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WO1997023600A1 - Vaccin conjugue contre les infections a bacteries gram negatif - Google Patents

Vaccin conjugue contre les infections a bacteries gram negatif Download PDF

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Publication number
WO1997023600A1
WO1997023600A1 PCT/US1996/019747 US9619747W WO9723600A1 WO 1997023600 A1 WO1997023600 A1 WO 1997023600A1 US 9619747 W US9619747 W US 9619747W WO 9723600 A1 WO9723600 A1 WO 9723600A1
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Prior art keywords
gram
pilin
pilo
dna
pili
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PCT/US1996/019747
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WO1997023600A9 (fr
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Peter Castric
Alan Cross
Jerald Sadoff
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United States Army Medical Research Materiel Command (Usamrmc)
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Priority to AU14619/97A priority Critical patent/AU1461997A/en
Publication of WO1997023600A1 publication Critical patent/WO1997023600A1/fr
Publication of WO1997023600A9 publication Critical patent/WO1997023600A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • Pseudomonas aeruginosa is recognized as a leading cause of life- threatening infection among compromised patient populations in hospitals. Cancer patients, and patients with burn wounds, cystic fibrosis, acute leukemia, organ transplants, and intravenous-drug addiction are particularly susceptible to acquiring a serious P. aeruginosa infection.
  • the most serious infections include pneumonia, septicemia, malignant-external otitis, and meningitis.
  • the mortality rate for such infections can exceed 50%, and is usually the highest for any bacterial pathogen (Cryz, S. J., Jr. In Pseudomonas aeruginosa as an Opporunistic Pathogen. Mario Campa et al., Eds. Plenum Press, New York, 1993, pp. 383-395. All documents cited herein infra and supra are hereby inco ⁇ orated by reference thereto).
  • aeruginosa is an opportunistic pathogen, therefore, a key component of this bacterium's pathogenicity is the ability of the microorganisms to adhere to epithelial cells of mucosal surfaces (E. H. Blackey, 1981, J. Infect. Dis. 143: 325-345).
  • the somatic pili of P. aeruginosa, protein filaments clustered around the flagellum and extending from the cell surface, have been implicated in adherence to host tissue (Ramphal et al., 1984, Infect. Immun. 44: 38-40; Woods et al., 1980, Infect. Immun. 29: 1 146-1 151). Pili are composed of monome ⁇ c subunits.
  • pilin which have a molecular weight of 15-18000 daltons (Frost and Paranchych, 1977, 7 Bacteriol. 131 - 259-269: Sastry et al , 1983, FEBS Lett 151:253-256; Sastry et al. .1985, J. Bacteriol 164 571-577) and are arranged in a helical fashion within the pilus fiber (Watts et al , 1983, Biochemistry 22: 3640-3646).
  • the pihns of P. aeruginosa are similar in structure and function to those produced by many Gram-negative bacteria such as species of the genera Dichelobacter (Elleman, T C.
  • pilin is encoded by a single chromosomal copy (Pasloske et al., 1985, FEBS Lett. 183: 408-412, Sastry et al., 1985, J Bacteriol 164: 571 -577), the pilA gene.
  • the nucleotide sequence of several P aeruginosa pilA genes is known and comparisons of deduced pilin primary structure and flanking DNA sequence have shown characte ⁇ stic variation which allows differentiation of P.
  • group I such as pih strain 1244
  • group II pihns PA 103, T2A, PAO, PAK pi ns
  • group I pilin determinants were relatively common (58/95 and include different strains of different immunotypes as determined by polyclonal antibody reaction) among clinical isolates.
  • P aeruginosa pih as a vaccine presents many advantages. Unlike previous hpopolysaccha ⁇ de-based Pseudomonas vaccines, pih produce minimal side effects and are well known to be lmmunogenic in humans, possibly due to antigen organization (Bachman, et al ,1993, Science 262 1448- 1451) brought about by subunit arrangement in the fiber.
  • Pi can be prepared easily in homogeneous form and methods described in this application allow large scale production of pi which are compatible with modern forms of vaccine delivery such as microencapsulation
  • the P aeruginosa pili can be engineered to act as "earners" for other protein epitopes. thereby conferring the advantages of pili to other antigens, DNA sequences coding for known protein epitopes (outer membrane proteins for example) could be used to replace known surface pilin epitopes by PCR methodologies.
  • the advantages of peptide technology can be applied to pilus vaccines Cocktails of peptide epitopes can be constructed to deal with antigenic vanation seen in serotype populations
  • the present invention relates to a pilus-based vaccine and is based on recent work showing that pih from group I strains of P aeruginosa, a clinically common group, are glycosylated and that pili glycosylation may be involved in specific or nonspecific adhesion to the host cell independent of pilin protein- mediated attachment (Cast ⁇ c, P. , 1995, Microbiology 141 1247- 1254)
  • Glycosylated pi are shown to produce high bronchial titers when delivered by the intranasal route Mice vaccinated with pure P aeruginosa strain 1244 pih in this manner are protected against respiratory challenge with P aeruginosa strain 1244 More specifically, the present invention relates to a new gene sequence, pilO, the product of which giycosylates the pi of P aeruginosa strains by adding the specific O-antigen components of the lipopolysaccharide (LPS) earned by that strain onto the pi resulting in antigenic glycosylated pili useful as a vaccine against infection with that strain. Nucleotide sequencing of a region downstream from the pilin structural gene (pilA) of P.
  • pilO the product of which giycosylates the pi of P aeruginosa strains by adding the specific O-antigen components of the lipopolysaccharide (LPS) earned by that strain onto the pi resulting in antigenic glyco
  • aeruginosa strain 1244 (a group I strain), revealed an open reading frame (ORF) potentially able to code for another protein
  • ORF open reading frame
  • This ORF, called pilO was flanked by a tRNA ,hr gene, which was followed by a transc ⁇ ptional termination sequence.
  • the tRNA lhr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P aeruginosa strains A 2200 base mRNA strand, which contained both the pilO and pilA transcripts, was prodcuced from this region, while a 650 base transcript containing only pilA was present in a 100-fold excess over the longer transcript.
