HK1081437B - Use of mycobacterial antigen mpb64 in the manufacture of a product for topical detection of active tuberculosis - Google Patents

Description

Application of mycobacterial antigen MPB64 in preparation of product for locally detecting active tuberculosis

Cross reference to related applications

This application is a divisional application of patent application No. 99803775.3 of the same name, original International application No. PCT/IB99/00215, International application date 1999, 2.4.d.

Technical Field

The present application relates to methods and compositions for detecting and diagnosing infectious diseases. In particular, the present invention relates to transdermal delivery systems or devices comprising mycobacterial antigens, wherein administration of the system or device stimulates an immune response sufficient for detection and diagnosis of active mycobacterial infection.

Background

Detection of infectious diseases is typically accomplished using assays that monitor immune responses. In many cases, however, these tests are cumbersome and often give inconsistent results. In addition, the lack of sophisticated laboratory equipment for individuals living in underdeveloped areas often reduces the effectiveness of the tests, and it is not reasonable that the incidence of infectious diseases may be higher in these areas. Accurate diagnosis and detection of infectious diseases is important not only for therapy, but also for prevention of morbidity and disease transmission. The need for sensitive and accurate detection methods has become particularly clear at present, especially in light of the increasing infection caused by mycobacteria and the like.

Mycobacterial infections are often manifested as diseases such as tuberculosis. Human infections caused by mycobacteria have been widespread since ancient times, and tuberculosis is still a cause of death today. Although morbidity has declined with increasing levels of living since at least the middle of the nineteenth century, mycobacterial infections still constitute a cause of morbidity and mortality in countries with limited medical resources, and can lead to overwhelming populations of widely spread diseases in immunocompromised patients. Despite efforts made by most health organizations worldwide, eradication, or near eradication, of mycobacterial disease has never been achieved. Almost one third of the world's population is infected with a Mycobacterium tuberculosis complex, commonly referred to as Tuberculosis (TB), with about eight million new patients each year and three million patients dying from TB each year.

TB has risen again over decades of decline. In the united states, up to ten million people are believed to be infected. It was reported that there were approximately 28000 new cases in 1990, which is 9.4% more than in 1989. Has increased by 16% from 1985 to 1990. Overcrowding of living conditions and shared spaces has contributed even more to the spread of TB, such as the increased incidence observed in the united states among prison inmates and homeless individuals in large cities.

About half of all Acquired Immune Deficiency Syndrome (AIDS) patients will be infected with mycobacteria, with TB being a particularly devastating complication. AIDS patients are at higher risk of clinical development of TB, and anti-TB treatment does not appear to be as effective in non-AIDS patients. Thus, infection often progresses to a fatal, widespread disease.

Mycobacteria other than mycobacterium tuberculosis are gradually found in opportunistic infections that afflict AIDS patients. Organisms from the M.avium complex (MAC) intracellular, particularly the fourth and eighth serotypes, account for 68% of mycobacterial isolates from AIDS patients. Enormous numbers of MACs (up to 1010 acid-fast bacilli per gram of tissue) were found, and consequently, the prognosis for infected AIDS patients is poor.

The World Health Organization (WHO) continues to support combat against TB, recommending active preventative measures, such as "extended procedures for immunization" (EPI), and active treatment compliance measures, such as "direct-observed short-term treatment" (DOTS). Diagnosis, treatment and prevention are equally important for the eradication of TB. Rapid detection of active TB patients is beneficial for early treatment, whereas with early treatment, cure rates are expected to be 90%. Therefore, early diagnosis is critical for combat against TB. In addition, therapeutic compliance not only ensures elimination of infection, but also reduces the emergence of drug resistant strains.

The emergence of drug-resistant mycobacterium tuberculosis is a very disturbing phenomenon. New TB cases demonstrated a rate of toleration of at least one standard drug that increased from 10% in the eighties of the twentieth century to 23% in 1991. Therefore, the compliance of the treatment regimen also plays a decisive role in the efforts to eliminate TB and prevent the emergence of drug-resistant strains.

