WO1996041171A1

WO1996041171A1 - Methods and articles of manufacture for diagnosis of mycobacterial infection

Info

Publication number: WO1996041171A1
Application number: PCT/US1996/010023
Authority: WO
Inventors: Fazal S. Raheman; Saraswathy V. Nochur; Peter J. Mione
Original assignee: Dynagen, Inc.
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1996-12-19

Abstract

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. 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 of 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.

Description

METHODS AND ARTICLES OF MANUFACTURE FOR DIAGNOSIS OF MYCOBACTERIAL INFECTION

Field of Invention

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. 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.

Background

The term "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. As the first tuberculin, the Koch formulation is often referred to as "old tuberculin" (OT). There are other types of tuberculins; however, only two are commonly used throughout the world. These are OT and "purified protein derivative" (PPD). PPD was first prepared by Florence Seibert by ammonium sulphate precipitation of M. tuberculosis culture filtrate. In 1939, 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. (Seibert, F.B. and Glenn, J.T., Amer. Rev. of Tuberculosis, Vol. 44:9, 1941). In 1959, The World Health Organization (WHO) sponsored the manufacture of another large batch of PPD, called PPD RT 23. Currently, 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.

Although it has been in use worldwide for more than three decades, PPD is far from being the ideal tuberculin antigen. A WHO document dated February 2, 1963 issued in connection with PPD states, "it is expected that this batch (PPD RT 23) will suffice until eventually a much better kind of tuberculin than PPD has been developed, and even after that time will be available in cases in which such a new kind of tuberculin is not adopted for some particular reason. Thus, RT23 may well be the last batch of PPD (for intradermal testing) ever to be prepared." The emergence of acquired immune deficiency syndrome (AIDS) has brought a resurgence of tuberculosis in the Western World. Individuals with AIDS are also susceptible to mycobacterial disease from organisms not normally pathogenic in humans. These "atypical" mycobacteria comprise M. avium. M. intracellulare. and M. kansasii. among others. Presently there are no diagnostic tests to distinguish between infection due to M. tuberculosis versus infection due to atypical mycobacteria.

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, however, is for a prolonged period of time, at least six months. Such long term treatment with isoniazid, unfortunately, is associated with liver disfunction and toxicity and other side effects. Infections caused by atypical mycobacteria, on the other hand, 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. Summary of the Invention

The present invention features a method and a kit for distinguishing mycobacterial infected individuals from uninfected individuals.

As used herein, the term "subject" refers to an individual or animal that is the object of clinical study.

As used herein, 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.

As used in the present application, 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. Thus, 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. As used herein, 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. Preferably, 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. In the event the second antigen preparation is an atypical antigen, a positive result at both sites suggests infection with an atypical mycobacterial species. In the event 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.

Preferably, 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. In the event that positive DTH reactions are observed only at the first and second sites, where the second site is a tuberculosis antigen, such results suggest either infection with M. tuberculosis or a mixed infection due to M. tuberculosis and atypical mycobacteria other than the species of mycobacterial from which atypical antigen is derived. In the event a positive DTH reaction is observed at all three sites where the second site is a tuberculosis antigen, such results suggest infection with M. tuberculosis and such atypical mycobacterial from which the atypical antigen was derived and possibly other atypical mycobacterium. In the event that positive DTH reactions are observed at the first site and the site ofthe atypical antigen, but not at the site ofthe tuberculosis antigen, such results suggest infection with the particular atypical mycobacterial species from which the atypical antigen was derived, or a mixed infection with the particular atypical mycobacterial species and another atypical mycobacterium, but not tuberculosis infection. A negative DTH reaction at the first site suggests the subject does not have, and has not been exposed to, mycobacterial antigens. Based on the activities ofthe various antigen preparations in large human populations, a positive DTH reaction is determined. A cut off induration diameter is determined for positive results in each antigen preparation. This cut off is determined for the pan-specific mycobacterial antigen versus the tuberculosis specific antigen, versus the antigen specific for the atypical mycobacterium. Thus, 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.

Preferably, the tuberculosis antigen is selected from the group consisting of MPT64/MPB64 or TB 66.