  • pilO is specific for strain 1244 pilin.
  • strain 1244 will produce strain PA103 pili (a group II pilin) from a plasmid carrying the cloned gene, but will not glycosylate the PA 103 pilin protein.
  • pilO is not specific for the O-antigen attached to the strain 1244 pilin. pilO will glycosylate strain 1244 pili with the LPS O-antigen repeating units of other P. aeruginosa strains.
  • strain PAK another group II strain
  • strain 1244 pilin gene carrying a plasmid retaining both the pilO gene and strain 1244 pilin gene
  • strain 1244 pili carrying the chemically and serologically distinctive strain PAK O-antigen.
  • the potential for O- antigen range of glycosylation is extremely wide and can extend to virtually any Gram-negative bacterium, P. aeruginosa and other species.
  • pilin glycosylation allows the design of vaccines specific for different strains of P. aeruginosa, or of Gram-negative bacteria, by the addition to the pilin core of the specific glycan produced in the bacteria for which a vaccine is desired and using the resulting pili as a vaccine.
  • Other vaccines presently being studied are composed of the LPS associated with the O-antigen.
  • the use of an LPS based vaccine has several serious drawbacks as compared to the use of glycosylated pili as a vaccine. Glycosylated pili contain the immunogenic portion (O-antigen) but not the toxic part (lipid A) of the LPS molecule resulting in a safer, better tolerated vaccine.
  • the range of protection of a cocktail of glycosylated pili will be broader than a cocktail of LPS due to the common epitopes of the pilus protein. Protection will be both pilus based and O-antigen based. LPS purification is time consuming, expensive and difficult. Glycosylated pili can be produced in large amounts by a new method described in this application involving the use of broth cultures instead of the commonly used agar cultures. Purification of glycosylated pili is quickly accomplished, inexpensive, and requires only common laboratory procedures.
  • the design of a pilus vaccine and understanding the immunological relationship among native pili is dependent on the ability to determine which parts of the pilus surface make up the B-cell epitopes.
  • the positions of the protein B-cell epitopes of the native strain 1244 pih were determined using the Geysen tethered peptide pin assay as described in Cast ⁇ c and Deal, 1994, supra Four epitope regions were revealed representing the portions of the pilin p ⁇ mary structure which occupy the surface of the pilus fiber. These sequences would be important in peptide vaccine design Two of these sequences, region 3 (SEQ ID NO 3) and region 4 (SEQ ID NO' 4) may also represent glycosylation recognition sequences according to recent results from peptide mapping of PilA.
  • the pilO protein is capable of glycosylating any protein which contains the pilin glycosylation recognition sequence.
  • the sequence can be incorporated in several ways, for example, using PCR methods, the pilin glycosylation recognition sequence can be inserted into a gene of a known surface protein; this engineered gene can be inco ⁇ orated into the chromosome of the host by gene replacement, and pilO hyperexpressed from a broad host range plasmid such as pPAC46 which grows and functions in nearly all Gram- negative bactena.
  • It is yet another object of the present invention to provide a method for the diagnosis of Gram-negative bacterial infection comprising the steps of: (i) contacting a sample from an individual suspected of having the disease with antibodies which recognize glycosylated pilin or an attached glycan of Gram-negative bacteria; and
  • aeruginosa in mammalian tissue or serum It is another object of the present invention to provide a diagnostic kit comprising antibodies against glycosylated pi ns of Gram-negative bacteria and ancillary reagents suitable for use in detecting the presence of Gram-negative bacteria in mammalian tissue of serum It is a further object of the present invention to provide a diagnostic kit compnsmg p ⁇ mers specific for the amplification of pilO sequences and ancillary reagents suitable for use in detecting the presence P aeruginosa in mammalian tissue or serum.
  • Figure 1 shows sequence homology of the region downstream from the pilO gene, with the region downstream of the pilA gene of P. aeruginosa strain PA103 (Johnson et al, 1986, 7. Biol. Chem. 261: 15703- 15708). Colons indicate base homology. Regions containing significant dyad symmetry are indicated by opposing arrows. The tRNA gene region is highlighted.
  • Figure 2 shows a comparison of the pilin gene regions of P. aeruginosa strain 1244 and strain PA 103. Potential transcriptional stops are indicated by the letter "t”. Restriction endonuclease sites are H. Hmdlll; N, Nhel: P, Pstl; S, Sphl.
  • Figure 3 is a Northern blot analysis of mRNA extracted from P. aeruginosa strain 1244.
  • A. Probes were prepared from restriction fragments of cloned DNA indicated in the Fig. 2 map as follows: Lane 1, Probe 1 (the Sphl-Nhel fragment); Lane 2, Probe 2 (the Nhel-Pstl fragment); Lane 3, Probe 3 (the Pstl-Hindlll fragment). Arrows indicate positions of E. coli strain UB 101 ribosomal RNA.
  • B Densitometric scan of lane 1 of panel A of this figure. Scanning was from bottom to top of the autoradiogram shown. Electrophoresis point of origin is marked by the arrow.
  • Figure 4 shows immunodetection of electrophoretically separated P. aeruginosa pilin.
  • A SDS-PAGE of pilin at 5°C. Lane 1 , 3 ⁇ g pilin produced by P. aeruginosa strain 1244N3(pPAC46), Lane 2, 3 ⁇ g pilin produced by P. aeruginosa strain I244N3(pPAC24). Molecular weights are given as times
  • P. aeruginosa strain 1244N3(pPAC24) Lane 2, 3 ⁇ g pilin produced by P. aeruginosa strain 1244N3(pPAC46).
  • the p ⁇ gradient was determined by focusing pl standards on an identical gel in the absence of octyl glucoside.
  • Figure 5 shows the detection of P. aeruginosa pilin carbohydrate by immunoblot. Lane 1 , 10 ⁇ g pilin produced by P. aeruginosa strain 1244N3(pPAC46), Lane 2, 10 ⁇ g pilin produced by P. aeruginosa strain 1244N3(pPAC24). Molecular weights are given as times 10 " ⁇ Arrows indicate positions of these pihns on an identical blot using monoclonal antibody 6-45 for detection.
