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WO2016196323A1 - Sérodiagnostic de la mélioïdose et de la morve en utilisant des polysaccharides - Google Patents

Sérodiagnostic de la mélioïdose et de la morve en utilisant des polysaccharides Download PDF

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Publication number
WO2016196323A1
WO2016196323A1 PCT/US2016/034732 US2016034732W WO2016196323A1 WO 2016196323 A1 WO2016196323 A1 WO 2016196323A1 US 2016034732 W US2016034732 W US 2016034732W WO 2016196323 A1 WO2016196323 A1 WO 2016196323A1
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Prior art keywords
polysaccharide
burkholderia
purified
subject
polysaccharides
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English (en)
Inventor
Paul James BRETT
Mary Nicole BURTNICK
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University of South Alabama
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University of South Alabama
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • melioidosis is a potentially fatal disease caused by the environmental Gram- negative bacterium Burkholderia pseudomallei.
  • the disease is endemic in tropical countries and continues to be a significant public health concern with increasing numbers of cases reported over the past century in many countries across the world.
  • Management of melioidosis in patients can be challenging due to the requirement of specific antibiotic treatment with ceftazidime or a carbapenem. In northeast Thailand, for example, the mortality rate of such patients is 40%, and melioidosis is the third most common cause of death from infectious diseases after acquired- immunodeficiency syndrome (AIDS) and tuberculosis.
  • AIDS acquired- immunodeficiency syndrome
  • B. pseudomallei typically infects people following exposure to contaminated soil and water by inoculation, inhalation or ingestion.
  • melioidosis The clinical spectrum of melioidosis is diverse, consisting of acute fulminant septicemia, subacute illness, chronic infection and subclinical disease. Melioidosis is often associated with a prolonged fever and bacteremia, and it can involve multiple organ infections. In acute forms, death can occur within 24-48 hours of the onset of symptoms.
  • the standard diagnostic method for melioidosis is bacterial culture followed by biochemical identification.
  • the culture method is specific, but it is limited by low sensitivity and takes several days before a result is available.
  • the current standard serology test for melioidosis is an indirect haemagglutination assay (IHA), which is widely used due to its simplicity to perform.
  • IHA indirect haemagglutination assay
  • Unfortunately the IHA has been reported to be unreliable in many studies, and it has a low specificity and limited shelf-life, because it is prepared using sheep red blood cells sensitized with crude B. pseudomallei antigen.
  • the IHA is prepared from viable bacterial cultures, the preparation of the assays in countries where B.
  • pseudomallei is categorized as a Biological Select Agent or Toxin ("select agent") or the equivalent thereof is neither desirable nor practical in light of the restrictions imposed on such select agents. Further, the IHA is poorly standardized, because different laboratories use different strains for antigen preparations.
  • OPS O-polysaccharide
  • LPS lipopolysaccharide
  • CPS 6-deoxyheptan capsular polysaccharide
  • the present disclosure pertains to a method for detecting, in a sample from a subject, antibodies to Burkholderia polysaccharides, the method comprising the steps of: obtaining a sample from a subject; incubating the sample with at least one Burkholderia polysaccharide; and detecting the formation of at least one antibody-polysaccharide complex comprising at least one purified Burkholderia polysaccharide, wherein formation of the at least one complex is substantially indicative of antibodies to at least one Burkholderia polysaccharide being present in the sample.
  • the at least one polysaccharide is selected from the group consisting of B.
  • the method comprises incubating a sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is a capsular polysaccharide.
  • the at least one purified Burkholderia polysaccharide is conjugated to a ligand.
  • the at least one purified Burkholderia polysaccharide is biotinylated.
  • the present disclosure pertains to a method for diagnosing glanders or melioidosis disease in a subject, the method comprising the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and diagnosing the subject with glanders or melioidosis when the formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides is detected.
  • the method comprises incubating a sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is a capsular polysaccharide.
