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WO1996041171A1 - Procedes et produits pour le diagnostic d'infections mycobacteriennes - Google Patents

Procedes et produits pour le diagnostic d'infections mycobacteriennes Download PDF

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Publication number
WO1996041171A1
WO1996041171A1 PCT/US1996/010023 US9610023W WO9641171A1 WO 1996041171 A1 WO1996041171 A1 WO 1996041171A1 US 9610023 W US9610023 W US 9610023W WO 9641171 A1 WO9641171 A1 WO 9641171A1
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WIPO (PCT)
Prior art keywords
antigen
subject
induration
site
atypical
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Application number
PCT/US1996/010023
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English (en)
Inventor
Fazal S. Raheman
Saraswathy V. Nochur
Peter J. Mione
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Dynagen, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Dynagen, Inc. filed Critical Dynagen, Inc.
Publication of WO1996041171A1 publication Critical patent/WO1996041171A1/fr

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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis

Definitions

  • This invention relates to the use of purified single protein antigens as challenge antigens for eliciting a cell mediated immune response as a means of detecting mycobacterial infection in humans or animals.
  • a cell mediated immune response is manifest in the subject as a delayed type hypersensitivity (DTH) skin reaction upon introduction by intradermal injection of purified single protein tuberculin antigens.
  • DTH delayed type hypersensitivity
  • the use of a combination of two or more single protein antigens with different species specificities permits the differential diagnosis of tuberculosis infection from infections due to atypical mycobacteria such as Mycobacterium avium. Mycobacterium intracellulare or Mycobacterium kansasii. and also permits classification of infected subjects as those at low risk for contracting active disease versus those at high risk for contracting active disease.
  • tuberculin refers to extracts of M. tuberculosis.
  • the first tuberculin was prepared by Robert Koch less than a decade after his discovery of M. tuberculosis in 1882.
  • the Koch formulation is often referred to as "old tuberculin” (OT).
  • OT old tuberculin
  • PPD purified protein derivative
  • PPD-S Seibert and others manufactured a large batch of PPD, referred to as PPD-S, which is still used as a PPD standard in the U.S.
  • PPD RT 23 The World Health Organization (WHO) sponsored the manufacture of another large batch of PPD, called PPD RT 23.
  • PPD RT 23 serves as the international standard.
  • PPD is widely used as a skin test antigen for the diagnosis of tuberculosis infection.
  • the most common mode of administration of PPD is the Mantoux technique.
  • the Mantoux technique consists of an intradermal injection of 0.1 mL of PPD on the volar surface of the forearm. A positive test results in an induration reaction which is measured and interpreted usually 48 hours after administration. Multiple puncture devices are also available.
  • PPD is a cocktail of many proteins and polysaccharides. Since the precise identity and concentration ofthe individual proteins is unknown, the manufacture of PPD has significant batch-to-batch variability. For the same reasons, PPD cannot be accurately characterized. Every newly prepared batch of PPD is compared with the standard PPD, and pretested in human volunteers prior to commercialization. The two PPD standards are also different from each other. On a weight basis, PPD RT23 has two and one-half times the biological activity of PPD- S.
  • Distinguishing infection caused by M. tuberculosis from infection caused by atypical mycobacteria has clinical significance.
  • Infections caused by M. tuberculosis are treated prophylactically.
  • the drug commonly used for such prophylactic treatment is isoniazid.
  • Prophylactic treatment with this drug is for a prolonged period of time, at least six months.
  • Such long term treatment with isoniazid is associated with liver disfunction and toxicity and other side effects.
  • Infections caused by atypical mycobacteria are normally not treated prophylactically. It therefore would be desirable to distinguish infection caused by M. tuberculosis versus infection caused by atypical mycobacterial to avoid unnecessary treatment with isoniazid and to avoid unwanted side effects and toxicities.
  • the present invention features a method and a kit for distinguishing mycobacterial infected individuals from uninfected individuals.
  • subject refers to an individual or animal that is the object of clinical study.
  • the term "antigen” or “tuberculin” refers to a substance that when introduced into a subject stimulates a cell mediated immune response.
  • a “pure” or “single” protein antigen is a material which provides a discrete band on gel electrophoresis and is approximately 75% or greater in purity.
  • a delayed type hypersensitivity (DTH) reaction is a cell mediated immune response induced in a subject due to the presence of a foreign antigen. With respect to mycobacterial antigens, these hypersensitivity reactions are well characterized and commonly assume the form of an induration at the site of administration ofthe antigen. Such induration reactions typically are circular and have a measurable diameter.
  • the DTH reaction typically manifests itself between 24 and 144 hours after the administration of an effective amount of antigen. Even more typically, the reaction manifests itself between 48 and 72 hours after administration, which is the typical time period for observing subjects for a DTH reaction.
  • a positive result is an induration having a diameter equal to or greater than a set or cutoff value. The cutoff value is determined by the comparison of indurations from healthy individuals, infected individuals, and individuals who exhibit DTH reactions due to vaccination.
