US20070128225A1 - Immunogenic sequences - Google Patents

Immunogenic sequences Download PDF

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US20070128225A1
US20070128225A1 US10/516,215 US51621503A US2007128225A1 US 20070128225 A1 US20070128225 A1 US 20070128225A1 US 51621503 A US51621503 A US 51621503A US 2007128225 A1 US2007128225 A1 US 2007128225A1
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tularensis
nucleic acid
seq
antigen
subspecies
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Joann Prior
Richard Prior
Paul Hitchen
Ann Dell
Richard Titball
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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  • the present invention relates to nucleic acid sequences, in particular genes that encode the enzymes which produce the O-antigen of Francisella tularensis , and their use as or in the production of vaccines and in diagnosis.
  • Francisella tularensis is a small Gram-negative coccobacillus, which causes the zoonotic disease Tularemia.
  • the genus Francisella contains two species: F. tularensis and Francisella novicida .
  • F. novicida be considered a subspecies of F. tularensis (Hollis D G, et al., J. Clin. Micro. 27: 1601-1608).
  • the closely related bacterium Yersinia philomiragia is now also considered a member of the genus Francisella , due to its high degree of relatedness at the DNA level.
  • tularensis other than novicida these are: subspecies tularensis , subspecies holarctica and subspecies mediaasiatica .
  • the subspecies tularensis and holarctica can be identified on the basis of virulence, citrulline ureidase activity and acid production from glycerol (Olsufjev N G, et al. (1959) J. Hyg. Epidemiol. Microbiol. Immunol. 3: 138-149.
  • Francisella tularensis subspecies mediaasiatica is predominantly found in central asian republics of the former USSR. Strains of this subspecies possess citrulline ureidase activity, and are able to ferment glycerol, but are less virulent than strains of F. tularensis subspecies tularensis in rabbit.
  • Tularemia is a disease occurring in the northern hemisphere; with cases frequently found in Europe, N. America, Asia, N. Russia and Japan. Rodents are thought to be the main reservoir of the bacteria, with ticks as one of the main vectors.
  • the lipopolysaccharide (LPS) of Gram-negative bacteria is the major component of the outer membrane.
  • the molecule is composed of 3 regions, lipid-A, which is embedded in the outer membrane and has a conserved structure between species, and two polysaccharides, the core oligosaccharide which can vary in complexity between species, and the O-antigen which is a very polymorphic structure (Kenne L, et al. (1983) Bacterial Polysaccharides The polysaccharides . Academic Press, pp. 287-362).
  • the LPS molecule is thought to be required by the bacteria for protection against serum killing (Whitfield C, et al, (1997) Mol. Micro. 23: 629-638) and cationic antimicrobial peptides (Groisman E A. (1994). Trends. Microbiol. 2: 444-449).
  • LPS from a subspecies holarctica strain appears to be less toxic than other Gram-negative LPS and its O-antigen contains rare sugars which are related in structure to those found in Pseudomonas aeruginosa 06 and Shigella dysenteriae type 7.
  • the applicants have now determined the genetic basis of O-antigen production in F. tularensis subspecies tularensis . Furthermore, they have established the efficacy of LPS from various F. tularensis strains as a vaccine.
  • nucleic acid which encodes a series of enzymes or enzyme fragments which, when expressed together in a cell, are able to produce an immunogenic moiety able to produce an immune response in an animal to which it is administered, which response is protective against Francisella tularensis infection, said nucleic acid encoding at least some of the enzymes of SEQ ID NOS 3-17, or modifications thereof.
  • modification refers to sequences of amino acids, which differ from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids.
  • Amino acid substitutions may be regarded as “conservative” where an amino acid is replaced with a different amino acid with broadly similar properties.
  • Non-conservative substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide.
  • modifications will be at least 60% identical, preferably at least 75% identical, and more preferably at least 90% identical to the base sequence.
  • the invention comprises a nucleic acid which encodes enzymes of SEQ ID NOS 3-17.
  • a preferred example of such a nucleic acid comprises SEQ ID NO 1 or a variant thereof.
  • the nucleic acid is of SEQ ID NO 1.
  • nucleic acid sequences means any substitution of, variation of, modification of, replacement of, deletion of, or the addition of one or more nucleic acid(s) from or to a polynucleotide sequence providing the resultant protein sequence encoded by the polynucleotide exhibits the similar properties as the protein encoded by the basic sequence.
  • the term therefore includes allelic variants, degenerate variants which encode similar proteins but vary only as a result of the degeneracy of the genetic code. It also includes a polynucleotide which substantially hybridises to the polynucleotide sequence of the present invention. Preferably, such hybridisation occurs at, or between low and high stringency conditions.
  • low stringency conditions can be defined as 3 ⁇ SSC at about ambient temperature to about 55° C. and high stringency condition as 0.1 ⁇ SSC at about 65° C.
  • SSC is the name of the buffer of 0.15M NaCl, 0.015M tri-sodium citrate. 3 ⁇ SSC is three times as strong as SSC and so on.
  • variants have 65% or more of the nucleotides in common with the polynucleotide sequence of the present invention, more typically 70%, preferably 75%, even more preferably 80% or 85% and, especially preferred are 90%, 95%, 98% or 99% or more identity.
