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WO2005026732A1 - Identification de phenotypes bacteriens fondee sur des anticorps - Google Patents

Identification de phenotypes bacteriens fondee sur des anticorps Download PDF

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Publication number
WO2005026732A1
WO2005026732A1 PCT/GB2004/003897 GB2004003897W WO2005026732A1 WO 2005026732 A1 WO2005026732 A1 WO 2005026732A1 GB 2004003897 W GB2004003897 W GB 2004003897W WO 2005026732 A1 WO2005026732 A1 WO 2005026732A1
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WIPO (PCT)
Prior art keywords
vaccine
sample
binding agent
lps
labelled
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2004/003897
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English (en)
Inventor
Rosa Rebecca Taylor
Margaret Gillian Hartley
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UK Secretary of State for Defence
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UK Secretary of State for Defence
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Filing date
Publication date
Application filed by UK Secretary of State for Defence filed Critical UK Secretary of State for Defence
Publication of WO2005026732A1 publication Critical patent/WO2005026732A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS
    • 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

  • the present invention relates to methods for determining the phenotype of gram-negative bacteria, which are useful, in particular in vaccine production and quality control.
  • a lipopolysaccharide (LPS) -rich outer membrane surrounds gram- negative bacteria.
  • the LPS molecules themselves are made up of three regions, a lipid A region, a core oligosaccharide, and an 0 side chain, which is highly diverse.
  • the phenotype of a bacterial strain and in particular the lipopolysaccharide phase variation, can vary, and as a result, the properties, of the strain, for example, its applicability as a vaccine, can be affected.
  • F. tularensis LVS The successful licensing and manufacture of F. tularensis LVS will require a number of quality control tests throughout the entire production process to ensure that batches of vaccine are safe, efficacious and have reproducible properties.
  • the efficacy of a vaccine needs to be assessed as far as possible, prior to administration. Once administered, even monitoring of a patient's antibody response to the vaccine may not give a clear idea of the level of protection afforded, due to the complexity and variety of the immune response. The best way to ensure protection is to ensure that appropriate doses of immunogen are administered initially.
  • An ideal vaccine would comprise 100% "blue" phenotype. However, the proportion of blue and grey colony types can vary in different batches of vaccine.
  • a method for confirming the presence or amount of a particular immunogenic phenotype of a gram negative bacteria in a sample comprising contacting a sample of said bacteria with a labelled binding agent which is specific for a lipopolysaccharide (LPS) of said particular phenotype of said bacteria, detecting the presence of a complex formed by said binding agent and LPS within the sample, and relating that to the presence or amount of said phenotype within the sample.
  • a labelled binding agent which is specific for a lipopolysaccharide (LPS) of said particular phenotype of said bacteria
  • the method of the invention is particularly suitable for testing the quality of a vaccine strain, such as F. tularensis, where the applicants now believe that the immunogenicity and the levels of protection afforded by the vaccine, is affected by lipopolysaccharide phase variation.
  • the binding agent is labelled with a visible label, and the presence of said complex is determined using flow cytometry.
  • the any cells that are of the target phenotype will bind to the labelled binding agent, and will provide a appropriate signal when passing through the flow cytometer.
  • Other cells will be distinguishable by the absence of label on them.
  • Particular labels are fluorescent labels such as fluorescein or the like.
  • the fluorescent label is suitably one which can assist in detection in a conventional flow cytometer.
  • the sample is also contacted with a second labelled binding agent that specifically binds an LPS of a different phenotype, and wherein the presence of a complex between said second labelled binding agent and LPS in a sample is also detected.
  • the second labelled binding agent carries a different label to the first labelled binding agent, so that this can be distinguished in the flow cytometer.
  • further labelled antibodies may be added to the sample to detect the presence of a variety of phenotypes .
  • fluorophores When looking at a range of variants, fluorophores should be selected such that their emission wavelength are suitably distinct, preferably with at least 50 nm difference in emission wavelength.
  • Such methods will provide a user with an objective, quantitative answer, which will be reproducible to accepted statistical standards. It is more accurate than the current microscope based method, and furthermore, is easier to perform, as the protocol could be followed by non-technical staff.