  • Figure 8 Major epitopes of P aeruginosa 1244 pilus as determined by the Geysen pin test.
  • the present invention relates to a DNA or cDNA segment which encodes pilO, a glycosylation sequence of pilA pilin core protein.
  • the sequence of the gene, specified in SEQ ID NO 1 was obtained by sequencing a region downstream from the P aeruginosa 1244 pilA gene
  • the sequenced gene fragment comp ⁇ sing 1386 nucleotides of open reading frame with a 60.1 % G+C content consistent with the general range given for P aeruginosa chromosomal DNA.
  • DNA or polynucleotide sequences to which the invention also relates include sequences of at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, most preferably at least about 15-20 nucleotides conesponding, l e., homologous to or complementary to, a region of the pilO nucleotide sequence
  • the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a sequence which is unique to the pilO gene Whether or not a sequence is unique to the pilO gene can be determined by techniques known to those of skill in the art.
  • the sequence can be compared to sequences in databanks, e.g., GenBank.
  • Regions from which typical DNA sequences may be denved include but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and/or non-translated regions.
  • the denved polynucleotide is not necessanly physically denved from the nucleotide sequence shown in SEQ ID NO:l, but may be generated in any manner, including for example, chemical synthesis or DNA replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the reg ⁇ on(s) from which the polynucleotide is derived.
  • sequences of the present invention can be used in diagnostic assays such as hybridization assays and polymerase chain reaction assays and for the discovery of other pilO sequences.
  • the present invention relates to a recombinant
  • DNA molecule that includes a vector and a DNA sequence as described above.
  • the vector can take the form of a plasmid such as any broad host range expression vector for example pMMB66EH and others known in the art.
  • the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs.
  • the host cell can be any Gram-negative bacteria for which glycosylated pilin is desired.
  • the vector containing the pilO gene is expressed in the bacteria and the product of the pilO gene is able to add subunits of the specific O-antigen of the host bacteria to the core pilin producing glycosylated pilins which can be isolated for use as a vaccine and in diagnostic assays.
  • the plasmid pPAC46 described below in Materials and Methods, containing both the pilA and pilO wherein the pilA gene of strain 1244 was placed downstream from the tac promoter (in such a way as to inactivate the pilA promoter) of the broad host range expression vector pMMB66EH (Furste, et al., 1986, Gene 48: 1 19-131) results in high levels of glycosylated pilus production by hyper-expression of the pilA gene.
  • Promoters other than the tac promoter include ⁇ P L promoter, T7 promoter, and MalE promoter. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning: A Laboratory Manual ( 1982) or DNA Cloning.
  • the DNA sequence can be present in the vector operably linked to a highly purified IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of glycosylated pilin.
  • the transformed or transfected host cells can be used as a source of DNA sequences described above.
  • the recombinant molecule takes the form of an expression system
  • the transformed or transfected cells can be used as a source of the protein described below.
  • the present invention relates to a pilO protein having an amino acid sequence corresponding to SEQ ID NO: 2 and encompassing 461 amino acids or any allelic variation thereof.
  • a polypeptide or amino acid sequence derived from the amino acid sequence in SEQ ID NO.2 refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 ammo acids, and more preferably at least 8-10 amino acids, and even more preferably at least 1 1-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.
  • a recombinant or denved polypeptide is not necessarily translated from a designated nucleic acid sequence, or the sequence in SEQ ID NO 1. it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system
  • the polypeptide can be fused to other proteins or polypeptides for example, MalE protein for transport into the pe ⁇ plasm or for secretion from the cell
  • the present invention relates to a method of producing glycosylated pilin which includes cultunng the above-described host cells, under conditions such that the DNA fragment is expressed and a glycan specific for such host cells is added to the core pilin protein producing glycosylated pilin.
  • the glycosylated pih can then be isolated using methodology well known in the art or by the new large scale production method described below
  • the glycosylated pih can be used as a vaccine for immunity against infection with the host bactena or as a diagnostic tool for detection of host bactenal infection.
  • the transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit adhesion of host bacteria to cells, such as host proteins or chemically derived agents or other proteins which may interact with the bactena to down-regulate or alter the expression of pilO or affect the ability of pi to adhere to cells.
  • a method for testing the effectiveness of a drug or agent capable of inhibiting the adhesion of the Gram-negative bactena being studies can be for example the adhesion assay of Deal and K ⁇ van, Met. Enzymol. 236: 346-353
  • the present invention relates to antibodies specific for the above-desc ⁇ bed glycosylated pih.
  • an antibody can be raised against the complete glycosylated pi or against a portion thereof
  • Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the pih (or polypeptide) of the present invention, or the specific O-glycan attached to the pilin, or a unique portion of the pilin.
  • Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986).
  • the protein or polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, thereby producing higher titers of neutralizing antibody when used as a vaccine.
  • proteins or polypeptides examples include any adjuvants or carriers safe for human use, such as aluminum hydroxide.
  • the present invention relates to a method of detecting the presence of Gram-negative bacterial infection or antibodies against Gram-negative bacteria in a sample.
  • a diagnostic assay can be constructed by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocellulose membrane), all or a unique portion of the glycosylated pili described above, for example the O-antigen, and contacting it with the serum of a person suspected of having a Gram-negative bacterial infection.
  • the presence of a resulting complex formed between glycosylated pili and antibodies specific therefor in the serum can be detected by any of the known methods common in the art, such as fluorescent antibody spectroscopy or coiorimetry. This method of detection can be used, for example, for the diagnosis and typing of Gram- negative bacterial infections.
  • the adhesion properties of the Gram-negative bacteria are critical to its virulence and central to the development of pathology. Consequently, the ability of an individual to reduce bacterial adhesion would determine the patient's ability to prevent infection or disease.
  • This may take the form of antibodies which inhibit the adhesion of pili to cells; this can be measured in an assay for the detection of glycosylated pili as described below. Such assays can be used to screen individuals after receiving a Gram-negative vaccine to measure the production of protective antibodies.