Preferably, 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. And, 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.

In the event that a positive DTH reaction is observed only at the first site, and the second antigen is a tuberculosis antigen, such results indicate that the subject is infected with a mycobacterial species which is not M. tuberculosis. In the event the second antigen is an atypical antigen, and a positive DTH reaction is observed only at the first site, such results suggest the subject is infected with a mycobacterial species other than the atypical mycobacterium from which the atypical antigen was derived.

In the event that positive DTH reactions are observed at the first and second sites and the second antigen is a tuberculosis antigen, such results suggest either infection with M. tuberculosis or a mixed infection due to M. tuberculosis and atypical mycobacteria.

A negative DTH reaction at the first site suggests lack of exposure to mycobacterial antigens. Preferably, the kit 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.

In the event that 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. In the event that 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. tuberculosis and the atypical mycobacterial species from which the third single protein antigen was derived, and possibly other atypical mycobacterial species as well. 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. Preferably, the tuberculosis antigen is selected from the group consisting of

MPT64/MPB64 or TB 66 antigens.

Preferably, the atypical antigen is specific for M. avium or M. avium complex Preferably, the atypical antigen is selected from the group consisting of 12kD and 43kD antigens. Preferably, the instructions specify that each ofthe antigen preparations is administered at least one inch apart on the volar surface ofthe left or right forearm.

Preferably, 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. Preferably, 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. Alternatively, 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. In the event an induration is present at the first and second sites, 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.

Preferably, 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:

- z. y

where 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.

Preferably, 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. Where 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. Preferably, 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. Preferably, 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. Based on the antigens used, 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. In the event an induration is present at the first and second sites, 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.

Preferably, 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. At relative concentrations such as those set forth in the Examples, 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.

Preferably, the instructions specifiy that each ofthe antigen preparations is administered at least one inch apart on the volar surface ofthe left or right forearm. Preferably, the instructions specify that the step of observing DTH reactions is performed

24 to 144 hours after the administration ofthe antigen preparations. More preferably, the observing step is performed 48 to 72 hours after administration. Preferably, 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. In the alternative, 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.

Other features and advantages ofthe present invention will be apparent from the following description which by way of illustration shows preferred embodiments to the present invention and the principles thereof in what is now considered to be the best mode to apply these principles.

Brief Description of he Drawings

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.

Detailed Description This invention will be described in detail with respect to a method and article of manufacture featuring single protein antigens. The articles of manufacture are described with respect to two kits. A first kit, generally designated by the numeral 11. is depicted in Figure 1. 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. In the event one or more antigens are lyophilized 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. kansasii . The delayed hypersensitivity reaction is in the form of an induration at the second site. Preferably, the kit 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.

Individuals skilled in the art will recognize that additional antigens to different mycobacterial species may be utilized in substitution or in addition to the third antigen preparation.

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. In the event that no induration is formed at the second site or that the induration diameter at the second site is below the cut off for the second antigen, such results suggest infection due to atypical mycobacteria such as M. avium. M. intracellulare. or M. kansasii. In the event that a third antigen is used which is specific to a particular atypical mycobacterial species, an induration with a diameter greater than the cut off for this third antigen at the third site would suggest infection due to the particular atypical mycobacterial species. A positive response at the first site and, the lack of induration or an induration diameter below the cut off for the third antigen at the third site would indicate infection due to M. tuberculosis or due to some other atypical mycobacterial species. A positive DTH reaction at all three sites would indicate mixed infections due at least to M. tuberculosis and the particular atypical mycobacterial species from which the third antigen was derived. In operation, 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. In the event that the third single protein antigen preparation is not deemed necessary, the kit 15 may contain only first vial 17, and second vial 19, and instructions 31. In the event that the single protein antigens are contained as lyophilized powders, 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.

A second kit, generally designated by the numeral 111 is depicted in Fig. 2. 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.

Where the first antigen is PPD and the second antigen is antigen 6, the diameter values are compared with the formula X/Y = Z. The letter X denotes the diameter ofthe indurant ofthe first site and Y denotes the diameter ofthe induration ofthe second site. At relative concentrations as set forth in the Examples, 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.