  • the term “active” means a pathological condition in which the subject is infected with mycobacteria and is clinically symptomatic for the disease.
  • the term “inactive” refers to a condition in which the subject may manifest a delayed type hypersensitivity to mycobacterial antigens without having clinical manifestations of mycobacterial disease.
  • inactive subjects include healthy individuals, infected or uninfected with mycobacteria, and who present no clinical symptoms ofthe disease, including vaccinated individuals who do not present any clinical symptoms ofthe disease.
  • the term “high risk” refers to a subject with active disease or has a high probability of developing active disease within a one month to one year period. A high probability is approximately greater than 15%.
  • the term “low risk” refers to a subject with no clinical manifestations ofthe disease or presently lacks exposure to the disease and is not likely to develop active disease within a one month to one year period without further exposure or other factors contributing to pathology.
  • the method ofthe present invention is suited for determining the presence of mycobacterial infection caused by one or more ofthe mycobacteria selected from the group consisting of M. tuberculosis complex, M. avium complex, M. kansasii. or any other mycobacterium that may be of clinical significance.
  • One embodiment ofthe present invention features a method for determining the causative agent of mycobacterial infection.
  • the method comprises the steps of administering a first antigen preparation by intradermal injection to a first site on the skin of a subject.
  • the first antigen preparation is cross reactive to all mycobacterial species.
  • this antigen is a single protein antigen.
  • the first antigen preparation is capable of eliciting a DTH reaction in the event the subject has a mycobacterial infection or has been exposed to antigens belonging to any mycobacterial species.
  • the DTH reaction is in the form of an induration.
  • the method further comprises the step of administering by intradermal injection to a second site on the skin ofthe subject a second antigen preparation.
  • the second antigen preparation is selected from one of a group of antigens consisting of tuberculosis antigens and atypical antigens.
  • Tuberculosis antigens are antigens produced by M. tuberculosis, but not by atypical mycobacteria such as M. avium. M. intracellulare. or M. kansasii.
  • the antigen is capable of eliciting a DTH reaction in the event the subject has been infected with M. tuberculosis, but not with atypical mycobacteria such as M . avium. M. intracellulare. or M. kansasii.
  • the delayed hypersensitivity reaction is in the form of an induration at the second site.
  • An atypical antigen is produced by an atypical mycobacterium of clinical significance such as M. avium. M. intracellulare. or M. kansasii. but not by M. tuberculosis.
  • the atypical antigen is capable of eliciting a delayed hypersensitivity reaction in the event the subject has been infected with the specific atypical mycobacterium such as M avium. M. intracellulare. or M. kansasii. but not if the subject was infected only with M. tuberculosis.
  • the method further comprises the step of observing the subject for the formation of indurations at the first and the second sites of administration. A positive DTH reaction at both sites suggests M.
  • tuberculosis infection in the event a tuberculosis antigen is the second antigen preparation.
  • a positive DTH reaction at the first site and a negative DTH reaction at the second site suggests possible atypical mycobacterial infection in the event the second antigen preparation is a tuberculosis antigen.
  • the second antigen preparation is an atypical antigen, a positive result at both sites suggests infection with an atypical mycobacterial species.
  • the second antigen preparation is an atypical antigen, a positive result at the first site and a negative result at the second site suggests infections with a mycobacterial species other than that from which the atypical antigen was derived.
  • the method comprises the step of administering by intradermal injection to a third site on the skin ofthe subject a third antigen preparation.
  • the third antigen preparation is selected from the group of antigens consisting of a tuberculosis antigen and an atypical antigen which is not the second antigen.
  • the method further comprises the step of observing the subject for the formation of indurations at each ofthe three sites of administration ofthe antigens. In the event that a positive DTH reaction is observed only at the first site, such results indicate that the subject is infected with a mycobacterial species which is not M. tuberculosis and not the atypical mycobacterium from which the atypical antigen was derived.
  • This cut off is determined for the pan-specific mycobacterial antigen versus the tuberculosis specific antigen, versus the antigen specific for the atypical mycobacterium.
  • DTH reaction at the first site with an induration diameter greater than the cut off value for the first antigen is indicative ofthe presence of mycobacterial infection.
  • a DTH reaction with an induration diameter greater than the cutoff value for tuberculosis antigen is indicative of infection due to M.tuberculosis.
  • a DTH reaction with induration diameter greater than the cut off value for atypical antigen is indicative of infection by the atypical mycobacterial species from which the antigen was derived.
  • a preferred first antigen preparation is selected from the group consisting of antigen 6 (also referred to as alpha antigen, antigen 85B, or MPT59), 12kD, 30kD, 32kD, 38kD, and multiple protein antigens, such as PPD and old tuberculin.
  • the tuberculosis antigen is selected from the group consisting of MPT64/MPB64 or TB 66.
  • the atypical antigen is selected from the group consisting of 12kD and 43kD.
  • a further embodiment ofthe present invention features a kit for determining the causative agent of mycobacterial infection.
  • the kit comprises a first antigen preparation that is cross- reactive among all mycobacteria, but is specific to mycobacteria.