  • Another mechanism to reduce adhesion of pili to cells is by down-regulating or altering the adhesion receptors present on the ceils.
  • Glycosylated pili can be used to measure the availability of cell receptors by contacting labeled glycosylated pili to target tissue either ex vivo or in vivo and measuring the degree to which labeled pili bind to target tissue.
  • Pili can be labeled by any detectable label known in the art such as a radionuclide, for example.
  • pili, glycosylated or unglycosylated can be used as receptor analogs to block the adhesion receptors present on host cells, thereby reducing or inhibiting the adhesion of bacteria to host cells. These pi can be administered in a mouthwash for example.
  • the present invention relates to a method of detecting the presence of Gram-negative bactena pili in a sample.
  • a diagnostic assay can be constructed by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocellulose membrane), antibodies specific for Gram-negative pi , and contacting it with serum or tissue sample of a person suspected of having Gram-negative bactenal infection.
  • a surface i.e. a solid support
  • a microtitration plate or a membrane e.g. nitrocellulose membrane
  • the presence of a resulting complex formed between pili in the serum and antibodies specific therefor can be detected by any of the known methods common in the art, such as fluorescent antibody spectroscopy or colo ⁇ metry.
  • the present invention relates to a diagnostic kit which contains glycosylated pili from a specific strain or species Gram-negative bacteria or several different strains and species of Gram-negative bactena and ancillary reagents that are well known in the art and that are suitable for use in detecting the presence of antibodies to Gram-negative bacteria in serum or a tissue sample.
  • Tissue samples contemplated can be monkey and human, or other mammals.
  • the present invention relates to DNA or nucleotide sequences for use in detecting the presence or absence of P. aeruginosa using the polymerase chain reaction (PCR).
  • the DNA sequence of the present invention can be used to design primers which specifically bind to the pilO DNA for the pu ⁇ ose of detecting the presence, absence, or quantitating the amount of P. earuginosa.
  • the p ⁇ mers can be any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18-25 nucleotides. Reagents and controls necessary for PCR reactions are well known in the art.
  • the amplified products can then be analyzed for the presence or absence of pilO sequences, for example by gel f ractionation, with or without hy ⁇ dization, by radiochemistry, and immunochemical techniques. This method can also be used for typing a Gram-negative bacterial infection.
  • the present invention relates to a diagnostic kit which contains PCR p ⁇ mers specific for pilO, and ancillary reagents that are well known in the art and that are suitable for use in detecting the presence or absence of P. aeruginosa in a sample using PCR.
  • Samples contemplated can be human or other mammals.
  • the present invention can be used to diagnose P. aeruginosa infection by using the DNA sequences for detecting the presence or absence of pilO in genomic DNA using hybridization assays such as Southern hybridization or the expression of pilO gene by northern hybridizations and other hybridization assays well known to a person with ordinary skill in the art.
  • the present invention relates to a vaccine for protection against Gram-negative bacterial infections.
  • the vaccine comprises glycosylated pili, or a portion thereof, from a specific strain or species of Gram- negative bacteria or preferably, a pool of glycosylated pili from different strains or species of Gram-negative bacteria to be used as a multivalent vaccine.
  • the vaccine can be prepared by inducing expression of a recombinant expression vector comprising pilA and pilO in a Gram-negative host(s) and purifying the glycosylated pili described above.
  • the purified solution is prepared for administration to mammals by methods known in the art, which can include filtering to sterilize the solution, diluting the solution, adding an adjuvant and stabilizing the solution.
  • the vaccine can be lyophilized to produce a vaccine against Gram-negative bacteria in a dried form for ease in transportation and storage.
  • the vaccine may be prepared in the form of a mixed vaccine which contains the glycosylated pili described above and at least one other antigen as long as the added antigen does not interfere with the effectiveness of the vaccine and the side effects and adverse reactions are not increased additively or synergistically.
  • the vaccine may be stored in a sealed vial, ampule or the like.
  • the present vaccine can generally be administered in the form of a liquid or suspension.
  • the vaccine is dissolved or suspended in sterilized distilled water before administration.
  • the vaccine may be administered orally, subcutaneously, intradermally or intramuscularly but preferably intranasally in a dose effective for the production of neutralizing antibody and protection from infection or disease.
  • the present invention relates to a method of reducing Gram-negative bacterial infection symptoms in a patient by administering to said patient an effective amount of anti-pili or anti-glycosylated pili antibodies, or pili (glycosylated or unglycosylated) as described above, or other agents capable of blocking pili function. Since glycosylated pili are involved in adhesion of bacteria to host cells, inhibiting or reversing the adhesion of bacteria may reduce or eliminate the development of Gram-negative bacterial infections.
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of the above compounds which is in the range of from about lpg kg to 10 mg kg (body weight of patient), although a lower or higher dosage may be administered.
  • the present invention provides a method for large scale production of pili. Pure, undenatured pili in milligram quantities are required for vaccine use. Current methods of pili purification present difficulties.
  • the present invention provides immunogenic epitopes from the pilA structural pilin gene representing possible glycosylation recognition sequences. These epitopes were mapped by standard peptide mapping techniques These epitopes include the amino acid sequences designated SEQ ID NO.3 and SEQ ID NO.4. These peptides can be generated HI any manner, including for example, chemical synthesis, or expression of a recombinant expression system.
  • the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants for example, thereby producing higher titers of anti-pi n antibodies for protection against P. aeruginosa infection
  • these peptides may be used with or without glycosylation as a vaccine or for the production of antibodies which can then be used to generate passive immunity against P aeruginosa infection.
  • strain 1244 peptide sequence conferring glycosylation can be inco ⁇ orated into other P. aeruginosa cell surface proteins of potential vaccine value using standard recombinant DNA methodologies.
