelleted by centrifugation at <NUM>×g for <NUM> and resuspended in <NUM> <NUM> Tris, <NUM> NaCl pH <NUM> (TBS). Cells were lysed using a highpressure cell homogeniser. <NUM>% SDS and <NUM>% Triton X-<NUM> were added and the lysed material incubated for <NUM> hr at <NUM> with mixing. The material was then centrifuged at <NUM>×g for <NUM>. Pellet was discarded before <NUM>µl of c-Myc sepharose (Thermo Scientific USA) was added. This was allowed to incubate O/N at <NUM> with mixing. The material was then centrifuged at <NUM>×g for <NUM> and the supernatant removed. <NUM> TBS was added with <NUM>% Tween. This was washed <NUM> times by pulsing at <NUM>,<NUM>×g. Protein elution was achieved by the addition of <NUM>µl 2XSDS loading buffer containing <NUM>µl DTT and boiled for <NUM> minutes. Western blot was carried out to visualise the result.

Claim 1:
A transcription cassette comprising:
i) a nucleic acid molecule comprising a nucleotide sequence that encodes an oligosaccharyltransferase polypeptide as set forth in SEQ ID NO: <NUM>; or
ii) a nucleic acid molecule comprising a nucleotide sequence that is degenerate to the nucleotide sequence set forth in SEQ ID NO: <NUM> or <NUM> and encodes an oligosaccharyltransferasepolypeptide comprising an amino acid sequence as set forth in SEQ ID NO: <NUM>,
wherein said nucleic acid molecule is operably linked to a promoter adapted for expression in a bacterial host cell.