  • Variants may comprise the basic sequence which has been modified to ensure that the codon usage is enhanced or optimised, as would be understood in the art, for a particular organism in which it is required that the sequence is expressed in a desired organism, for example a prokaryotic cell such as E. coli .
  • This may involve modifying the content of particular nucleotides, for instance changing the percentage of G and C present in the sequence, to suit that usually found in genes which are highly expressed in the target organism.
  • particular variants of SEQ ID NO 1 are synthetic variants, engineered to remove codons rarely found in highly expressed genes from common expression hosts such as E. coli and, at the same time, avoid the introduction of codons rarely found in genes coding for O-antigens.
  • codons for the amino acids arg, leu, ile, gly and pro are changed to CGT or CGC (arg), CTG, CTT or CTC (leu), ATC or ATT (ile), GGT or GGC (gly), and CCG CCA or CCT (pro), thus eliminating rare codons.
  • BESTFIT When comparing nucleic acid sequences for the purposes of determining the degree of identity, programs such as BESTFIT and GAP (both from Wisconsin Genetics Computer Group (GCG) software package). BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and fins the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length.
  • SEQ ID NO 1 comprises a series of genes which encode a number of enzymes which are shown hereinafter in FIG. 5 and SEQ ID NOs 3-17.
  • any variants of SEQ ID NO 1 encode enzymes of SEQ ID NOS 3-17 or modifications of these.
  • fragment used in relation to amino acid sequences refers to any portion of the given amino acid sequence which has the same activity as the complete amino acid sequence. Fragments will suitably comprise at least 20 and preferably at least 50 consecutive amino acids from the basic sequence.
  • fragments is also used in relation to nucleic acid sequences. Fragments of SEQ ID NO 1 may have applications in diagnostics, and these form a further aspect of the invention. For diagnostic purposes, fragments may be quite short, for example from 5-30 bases, which may be used as primers or probes. Particular characteristic regions of SEQ ID NO 1 from which suitable fragments for diagnostic purposes may be identified are elucidated hereinafter. Fragments which are useful in therapy would generally be expected to be longer, for example from 600-17,000 bases long.
  • SEQ ID NO 41 A region of genome of the F. tularensis strain Schu 24 (subspecies tularensis ) which includes SEQ ID NO 1, and which is responsible for expression of the set of enzymes necessary for constructing the polysaccharide, has been identified. It is shown hereinafter in FIG. 6 as SEQ ID NO 41. This sequence includes a number of genes including a series of genes that encode the enzymes illustrated in FIG. 5 hereinafter as SEQ ID NOS 3-20. Putative functions were applied to these genes by comparison with known sequences as illustrated in Table 1. TABLE 1 Gene SEQ ID F.
  • tularensis product NO protein size (aa) Putative function 2 Transposase 247 Hypothetical protein Transposase 3 WbtA 578 Sugar epimerase 4 WbtB 205 Galactosyl transferase Glycosyl transferase 5 WbtC 263 UDP-glucose 4-epimerase 6 WbtD 363 Sugar transferase 7 WbtE 436 LPS biosynthesis Dehydrogenase 8 WbtF 323 C 4-epimerase 9 Wzy 409 Membrane protein/O-antigen polymerase 10 WbtG 366 Transferase 11 WbtH 628 Asparagine synthetase 12 WbtI 360 Sugar transaminase/ perosamine synthetase 13 WbtJ 241 Formyl transferase 14 Wzx 495 o-antigen flippase 15 WbtK 286 Glycosyl transferase 16 WbtL 294 Glucose
  • proteins illustrated as SEQ ID NOS 3-17 are believed to be involved in O-antigen biosynthesis.
  • the O-antigen itself has applications both in diagnostics and as a prophylactic or therapeutic vaccine.
  • nucleic acids of the invention When expressed together in a host cell, they will result in the construction of an antigen that produces an immune response in an animal including a human, which is protective against infection by F. tularensis . Thus they may be used in the production of prophylactic or therapeutic vaccines.
  • the nucleic acid may be included in a vector such as a live viral vaccine, for instance, adenovirus vector or vaccinia, or in a plasmid to form so-called “naked DNA” vaccines, or preferably in a bacterial vector such as attenuated Salmonella species.
  • the nucleic acid will be under the control of suitable control elements such as promoters, signal sequences, enhancers and the like, as would be understood in the art.
  • the nucleic acid is expressed either within the cells of the patient to whom the vaccine is administered, or in the case of bacterial vectors, within the host cell itself. As a result a series of enzymes are produced which are able to construct the protective O-antigen in situ.
  • the vector is suitably combined with a pharmaceutically acceptable carrier in a vaccine formulation.
  • a pharmaceutically acceptable carrier depends upon the type of vector being used, as would be understood in the art.
  • the vaccine comprises a recombinant Salmonella
  • it is suitably in the form of a composition which is suitable for oral administration.
  • the nucleic acid may be included in an expression vector which is used to transform a host cell.
  • Suitable host cells are prokaryotic or eukaryotic cells, but are preferably prokaryotic cells such as E. coli .