  • cell surface proteins include flagella and outer membrane proteins such as F and P
  • This recognition sequence could be likewise inco ⁇ orated into surface proteins of vaccine potential from other bacteria such as Bordetella pertussis, Francisella species, Neissena meningiditis, Neisseria gonorrhoeae, Vibrio cholerae, Brucella species, certain strains of Escherichia coli, and Yersima species. Expression of pilO from a plasmid in such an organism would result in the addition of the organisms specific O-antigen to this protein
  • P aeruginosa 1244 Bacterial strains and culture conditions.
  • P aeruginosa 1244 a clinical isolate which has been used in pilus-mediated adhesion studies (Ramphal et al , 1984, Infect Immun. 44 38-40), was provided by A T. McManus, U.S. Army Institute of Surgical Research, San Antonio, Texas.
  • P aeruginosa strain 1244N3 a mutant which is unable to make pih or produce pilin (Ramphal et. al., 1991 , Infect. Immun. 59: 1307- 131 1 ) due to an inactivated rpoN gene, was furnished by S. Lory, University of Washington. Cultures were grown on LB medium (Sambrook et al., 1989, Molecular Cloning: a Laboratory Manual, vol. I , 2nd edn. Cold Spring Harbor, N.Y.-
  • Antibiotic concentrations in selective media used for P. aeruginosa were as follows: carbenicillin (Cb), 500 ⁇ g ml " '; tetracycline (Tc), 50 ⁇ g ml " '
  • Antibiotics used with E. coli were: ampicillin (Ap), 75 ⁇ g ml "1 ; spectmomycin (Sp), 25 ⁇ g ml "1 ; tetracycline (Tc), 10 ⁇ g ml "1 Nucleotide sequencing. The Sanger dideoxy method (Sanger et al., 1977, Proc Natl. Acad. Sci.
  • the template was single-stranded M 13 subclones of phage Lambda or plasmid clones (Cast ⁇ c et al., 1989, Mol. & Gen. Genet. 216: 75-80) containing DNA downstream from the pilA gene. Synthetic primers, derived from known sequences, or universal primers were employed.
  • Plasmid constructs The plasmid pPAC46, which contained both pilA and pilO, was constructed by Iigating the Sphl-HindUl plasmid fragment containing the pilA and adjacent DNA (Fig. 2) of pPAC202 (Cast ⁇ c et ⁇ l., 1989, supra) to plasmid pUC 18 digested with these same enzymes
  • the Ec ⁇ RI-H/rtdi ⁇ fragment of this construct, pPAC124 was ligated with the broad host range expression vector pMMB66E ⁇ ( Ap r , l ⁇ c ; Furste et ⁇ l , 1986, supra) which had been digested with the same restriction enzymes
  • the Ec ⁇ RI-Hmdlll fragment of this construct which contained only the pilA gene, was ligated with pMMB66 ⁇ digested with the same restnction enzymes.
  • the pilA gene of both pPAC24 and pPAC46 was under control of the vector t ⁇ c promoter. Since the Sphl site used in plasmid construction is located within the pilA promoter neither pPAC24 nor pPAC46 were able to express the pilin structural gene using the P. aeruginosa promoter. Plasmid DNA was introduced into E.
  • Probe DNA isolated from pPAC24 or pPAC46. was labelled by nick translation using [ ⁇ - 12 P] dCTP. Prehybridization, hybridization, and washing steps were as described by Sambrook et al., 1989. Detection of hybridization was by autoradiography. Densitometric scanning was carried out using an LKB Ultroscan laser densitometer.
  • the pili remaining in the supernatant fluid were purified by a variation of the method of Silipigni-Fusco ( 1987, Studies in the role of somatic pili as virulence and immunity factors in the pathogenicity of Pseudomonas aeruginosa. Ph.D. thesis, University of Pittsburgh, USA). In this procedure the supernatant fluid was made 0.1 M with respect to MgCl,, and stored on ice for 60 min. The resulting precipitate, which contained the pilus fibres, was removed by centrifugation at 12,000 g for 15 min.
  • Pilin analysis N-terminal amino acid sequence analysis and amino acid analysis was performed on purified pilin from strain 1244N3(pPAC24). This protein was subjected to PAGE (15%T) which was followed by electroblotting to polyvinylidene difluoride paper. Preliminary staining showed that pilin was the major protein present and that it was well separated from the minor contaminants present. Pilin bands were excised and subjected to gas- phase sequencing and to amino acid analysis (University of Pittsburgh Peptide Facility).
  • Pilin immunoblot analysis Pilins were separated by PAGE ( 12.5%T in the presence of either 0.1 % SDS at 5°C or 30.0 mM octyl glucoside at 15°C) or subjected to isoelectric focusing (pH 3.0 to 9.5) in the presence of 30.0 mM octyl glucoside at 15 C C using the LKB-Pharmacia Phastsystem. Proteins were transferred to nitrocellulose paper by diffusion blotting, pilin adsorbed to the paper was detected using anti-pihn monoclonal antibody 6-45 (Saiman et al., 1989, Immun. 57 2764-2770) as previously described (Cast ⁇ c et al , 1989, supra) The presence of pi n-bound sugar residues was detected by first denvatizing purified pih using the DIG Glycan Detection kit of
  • Boeh ⁇ nger Mannheim Biochemica Indianapolis. This protein was subjected to SDS-PAGE (12.5%T) using the BioRad MiniProtean II system in which temperature was neither controlled nor monitored. Pilin was electroblotted to nitrocellulose paper, probed with an alkaline phosphatase- labelled antibody specific for the hapten used in pi n de ⁇ vatization, and developed as described by Boeh ⁇ nger Mannheim Biochemica (Indianapolis)
  • Electron microscopy Cells to be analyzed by electron microscopy were grown on LB agar plates at 37°C for approximately eighteen hours and suspended in phosphate buffered saline. The cells were coated on 200 mesh formvar-coated copper grids, subjected to staining with 1 % uranyl acetate, and viewed in a Philips 201 electron microscope.