  • the method can be carried out in one reaction pot and doesn't require the growing up of colonies (which may add to inaccuracies) . It is also much faster, as the analysis of a vaccine strain using the method of the invention may take only approximately 2 hours as compared to about 3 days using a conventional microscope method
  • the binding agent is an i munoglobulin, such as an antibody or a binding fragment thereof.
  • Antibodies or binding fragments thereof may be polyclonal or monoclonal, and these may be produced using conventional methods .
  • polyclonal antibodies may be generated by immunisation of an animal (such as a rabbit, rat, goat, horse, sheep etc) with the toxin or immunogenic subunits or fragments thereof, to raise antisera, from which antibodies may be purified.
  • an animal such as a rabbit, rat, goat, horse, sheep etc
  • toxin or immunogenic subunits or fragments thereof to raise antisera, from which antibodies may be purified.
  • Monoclonal antibodies may be obtained by fusing spleen cells from an immunised animal with hybrido a cells, and selecting cells which secrete suitable antibodies .
  • Antibody binding fragments include F(ab') 2 , F(ab) 2 , Fab or Fab' fragments, as well as single chain (sc) antibodies, FV, VH or VK fragments, but they may also comprise deletion mutants of an antibody sequence.
  • Acronyms used here are well known in the art . They are suitably derived from polyclonal or monoclonal antibodies using conventional methods such as enzymatic digestion with enzymes such as papain or pepsin (to produce Fab and F(ab') 2 fragments respectively). Alternatively, they may be generated using conventional recombinant DNA technology.
  • the labelled binding agent is a labelled monoclonal antibody.
  • the method is particularly applicable to the analysis of strains of gram-negative bacteria intended for use as a vaccine.
  • the gram-negative bacteria is Francisella tularensis
  • this may therefore comprise F. tularensis live vaccine strain (LVS) .
  • the method can be used to detect and/or quantitate the amount of "blue" phenotype, which has mutated to "grey" phenotype in a sample, to check its suitability as a vaccine.
  • the or each labelled binding agent is specific for an 0 side chain of a lipopolysaccharide.
  • the method of the invention will suitably be carried out by suspending a dry batch of bacterium, and in particular a dry live vaccine strain, in a suitable medium or solvent such as phosphate-buffered saline (PBS) . After a suitably equilibration time, for instance, about 30 minutes, the sample may be diluted to the required concentration.
  • a suitable medium or solvent such as phosphate-buffered saline (PBS)
  • one or more labelled binding partners as described above, and in particular, fluorescently labelled antibodies is added to the sample.
  • One specific antibody in particular, an anti-LPS antibody is added for each phenotype to be detected, but where there are more than one, each one is differently and distinguishably labelled.
  • the mixture is then incubated to allow antibody-antigen interaction to take place, for example at temperatures of from 5-45 a C, such as about 37°C. Generally incubation will be carried out in the dark, for example for a period of from 5 minutes to 1 hour, and generally for about 15 minutes .
  • the mixture is then added to a flow cytometer by dilution with carrier fluid.
  • the flow cytometer may be a standard laboratory cytometer, provided with suitable excitation laser sources . Each cell passes sequentially in front of a focussed light beam, and the optical characteristics of the cell are determined.
  • the flow cytometer can be set to produce two dimensional charts of side vs forward scatter of the light beam, to sort the cells on the basis of size. Forward scatter vs fluorescence intensity will allow separation quantitation of labelled cells . This latter operating mode is particularly suitable in the context of the present invention.
  • the labelled bacteria can be analysed by 2 channel FACS analysis.
  • the data from this analysis will provide a rapid readout of the proportion of the two respective phenotypes, for example, blue and grey bacteria, in the sample vaccine.
  • the test described above can be automated to judge vaccine strains acceptable or otherwise.
  • the original 1955 Soviet vaccine control procedures stated that the 1 immunogenic ' (i.e. blue) type 'must constitute 20 to 30% of the total number of organisms in the vaccine' . It is thought that if the LVS vaccine lots can be shown to contain 80% blues or above, they will be considered acceptable.
  • the viability of the strain can be tested using conventional methods.
  • the invention further provides a quality control method for determining the efficacy or immunogenicity or quality of a live vaccine, said method comprising subjecting said vaccine to a method as described above, and detecting the amount of immunogenic phenotype in the vaccine sample.
  • the live vaccine sample is a vaccine strain of F. tularensis .
  • kits for carrying out a method as described above comprising a labelled binding agent which is specific for a lipopolysaccharide of a particular phenotype of a gram- negative bacteria, and in particular a fluorescently labelled antibody which is specific for an 0 side chain of a vaccine strain of gram-negative bacteria such as F. tularensis LVS, as outlined above.
  • LPS LPS-associated antigens