  • the nucleic acid used is a synthetic variant of SEQ ID NO 1, optimised for expression in the particular host cells. The protective O-antigen can then be recovered from these cells after culture thereof.
  • a method of preparing a prophylactic or therapeutic vaccine comprises transforming a host cell with a nucleic acid of the invention, culturing said host cell, and recovering a moiety which produces a protective immune response against F. tularensis therefrom.
  • Vaccines of this type will suitably be in the form of a pharmaceutical composition, in which the antigen is combined with a pharmaceutically acceptable carrier, as would be understood in the art.
  • compositions of the invention may be in a form suitable for oral use, for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosin.
  • inhalation for example as a finely divided powder or a liquid aerosol
  • insufflation for example as a finely divided powder
  • parenteral administration for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosin.
  • compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
  • the amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration.
  • a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.
  • Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.
  • the size of the dose for therapeutic or prophylactic purposes of moiety of the invention will naturally vary according to the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.
  • the invention provides recombinant O-antigen of F. tularensis which is obtainable from a host cell which expresses proteins of SEQ ID NO 3-17, or modifications thereof.
  • the applicants' realisation of the sequence of the O-antigen sequence provides the possibility that this sequence can form the basis of diagnostic tests, to determine whether a patient has an F. tularensis infection.
  • samples such as blood or saliva samples may be taken from the patient and the presence of SEQ ID NO 1 or variants thereof detected.
  • Specific detection methods are well known in the art, and may include amplification procedures such as the polymerase chain reaction (PCR) and/or other detection methods using for example labelled probes that hybridise to the target sequence and particularly SEQ ID NO 1. Primers and probes of this kind form a further aspect of the invention.
  • PCR polymerase chain reaction
  • primers and probes it may also be possible to allow differentiation between strains of F. tularensis infection.
  • primers comprising SEQ ID NOS 21 and 22 and 35 and 36 set out hereinafter will allow distinction between strains of F. tularensis subspecies tularensis , and F. tularensis subspecies holarctica as described below.
  • this possibility arises because of differences in the downstream sequence, and in the latter, because of differences in deletions in the flanking transposase sequence. Consequently, analysis using primers or probes based upon these regions may be used to determine whether any particular strain is F. tularensis subspecies tularensis or otherwise.
  • LPS extracted from F. tularensis strain Schu S4 was shown to have a characteristic ladder pattern after gel electrophoresis. However, the LPS was difficult to stain and required additional oxidation in order to visualise the O-antigen bands. This may suggest that the sugars in the O-antigen of F. tularensis strain Schu S4 are not oxidised in the same way as the O-antigen sugars found in most other bacteria.
  • the F. tularensis strain Schu S4 O-antigen gene cluster contained 15 genes, the putative functions of which was assigned (see Table 1 above) based on the BLAST results and structural information about the sugars contained in the O-antigen. Genes within the cluster are likely to be responsible for the production of the O-antigen molecule as well as the transportation of the molecule out of the bacterial cell.
  • O-antigen polymerase wzy
  • wzy-independent O-antigen polymerase
  • wzy-independent the polymerase
  • wzx flippase
  • wzz chain length determinant
  • wzy-independent a different set of proteins are involved in the transportation and polymerisation of the LPS molecule.
  • the transporter is ATP driven and composed of two proteins wzt and wzm that belong to the ABC-transporter family.
  • the TMHMM analyses of the putative O-antigen flippase (Wzx) and polymerase (Wzy) proteins supported their assigned functions based on sequence similarity.
  • the predicted numbers of trans-membrane helices for the F. tularensis proteins of 14 and 11 for Wzx and Wzy respectively are similar to those predicted for other bacteria, in which these cytoplasmic membrane proteins have been predicted to have around 10-12 trans-membrane helices.
  • the prediction of 2 large periplasmic domains for the F. tularensis Wzy protein is consistent with the two large periplasmic domains of the Shigella flexneri Wzy protein.
  • wbtM which has a putative promoter of its own.
  • manC and manB Downstream of the second transposase are manC and manB, which also have their own putative promoter and are probably not involved in biosynthesis of the O-antigen as mannose was not found to be part of the structure of the F. tularensis O-antigen, nor is it one of the intermediate products required for its synthesis.
  • the two genes manC and manB may once have been involved in biosynthesis of the O-antigen in an ancestor of F. tularensis .
  • the presence of transposases flanking the O-antigen biosynthetic gene cluster wbtA to wbtM suggests this cluster may have been horizontally acquired, perhaps replacing an ancestral polysaccharide gene cluster.
  • the O-antigen gene cluster appears to be present in all subspecies tularensis and B strains screened. However, there is at least one difference between the clusters in subspecies tularensis and B strains within a region containing a transposase. BLAST analysis using the partially deleted transposase has revealed possibly over 50 copies of it in the F. tularensis Schu S4 genome. It is possible that the insertion sequence originated in the F. tularensis genome from S. pneumonia and was copied randomly within the genome. The open reading frames flanking the insertion sequence have no significant homology within the F. tularensis genome, suggesting that these genes were not imported to this locus with the insertion.
  • the invention provides LPS obtainable from F. tularensis subspecies tularensis for use as a vaccine against infection by F. tularensis .