  • the antigen preparation is capable of being administered to a subject for intradermal injection to a first site on the skin of the subject and capable of eliciting a DTH reaction in the event the subject has a mycobacterial infection.
  • the DTH reaction is in the form of an induration.
  • the kit further comprises a second antigen preparation.
  • the second antigen preparation is selected from the group of antigens consisting of a tuberculosis antigen and atypical antigen.
  • the second antigen preparation is capable of being administered to a subject by intradermal injection to a second site on the skin of the subject.
  • the tuberculosis antigen is specific to M. tuberculosis and does not elicit a reaction due to infection with atypical mycobacteria such as M. avium. M. kansasii. or M. intracellulare.
  • the tuberculosis antigen is capable of eliciting a DTH reaction in the event the subject has an infection caused by M. tuberculosis.
  • the DTH reaction is in the form of an induration.
  • a atypical antigen is an antigen specific to atypical mycobacterium such as M. avium. M. intracellulare. or M. kansasii. but not M. tuberculosis.
  • the atypical antigen is capable of eliciting a DTH reaction in the event the subject has an infection caused by the particular atypical mycobacterium, such as M. avium.
  • the DTH reaction is in the form of an induration.
  • the kit further can include instructions for observing the subject for the formation of induration at all sites of administration ofthe antigen preparations.
  • a positive DTH reaction for the first antigen preparation at the first site is indicative of infection due to any mycobacterial species.
  • a positive DTH reaction for the second antigen preparation at the second site is indicative of infection with or exposure to M. tuberculosis in the event the second antigen is tuberculosis antigen.
  • a positive DTH reaction for the second antigen preparation at the second site is indicative of infection by the specific atypical mycobacterial species from which the antigen was derived, in the event the second antigen is atypical antigen.
  • kits further comprises a third antigen preparation for administration intradermally to a third site of a subject.
  • the third antigen preparation is selected from the group of antigens consisting of tuberculosis antigen and atypical antigen which is not the second antigen.
  • a first antigen, a tuberculosis antigen and an atypical antigen are administered to a subject, and positive DTH reactions are observed at the first site and the site of the atypical antigen but not at the tuberculosis antigen site, such results suggest infection with the particular atypical mycobacterial species from which the antigen was derived, or a mixed infection with the particular atypical mycobacterial species and another atypical mycobacterium, but not tuberculosis infection.
  • a positive DTH reaction is observed at the sites of a first antigen, a tuberculosis antigen and an atypical antigen, such results indicate a mixed infection due to M.
  • a positive DTH reaction at the site ofthe first antigen preparation and a negative DTH reaction at the site ofthe tuberculosis antigen and the atypical antigen suggests an infection with an atypical mycobacterial species other than such species from which atypical antigen is derived.
  • a positive DTH reaction at the site ofthe first antigen and at the site ofthe tuberculosis antigen, but not at the atypical antigen site suggests infection with M. tuberculosis or a mixed infection of M. Tuberculosis and an atypical mycobactial species other than the species from which the atypical antigen was derived.
  • a negative DTH result at the first antigen preparation site suggests that the subject has not been exposed to mycobacterial antigens.
  • a preferred first antigen preparation is selected from the group consisting of antigen 6 (also referred to as alpha antigen, antigen 85B, or MPT59), 12kD, 30kD, 32kD, 38kD, or PPD.
  • antigen 6 also referred to as alpha antigen, antigen 85B, or MPT59
  • 12kD also referred to as alpha antigen, antigen 85B, or MPT59
  • 12kD also referred to as alpha antigen, antigen 85B, or MPT59
  • 12kD also referred to as alpha antigen, antigen 85B, or MPT59
  • 12kD also referred to as alpha antigen, antigen 85B, or MPT59
  • 12kD also referred to as alpha antigen, antigen 85B, or MPT59
  • 30kD also referred to as alpha antigen, antigen 85B, or MPT59
  • 32kD also referred to as 32kD, 38kD, or PPD
  • PPD tubercul
  • the atypical antigen is specific for M. avium or M. avium complex
  • the atypical antigen is selected from the group consisting of 12kD and 43kD antigens.
  • the instructions specify that each ofthe antigen preparations is administered at least one inch apart on the volar surface ofthe left or right forearm.
  • the instructions specify that the step of observation for the DTH reactions is 24 to 144 hours after the administration of all the antigen preparations. More preferably, the step of observing is performed 48 to 72 hours after administration.
  • the kit comprises suitable packaging and containment vessels for two or three single protein antigens.
  • the kit may comprise single protein antigens in solution contained within separate vessels until used.
  • antigens may be lyophilized and contained in separate suitable vessels for reconstitution. In the event that the antigens are in the lyophilized form, one, two or three separate diluents or buffers or solutions may be provided in suitable vessels for reconstitution ofthe antigens.
  • a further embodiment ofthe present invention features a method for identifying mycobacterial infection in a subject, and determining whether such infection, if present, represents a low risk or high risk for active disease.