  • the phage sensitivity test was performed by streaking a loop of pilus- specific phage PE69 on an LB agar plate This was cross-streaked with diluted P aeruginosa 1244 strains to be tested Phage sensitivity was inte ⁇ reted from me absence of growth at the cross-streak junction Twitching (Hen ⁇ chsen, 1983, Annu Rev Microbiol 37 81 -93) was determined by streaking strains of interest on a well dried LB agar plate where motility was scored as spreading growth at between 48 and 72 hours at 37°C E MP E 1
  • the nucleotide sequence downstream from the P aeruginosa strain 1244 pilA gene contains a potential transc ⁇ ptional termination structure situated between positions 626 to 708 (noted previously in Cast ⁇ c et al , 1989, supra)
  • a ribosome-bmding site (GGAG) is seen at position 717 followed closely by three start codons, the first of which is located immediately in frame with a stop codon (TGA).
  • the second and third, GTG and ATG are in frame with each other and begin an open reading frame (referred to hereafter as pilO) which extends 1383 base pairs, using the ATG codon, to position 21 14 Codon usage of the 461 codons of the pilO gene is consistent with that of most other P aeruginosa genes (West & Iglewski, 1988, Nucleic Acids Res. 16: 9323-9335). However, significant differences are seen when it is compared with the adjacent pilA gene or with the pilA genes of other P. aeruginosa strains (West & Iglewski, 1988, supra; Castric & Deal, 1994, supra).
  • the arg codons, CGT and GCG appear at rates of 26/31 and 2/31 in the pilA gene, but occur at rates of 7/31 and 1 1/31, respectively, in the pilO gene.
  • the G+C% content of pilO (60.1%) is consistent with the general range given for P. aeruginosa chromosomal DNA (West & Iglewski, 1988), but quite different from the value of 52.2% determined for the strain 1244 pilA gene.
  • a search of the Genbank database revealed no sequences with significant homology to the pi 10 gene.
  • a tRNA" gene which contains an anticodon recognizing the rare codon ACG, can be seen just downstream of the pilO gene between positions 2166 and 2241, and is followed, between positions 2245 and 2282, by a potentially bidirectional transcriptional stop containing a 17 bp loop stem.
  • a tRNA gene and transcriptional stop region have been noted previously (Hobbs et al., 1988, Gene 62: 219-227) as occurring immediately downstream from the P. aeruginosa strain PAK pilA gene, and may also be seen in the analogous region of the pilA genes of P. aeruginosa strains PAO and PA 103 (Johnson et al., 1986, supra).
  • a homology comparison Fig.
  • RNA 2 RNA 1
  • RNA 1 RNA 2200 base
  • the smaller transcript was present in approximately a 100-fold excess over the larger mRNA as determined by densitometric scanning.
  • the transcription startpoint may be presumed to begin between positions 150 and 160, suggesting that the larger transcript extends into the pilO region.
  • the intermediate-sized RNA fragments might be formed by early termination, due to the loop structure refened to earlier, or might be degradation products of the larger species.
  • Probe 2 which was composed of pilO DNA, hybridized only with the larger fragment and the intermediate pieces suggesting that the origin of the 2200 base mRNA is in the pilA region and that synthesis of this molecule extends downstream through the pilO gene. This was confirmed by the reaction of probe 3 which was composed of DNA from the latter part of the pilO gene and the tRNA-transcriptional stop region. This probe hybridized with the larger fragment but with neither the pilA transcript nor the intermediate fragments. This probe also hybridized with a very small RNA population, presumably tRNA produced by the p ⁇ /0-associated gene as well as with cross-reacting species. Overall these results indicate that pilA, pilO and probably the adjacent tRNA gene are part of a single transcriptional unit which utilizes the pilA promoter.
  • Fig. 4 shows that pilin from strain 1244N3(pPAC46) reacted with this antibody indicating the presence of sugar moieties, while pilin from strain 1244N3(pPAC24) gave no reaction.
  • Pili were dissociated into pilin monomers and dimers in the presence of 30.0 mM octyl glucoside (Watts et al, 1982a, Can. J. Biochem. 60: 867-872; Watts et al, 1982b, J. Bacteriol. 151: 1508- 1513; Watts et l, 1983, Biochemistry 22: 687-691), subjected to isoelectric focusing, then blotted to nitrocellulose paper, and probed with pilin-specific monoclonal antibodies.
  • the pl of mature strain 1244 pilin is predicted from the pilA gene sequence (assuming that the amino-terminus is blocked and the two cysteines form a disulfide) to be 7.00.
  • Carbenicillin 200 ⁇ g/ml], Tetracyline [50 ⁇ g/ml], and isopropyl thiogalactoside [IPTG, 5 mM] was inoculated with 25 ml of an overnight culture of Pseudomonas aeruginosa 1244N3(pPAC46) grown with the same medium (minus the IPTG). This host strain is RpoN " .
  • the inoculated medium was incubated at 37C with rotary agitation 250 ⁇ m for 7 hours.
  • the overnight starter culture was grown under the same conditions in a 125 ml Erlenmyer flask.
  • This material was suspended with 50 ml 10.0 mM Tris/HCl, pH 7.6 containing 20% sucrose and centrifuged (4,200 xg for 15 min). The precipitate was discarded and the supernatant fluid was reprecipitated with PEG and MgCl 2 as described above, and stored ovemight at 4C. The light grey-tan precipitate (containing the pili) was removed by centrifugation (13,200 x g for 30 min) and suspended with 25 ml 10.0 mM Tris/HCI, pH 7.6 containing 20% sucrose.
  • ICR mice 25-30 g were immunized with P. aeruginosa strain 1244 pili at a concentration of 5 mg/mouse/dose diluted in physiologic saline.
  • the following immunization schedules were used to determine antibody response: i.n./i.n., s.c./s.c, i.p./i.p., i.n./s.c, s.c./i.n., i.n./i.p., i.p./i.n..
  • the time interval between the doses was 7 days.
  • mice were anesthetized i.p.
  • mice were sacrificed by CO 2 inhalation to obtain serum samples via cardiac puncture as well as bronchoalveolar lavage (BAL) samples for antibody titers.
  • BAL samples lungs were washed once with 1 ml of sterile physiologic saline via a 25 G hypodermic needle inserted into the trachea.