  • Vaccine compositions containing LPS from F. tularensis subspecies tularensis are also novel and form a further aspect of the invention. These will comprise pharmaceutically acceptable carriers as described above.
  • FIG. 1 SDS-PAGE analysis of LPS isolated from E. coli strain K325, 1.25 ⁇ g (track 1) and F. tularensis strain Schu S4, 50 ⁇ g (track 2).
  • FIG. 2 The genetic organisation of the O-antigen gene cluster in F. tularensis strain Schu S4. The G+C content of the O-antigen cluster is shown in the upper panel.
  • FIG. 3 Schematic structure of an O-antigen subunit of F. tularensis strain Schu S4 and the assignment of putative functions to the O-antigen gene cluster genes. A single O-unit is shown with sugar residues and glycosidic linkages indicated.
  • FIG. 4 Shows the region of the genome the nucleic acid sequence of the F. tularensis genome which encodes all the proteins shown in FIG. 5 .
  • FIG. 5 Shows the amino acid sequences of proteins encoded by SEQ ID NO 1, as well as a number of flanking gene sequences,
  • FIG. 6 Shows the nucleic acid sequence (SEQ ID NO 1) which encodes the enzymes necessary for O-antigen production.
  • Bacterial strains used in this study are shown in Table 2 and were cultured at 37° C. on BCGA agar for 48 hrs. TABLE 2 Species and Strain Subspecies F. tularensis Schu4 tularensis F. tularensis 199 tularensis F. tularensis 230 tularensis F. tularensis 041 tularensis F. tularensis LVS holarctica F. tularensis 200 holarctica F. tularensis 025 holarctica F. tularensis 075 holarctica F. tularensis HN63 holarctica F. tularensis 147 mediaasiatica LPS Purification
  • LPS was purified from F. tularensis strain Schu S4 using a hot-phenol and water extraction method (Westphal O, et al. (1965). Methods in Carbohydrate Chemistry 5: 83-91).
  • Glycine gel electrophoresis was performed according to the method of Laemmli (Laemmli U K. (1970). Nature 227: 680-685.) using a 12.5% separating gel with a 4.5% stacking gel. Ten ⁇ l of each sample were electrophoresed for approx 2 h at 100 mV in the Mini-protean II slab system (Biorad).
  • the sequence encoding the O-antigen biosynthetic cluster was identified and extracted from the Known protein sequences (obtained from GenBank) involved in the biosynthesis of the O-antigen of other bacteria were used to probe the F. tularensis Schu S4 partial genome sequence (Prior R G, et al. (2001) Journal of applied microbiology 91: 614-620), available at http://artedi.ebc.uu.se/Projects/Francisella/, using TBLASTN (Altschul S F. et al. (1997) Nucleic acids research 25: 3389-3402).
  • the contig containing the putative O-antigen gene cluster was extracted and subsequently analysed using the annotation tool Artemis (http://www.sanger.ac.uk/Software/Artemis). This allowed visualization of BLASTN, BLASTX and BLASTP searches, GC content and other analyses performed on the sequence and the predicted proteins.
  • DNA was prepared from the F. tularensis strains shown in table 1, by phenol extraction, as described by Karlsson et al 2000 ( Microb. Comp. Genom. 5: 25-39).
  • Ten pairs of overlapping PCR primers were designed to amplify the whole of the putative O-antigen gene cluster in approximate 2 kilobase segments using the DNAstar program PrimerSelect.
  • the primers were designed with annealing temperatures ranging from 42 to 59° C., although all were used successfully at 49° C.
  • PCR amplification using each pair of primers with each template DNA was carried out in the following mixture: 1 ⁇ PCR buffer (including 1.5 mM MgCl 2 ), 0.2 mM deoxynucleoside triphosphates (dNTPs), 2.5 mM forward primer, 2.5 mM reverse primer, 2.0 ⁇ l template DNA, 0.5 U Taq polymerase and filtered sterile water to a final volume of 20 ⁇ l.
  • the reaction mixtures were incubated at 90° C. for 1 min and then cycled at 90° C. for 1 min, 49° C. for 1 min and 72° C. for 2 min 25 s for 30 cycles, with a final incubation at 72° C. for 10 min.
  • PCR products were visualised on 0.5% agarose gels, with ethidium bromide staining.
  • PCR buffer, dNTPs, and polymerase were from Roche.
  • PCR primers were synthesised by MWG-Biotech.
  • PCR products amplified from Schu S4, HN63 and LVS DNA using primer pair 8 were cloned into pGEM-T easy (Promega) for sequence analysis.
  • Ligated DNA was transformed in E. coli JM109 chemically competent cells (Promega) and putative clones were screened using both colony PCR and digestion with restriction endonucleases. All DNA manipulations, including ligations, transformations, colony PCR, restriction endonuclease digestions and agarose gel electrophoresis were carried out according to methods described by Sambrook et al (1987) Molecular cloning: A laboratory manual. Cold Spring Harbor, N.Y.).
  • the three constructs were sequenced at Oswel by the dideoxynucleotide chain-termination method (Sanger F, et al. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467) using universal primers. Each sequence was compared and the BLAST (Altschul S F, et al. (1990) Basic local alignment search tool. J. Mol. Biol. 215: 403-410) function of the ARTEMIS software package was used for homology searches in the locally held GenBank databases to identify the functions of the differential regions of DNA.