  • the method comprises the steps of administering to a subject by intradermal injection to a first site on the subject's skin, an effective amount of a first antigen.
  • the first antigen preparation is selected from the group consisting of multiple protein derivative of M. tuberculosis or a single protein tuberculin.
  • the first antigen preparation is capable of eliciting a DTH reaction in the event the subject has a mycobacterial infection.
  • the DTH reaction is in the form of an induration reaction, having a measurable diameter at the site of administration.
  • the method further comprises the steps of administering to the subject by intradermal injection to a second site on the subject's skin an effective amount of a second antigen preparation.
  • the second antigen preparation is a single protein antigen capable of eliciting a DTH reaction in the event the subject has a mycobacterial infection.
  • the delayed hypersensitivity reaction is in the form of an induration reaction having a measurable diameter at the second site.
  • the method further comprises the step of comparing the diameter ofthe induration at the first site and the diameter ofthe induration at the second site to a characteristic value for such antigen preparations to determine whether the subject is at high risk or low risk for developing the disease.
  • the diameter ofthe induration at the first site is compared with the diameter of the induration at the second site in accordance with the following formula:
  • X is the width ofthe induration at the first site and Y is the width ofthe induration at the second site and Z is a numerical value determined by X and Y.
  • Values of Z are characteristic for each specific first and second antigen preparations selected for use in this embodiment. A value of Z greater than a characteristic value for active disease suggests the individual is at high risk or has active mycobacterial disease.
  • the first mycobacterial antigen preparation is PPD and the second mycobacterial antigen preparation is antigen 6.
  • a value of Z less than the characteristic value for active disease suggests the subject is at low risk.
  • the first antigen preparation is PPD and the second antigen preparation is antigen 6, at relative concentrations as set forth in the Examples, a Z value greater than 1.05, plus or minus two standard deviations, suggests active mycobactria infection and values less than 1.05, plus or minus two standard deviations, suggest exposure to mycobacterial antigens without active mycobacteria infection.
  • the subject is observed 24-144 hours after the administration ofthe first and second antigen preparation. Even more preferably, the subject is observed 48-72 hours after administration.
  • the first and second antigen preparation may overlap in proteins. That is, a single protein antigen may be a component of PPD.
  • the second antigen preparation is a single protein, provided such protein comprising the first and second antigen preparations are different.
  • a further embodiment ofthe present invention features a kit for identifying mycobacterial infection in a subject, and determining whether such infection, if present, represents a low or high risk for active disease.
  • the kit comprises an effective amount of a first antigen preparation.
  • the first antigen preparation comprises a multiple protein derivative of M tuberculosis or a single protein tuberculin for intradermal injection to a first site on the skin of a subject.
  • the first antigen preparation is capable of eliciting a DTH reaction in the event the subject has a mycobacterial infection.
  • the DTH reaction is in the form of an induration reaction, having a measurable diameter at the site of administration.
  • the kit further comprises an effective amount of a second antigen preparation for administration to the subject at a second site by intradermal injection.
  • the second antigen preparation is an antigen and capable of eliciting a delayed hypersensitivity reaction in the event the subject has a mycobacterial infection.
  • the DTH reaction is in the form of an induration reaction observed at 48-72 hours having a measurable diameter at the second site.
  • the kit further comprises instructions for observing the subject for the presence of DTH reactions at the first and second sites.
  • An induration diameter above the cutoff for each ofthe antigens at the respective sites of administration would be indicative ofthe presence of mycobacterial infection.
  • the induration diameters at the two sites will be such that the induration due to one ofthe antigens will measure significantly lower in individuals at high risk for active disease compared to healthy infected patients at low risk for active disease.
  • the kit further comprises the instructions for measuring the diameter ofthe induration at the first site and the diameter ofthe induration at the second site. The induration diameters ofthe two antigens are compared with characteristic values representing infected individuals at low risk for active disease versus infected individuals at high risk of active disease.
  • the first antigen is PPD and the second antigen is antigen 6.
  • Such antigens give rise to indurations diameters of a characteristic value, such that the ratio X/Y, where X and Y are as previously described equal a value Z.
  • values of Z greater than 1.05 suggest active disease or high risk of active disease.
  • Z values less than 1.05 suggest low risk of active disease.
  • the instructions specifiy that each ofthe antigen preparations is administered at least one inch apart on the volar surface ofthe left or right forearm.
  • the instructions specify that the step of observing DTH reactions is performed
  • the kit comprises suitable packaging and containment vessels for the first and second antigens.
  • the kit may comprise the first and second antigen preparations in solution contained within separate suitable vessels until used.
  • the antigen preparations may be lyophilized and contained in suitable vessels for reconstitution. In the event that the antigen preparations are in the lyophilized form, diluents, buffers and solutions may be provided in suitable vessels for reconstitution ofthe preparations.
  • the present inventions feature single protein antigens which are well characterized and can be reproduced accurately to provide consistent results.
  • the single protein antigens are capable of consistent biological activity per unit mass of antigen. There are minimal batch to batch variations.