  • Antibody titers were determined by means of an ELISA using 96-well plates coated with P. aeruginosa 1244 pili at a concentration of 2 mg/ml (50 ml/well, 4 ° C, over night [O/N]). After blocking and washing, samples were serially diluted (2-fold) in duplicate in blocking buffer and incubated O/N at 4 ° C.
  • Bound antibodies were detected using goat-anti-mouse-IgG/alkaline phosphatase labelled, goat-anti-mouse-IgM alkaline phosphatase labelled, and goat-anti-mouse-IgA/alkaline phosphatase labelled (Incubation O/N, 4 C); plates were developed with p-Nitrophenylphosphate in diethanolamine buffer and read in a Biotek ® Ceres 900 C ELISA reader. ELISA titers are defined as log l0 of the dilution which gave a change of A ⁇ of 0.200 after 30 min.
  • P. aeruginosa 1244 was grown to mid-log phase in trypticase soy broth for 4 h at 37 ° C. After washing the bacteria with physiologic saline, the OD 6j0 was adjusted to 0.480 which corresponded to an estimated CFU count of 2 x lOVml.
  • Mice were challenged i.n. using 2 different doses (LD 100 , LD 5 ) at day +7 after the second dose of pili. Anesthesia was done as described above, and bacteria were delivered in a final volume of 50 ml. Mortality and body weight were monitored daily for 14 days. Control mice received physiologic saline only. Time to recovery in sublethal challenge experiments was defined as number of days until the baseline body weight was regained or (if animals did not reach their baseline body weight) as number of days until stabilization of body weight (3 consecutive days with weight change
  • the antigenicity of the pilin glycan was studied.
  • the isolated glycan competes with pure pili (Fig 7) for reaction with monoclonal 1 1.14 (an antibody which reacts with both strain 1244 LPS and pili).
  • monoclonal 1 1.14 an antibody which reacts with both strain 1244 LPS and pili.
  • These results show that pilin glycans are relatively small (probably mono and di-saccharides). Small size indicates that (since glycosylation appears to change the pilin molecular weight by a value of 1200) several copies of these glycans exist on the pilus surface.
  • a certain amount of heterogeneity among the glycans is likely and the pilin glycan contains an epitope which is also found on the strain 1244
  • the plasmids described above (which contain either the pilA gene alone (pPAC24) or both the pilA and pilO genes (pPAC46) can easily be moved (by conjugal mating) into various P. aeruginosa strains where these pilin genes can be expressed through IPTG stimulation of the tac promoter.
  • the 1244 pilin produced had the apparent molecular weight predicted by the gene structure.
  • the 1244 pilin produced appeared to be modified as indicated by apparent molecular weight. If these modified pilins contain glycan composed of host cell LPS O-antigen building blocks it is potentially possible to detect this material using O-antigen type- specific antisera. Experiments exploring this possibility are as follows.
  • Pihn produced from plate cultures (grown in the presence of IPTG) was subjected to isoelectric focusing, followed by diffusion blotting to PVDF paper. These blots were then probed with monoclonal antibodies giving the results seen in Table 8 (numbers m parentheses are approximate pi's)
  • Vaccine design requires knowledge of epitope structure. While B- and T-cell epitopes of P. aeruginosa PAO and PAK pili have been determined in general terms (Watt, et al., 1983, Infect. Immun. 42: 1 13- 121 ; Sastry et al, 1985, Can. J. Biochem. Cell. Biol. 63: 284-291), nothing is known of the common pilus type produced by strain 1244. Analysis of this pilus was carried out using the Geysen test (Carter, 1994, In: Methods in Molecular Biology.
  • Regions 1 and 4 were determined to be immunodominant based on frequency and intensity of reaction. Since the anti-pilus sera were raised against native pili these epitope regions represent portions of the pilin primary structure which occupy the pilus fiber surface, and so are key targets in peptide vaccine design.
  • a peptide In order for a peptide to provide a protective response it must occupy a part of the pilus surface which is involved in the two activities of the pilus critical to pathogenesis: a)adhesion tothe host cell, and b) retraction of the pilus fiber (which insures locking on of the pathogen to the host cell surface). Since both activities are required for colonization and, interference of either by the immune response would be protective. Since region 1 (SEQ ID NO: 5) is a potential protective epitope, it is important to note that it contains a sequence (LKT— E) common to most P. aeruginosa pilins (Castric and Deal, 1994, supra).
  • Anti- 1244 pilus monoclonal 5.44 also maps to this region (Castric and Deal, 1994, supra). This monoclonal reacts (Western blot) with pilins from strains which only contain this sequence similarity indicating that these residues form the core of this epitope region.
  • the carboxy-proximal portion, roughly analogous to region 4 of 1244 pilin, of the strain PAO and PAK pilins house the host receptor binding site (Lee et al. , 1989, Molec. Microbiol. 3: 1493- 1499; Doig et al , 1989, Can. J. Microbiol 35: 141- 1 145; Doig et al. , 1990, Infect. Immun.
  • Anti- 1244 pilus monoclonal 6.45 maps to region 4 of 1244 pilin [Castric and Deal, 1994, supra] and blocks adhesion to bovine tracheal cells (Saiman et al. , 1990, J. Infect. Dis. 161:541-548) indicating that region 4 contains the host binding site and so is a prime vaccine target.
  • results presented in this paper suggest that pilin from P. aeruginosa 1244 also is glycosylated.
  • the composition of the pilin-associated material remains to be determined, however several candidates, including the acidic moieties of the lipopolysaccharide core, alginic acid subunits, as well as other acidic elements or phosphorylated compounds, must be considered.
  • Limitations of SDS-PAGE prevent reliable molecular weight determinations of glycosylated proteins, a test which must await the use of a more accurate technique such as mass spectrometry.
  • Linkage of glycosylated proteins is usually via the reducing end of an oligosaccharide sugar through an O- or N-linkage to the hydroxyl group of serine (or threonine) or the amide group of asparagine (Montreuil et al, 1986, In Carbohydrate Analysis, a Practical Approach, pp. 143-204. Edited by M. F. Chaplin & J. F. Kennedy. Oxford: IRL Press).