  • the hot phenol-water extraction method was used to purify LPS from 2.2 g of freeze dried F. tularensis strain Schu S4. This resulted in 7 mg of LPS, which is a yield of 0.3%.
  • the LPS was difficult to visualise after SDS-PAGE and silver staining.
  • the oxidation step was increased from 5 min to 10 min to visualise a ladder pattern ( FIG. 1 ).
  • the F. tularensis O-antigen biosynthetic gene cluster was found to be 17 Kb in length and contain 15 genes putatively identified as being involved in O-antigen biosynthesis, flanked by two transposases ( FIG. 2 ). Possible promoter sites were identified just upstream of the genes wbtA and wbtM. Downstream of the second transposase are located the genes manC and manB, with a possible promoter just upstream of manC.
  • FIG. 2 also shows the G+C content plot of the cluster using a window size of 500 bases.
  • the overall G+C content of this region of the genome at 31.27% is slightly lower than the genome average of approximately 33%.
  • the plot shows that the central section of the cluster, from wzy to wbtK, generally has an even lower G+C content.
  • genes for the transcription termination factor rho and thioredoxin Downstream from manC, on the opposite strand, are located the genes for the transcription termination factor rho and thioredoxin. In E. coli both of these genes are also found flanking one end of a polysaccharide biosynthetic gene cluster—that of the enterobacterial common antigen.
  • the O-antigen repeat unit of F. tularensis is shown in FIG. 3 , together with the putative role of the genes involved in O-antigen biosynthesis. Based on their homology to other LPS and sugar biosynthetic genes, in particular P. aeruginosa serotype O6 which expresses a similar O-antigen repeat structure (Knirel Y A, et al. (1985) Eur. J. Biochem. 150: 541-550), the putative role of the gene products have been assigned.
  • WbtF shows homology to UDP-glucose 4-epimerases, including WbpP and VipB
  • WbtE shows homology to WbpO and VipA
  • WbtH produces significant alignments with glutamine amidotransferases, including WbpS of P. aeruginosa serotype O6, which may putatively be involved in the formation of the GalNAcAN amido group.
  • Biosynthesis of the fourth sugar, 4N-formyl-quinovosamine most likely involves WbtI, WbtJ, WbtL and WbtM. Sequence homology suggests that WbtL may be involved in the formation of the activated sugar dTDP-D-Glucose with WbtM functioning as a dTDP-D-Glucose 4,6-dehydratase.
  • WbtI is proposed to be involved in Qui4NFm amination since it shows homology to RfbE, a perosamine synthetase.
  • WbtJ is likely to be responsible for the addition of the N-formyl moiety, showing significant homology to formyltransferases.
  • glycosyltransferases are required to form the oligosaccharide units of the O-antigen repeat. Four glycosyltransferases would be necessary for the synthesis of each O-antigen unit in F. tularensis .
  • WbtB is proposed to mediate the addition of QuiNAc to undecaprenyl phosphate (Und-P) to initiate O-antigen biosynthesis.
  • WbtD and WbtG are probable GalNAcAN transferases, possibly involved in the addition of the two consecutive GalNAcAN residues onto the O-antigen unit. WbtD shares homology to WbpU of P.
  • aeruginosa strain O6 proposed to transfer 2-formamido-2-deoxy-D-galactouronamide (GalNFmAN) onto QuiNAc (Belanger M, et al. (1999). Microbiology 145: 3505-3521).
  • WbtG is homologous to WbpT of P. aeruginosa , thought to be involved in addition of GalNAcA to GalNFmAN.
  • WbtK is probably the fourth glycosyltransferase, which adds 4,6-dideoxy-4-formamido-D-glucose (QuiNA4Fm) to complete the tetrasaccharide O unit.
  • the O-antigen repeat units are translocated to the periplasmic face of the inner membrane via Wzx, a transporter/flippase. Wzy then acts as an O-antigen repeat unit polymerase.
  • the F. tularensis Wzx protein had a predicted 14 trans-membrane helices, with both termini on the cytoplasmic side of the membrane.
  • the F. tularensis Wzy protein had a predicted 11 trans-membrane helices, with the amino terminus predicted to be on the cytoplasmic side of the membrane, and the carboxy terminus on the periplasmic side. Additionally, the Wzy protein was predicted to have two large periplasmic domains from amino acids 142-178 and 268-327.
  • Primer pair 1 covering the start of the gene cluster, had to be designed to amplify a 4.8 Kb region due to lack of suitable priming sites upstream of the cluster because of the presence of an insertion element found many times in the F. tularensis Schu S4 genome.
  • This primer pair 1 produced the relevant size product for F. tularensis Schu S4, but when used on subspecies holarctica , strain LVS, did not produce a product.
  • this primer pair may have particular applications in diagnostics where distinction between F. tularensis subspecies tularensis and F. tularensis subspecies holarctica is required. Where samples containing DNA from the former is present, a PCR using primer pair 1 will generate a product, which would not be present in the second case.
  • the PCR using primer pair 8 revealed a difference in size between subspecies tularensis strains and subspecies holarctica and subspecies mediaasiatica .