  • the single protein antigens also provide high sensitivity for the detection of mycobacterial infection.
  • the single protein mycobacterial antigens provide clear and discreet induration reactions with minimal erythema for ease of interpretation.
  • Embodiments ofthe present invention allow sufficient specificity to differentiate between one or more ofthe following situations: tuberculosis infection versus infections due to atypical mycobacteria; exposure versus active disease; dormant low risk infection versus risk of activation; and a favorable versus unfavorable prognosis.
  • Fig. 1 depicts a kit embodying features ofthe present invention
  • Fig. 2 depicts a kit comprising features ofthe present invention
  • Fig. 3 depicts in graphical form a comparison of ratios of PPD indurations to single protein antigen indurations in active diseased subjects versus healthy, infected subjects.
  • Kit 11 is intended for use in identifying the causative agent of mycobacterial infection.
  • Kit 11 is comprised ofthe following major components, package 15, first vial 17, second vial 19, third vial 21 and instructions 31.
  • Package 15 may take many forms. Individuals skilled in the art may modify the structure to suit particular needs. As depicted, package 15 is a box-like structure for holding first vial 17, second vial 19, third vial 21 and instructions 31.
  • First vial 17 comprises at least one dose and preferably several doses of an effective amount of a first antigen preparation.
  • the antigen preparation is selected from a group consisting of multiple protein derivatives and single protein antigen derived from mycobacteria.
  • a preferred multiple protein antigen is PPD.
  • a preferred first single protein antigen is antigen 6.
  • the first vial 17 may also contain salts and buffers to make solutions isotonic.
  • the first vial 17 may also comprise preservatives and surfactants.
  • the first antigen preparation is held in solution or is held as a lyophilized powder for reconstitution. Individuals skilled in the art are knowledgeable regarding the reconstitution of such powders.
  • the kit may comprise additional vials (not shown) containing diluent, buffer or solutions for reconstitution.
  • the first antigen preparation is capable of being administered to a subject for intradermal injection to a first site on the skin ofthe subject.
  • the antigen preparation is capable of eliciting a delayed hypersensitivity reaction in the event the subject has mycobacterial infection.
  • the hypersensitivity reaction is in the form of an induration.
  • the second vial 19 contains a second antigen preparation.
  • the second antigen preparation is selected from one of a group of antigens which are tuberculosis specific antigens.
  • the second antigen preparation is capable of being administered to a subject by intradermal injection to a second site on the skin ofthe subject.
  • the tuberculosis specific antigen is an antigen specific to Mycobacterium tuberculosis that is capable of eliciting a DTH reaction in the event the subject has an infection caused by Mycobacterium tuberculosis.
  • the tuberculosis antigen does not produce a delayed hypersensitivity reaction to individuals having infection or exposure to atypical mycobacteria such as Mycobacterium avium. M. intracellulare. or M.
  • kits further comprises a third antigen preparation.
  • This third antigen preparation is contained in third vial 21.
  • the third antigen preparation is selected from the group of atypical antigens.
  • the atypical antigen is an antigen specific to Mycobacterium avium. M. intracellulare. or M. kansasii.
  • the atypical antigen is capable of eliciting a DTH reaction in the event the subject has infection caused by atypical mycobacteria such as Mycobacterium avium. M. intracellulare. or M. kansasii. but not by Mycobacterium tuberculosis.
  • the delayed hypersensitivity reaction is the form of an induration at the third site.
  • the instructions 31 direct the health practitioner to monitor the subject for the presence of delayed hypersensitivity reactions 24 to 144 hours after administration of antigens, and most preferably 48 to 72 hours after administration.
  • the instructions further instruct the health practitioner that an induration with a diameter greater than the established cut off for the first antigen at the first site is indicative of mycobacterial infection.
  • An induration with a diameter greater than the established cut off for the second antigen at the second site is indicative of infection with M. tuberculosis or of a mixed infection due to M. tuberculosis and an atypical mycobacterium.
  • the first antigen preparation is withdrawn from first vial 17 and administered to the subject at a first site by intradermal injection with a hypodermic needle.
  • the second antigen preparation is withdrawn from second vial 19 and administered to the subject at a second site by intradermal injection with a second hypodermic needle.
  • a third antigen preparation is withdrawn from third vial 21 administered to the subject at a third site by intradermal injection by a third hypodermic needle.
  • the kit 15 may contain only first vial 17, and second vial 19, and instructions 31.
  • the health practitioner reconstitutes the antigens using the diluent or buffer or solution contained in additional vials (not shown).
  • the subject is then monitored for the formation of a delayed hypersensitivity reaction at any ofthe three sites.
  • the formation of induration reactions at the three sites is evaluated in accordance with the instructions 31.
  • Kit 111 is intended for use in identifying subjects with mycobacterial infection and classifying them as being at low versus high risk for active disease.
  • Kit 111 is comprised ofthe following major components, package 115, first vial 117, second vial 119 and instructions 125.
  • Package 115 may take many forms. Individuals skilled in the art may modify the structure to suit particular needs. As depicted, package 115 is a boxlike structure for holding first vial 117, second vial 119 and instructions 125.