  • the residues in the region of N-linked moieties have the characteristic consenus sequence N-X-S (or T). While this sequence may be found in the N. meningitidis pilin primary structure (Virji et al, 1993), it is absent in P. aeruginosa strain 1244 pilin, suggesting the presence of an N- linkage, a different sequence specificity, or the utilization of an alternative method of attachment.
  • N-X-S or T
  • pili from P. aeruginosa strains PAO and PAK contained no sugar residues. Since the primary structure of pili from strain 1244 is distinctive when compared to those of strains PAO and PAK (Castric & Deal, 1994), pilus modification may represent a strain difference. P. aeruginosa strains producing pili antigenically related to those of strain 1244 are common among clinical isolates (Castric & Deal, 1994). Thus, pilin glycosylation by this bacterium could be useful in clinical identification. Likewise, demonstration of the presence of the pilO gene could be of diagnostic value.
  • pilO gene is part of the pilA transcriptional unit.
  • the pilA and pilO genes are present in the form of an operon, two major transcription products are generated (an individual pilA message and a pilAlpilOltRNA 0 " transcnpt) which are present in a ratio of about one hundred to one.
  • pi n is a major cell protein, while PilO would likely be required in only catalytic or regulatory amounts.
  • the difference in transcription levels could be the result of premature termination of message synthesis or relative instability of the transcript corresponding to the pilO region.
  • the role of translation efficiency should also be considered a factor in differential gene expression since the beginning of the pilO message contains a short loop region which includes the start codon.
  • Such a structure has been suggested to be able to significantly reduce translation (Gold, 1988, Annu. Rev. Biochem. 57: 199- 233), and may function to further control expression of a gene coding for a product required in small amounts when it shares a promoter with a gene coding for a product needed in large amounts.
  • the pilO gene product is predicted to code for a protein with a molecular weight of 50.862 using as start codon the ATG beginning at position 729 (Fig. 1). Hydropathy profile (Kyte & Doolittle, 1982, J. Mol. Biol. 157: 105- 132) of the primary structure indicates that PilO contains nine hydrophobic regions which are flanked by clusters of charged residues. Secondary structure prediction (Chou & Fassman, 1974, Biochemistry 13: 21 1-221) suggests that large portions of these hydrophobic regions are composed of ⁇ -structure which are of adequate length to span the 3.0 nm membrane lipid core.
  • CTGTGGCTGG CCCTCGTTTC GGTCCTGGTC TTGGCCCAGG 1120 TGGACGGCGT GTTCGTCATG CCCTTCACCC AGACCGTATT 1160
  • Trp Leu Val Met Leu Leu Leu Leu Ala lie Gin Trp Cys lie Ser Phe

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Abstract

L'invention concerne un vaccin à large spectre contre les bactéries Gram négatif, constitué d'un conjugué biologique glycane-pilus. Le noyau du conjugué est un type de pilus commun auquel est attaché in vivo le glycane choisi. L'accumulation de ces bioconjugués produit un vaccin plurivalent. Ces pili donnent des taux bronchiques élevés quand ils sont administrés par voie intra-nasale. Les souris ainsi vaccinées avec des pili de la souche 1244 de P. aeruginosa glycosylée pure sont protégées contre les tests de provocation respiratoires réalisés avec la souche 1244 de P. aeruginosa. L'invention concerne également une séquence d'ADN et d'acides aminés d'un gène nouveau, pilO, capable de glycosyler la piline de bactérie Gram négatif, ainsi que ses utilisations.
PCT/US1996/019747 1995-12-22 1996-12-19 Vaccin conjugue contre les infections a bacteries gram negatif WO1997023600A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003071877A1 (fr) * 2002-02-27 2003-09-04 Duquesne University Of The Holy Ghost Compositions et procedes destines a provoquer une reponse immunitaire contre les infections bacteriennes a gram negatif
WO2013023296A1 (fr) 2011-08-12 2013-02-21 The Governors Of The University Of Alberta Procédé de diagnostic d'infections bactériennes utilisant des glycoprotéines bactériennes
US9052314B2 (en) * 2013-03-14 2015-06-09 Silver Lake Research Corporation Biomarkers for detecting the presence of bacteria

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MASTERS THESIS. DUQUESNE UNIVERSITY, 1993, DAVIS S.A., "Comparison of Heterologous Pili Production in Pseudomonas Aeruginosa PA103 and 1244 and Development of a Method for Purification of PA103 Pili", pages 13-47. *
MASTERS THESIS. DUQUESNE UNIVERSITY, 1995, RADHAKRISHNAN L., "Identification of Stimuli that Affect the Expression of PilA, the Pilin Structural Gene in Pseudomonas Aeruginosa 1244", page 10. *
MICROBIOLOGY, 1995, Vol. 141, No. 5, CASTRIC P., "PilO, a Gene Required for Glycosylation of Pseudomonas Aeruginosa 1244 Pilin", pages 1247-1254. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003071877A1 (fr) * 2002-02-27 2003-09-04 Duquesne University Of The Holy Ghost Compositions et procedes destines a provoquer une reponse immunitaire contre les infections bacteriennes a gram negatif
US7135175B2 (en) 2002-02-27 2006-11-14 Duquesne University Of The Holy Ghost Compositions and methods for eliciting an immune response to gram-negative bacterial infections
WO2013023296A1 (fr) 2011-08-12 2013-02-21 The Governors Of The University Of Alberta Procédé de diagnostic d'infections bactériennes utilisant des glycoprotéines bactériennes
AU2012297533B2 (en) * 2011-08-12 2016-02-25 Instituto De Investigaciones Biotecnologicas-Instituto Tecnologico De Chascomus Consejo De Investigaciones Cientificas Y Tecnicas, Universidad Nacional De San Martin Method of diagnosing bacterial infections using bacterial glycoproteins
US9052314B2 (en) * 2013-03-14 2015-06-09 Silver Lake Research Corporation Biomarkers for detecting the presence of bacteria

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