  • the at least one purified Burkholderia polysaccharide is conjugated to a ligand.
  • the at least one purified Burkholderia polysaccharide is biotinylated.
  • the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein.
  • the at least one purified polysaccharide is attached or immobilized to a solid support.
  • the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.
  • the present disclosure pertains to a kit comprising at least one Burkholderia polysaccharide.
  • the at least one Burkholderia polysaccharide comprises a mixture of purified O-polysaccharide and capsular polysaccharide.
  • the kit includes a labeling reagent capable of binding to an antibody that recognizes the at least one purified Burkholderia polysaccharide.
  • the at least one purified Burkholderia polysaccharide is attached to a solid support.
  • the solid support is selected from the group consisting of a resin, a membrane, a bead, or a microtiter plate well.
  • the labeling reagent is a protein conjugated to a detectable label.
  • the detectable label is utilized in combination with a solid support that is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.
  • Fig. 1 depicts the agglutination of antigen-coated latex beads following incubation with antigen-specific immune serum.
  • Fig. 2 depicts the conjugation of chemically activated B. pseudomallei OPS and CPS antigens to amine-derivatized latex beads.
  • FIG. 3 depicts IHA titers of melioidosis patients (A), healthy Thai donors (B), and healthy U. S. donors (C).
  • Fig. 4 depicts positive and negative results of OPS-latex agglutination test (A) and CPS-latex agglutination test (B) of all serum samples compared to IHA titers.
  • the present disclosure generally pertains to methods for the serodiagnosis of melioidosis and glanders in a subject.
  • the method for the serodiagnosis of melioidosis and glanders in a subject comprises the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and diagnosing the subject with glanders or melioidosis when the formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides is detected.
  • the at least one purified Burkholderia polysaccharide may comprise O-polysaccharide, capsular polysaccharide, or any combination thereof.
  • detectable label means a chemical compound that can be detected either directly or indirectly by visual or instrumental means.
  • a detectable label may consist of a molecule that itself produces a signal that can be detected, such as a fluorescent, chemiluminescent or radioactive signal.
  • the signaling label may comprise a molecule that requires reaction with another molecule to generate a signal that can be detected.
  • Detectable labels also include compounds that can be detected visually, for example, colored dyes.
  • sample means a biological sample collected from a subject.
  • sample examples include, but are not limited to, whole blood, serum, plasma, saliva and other biological samples known in the art.
  • the term "subject” means any and all organisms. “Subject” may refer to a human or any other animal. “Subject” may also refer to a fetus. Burkholderia polysaccharide can be naturally occurring or synthetic. Suitable methods of purification of a Burkholderia polysaccharide include, but are not limited to, chemical and detergent extraction of whole Burkholderia cells pellets and precipitation from Burkholderia cell culture supernatants, as known to those skilled in the art.
  • Burkholderia polysaccharide comprises at least one purified, biotinylated Burkholderia polysaccharide.
  • Biotinylation enables immobilization or attachment of a Burkholderia polysaccharide to a solid support. Suitable methods of activation for biotinylation include, but are not limited to, oxidation and cyanylation.
  • Burkholderia polysaccharide comprises at least one purified Burkholderia polysaccharide that is covalently linked to a peptide or protein.
  • Covalent linking enables immobilization or attachment of a Burkholderia polysaccharide to a solid support.
  • Suitable methods of activation for covalent linking include, but are not limited to, oxidation and cyanylation.
  • Suitable peptides are known to those skilled in the art.
  • Suitable proteins include, but are not limited to, bovine serum albumin, OVA, bacterial proteins or toxoids, mammalian proteins, and plant proteins.