  • First vial 117 comprises at least one dose and, preferably, several doses of an effective amount of a first antigen preparation.
  • the first antigen preparation is selected from the group consisting of multiple protein derivatives and single protein antigens capable of eliciting a delayed hypersensitivity reaction in a subject to whom it is administered.
  • the hypersensitivity reaction is in the form of an induration having a diameter.
  • a preferred first antigen preparation is PPD.
  • the first vial 117 may also contain salts to buffer and make solutions isotonic.
  • the first vial 117 may also comprise preservative and surfactants.
  • the first antigen is held in solution or is held as a lyophilized powder for reconstitution. Individuals skilled in the art are knowledgeable regarding the reconstitution of such powders.
  • the second vial 119 comprises at least one and, preferably, several doses of an effective amount of a second antigen preparation.
  • the second antigen preparation is held in solution or is held as a lyophilized powder for reconstitution. Individuals skilled in the art are knowledgeable regarding the reconstitution of such powder.
  • the second vial may also contain salts to buffer and make the solution isotonic.
  • the second vial 119 may also comprise preservatives and surfactants.
  • the second antigen preparation is a single protein antigen capable of eliciting a delayed hypersensitivity reaction in a subject to whom it is administered.
  • the hypersensitivity reaction is in the form of an induration having a diameter.
  • a preferred second antigen preparation is antigen 6.
  • Instructions 125 direct the health practitioner to observe the subject for the presence of induration at the first and second sites 24-144 hours after administration ofthe antigens and, more preferably, 48-72 hours after administration ofthe antigens. Such induration, if present with a diameter greater than the established cut off values for each ofthe antigens, is indicative of mycobacterial infection.
  • the instructions further comprise a directive to measure the diameters ofthe induration at the first and second sites.
  • the instructions indicate that the diameters ofthe indurations at the first and second sites are to be compared, and either based on the specific diameter values or on the ratio ofthe two induration diameters which have characteristic values for active and inactive disease, it can be determined as to whether the infected subject is at low risk or high risk for contracting active disease.
  • the letter X denotes the diameter ofthe indurant ofthe first site and Y denotes the diameter ofthe induration ofthe second site.
  • values of Z about 1.05 and above suggest active infection or high risk. Values less than about 1.05 suggest low risk of active infection.
  • a health practitioner reconstitutes the first antigen contained in first vial 117 and the second antigen contained in the second vial 119 if such antigens were contained in the first vial 117 and second vial 119 as lyophilized powders.
  • the kit comprises solutions for reconstitution in one or more additional vials (not shown).
  • the health practitioner withdraws an effective amount ofthe antigen from the first vial 117 with a hypodermic needle.
  • This effective amount of antigen is injected intradermally to a first site on the skin of a subject.
  • An effective amount of a second mycobacterial antigen is withdrawn from second vial 119 with a second hypodermic needle.
  • the effective amount of a second mycobacterial antigen is administered intraderrnally to a second site on the skin of a subject.
  • the instructions 125 direct that the first and second sites on the volar surface ofthe left or right forearm be separated by approximately one or more inches.
  • the instructions direct the health practitioner to monitor the subject for the formation of indurations 24 to 144 hours after administration, and most preferably, 48 to 72 hours after administration. The appearance of induration reactions above the respective cut off values for each ofthe antigens at the first and second sites would be indicative of mycobacterial infection.
  • the instructions 125 direct the health practitioner to measure the diameters ofthe indurations, if present, at the first and second sites.
  • the instructions 125 further direct the health practitioner to compare the diameter ofthe induration at the first site to the diameter ofthe induration at the second site, and either based on the specific diameter values or on the ratio of the two induration diameters, the results are assessed to determine whether the infected subject is at low risk or high risk for contracting active disease.
  • Example 1 Making Antigen 6 Mycobacterium tuberculosis, strain H37Ra, was grown on synthetic liquid medium of
  • the preparation was concentrated by lyophilization and then reconstituted at 15-20 mL of 0.01 M sodium phosphate pH 8.0. Once again, this preparation was applied to a column under the same chromatographic conditions. Fractions that elute with an absorbance at 280 nm that is greater than that at 260 nm were pooled. The final preparation, 30,000 daltons antigen, was dialyzed against distilled water and lyophilized for storage in 200 ⁇ g concentrations.
  • Immunoelectrophoresis was performed as described by Janicki et al, "A reference system for antigens of Mycobacterium tuberculosis.” Am. Rev. Respir. Pis. 104:602-604 (1972). Reference antisera and culture filtrate antigen were supplied by Anna Tsang, National Jewish Hospital, Denver, Colo. Crossed immunoelectrophoresis with intermediate gels was performed on 5 x 7 cm glass plates as described by Closs et al., "The antigens of Mycobacterium boyjs, strain BCG. studied by crossed immunoelectrophoresis: a reference system," Scand. J. Immunol. 12:249-63 (1980).