In operation, 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. Preferably, the kit comprises solutions for reconstitution in one or more additional vials (not shown).

After reconstitution, or in the event the first antigen preparation and second antigen preparation are contained as solutions within the first vial 117 or second vial 119, 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. In addition, 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.

The advantages and features ofthe present invention will be further highlighted by reference to the following examples.

Example 1 - Making Antigen 6 Mycobacterium tuberculosis, strain H37Ra, was grown on synthetic liquid medium of

Proskauer - Beck. The cells were separated by filtration and the culture filtrate was sterile filtered through 0.22 micron filters and dialyzed against distilled water for 48 hours at 4°C with many changes of dialysate.

Dry ammonium sulfate was added to the culture filtrate slowly with constant stirring at 4°C overnight, reaching 50% ammonium sulfate saturation. The culture filtrate was centrifuged at 15,000 rpm for 20-30 minutes to recover the precipitate. The precipitate was reconstituted in distilled water and dialyzed against 0.01 M sodium phosphate, pH 8.0 for 48 hours at 4°C with many changes of dialysate. A DEAE-cellulose column was prepared by packing a 33 x 2.5 cm column uniformly with diethylaminoethyl-cellulose (DEAE) in 0.1 N NaOH. Approximately 20 mg of ammonium sulfate preparation in 5 mL buffer was applied with a graduated pipette to the top of the column. Approximately 150-200 mL of 0.01 M sodium phosphate buffer was added to the column before applying a linear gradient, starting with 100%) of 0.01 M sodium phosphate, pH 8.0 and ending with 100%) of 0.3 M sodium phosphate pH 5.6. The 30,000 daltons antigen starts to elute from the column at 0.07 M concentration. Four to five mL fractions were collected and the absorbance was determined by a spectrophometer at 260 nm and 280 nm. Fractions having an absorbance at 280 nm that was greater than that at 260 nm were pooled. Fractions resulting from 5-6 runs were pooled and dialyzed against distilled water. 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 μg concentrations.

Antigen characterization. SDS-PAGE was performed as described by LaemmLi U.K., "Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4," Nature 227:680-685 (1970). Gels were stained with Coomassie blue dye. Molecular weights were estimated by the method of Shapiro et al, "Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels," Biochem. Biophys. Res. Commun. 28:815-820 (1967). Immunoblotting was performed as described by Towbin et al, "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications," Proc. Natl. Acad. Sci. USA 76:4350-4354 ( 1979). For this procedure, monoclonal antibodies TB-C-25, -26, -27, -28, -29 and -30 were used as hybridoma cell culture supernatants or were precipitated with ammonium sulfate at 50%> saturation from ascitic fluid and then used in concentrations of 0.04 to 0.10 mg/mL. Alkaline phosphate-conjugated rabbit anti-goat IgG (Sigma Chemical Co., St. Louis, Mo.) and goat anti-mouse immunoglobulins (Organon Teknika Corp., West Chester, Pa.) were used as secondary antibodies, and the blots were developed by using the ProtoBlot Western Blot Alkaline Phosphatase System (Promega Corp., Madison, Wis).

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. May 1994, 2058-2064. Methods of making five antigens, 17 kDa, 24 kDa, 38 kDa, 17-19 kDa, and 32-33 kDa, are disclosed and taught in an article by Haslov, K., et al., "Comparison ofthe Immunological Activity of Five Defined Antigens from Mycobacterium tuberculosis in Seven In Bred Guinea Pig Strains. The 38-kDa Antigen is Immunodominant", Scand J. Immunology. 31, 503-514 (1990). Methods of making 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.