  • the presence of LPS in a vaccine such as a sub-unit vaccine may be unwanted for other reasons.
  • the LPS of many types of gram-negative bacteria are toxic to animals. This applies to for example, the LPS of E. coli, which may be used to express recombinant sub-unit vaccines .
  • the labelled binding agents described above which are specific for these unwanted LPS, may be used in detection methods as described above, to readily and rapidly detect LPS contaminants in vaccines .
  • the invention provides a method for detecting LPS in a vaccine such as a sub-unit vaccine, said method comprising contacting a sample of said vaccine with a labelled specific binding agent for LPS, and detecting any complex formed between the complex and vaccine particles.
  • Figure 1 shows a Western blot of F. tularensis LVS, blue, grey and cap " and F. novicida proteinase K digests probed with the anti-F. tularensis monoclonal antibody used in the test.
  • the LVS control preparation was not treated with proteinase K.
  • the characteristic ladder associated with binding of antibody to the 0-antigen of LPS is seen in the blue and LVS samples only.
  • Figure 2 shows a Western blot of F. tularensis LVS, blue and grey (harvested from vial f or BCGA plates*) and F. novicida, proteinase K digests, probed with anti-F. novicida monoclonal antibody (FnMab: upper blot) or anti-F. tularensis monoclonal antibody (FtMab: lower blot) .
  • Figure 3 shows the results of a FACS display from blue and grey phenotypes cultured on BCGA.
  • Cells were stained with FITC- labelled anti-F. tularensis monoclonal antibody.
  • Display (3a) shows cells sorted by size. The fluorescence with region Rl from a mixed preparation is shown in is shown by density plot (3b) .
  • (3c) and (3d) show the staining pattern of individual blue and grey phenotypes, respectively.
  • Figure 4 is a graph showing the percentage of F. tularensis LVS blue phenotypes in NDBR lots of LVS vaccine estimated by the test.
  • FtMab and FnMab monoclonal antibodies against F. tularensis and F. novicida
  • the LVS blue cultures provided were 'mostly' , but not necessarily 100% blue. This could not be assessed by culture as grey colony types take longer to form visible colonies on BCG agar.
  • F. tularensis HN63 and Schu4 were also tested, as was LVS capsular-deficient strain (cap " ) was as described by SANDSTROM, G., et al. (1988). Infection and Immunity 56, 1194-1202.
  • Heat-killed cells or LPS preparations (proteinase-K treated heat killed cells) in PBS were run on SDS-PAGE at 5 ⁇ g protein per lane, followed by Western blot. The blots were probed with antibody and detection used the ECL plus kit (Amersham Pharmacia) .
  • Antibodies were used at 1/1000 and goat anti-mouse IgG 3 conjugate at 1/40,000.
  • the anti-F. tularensis monoclonals were directed against the LPS 0 side chain present on the blue variant, an example of which is shown in figure 1. They also reacted with SCHU4 (weakly) and HN63 (data not shown) ; however, none of these antibodies reacted with LVS grey and cap- strains, or F. novicida .
  • Example 2 FACS Analysis A Becton Dickinson FACScan flow cytometer was used to devise a rapid test .
  • the selected antibody was produced by a cultured cell line, purified by dialysis and conjugated to FITC. 45 ⁇ l of cells at 1 xlO 5 dilution were mixed with FITC-Iabelled antibody, incubated for 15 minutes at 37°C in the dark, then diluted in pre-filtered Isoton II fluid. This mixture was vortexed and analysed by FACS producing a count of labelled and non-labelled particles of the appropriate size.
  • the BD CellQuestTM Pro (version 4.0.1) was used for analysis.
  • the typical presentation of a cell population is shown in figure 3.
  • the left hand figure (3a) is a density plot of the size profile of all cells measured by forward and side scatter.
  • the right hand picture (3b) represents the fluorescence from an artificially mixed population of blue and grey phenotypes within the ringed region (Rl: operator set). This becomes the chosen area for analysis .
  • the lower two displays show the fluorescence profile of the individual blue and grey cultures (c and d) . Division between the upper (blue) population and the lower (grey) population is set by the operator. The software will then express these populations as a percentage of the total in the Rl area.
  • Tests carried out on the frozen vials of separately provided grey and blue batches reflected the total amount of each (data not shown) .
  • Example 3 Test of vaccine vials NDBR Vaccine lots 2, 4 and 11 (stored at -20°C) were reconstituted in 2ml of sterile distilled water and left at room temperature for at least half an hour prior to dilution and testing by FACS as described in Example 2. All dilutions were made in PBS.
  • Tests were carried out on NDBR 101 lots 2, 4 and 11 with cells at concentrations of 10 6 and 10 7 per test (in 45 ⁇ l) .
  • Figure 4 is a summary of the tests carried on out these vaccine lots. Error bars are SEM. There was a small difference in estimates when the staining of 10 6 and 10 7 cells were compared.
  • the NDBRIOI batch of LVS vaccine contained 99% blue and 1% grey colonies (WAAG, D.M., et al. (1992). Journal of Clinical Microbiology 30, 2256-2264; WAAG, D.M., et al. (1996). FEMS Immunology and Medical Microbiology 13, 205-209). These figures are reflected in our results.