  • Polyvalent rabbit anti-BCG immunoglobulin (lot 063) was provided by DAKO Immunoglobulins, Copenhagen, Denmark. Production of anti-BCG85 antisera K1019A and K1032B, which react with the three components of this complex, is described elsewhere. Characterization ofthe specificity of monoclonal antibodies by crossed immunoelectrophoresis was performed by comparing the precipitation pattern in the top gel in two plates containing (1) BCG culture filtrate plus saline solution or (2) BCG culture filtrate plus the monoclonal antibody in the circular antigen well or (3) rabbit anti-mouse immunoglobulin in the intermediate gel as described previously.
  • Methods of making the antigen TB66 (66-kDa of M. tuberculosis) are disclosed and taught in an article by Deshpande, R.G., et al. "Skin Reactivity and Fibronectin-Binding Property of TB66 (66-kDa protein of Mycobacterium tuberculosis)". J. Med. Microbiol. Vol. 41 (1994) 378-382.
  • Methods of making the antigen MPT 64 of M. tuberculosis are disclosed and taught in an article by Oettinger, T., et al. "Cloning and B-Cell-Epitope Mapping of MPT64 from Mycobacterium tuberculosis H37RV", Infection and Immunity.
  • MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27kDa), and MPT64 (26 kDa) are disclosed and taught in an article by Rambukkana, A., et al., "Heterogeneity of Monoclonal Antibody Reactive Epitopes on Mycobacterial 30-Kilodalton-Region Proteins and the Secreted Antigen 85 Complex and Demonstration of Antigen 85B on the Mycobacterium leprae Cell Wall Surface", Infection and Immunity. (Dec. 1992), Vol. 60, 1, 5172-5181.
  • MPB59, MPB64, MPB70, 19 kDa and 12 kDa are disclosed and taught in an article by Fifis, T., et al., "Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate", Infection & Immunity. 59(3) : 800-7, (1991 Mar.).
  • Example 2 Five male guinea pigs of albino strain were sensitized by using heat killed M- tuberculosis suspended in Marcol-52. Five guinea pigs, matched for sex and body weight, and which were never exposed to mycobacterial antigens, were used as control animals. Test animals and control animals were tested after six weeks for delayed hypersensitivity reaction to PPD and antigen 6. A 0.1 mL volume dose was administered intradermally to each animal. The induration diameter at the site of injection was measured after 24 hours. PPD was tested in 1, 2, and 4 T.U.
  • the controls were negative for skin induration.
  • the results for test animals are expressed as mean of five animals are set forth in Table 1.
  • Example 3 highlights a delayed hypersensitivity response to antigen 6 and PPD in guinea pigs sensitized with live M- tuberculosis.
  • the experimental design was similar to that in
  • Example 1 except that the animals were sensitized by challenging the animals with aerosols of live M- tuberculosis H 37 R V .
  • One and four T.U. doses of PPD were compared with a 100 ng dose of antigen 6 in five infected and two control guinea pigs.
  • the mean indurations are set forth in
  • Example 4 illustrates the lack of specificity of antigen 6 for M tuberculosis complex.
  • Six groups of five guinea pigs each were sensitized with various microorganisms. One group was sensitized using heat killed M- bovis. A second group was sensitized using heat killed M- kansasii. A third group was sensitized using heat killed M- intracellulare. A fourth group was sensitized using heat killed M- avium. A fifth group of guinea pigs were sensitized with Escherichia coli. A sixth group was sensitized with Candida albicans. The fifth and sixth groups were used to test cross reactivity with non- mycobacterial species. A seventh group of five guinea pigs were not sensitized with any organism for use as negative controls. The results are set forth in Table 3.
  • Antigen 6 100 ng 12.5 3.5 13.0 10.0
  • Induration reactions are the mean of reactions from five guinea pigs.
  • the human cell mediated immune response to antigen 6 was evaluated by conducting a leukocyte migration inhibition (LMI) assay on fresh blood samples collected from ten tuberculosis patients. Briefly, 10-20 mL blood was drawn in heparin vials from each patient. The sample was immediately processed to separate the leukocytes. Capillaries were charged with the leukocytes and placed in a specially designed migration chamber enriched with RPMI 1634 cell culture medium. The chambers were sealed and incubated in humid at 37oC for minimum 18 hours. After the incubation, the area of migration was measured and results expressed as migration ratio (M.R.).
  • LMI leukocyte migration inhibition
  • Ratio values less than 0.8 were considered positive. Ratio values greater than 0.8 were considered negative.
  • antigen 6 is capable of generating a cell mediated immune response in humans exposed to tuberculosis infection.
  • guinea pigs were sensitized by a parenteral route in groups of five animals each using M. tuberculosis. M. bovis. M. kansasii. M. intracellulare. and M. avium. respectively for each ofthe groups.
  • An unsensitized group of five guinea pigs was used as a control.
  • the animals were tested for delayed hypersensitivity reaction using Purified Protein Derivative (PPD) in 1 T.U. and 4 T.U. doses, antigen 6 in 100 ng dose and antigen MPB64 in 200 ng and 400 ng doses.