  • Subspecies tularensis strains show a deletion of 303 nucleotides when compared to subspecies holarctica strains (including LVS) and subspecies mediaasiatica .
  • Cloning and sequence analysis of this region from the subspecies tularensis strain Schu S4 the subspecies holarctica strain HN63 and LVS has shown that the deletion in Schu S4 occurs at the beginning of a putative transposase that is similar to IS630-spn 1 transposase ORF 1 of Streptococcus pneumoniae.
  • primer pair 8 may also be particularly useful in distinguishing between strains of F. tularenis .
  • a separation of the products on the basis of size, for example on a gel, should reveal distinguishable differences therebetween.
  • LPS was purified from F. tularensis strain Schu S4 or from strain LVS using a hot-phenol and water extraction method mentioned above in Example 1.
  • F. tularensis strain LVS or strain Schu S4 LPS The ability of F. tularensis strain LVS or strain Schu S4 LPS to protect BALB/c mice from a F. tularensis was determined by immunizing groups of six female BALB/c mice by the i.p. route with the purified LPS obtained. On each dosing occasion, mice were given 50 ⁇ g of LPS in phosphate buffered saline (PBS). The mice received three immunizations, each 7 days apart.
  • PBS phosphate buffered saline
  • mice were challenged i.p. with F. tularensis LVS (1 ⁇ 10 5 CFU) 21 days after the last immunization. All control animals died after challenge. Mice which had been immunised LPS isolated from the LVS strain were protected from death. Mice which had been immunised with LPS from either the SchuS4 or LVS strain showed and extended time to death. At a challenge dose of 10 cfu animals immunised with SchS4 LPS survived for an average of 64 hours (with 99% confidence) longer than the unimmunised controls.

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Cited By (5)

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US20090087456A1 (en) * 2005-09-07 2009-04-02 James Edward Eyles Adjuvanted vaccine
US20100021501A1 (en) * 2006-07-25 2010-01-28 Michell Stephen Lloyd Live vaccine strains of francisella
WO2012051199A3 (fr) * 2010-10-12 2012-06-07 Monsanto Technology Llc Plante et semence de soja correspondant à l'événement transgénique mon87712 et procédé pour les détecter
US8198430B2 (en) 2002-05-31 2012-06-12 The Secretary Of State For Defence Immunogenic sequences
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Families Citing this family (48)

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Publication number Priority date Publication date Assignee Title
US20040121309A1 (en) 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in blood, bodily fluids, and bodily tissues
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
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US20030027135A1 (en) 2001-03-02 2003-02-06 Ecker David J. Method for rapid detection and identification of bioagents
US7226739B2 (en) 2001-03-02 2007-06-05 Isis Pharmaceuticals, Inc Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations
US7217510B2 (en) 2001-06-26 2007-05-15 Isis Pharmaceuticals, Inc. Methods for providing bacterial bioagent characterizing information
US7172868B2 (en) 2002-08-01 2007-02-06 The Regents Of The University Of California Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis
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US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
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US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
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US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
JP4810533B2 (ja) 2004-05-24 2011-11-09 アイビス バイオサイエンシズ インコーポレイティッド ディジタルスレショルド化による選択的イオン濾過作用を用いた質量分光測定法
US20050266411A1 (en) 2004-05-25 2005-12-01 Hofstadler Steven A Methods for rapid forensic analysis of mitochondrial DNA
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
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US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
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WO2011115840A2 (fr) 2010-03-14 2011-09-22 Ibis Biosciences, Inc. Recherche de parasites par le biais de la recherche d'endosymbiotes
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3148120A (en) * 1952-01-24 1964-09-08 Wander Ag Dr A Hot aqueous phenol extraction of gramnegative bacterial lipopolysaccharides
US5066596A (en) * 1984-02-01 1991-11-19 Enterovax Limited Bacterial strains harboring cloned genes controlling vibrio cholerae o-antigen biosynthesis
US6444804B1 (en) * 1997-05-02 2002-09-03 Joseph S. Lam Proteins involved in the synthesis and assembly of core lipopolysaccharide of Pseudomonas aeruginosa
US6444210B1 (en) * 1996-07-03 2002-09-03 Her Majesty the Queen in right of Canada, as represented by the Minister of National Defence of Her Majesty′s Canadian Goverment Bacterial and synthetic polysaccharide immunomodulators that enhance general immunity
US20070066801A1 (en) * 2003-07-14 2007-03-22 Engler Jeffrey A Identification of reagents for the diagnosis and study of francisella

Family Cites Families (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US644804A (en) 1899-06-01 1900-03-06 Peter Francis Mccaffrey Acetylene-gas lamp.