Methods of making the antigens MPT32, MPT45, MPT51, MPT53, and MPT63, and MPT46 are taught and disclosed in an article by Nagai, S., et al., "Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis". Infection and Immunity. 59 (1): 372-82, (1991 Jan). Methods of making the antigens MPT44, MPT59, MPT45, MPT51, MPT64 are taught and disclosed in an article by Rambukkana, A., et al., "Identification and characterization of epitopes showed between the mycobacterial 65- kilodalton heat shock protein and the actively secreted antigen 85 complex: their in situ expression on the cell wall surface of Mvcobacteium leprae". Infection and Immunity. 60(11): 4517-27, 1992, Nov. Methods of making antigen 85 are disclosed and taught in an article by Wiker, HG. et al., "Quantitative and qualitative studies on the major extracellular antigen of

Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG", American Review of

Respiratory Disease. 141 (4 Pt 1) : 830-8, (1990 Apr.).

Methods of making BCG antigens 60, 63, 82, 89, 23, 27, 71, 84 are disclosed and taught in an article by Harboe M., "Antigens of PPD, old tuberculin, and autoclaved Mycobacterium bovis

BCG studied by crossed immunoelectrophoresis". Review of Respiratory Disease. 124(1) : 80-7,

(1981 Jul.).

Methods of making the antigen MPB64, 24 kDa protein and 38 kDa protein are disclosed and taught in an article by Andersen, A.B. et al., "MPB 64 possesses 'tuberculosis-complex'- specific B- and T-cell epitopes", Scandinavian Journal of Immunology. 34(3) : 365-72, (1991

Sep.).

Methods of making the antigen MPT51, 85 A, 85B, 85C, and MPT64 are disclosed and taught in an article by Wiker, H.G. et al., "A family of cross-reacting proteins secreted by

Mycobacterium tuberculosis", Scandinavian Journal of Immunology. 36(2) : 307-19, (1992 Aug.).

Methods of making the antigens MPB59, MPB64, MPB70, MPB8 and 85B are disclosed and taught in an article by Wiker, H.G., et al., "MPB59, a widely cross-reacting protein of

Mycobacteium bovis BCG," International Archives of Allergy & Applied Immunology. 81(4) :

307-14, (1986). Methods of making the antigens 32 kDa, 30 kDa, 24 kDa, 22 kDa, 19 kDa, 12 kDa, p.32,

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.).

Methods of making the 43kDa antigen are disclosed and taught in an article by Deshpande, R.G., et al. "Isolation of 43kDa protein from M. Tuberculosis H37RV and its identification as a pyridine nucleotide transhydroxylase," J. of Applied Biology (1994) Vol. 77, 639-643.

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.

(Tuberculin Unit) doses and antigen 6 was tested in 100 ng, 200 ng, and 400 ng doses.

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.

TABLE 1 Delayed Hypersensitivity Reactions to PPD and Antigen 6 in Guinea Pigs Infected With

Heat-killed M. tuberculosis

Mean Induration Reaction

Antigen Dose ( mm) PPD 1 T.U. 9.8 PPD 2 T.U. 10.8 PPD 4 T.U. 12.3 Antigen 6, 100 ng 12.8 Antigen 6, 200 ng 13.8 Antigen 6, 400 ng 15.00

The results suggest that skin indurations caused by antigen 6 are a measure of cell mediated immunity to mycobacteria and a means to diagnose tuberculosis infection.

Example 3

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 H₃₇R_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

Table 2.

TABLE 2 Delayed Hypersensitivity Reactions to PPD and Antigen 6 in Guinea Pigs Infected With Live M. tuberculosis

Mean Transverse Induration

Antigen Dose (mm)

PPD 1 T.U. 6.7 PPD 4 T.U. 16.1 Antigen 6, 100 ng 13.6

The results suggest that skin indurations caused by antigen 6 are a measure of cell 0 mediated immunity to mycobacteria and a means to diagnose tuberculosis infection. Example 4

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.

TABLE 3 Delayed Hypersensitivity Reactions to PPD and Antigen 6 in Guinea Pigs Infected With Various Bacteria

Mean Induration fmm] in Guinea Pigs* Sensitized With**: Antigen Doses M. bovis M. kansasii M. intracellulare M.avium

PPD 1 T.U. 7.4 7.25 5.25 4.8

PPD 4 T.U. 13.8 13.5 12.5 10.0

Antigen 6, 100 ng 12.5 3.5 13.0 10.0

* Induration reactions are the mean of reactions from five guinea pigs.