  • Burkholderia polysaccharide comprises at least one purified Burkholderia polysaccharide that is attached or immobilized to a solid support. Attachment or immobilization of a Burkholderia polysaccharide to a solid support may facilitate use in a detection assay. Suitable methods of immobilization are known in the art. In certain embodiments, covalent linking is used for immobilization. In other embodiments, non-covalent linking is used for immobilization. Suitable operating conditions for attachment or immobilization of a Burkholderia polysaccharide are known in the art. Suitable types of solid supports include, but are not limited to, a resin, a membrane, a bead, and a microtiter plate well.
  • Suitable resins include, but are not limited to, agarose, acrylamide, and sepharose.
  • Suitable membranes include, but are not limited to, nitrocellulose and polyvinylidene fluoride.
  • Suitable beads include, but are not limited to, latex, magnetic, and polylactic-co- glycolic acid.
  • the present disclosure pertains to a method for detecting, in a sample from a subject, antibodies to Burkholderia polysaccharides, the method comprising the steps of: obtaining a sample from a subject; incubating the sample with at least one Burkholderia polysaccharide; and detecting the formation of at least one antibody-polysaccharide complex comprising at least one purified Burkholderia polysaccharide, wherein formation of the at least one complex is substantially indicative of antibodies to at least one Burkholderia polysaccharide being present in the sample.
  • Suitable methods for detecting such complex formation include agglutination, colorimetric development, chemiluminescence, fluorescence and other techniques known in the art.
  • the at least one polysaccharide is selected from the group consisting of B. pseudomallei , Burkholderia mallei, and Burkholderia thailandensis , or a near-neighbor species, or is chemically synthesized.
  • the method comprises incubating the sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is a capsular polysaccharide.
  • the at least one purified Burkholderia polysaccharide is conjugated to a ligand.
  • the at least one purified Burkholderia polysaccharide is biotinylated. In certain embodiments, the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein. In certain embodiments, the at least one purified polysaccharide is attached or immobilized to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.
  • Figure 1 depicts an embodiment of the presently disclosed method, wherein at least one latex bead acts as a solid support for at least one purified Burkholderia polysaccharide, which, in the presence of antibodies specific for the least one Burkholderia polysaccharide, forms an antibody-bead complex.
  • the present disclosure pertains to a method for diagnosing glanders or melioidosis disease in a subject, the method comprising the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and detecting formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides, wherein formation of the at least one complex is substantially indicative of the subject having been exposed to B. pseudomallei, B. mallei, B. thailandensis or a near-neighbor species.
  • Suitable methods for detecting complex formation include agglutination, colorimetric development, chemiluminescence, fluorescence and other techniques known in the art.
  • the method comprises incubating a sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O- polysaccharide and at least one is a capsular polysaccharide.
  • the at least one purified Burkholderia polysaccharide is conjugated to a ligand.
  • the at least one purified Burkholderia polysaccharide is biotinylated.
  • the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein. In certain embodiments, the at least one purified polysaccharide is attached or immobilized to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.
  • the present disclosure pertains to a kit comprising at least one purified Burkholderia polysaccharide and a labeling reagent capable of binding to an antibody that recognizes one or more purified Burkholderia polysaccharides.
  • the at least one polysaccharide is selected from the group consisting of B. pseudomallei, B. mallei, and B. thailandensis, a near- neighbor species, or are chemically synthesized.
  • the at least one purified Burkholderia polysaccharide is immobilized or attached to a solid support.
  • the solid support can be a resin, a membrane, a bead, a microtiter plate well or other solid support known in the art.
  • the labeling reagent is a protein conjugated to a detectable label.
  • IHA antigens prepared from two clinical B. pseudomallei isolates, strains 199a and 207a were pooled and used to sensitize sheep red blood cells as described in Wuthiekanun V, et al. J Clin Microbiol 44:239 (2006). Before testing, the serum (50 ⁇ ) was inactivated at 56°C for 30 min, followed by pre-adsorption with 10% non- sensitized sheep red blood cells in PBS pH 7.2. 25 ⁇ of 1% sensitized and non- sensitized cell controls were incubated with 50 ⁇ of two-fold serial dilutions of each serum sample in 96-well plates as described in Wuthiekanun V, et al. Am J Trop Med Hyg 74: 1074 (2006). The results were read at the highest antibody titer that showed agglutination after incubating at room temperature for 2 hours. Positive IHA results were determined at a cut-off dilution > 1 : 160.