US4518310A (en) 1982-09-03 1985-05-21 Tech Development Inc. Apparatus for preventing overspeed of a turbine drive means
EP0250614A1 (fr) 1986-06-23 1988-01-07 Serum Und Impfinstitut Und Institut Zur Erforschung Der Infektionskrankheiten Schweiz. Fragments d'ADN provenant de nucléotides chromosomiques codant pour des synthétases glycidiques et des transférases glycosyliques
AU616662B2 (en) 1987-04-27 1991-11-07 Commonwealth Scientific And Industrial Research Organisation Peptide production by protein engineering
WO1992013871A1 (fr) 1991-01-31 1992-08-20 Washington University Polypeptides et polynucleotides utiles pour le diagnostic et le traitement de neisseria pathogene
AU3416693A (en) 1991-12-18 1993-07-19 State Of Oregon Acting By And Through The Oregon State Board Of Higher Education On Behalf Of Oregon State University, The Antigenic preparations that stimulate production of antibodies which bind to the pili of type IV piliated bacteria
DE4336530C1 (de) * 1993-10-26 1995-04-13 Max Planck Gesellschaft Rekombinante PilC-Proteine, Verfahren zu ihrer Herstellung und ihre Verwendung
US5951987A (en) * 1995-08-22 1999-09-14 Her Majesty The Queen As Represented By The Minister Of National Defence Of Her Majesty'canadian Government Polysaccharide vaccine to enhance immunity against brucellosis
CA2175435A1 (fr) * 1996-04-30 1997-10-31 Joseph Lam Proteines intervenant dans la synthese et la fixation de l'antigene o chez pseudomonas aeruginosa
GB2321103A (en) 1997-01-11 1998-07-15 Secr Defence Poxvirus antigen detection and recombinant expression vector
US6406705B1 (en) * 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
IL132327A0 (en) * 1997-04-11 2001-03-19 Univ Louisiana State Attenuated invasive vaccines against fish pathogens
US6261568B1 (en) * 1997-06-11 2001-07-17 Institut Pasteur Attenuated recombinant mycobacteria useful as immunogens or as vaccine components
US6444445B2 (en) * 1998-01-22 2002-09-03 The United States Of America As Represented By The Secretary Of The Army Live vaccine against Brucellosis
FR2782721B1 (fr) * 1998-09-01 2000-11-03 Inst Nat Sante Rech Med Nouveaux composes phosphohalohydrines, procede de fabrication et applications
JP2003511421A (ja) 1999-10-12 2003-03-25 ナショナル リサーチ カウンシル オブ カナダ 細胞毒性ｔリンパ球（ｃｔｌ）応答を誘発し、そして細胞内病原体及び癌に対してワクチン接種された宿主を保護するための無細胞ワクチンのための免疫調節キャリヤーとしてのアルキオソーム
US6552006B2 (en) * 2000-01-31 2003-04-22 The Regents Of The University Of California Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen
EP1309348B1 (fr) * 2000-02-11 2009-01-07 U.S. Army Medical Research Institute of Infectious Diseases Department of the Army Anticorps monoclonaux a vocation prophylactique et therapeutique contre vaccinia virus antigene
GB0021008D0 (en) 2000-08-26 2000-10-11 Secr Defence Protein and nucleic acid sequence
DK1379550T3 (da) 2000-12-21 2009-07-06 Us Gov Health & Human Serv Et kimært protein omfattende non-toxisk pseudomonas exotoxin A og en type IV pilin sekvenser
EP1408984B1 (fr) * 2001-07-20 2008-10-22 BioAgency AG Composes organophosphores pour l'activation de cellules t gamma/delta
FR2833266B1 (fr) * 2001-12-11 2004-10-22 Mayoly Spindler Lab Nouveaux derives phosphonates, leur procede de preparation, leur utilisation comme modulateurs de l'activite des lymphocytes tgamma9 delta2
WO2003068151A2 (fr) 2002-02-11 2003-08-21 Alexion Pharmaceuticals, Inc. Anticorps humains utilises comme agent therapeutique contre la vaccine ou la variole
GB0212666D0 (en) 2002-05-31 2002-07-10 Secr Defence Immunogenic sequences
WO2004004654A2 (fr) 2002-07-03 2004-01-15 Cambridge Scientific, Inc. Vaccins destines a induire l'immunite des muqueuses
RU2240822C2 (ru) 2002-07-17 2004-11-27 Научно-исследовательский институт микробиологии Министерства обороны Российской Федерации Способ получения противотуляремийной гипериммунной сыворотки и способ получения диагностикума эритроцитарного туляремийного иммуноглобулинового сухого
AU2003304107B2 (en) 2002-11-01 2008-05-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of preventing infections from bioterrorism agents with immunostimulatory CpG oligonucleotides
WO2005013918A2 (fr) 2003-02-28 2005-02-17 The Trustees Of The University Of Pennsylvania Compositions, methodes et kits de vaccins a base de sous-unite de poxvirus
GB0307089D0 (en) 2003-03-27 2003-04-30 Secr Defence Live vaccine strain
US20070025960A1 (en) 2003-05-16 2007-02-01 University Of Maryland Biotechnology Institute Bisphosphonates for prophylaxis and therapy against bioterrorism agents
BRPI0417088A (pt) 2003-12-02 2007-03-13 Innate Pharma ativador de célula t gama delta, composição farmacêutica, uso do ativador de célula t gama delta, composição de vacina, métodos para preparar um composto, e para ativar uma célula t gama delta, célula t gama delta, e, uso da mesma