** The guinea pigs sensitized with heat killed Escherichia coli and Candida albicans and the control group unsensitized with any organism did not show any activity.

The results suggest that skin test reaction attributable to antigen 6 is a measure of cell mediated immunity to the genus Mycobacterium and a means to diagnose mycobacterial infection.

Example 5

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.).

Mean Area of Migration in the Presence of Antigen in Chamber M.R.=

Mean Area of Migration in Control Chamber

TABLE 4

In Vitro Leukocyte Migrati ion Inhibition

PPD SPT™ I

Patient 5 TU/0.5 mL 100 ng/0.5 mL 10 ng/0.5 mL

1 0.68

2 0.17 0.13 1.00

3 0.36 0.45 1.00

4 0.90 1.00 0.80

5 - 0.55 -

6 0.80 0.50 0.60

7 0.51 1.30 0.50

8 0.50 1.18 0.62

9 0.40 0.79 0.50

10 0.75 0.67 0.50

Ratio values less than 0.8 were considered positive. Ratio values greater than 0.8 were considered negative.

As can be seen from the data, it is clear that antigen 6 is capable of generating a cell mediated immune response in humans exposed to tuberculosis infection.

Example 6

Twenty-five (25) 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. After six weeks, 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. 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:

TABLE 5 Delayed Hypersensitivity Reactions to PPD, Antigen 6, and MPB64 in Guinea Pigs Infected With Various Mycobacteria

Induration Size (in mm) in Guinea Pigs Sensitized With Antigen Dose M. tuberculosis M. bovis M. kansasii M. intracellulare M. avium

PPD 1 T.U. 6.7 7.4 7.3 5.3 4.8 4 T.U. 16.1 13.8 13.5 12.5 10

MPB64

400 ng 10.2 3.3 1.0 1.0 1.0

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.

Mean induration diameters in healthy contacts of TB patients were not significantly different between PPD and antigen 6. But induration diameters in the patients with active tuberculosis were significantly lower with antigen 6 as compared to 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).

These data are represented in Fig. 3. 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.

In Fig. 3, individuals infected with actual disease are indicated with a blacked-out or solid bar. Individuals infected without active disease are indicated with a vertically striped bar. Individuals without active disease did not have a PPD:antigen 6 induration ratio greater than 1.05. Individuals with active disease had induration ratios greater than 1.05. These results are set forth in Table 6 below:

TABLE 6

Delayed Hypersensitivity Reactions in Humans in Bacteriologically Confirmed Tuberculosis Cases and Healthy Contacts

Induration Reactions (in mml in TB Patients Tested With

Subject Code 5T.U.PPD 100 ng Antigt 2n 6

48 Hours 72 Hours 48 Hours 72 Hours

P-l 20 21 11 14*

P-3 19 NR 13* NR

P-4 0 0 0 0

P-5 18 23 14 14

P-6 16 16 10 10

P-7 15 16 14 8

P-8 0 0 0 0

P-9 20 18 13* 12*

P-10 15 17 11 9

P-l l 17 17 14* 12*

Mean ± SD (reactors only) 17.5+2.1 12.5+1.6

Mean PPD/ Antigen 6 ratio = 1.4

Induration reactions (mm) in Healthy Contacts Tested With: Subject Code 5T.U.PPD lOO ng Antigi en 6

48 Hours 72 Hours 48 Hours 72 Hours

C-l 16 16 17 14* C-2 15 15 17 16* C-3 17 13 21 16 C-4 17 16 15 15* C-5 11 12 12 9 C-6 13 14 14 11 * C-7 3 NR 0 NR C-9 14 NR 13* NR C-10 22 NR 16* NR

C-l l 14 NR 10.5 NR

Mean±SD (reactors only) 15.4+3.1 15.1+3.1 MeanPPD/Antigen 6 ratio = 1.02

* Well-defined induration. NR = Not Recorded.

These results suggest that while the ratio of PPD to SPT induration diameters are similar in the infected healthy contacts of tuberculosis patients, there is a significant difference in the induration diameters between PPD and antigen 6 among the tuberculosis patients. This aspect of the invention may be used to distinguish individuals with active disease versus healthy individuals who have mycobacterial infection.