Although more than 37 species of mycobacteria have been identified, more than 95% of all human infections are caused by six species of mycobacteria: mycobacterium tuberculosis, M.intracellulare, M.kansasii, M.fortuitum, M.cheloniae, and M.leprae. The most common human mycobacterial disease is Tuberculosis (TB), and the Mycobacterium species responsible for tuberculosis include Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum (Merck Manual 1992). Infection usually begins with the inhalation of infectious particles, which are able to reach the terminal pathways within the lungs. After phagocytosis by alveolar macrophages, the bacilli are able to replicate freely, eventually destroying the phagocytic cells. A cascade effect ensues in which phagocytes are destroyed, leading to the migration of additional macrophages and lymphocytes to the site of infection, where they are also eventually eliminated. In The initial stage, The disease is further spread by infected macrophages as they migrate to regional lymph nodes and into The blood stream and other tissues such as bone marrow, spleen, kidney, bone and central nervous system (see Murray et al, medical microbiology, The C.V.Mosby Company 219-230 (1990)).

The factors responsible for mycobacterial toxicity are not clear. Many studies suggest that cell wall lipids and bacterial surfaces are factors that contribute to colony morphology and toxicity. Evidence suggests that C-mycosides on the surface of certain mycobacterial cells have an important role in promoting survival of organisms within macrophages. For other mycobacteria, trehalose-6, 6' -dichotomanate is suggested, which is a cord factor.

The correlation between colony morphology and toxicity is particularly evident in M.avium. Mycobacterium avium exists in several prominent colony forms. Bacilli that grow as clear or rough colonies on conventional laboratory media can multiply within macrophages in tissue culture, are toxic after injection into susceptible mice, and are resistant to antibiotics. Rough or transparent bacilli maintained on laboratory media often spontaneously exhibit opaque colony morphology where they fail to grow within macrophages, are non-toxic to mice, and are highly susceptible to antibiotics. The differences in colony morphology between clear, rough and opaque strains of M.avium are almost certainly caused by the presence of a glycolipid that coats the surface of both clear and rough organisms and serves to protect the capsule. This capsule or coating, which is composed primarily of C-mycosides, apparently protects the virulent M.avium organism from lysozyme and antibiotics. In contrast, the non-toxic opaque form of M.avium contains little C-mycoside lipids on its surface. Both resistance to antibiotics and resistance to killing by macrophages have been considered as glycolipid barriers on the surface of mycobacterium avium.

Diagnosis of mycobacterial infection is confirmed by the isolation and identification of the pathogen, although routine diagnosis is based on sputum smears, chest X-ray examination (CXR) and clinical symptoms. Isolation of mycobacteria on the medium takes up to four to eight weeks. Species identification further took two weeks. There are several other techniques for detecting mycobacteria, such as Polymerase Chain Reaction (PCR), direct test for Mycobacterium tuberculosis or amplified direct test for Mycobacterium Tuberculosis (MTD), and assays that utilize radioactive labels.

One diagnostic test that is widely used to detect infections caused by mycobacterium tuberculosis is the tuberculin skin test. Although various versions of skin tests are available, one of the two tuberculin antigen formulations is commonly used: old Tuberculin (OT) or Purified Protein Derivative (PPD). The antigen preparation is injected intradermally into the skin, or is administered topically and then delivered invasively into the skin using a multi-pronged inoculator (Tine test). There are several problems with skin test diagnostic methods. For example, the tone test is generally not recommended because the amount of antigen injected into the intradermal layer cannot be precisely controlled (see Murray et al, medical microbiology, the C.V.Mosby Company 219. 230 (1990)).

Although tuberculin skin tests are widely used, they usually take 2-3 days to produce results, and many times the results are inaccurate, because false positives are sometimes seen in healthy subjects who have been exposed to mycobacteria. In addition, misdiagnosis often occurs because not only positive results are observed in active TB patients, but also positive results are observed in BCG-vaccinated persons and persons who have been infected with mycobacteria but have not developed disease. Therefore, it is difficult to distinguish active TB patients from others, such as household TB contacts, by the tuberculin skin test. Furthermore, the tuberculin test usually causes cross-reactivity in individuals infected with other mycobacteria (MoTT) than M.tuberculosis. The currently available diagnoses made with skin tests often cause errors and mistakes.