PT1697403E (pt) 2003-12-23 2010-05-10 Nationwide Childrens Hospital Pili de tipo iv de haemophilus influenzae
US7592326B2 (en) * 2004-03-15 2009-09-22 Karaolis David K R Method for stimulating the immune, inflammatory or neuroprotective response
WO2005102385A1 (fr) * 2004-04-26 2005-11-03 Innate Pharma Composition adjuvante et methodes d'utilisation correspondantes
WO2006060710A2 (fr) * 2004-12-02 2006-06-08 Becton, Dickinson And Company Preparations de vaccins destinees a une administration intradermique, contenant des adjuvants et des agents antigeniques
WO2006067635A2 (fr) 2004-12-20 2006-06-29 Innate Pharma S.A. Utilisation d'activateurs de lymphocytes g? t comme adjuvants de vaccins
US7683045B2 (en) * 2005-03-22 2010-03-23 Innate Pharma S.A. Class of γδ T cells activators and use thereof
CA2502999A1 (fr) 2005-04-20 2006-10-20 National Research Council Of Canada Souche de tularensis f. mutante et utilisations
GB0511722D0 (en) 2005-06-08 2005-07-13 Secr Defence Live and subunit vaccines
US20090087456A1 (en) * 2005-09-07 2009-04-02 James Edward Eyles Adjuvanted vaccine
GB0519161D0 (en) * 2005-09-20 2005-10-26 Secr Defence Adjuvanted vaccine
AU2006338607A1 (en) 2006-02-23 2007-08-30 Oregon Health & Science University Attenuated Francisella and methods of use
WO2008012538A2 (fr) 2006-07-25 2008-01-31 The Secretary Of State For Defence Souche de vaccin vivant
GB2445028A (en) 2006-12-21 2008-06-25 Secr Defence Inactivated Francisella vaccine
GB2444903A (en) 2006-12-21 2008-06-25 Secr Defence Live Francisella vaccine, inactivated at gene FTT1564
US8012466B2 (en) * 2008-01-31 2011-09-06 University Of Iowa Research Foundation Immunogenic compositions for activating γδ T cells
EP2123285A1 (fr) 2008-05-21 2009-11-25 INSERM (Institut National de la Santé et de la Recherche Médicale) Phosphoantigènes nucléosidiques à utiliser pour la thérapie liée à la cellule T VGAMMA9VDELTA2
US7588744B1 (en) * 2008-12-08 2009-09-15 Layne Christensen Company Method of recovering phosphate for reuse as a fertilizer
GB0901411D0 (en) 2009-01-29 2009-03-11 Secr Defence Treatment
GB0906234D0 (en) 2009-04-14 2009-05-20 Secr Defence Vaccine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3148120A (en) * 1952-01-24 1964-09-08 Wander Ag Dr A Hot aqueous phenol extraction of gramnegative bacterial lipopolysaccharides
US5066596A (en) * 1984-02-01 1991-11-19 Enterovax Limited Bacterial strains harboring cloned genes controlling vibrio cholerae o-antigen biosynthesis
US6444210B1 (en) * 1996-07-03 2002-09-03 Her Majesty the Queen in right of Canada, as represented by the Minister of National Defence of Her Majesty′s Canadian Goverment Bacterial and synthetic polysaccharide immunomodulators that enhance general immunity
US6444804B1 (en) * 1997-05-02 2002-09-03 Joseph S. Lam Proteins involved in the synthesis and assembly of core lipopolysaccharide of Pseudomonas aeruginosa
US20070066801A1 (en) * 2003-07-14 2007-03-22 Engler Jeffrey A Identification of reagents for the diagnosis and study of francisella

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8198430B2 (en) 2002-05-31 2012-06-12 The Secretary Of State For Defence Immunogenic sequences
US20090087456A1 (en) * 2005-09-07 2009-04-02 James Edward Eyles Adjuvanted vaccine
US20100021501A1 (en) * 2006-07-25 2010-01-28 Michell Stephen Lloyd Live vaccine strains of francisella
US8323664B2 (en) 2006-07-25 2012-12-04 The Secretary Of State For Defence Live vaccine strains of Francisella
US8790910B2 (en) 2006-07-25 2014-07-29 The Secretary Of State For Defence Live vaccine strain
US8609108B2 (en) 2009-04-14 2013-12-17 The Secretary Of State For Defence Gamma-glutamyl transpeptidase attenuated Francisella
WO2012051199A3 (fr) * 2010-10-12 2012-06-07 Monsanto Technology Llc Plante et semence de soja correspondant à l'événement transgénique mon87712 et procédé pour les détecter
US9493786B2 (en) 2010-10-12 2016-11-15 Monsanto Technology Llc Soybean plant and seed corresponding to transgenic event MON87712 comprising a B-box zinc finger protein 32, and methods for detection thereof
US10053704B2 (en) 2010-10-12 2018-08-21 Monsanto Technology Llc Soybean plant and seed corresponding to transgenic event MON87712 and methods for detection thereof
US10604765B2 (en) 2010-10-12 2020-03-31 Monsanto Technology Llc Soybean plant and seed corresponding to transgenic even MON87712 and methods for detection thereof
US10696976B2 (en) 2010-10-12 2020-06-30 Monsanto Technology Llc Soybean plant and seed corresponding to transgenic event MON87712 and methods for detection thereof

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