Various details ofthe present invention may be changed without departing from the scope ofthe invention. The foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

The invention is defined by the following claims.

Claims

1. A method of identifying mycobacterial infection in a subject and determining the causative agent ofthe mycobacterial infection comprising:

(a) administering to a subject, by intradermal injection to a first site on the skin of said subject first antigen preparation selected from the group consisting of multiple protein derivative and a first single protein antigen, said first antigen preparation capable of eliciting a delayed hypersensitivity reaction in the event said subject has a mycobacterial infection, said delayed hypersensitivity reaction in the form of an induration at said first site, said induration having a diameter, which diameter above a characteristic value for such first antigen constituting a positive response and below such value constituting a negative response;

(b) administering to a subject, by intradermal injection to a second site on the skin of said subject, a second antigen preparation, said second antigen preparation selected from the group of antigens consisting of a tuberculosis antigen and an atypical antigen, said tuberculosis antigen specific to M tuberculosis, but not specific to atypical mycobacteria such as Mycobacterium avium. M.intracellulare. or M. kansasii. said tuberculosis antigen capable of eliciting a delayed hypersensitivity reaction in the event said subject has an infection caused by M. tuberculosis, but not by M. avium. M. intracellulare. or M. kansasii. said delayed hypersensitivity reaction in the form of an induration, said atypical antigen consisting of an antigen specific to atypical mycobacteria such as M_ avium. M.intracellulare. or M.kansasii. but not Mycobacterium tuberculosis, said atypical antigen capable of eliciting a delayed hypersensitivity reaction in the event said subject has an infection caused by the specific atypical mycobacterium such as M. avium. M.intracellulare. or M. kansasii. from which said atypical antigen was derived, but not Mycobacterium tuberculosis, said delayed hypersensitivity reaction in the form of an induration, said induration having a diameter, which diameter above a characteristic value for such first antigen constituting a positive response and below such value constituting a negative response;

(c) observing said subject for the formation of an induration at said first site and an induration at said second site, the presence or absence of one or more induration indicating a positive or negative response are indicative ofthe following: (i) a positive response at said first site is indicative ofthe presence of mycobacterial infection, or exposure to mycobacteria antigens, a negative response indicates that the subject is free of mycobacterial infection or exposure;

(ii) (a) in the event said second antigen is a tuberculosis antigen, a positive response at said second site, is indicative of infection with Mycobacterium tuberculosis, and a negative response at the second site and a positive response at said first side is indicative of infection with atypical mycobacterial species; and

(ii) (b) in the event said antigen is a typical antigen, a positive response at siad second site is indicative of infection or exposure with the atypical mycobacterial species from which the atypical antigen was described, and, a negative response at the second site with a positive response at the first site is indicative of infection with M. tuberculosis or with atypical mycobacteria different from the species from which the third antigen was derived.

The method of claim 1 wherein said second antigen is a tuberculosis antigen.

The method of claim 1 wherein said second antigen is an atypical antigen.

4. The method of claim 1 further comprising a third antigen preparation, said third antigen selected from the group consisting of tuberculosis antigen and atypical antigen which is not said second antigen, said third antigen preparation administered to said subject by intradermal injection to a third site, said step of observing, in the event said three sites are positive indicative of a mixed infection of M. tuberculosis and the atypical mycobacterial species from which said atypical antigen was derived, and a negative response at the site ofthe atypical antigen with a positive response at the site ofthe tuberculosis antigen suggesting an infection with the atypical mycobacteria from which atypical antigen is derived, and a positive response at the site of tuberculosis antigen and a negative response at the site of said atypical antigen suggesting infection within M. tuberculosis but not the atypical mycobacterial from which atypical antigen is derived.

5. The method of claim 1 wherein said first mycobacterial antigen preparation is selected from a group of antigens consisting of antigen 6, purified protein derivative, 12kD, 30kD, and 38kD.

6. The method of claim 1 wherein said tuberculosis antigen is selected from the group consisting of MPB64/MPT64 and TB66.