  • PPD Purified Protein Derivative
  • antigen 6 in 100 ng dose
  • antigen MPB64 in 200 ng and 400 ng doses.
  • the induration reaction to these antigens was measured 24 and 48 hours after the intradermal injections. The results are set forth in Table 5 below:
  • Antigen 6 100 ng 13.6 12.5 3.5 13.0 10.0
  • the results are the mean induration diameters in each group of animals.
  • the control group did not show any reactions.
  • PPD reacted significantly in all the groups.
  • Antigen 6 showed significant reactions in all the groups except M. kansasii (mild reaction), thus indicating the cross-reactivity of this antigen among the mycobacterial species.
  • Antigen MPB64 showed significant reaction only in the M. tuberculosis group, indicating its specificity to M tuberculosis.
  • Example 7 Ten (10) bacteriologically confirmed cases of tuberculosis and 10 healthy volunteers who had long-term exposure to tuberculosis infection were recruited.
  • the tuberculosis cases were the indoor patients of TB Group of Hospitals, Sewri, Bombay, India.
  • the healthy volunteers were the attendants working in the hospital wards and mostly living in the hospital campus.
  • Each study subject was administered intradermal injections of 5 T.U. PPD and 100 ng Antigen 6 each on the volar surface of their forearms.
  • the induration diameters were read after 48 and 72 hours. Except one patient and one healthy contact all ofthe subjects gave positive reactions (induration diameters greater than or equal to ten mm) to both the antigen preparations.
  • the tuberculin positive subjects were further analyzed to compare the reaction characteristics of PPD and Antigen 6. Antigen 6 reactions were generally better defined than PPD.
  • PPD Antigen 6 ratios were calculated to investigate if there is any statistical significance in these two types of tuberculin for responders with active tuberculosis versus healthy contacts.
  • the active tuberculosis group had significantly high mean ratios compared to healthy contacts (p ⁇ 0.01).
  • Fig. 3 is a graphical representation ofthe ratios.
  • X/Y Z where X is the diameter of induration formed by PPD and Y is the diameter of induration formed by antigen 6.

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Abstract

La présente invention concerne l'utilisation d'antigènes purifiés de protéines isolées, comme antigènes de réaction pour induire une réaction immunitaire à médiation cellulaire, en tant que moyen de détection d'une infection mycobactérienne chez l'homme ou l'animal. Une réaction immunitaire à médiation cellulaire est manifeste chez le sujet, en tant que réaction cutanée à hypersensibilité retardée, après l'introduction, par injection intradermique, d'antigènes purifiés de tuberculine de protéine isolée. L'utilisation d'une combinaison de deux, ou plus, antigènes de protéines isolées ayant différentes particularités d'espèces permet un diagnostic différencié de l'infection tuberculeuse par opposition aux infections dues à des mycobactéries atypiques telles que Mycobacterium avium, Mycobacterium intracellulaire ou Mycobacterium kansasii, et elle permet aussi de classer les individus infectés en distinguant ceux qui courent un faible risque de contracter une maladie évolutive de ceux pour qui le risque est important.
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WO1992021697A2 (fr) * 1991-05-24 1992-12-10 Medical Research Council Peptides diagnostiques derives d'antigenes de la m.tuberculose
WO1995001440A1 (fr) * 1993-07-02 1995-01-12 Statens Seruminstitut Test cutane de diagnostic de la tuberculose

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992021697A2 (fr) * 1991-05-24 1992-12-10 Medical Research Council Peptides diagnostiques derives d'antigenes de la m.tuberculose
WO1995001440A1 (fr) * 1993-07-02 1995-01-12 Statens Seruminstitut Test cutane de diagnostic de la tuberculose

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MEDLINE ABSTRACT, Accession No. 93323428, issued May 1993, E. SHIGETO et al., "Tuberculin Sensitivity to Purified Protein Derivatives from Mycobacterium Other Than Tuberculosis (PPD-B, PPD-Y, PPD-F and PPD-C) and PPDs Among Patients with Mycobacteriosis". *
SOUTHERN MEDICAL JOURNAL, USA, Volume 71, No. 8, issued August 1978, A.E. PITCHENIK et al., "PPD-Tuberculin and PPD-Battey Dual Skin Testing of Hospital Employees and Medical Students", pages 917-918. *
SOUTHERN MEDICAL JOURNAL, USA, Volume 80, No. 1, issued January 1987, H.M. VANDIVIERE et al., "Atypical Mycobacteria Causing Pulmonary Disease: Rapid Diagnosis Using Skin Test Profiles", pages 5-7. *
THE AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, Volume 30, No. 5, issued September 1981, P. BROWN et al., "Mycobacterial and Fungal Skin Sensitivity Patterns Among Remote Population in Papua New Guinea and in the New Hebrides, Solomon and Caroline Islands", pages 1085-1093. *
TUBERCLE, Volume 72, issued 1991, L.O. LARSSON et al., "Sensitivity to Sensitins and Tuberculin in Swedish Children", pages 37-42. *

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