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Abstract

L'invention concerne un procédé permettant de confirmer la présence ou la quantité d'un phénotype immunogène particulier d'une bactérie gram-négatif dans un échantillon. Ce procédé consiste : à mettre en contact un échantillon de ladite bactérie avec un agent de liaison marqué qui est spécifique d'un lipopolysaccharide (LPS) dudit phénotype particulier de ladite bactérie ; à détecter la présence d'un complexe formé par ledit agent de liaison et le LPS à l'intérieur de l'échantillon, en particulier, au moyen d'une technique de type cytométrie en flux ; et à associer la présence de ce complexe à la présence ou la quantité dudit phénotype à l'intérieur de l'échantillon. Ce procédé est particulièrement approprié pour le contrôle de la qualité de vaccins de type F.tularensis LVS.
PCT/GB2004/003897 2003-09-13 2004-09-13 Identification de phenotypes bacteriens fondee sur des anticorps Ceased WO2005026732A1 (fr)

Applications Claiming Priority (2)

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GB0321508A GB0321508D0 (en) 2003-09-13 2003-09-13 Antibody-based identification of bacterial phenotypes
GB0321508.4 2003-09-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10882899B2 (en) 2018-07-04 2021-01-05 The Israel Institute of Biological Research (IIBR) Anti Francisella tularensis (FT) antibodies
EP3634996A4 (fr) * 2017-06-06 2021-03-03 InDevR, Inc. Analyse simultanée in vitro de la puissance d'un vaccin et de la concentration de toxines

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5190860A (en) * 1989-10-30 1993-03-02 The Texas A & M University System Differential diagnostic assay for brucellosis
CA2078162A1 (fr) * 1992-09-14 1994-03-15 Raymond Tsang Reactifs monoclonaux antisalmonelles specifiques et methode serologique unique pour la detection de differents serotypes courants de salmonelles et d'organismes semblables
EP0990902A1 (fr) * 1997-06-06 2000-04-05 Mitsubishi Chemical Corporation Procede servant a detecter une infection par escherichia coli enterohemorragique
US6524793B1 (en) * 1995-10-11 2003-02-25 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5190860A (en) * 1989-10-30 1993-03-02 The Texas A & M University System Differential diagnostic assay for brucellosis
CA2078162A1 (fr) * 1992-09-14 1994-03-15 Raymond Tsang Reactifs monoclonaux antisalmonelles specifiques et methode serologique unique pour la detection de differents serotypes courants de salmonelles et d'organismes semblables
US6524793B1 (en) * 1995-10-11 2003-02-25 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
EP0990902A1 (fr) * 1997-06-06 2000-04-05 Mitsubishi Chemical Corporation Procede servant a detecter une infection par escherichia coli enterohemorragique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
APPELMELK BEN J ET AL: "Antigenic and immunogenic differences in lipopolysaccharides of Escherichia coli J5 vaccine strains of different origins", JOURNAL OF GENERAL MICROBIOLOGY, vol. 139, no. 11, 1993, pages 2641 - 2647, XP001182942, ISSN: 0022-1287 *
DATABASE WPI Section Ch Week 200323, Derwent World Patents Index; Class B04, AN 1997-236023, XP002304479 *
ELLIS J ET AL: "Tularemia", CLINICAL MICROBIOLOGY REVIEWS, WASHINGTON, DC, US, vol. 15, no. 4, October 2002 (2002-10-01), pages 631 - 646, XP002295463, ISSN: 0893-8512 *
POXTON I R: "Antibodies to lipopolysaccharide", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 186, no. 1, 12 October 1995 (1995-10-12), pages 1 - 15, XP004021134, ISSN: 0022-1759 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3634996A4 (fr) * 2017-06-06 2021-03-03 InDevR, Inc. Analyse simultanée in vitro de la puissance d'un vaccin et de la concentration de toxines
US10882899B2 (en) 2018-07-04 2021-01-05 The Israel Institute of Biological Research (IIBR) Anti Francisella tularensis (FT) antibodies
IL267823B1 (en) * 2018-07-04 2023-07-01 The Israel Institute Of Biological Res Iibr Antibodies against Francisella tolerensis
IL267823B2 (en) * 2018-07-04 2023-11-01 State Of Israel Israel Institute For Biological Res Antibodies against Francisella tolerensis

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