7. The method of claim 1 wherein said atypical antigen is selected from a group consisting of 12kD and 43kD.

8. The method of claim 1 wherein said first and antigen preparations is administered at least one inch apart on the volar surface of said subject's left or right forearm.

9. The method of claim 1 wherein said step of observing the first and second sites is performed 24 to 144 hours after the administration ofthe antigens.

10. The method of claim 1 wherein said step of observing the first and second sites is performed 48 to 72 hours after administration ofthe antigens.

11. A kit for identifying mycobacterial infection in a subject, and determining the causative agent of mycobacterial infection comprising:

(a) an effective amount of a first antigen preparation for administration by intradermal injection to a first site on the skin of said subject, said first antigen preparation selected from multiple protein derivatives and single protein antigens capable of eliciting a delayed hypersensitivity reaction in the event said subject has a mycobacterial infection, said hypersensitivity reaction in the form of an induration at said first site, said induration having a diameter, which diameter above a characteistic value is a positive response and below said value is a negative response; (b) an effective amount of a second antigen preparation for administration by intradermal injection to a second site on the skin of said subject, said second antigen preparation selected from the group consisting of a tuberculosis antigen and an atypical antigen, said tuberculosis antigen specific to M. tuberculosis, but not specific to atypical mycobacteria such as Mycobacterium avium. M.intracellulare. or M. kansasii. said tuberculosis antigen capable of eliciting a delayed hypersensitivity reaction in the event said subject has an infection caused by M. tuberculosis, but not M. avium. M. intracellulare. or M. kansasii said delayed hypersensitivity reaction in the form of an induration, said induration having a diameter, said atypical antigen consisting of an antigen specific to atypical mycobacteria such as M. avium. M.intracellulare. and M.kansasii. but not Mycobacterium tuberculosis, said antigen capable of eliciting a delayed hypersensitivity reaction in the event said subject has an infection caused by a atypical mycobacterium such as M. avium. M.intracellulare. or M. kansasii. but not Mycobacterium tuberculosis, said delayed hypersensitivity reaction in the form of an induration said induration having a diameter, which diameter above a characteristic value is a positive response and below said value is a negative response;

(c) instructions for observing said subject for the formation of an induration at said first site and an induration of said second site, and determining whether such indurations are a positive response or negative response

(i) which positive response at said first site is indicative ofthe presence of mycobacterial infection, which positive response at said second site is indicative of infection with Mycobacterium tuberculosis, in the event said second antigen preparation is tuberculosis antigen, and is indicative of infection with the atypical mycobacterial species such as M. avium. M.intracellulare or M. kansasii if said second antigen is atypical antigen;

(ii) which positive response at said first site and negative response at said second site is indicative of infection with atypical mycobacteria other than M. tuberculosis, in the event said second antigen preparation is tuberculosis antigen, and which negative response is indicative of infection with

M .tuberculosis or with atypical mycobacteria different from the species from which atypical antigen was derived in the event atypical antigen is said second antigen preparation.

(iii) which negative response at said first site suggests an absence of mycobacterial infection and lack of exposure to mycobacterial antigens.

12. The kit of claim 11 further comprising a third antigen preparation, said third antigen selected from the group consisting of tuberculosis antigen and atypical antigen which is not second antigen and said instruction further direct for observing for the formation of an induration at said first site and an induration at said second site and an induration at said third site, and determining whether such induration is a positive response or a negative response, a positive response at all three sites is indicative of a mixed infection due to M. tuberculosis and the atypical mycobacterium from which said third antigen was derived.

13. The kit of claim 11 wherein said first antigen preparation is selected from a

J 5 group of antigens consisting of antigen 6, purified protein derivative, 12kD, 30kD, and 38kD.

14. The kit of claim 11 wherein said tuberculosis antigens is selected from the group consisting of MPB64/MPT64 and TB66.

10 15. The kit of claim 11 wherein said atypical antigen is selected from a group of antigens consisting of 12kD and 43kD.

16. The kit of claim 11 wherein each ofthe antigen preparations is administered at least one inch apart on the volar surface of said subject's left or right forearm.