  • the OPS- and CPS-latex agglutination tests were initially evaluated for sensitivity and specificity using pooled serum from five melioidosis patients or five healthy Thai donors.
  • 10 ⁇ of OPS-latex beads, CPS-latex beads, or uncoated latex beads were separately mixed with an equal volume of each serum sample on a glass slide and rotated at room temperature for 5 minutes.
  • serum was used at a 1 : 10 dilution (in PBS) for the OPS-latex test and undiluted for the CPS-latex test and uncoated latex beads.
  • the agglutination results were observed by eye against a black card and recorded as either positive (agglutination) or negative (no agglutination).
  • the range for the melioidosis patients was 0 to > 1 : 10,240 (median 1 :320, IQR 1 :80-1 : 1,280) and for the healthy Thai donors was 0 to 1 :5,120 (median 1 :40, IQR 1 : 10-1 :320).
  • the IHA titers of the healthy U.S. donors were less than 1 :80 in all cases and ranged between 0 and 1 :40 (median 0, IQR 0).
  • CPS-latex and OPS-latex were both 97.8% (88 of 90 healthy U.S. donors) and the IHA was 100%. Only two weakly positive reactions with either the CPS-latex (IHA titer 1 :20 for both sera) or OPS-latex (IHA titer 1 : 10 for both sera) were observed for the healthy U.S. donor samples.
  • IHA titers are in agreement with the polysaccharide-based latex agglutination tests
  • Both the OPS-latex and CPS-latex testing methods disclosed herein provide results that are comparable to the IHA test, which is currently the most commonly used serological test for melioidosis.
  • Benefits of the Burkholderia polysaccharide-based latex agglutination tests disclosed herein include that they have a longer shelf-life than the IHA, are inexpensive, standardized and simple to perform. As such, the methods disclosed herein may be valuable diagnostic tests for rapidly detecting B. pseudomallei infections.

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Abstract

La présente invention concerne d'une manière générale des procédés de sérodiagnostic de la mélioïdose et de la morve chez un sujet. Dans un mode de réalisation indicatif, le procédé de sérodiagnostic de la mélioïdose et de la morve chez un sujet comprend les étapes de : obtention d'un prélèvement d'un sujet; incubation du prélèvement issu du sujet avec au moins un polysaccharide de Burkholderia; et le diagnostic que le sujet est atteint de morve ou de mélioïdose lorsque la formation d'au moins un complexe anticorps-polysaccharide comprenant au moins l'un des polysaccharides de Burkholderia purifié est détectée. Le au moins un polysaccharide de Burkholderia purifié peut comprendre un O-polysaccharide, un polysaccharide de capsule, ou n'importe quelle combinaison de ces derniers.
PCT/US2016/034732 2015-05-29 2016-05-27 Sérodiagnostic de la mélioïdose et de la morve en utilisant des polysaccharides Ceased WO2016196323A1 (fr)

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CN113501888A (zh) * 2021-06-25 2021-10-15 中国人民解放军陆军军医大学 一种类鼻疽菌多糖及其制备方法和应用
CN116063582A (zh) * 2022-09-21 2023-05-05 中国人民解放军陆军军医大学 血清型b型类鼻疽菌的特异性o-抗原及其提取方法和应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113501888A (zh) * 2021-06-25 2021-10-15 中国人民解放军陆军军医大学 一种类鼻疽菌多糖及其制备方法和应用
CN116063582A (zh) * 2022-09-21 2023-05-05 中国人民解放军陆军军医大学 血清型b型类鼻疽菌的特异性o-抗原及其提取方法和应用

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