15

17. A method of identifying mycobacterial infection in a subject, and determining whether such infection, if present, presents the subject with a low risk or a high risk of contracting active disease, comprising the steps of:

(a) administering to said subject, by intradermal injection to a first site on 20 said subject's skin, an effective amount of a first antigen preparation, said first antigen preparation being capable of eliciting a delayed hypersensitivity reaction in the event said subject has a mycobacterial infection, said delayed hypersensitivity reaction in the form of an induration having a diameter at said first site;

(b) administering to a subject by intradermal injection to second site on said 25 subject's skin, an effective amount of a second antigen preparation for intradermal injection to a second site on said subject's skin, said second antigen preparation capable of eliciting a delayed hypersensitivity reaction in the event said subject has a mycobacterial infection, said delayed hypersensitivity reaction in the form of an induration having a diameter at said second site; 30 (c) observing said subject for presence of an induration at said first and second sites, said indurations, if present with diameters above the cut off values for the respective antigens, is indicative ofthe presence of mycobacterial infection; and, (d) measuring the diameters of induration at both sites and comparing the induration diameter ofthe first antigen preparation to the induration diameter elicited by the second antigen preparation, such results determining whether the subject is at low versus high risk for contracting active disease.

18. The method of claim 17 wherein said first antigen is a multiple protein derivative.

19. The method of claim 18 wherein said multiple derivative is purified protein derivative.

20. The method of claim 17 wherein said second antigen is antigen 6.

21. The method of claim 17 wherein each of the antigen preparations is administered at least one inch apart on the volar surface of said subject's left or right forearm.

22. The method of claim 17 wherein said step of observing the sites of administration ofthe antigens is performed 24 to 144 hours after the administration ofthe antigens.

23. The method of claim 17 wherein said step of observing the sites of administration ofthe antigens is performed 48 to 72 hours after administration ofthe antigens.

24. The method of claim 17 wherein said comparison is made in accordance with the ratio X/Y = Z wherein X is the induration diameter of said first antigen and Y is the induration diameter of said antigen and Z is a characteristic value representing a cutoff between high risk and low risk of contracting active disease.

25. A kit for identifying mycobacterial infection in a subject, and determining whether such infection, if present, places the subject at a low risk or a high risk of contracting active disease, comprising: (a) an effective amount of a first antigen preparation for administration by intradermal injection to a first site on said subject's skin, said first antigen preparation being capable of eliciting a delayed hypersensitivity reaction in the event said subject has a mycobacterial infection, said delayed hypersensitivity reaction in the form of an induration at said first site, which induration has a diameter;

(b) an effective amount of a second antigen preparation for administration by intradermal injection to second site on said subject's skin, said antigen capable of eliciting a delayed hypersensitivity reaction in the event said subject has a mycobacterial infection, said delayed hypersensitivity reaction in the form of an induration having a diameter at said second site, said induration having a diameter;

(c) instructions for observing said subject for presence of an induration at said first and second sites, said indurations, if present with diameters above the cut off values for the respective antigens, is indicative ofthe presence of mycobacterial infection; and

(d) instructions for measuring the diameters ofthe induration at both sites and comparing the induration diameters ofthe first antigen preparation to the induration diameter elicited by the second antigen preparation, such results determining whether the subject is at low versus high risk for contracting active disease.

26. The kit of claim 25 wherein said comparison is performed in accordance with the ratio X/Y = Z wherein X is the induration diameter of said first antigen and Y is the induration diameter of said antigen and Z is compared to a characteristic value representing a cut off between high risk and low risk of contracting active disease.

27. The kit of claim 25 wherein said second antigen preparation is a single protein antigen.

28. The kit of claim 27 wherein said single protein antigen is antigen 6.

29. The kit of claim 25 wherein said first antigen is a multiple protein derivative.

30. The kit of claim 29 wherein said multiple protein derivative is a purified protein derivative.

31. The kit of claim 26 wherein said first antigen preparation is purified protein derivative and said second antigen preparation is antigen 6, said value of Z is compared to a value of approximately 1.05, plus or minus two standard deviations, said value of Z below said characteristic value representing low risk and values above said characteristic value representing high risk.