There is a need for effective assays for detecting the presence of mycobacterial infections. In particular, a test that does not require invasion of the skin surface of the subject will reduce exposure to health care personnel testing the body fluids of the subject and reduce the risk of infection by other infectious agents that may be present in the subject. In addition, an assay that is easy to perform and has positive or negative results that are easy to determine is necessary to monitor compliance with treatment regimens for highly infectious diseases such as tuberculosis, particularly for individuals who are homeless, prison inmates, schoolchildren, and senior citizens.

There is also a need for an inexpensive and accurate method for distinguishing between persons who are active in the disease and those who have only been immunologically exposed to infectious agents but not in the active phase (e.g. those previously infected with mycobacteria), or who have been vaccinated with BCG. Furthermore, there is no known method for monitoring the effect of drug therapy in persons infected with mycobacteria, such as tests that can distinguish between active tuberculosis and other stages of healing or pre-exposure. Furthermore, there is a need for tests that can be easily performed on children who are particularly afraid of the skin tests currently used that involve needles or skin penetration. Such tests are particularly desirable for monitoring AIDS patients who are highly susceptible to mycobacterial infections. In addition, tests that are easy to perform and have positive or negative results that are easy to determine are necessary when monitoring diseases such as tuberculosis in homeless persons or prison inmates.

Summary of The Invention

The present invention comprises methods and compositions for the detection of infectious diseases. According to a preferred embodiment of the present invention, a transdermal delivery system or device is provided, such as a patch containing a mycobacterial antigen composition. The patch is applied to the skin and removed after a predetermined time. The skin is then examined for an immunogenic response to the antigen present in the patch.

Unlike prior art methods, the diagnostic methods and compositions provided herein are highly sensitive and specific. Most importantly, the diagnostic methods and compositions of the present invention are particularly effective in detecting mycobacterium tuberculosis infection in active tuberculosis patients, thereby eliminating the possibility of misdiagnosing individuals who have been vaccinated or who have been exposed to the organism without disease manifestation.

The diagnostic methods described herein include the topical administration of compositions comprising mycobacterial antigens including, but not limited to, MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80, or MPB83 for transdermal delivery and subsequent detection of immunogenic responses. The antigens may be used alone or in combination. It is particularly preferred to administer topically an antigen composition comprising MPB 64. The present invention is directed to any antigen characterized by MPB64, which is characterized by the presence of delayed-type hypersensitivity to the antigen in the case of active tuberculosis disease, but not in response to non-exposure to mycobacteria, or exposure to vaccines or other inactive tuberculosis states.

Accordingly, it is an object of the present invention to provide methods and compositions for the detection of infectious diseases.

It is another object of the present invention to provide methods and compositions for active tuberculosis detection.

It is another object of the present invention to provide methods and compositions for active tuberculosis detection by topical administration of antigen compositions for transdermal delivery.

It is another object of the present invention to provide methods and compositions for the detection of mycobacterial infections.

It is yet another object of the present invention to provide methods and compositions for the detection of active disease caused by mycobacterial species including Mycobacterium tuberculosis complex, M.intracellulare, M.kansasii, M.fortuitum, M.cheloniae, M.leprae, M.africanum, and M.microti.

It is another object of the present invention to provide methods and compositions for the detection of active disease caused by Mycobacterium tuberculosis.

It is also an object of the present invention to provide methods and compositions for the detection of active disease caused by mycobacterium bovis.

It is another object of the present invention to provide methods and compositions for the immunological detection of mycobacterial infections using topical application without the need for invasive procedures.

It is yet another object of the present invention to provide sensitive diagnostic methods and compositions for the detection of active disease caused by mycobacteria, wherein the antigen composition is topically applied and transdermally delivered, followed by examination of the skin for immunogenic response.

It is another object of the present invention to provide methods and compositions for the detection of active disease caused by mycobacteria, wherein the topically administered mycobacterial antigen composition comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB 83.

It is a further object of the present invention to provide methods and compositions for the detection of active disease caused by mycobacteria wherein the topically administered mycobacterial antigen composition comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83, wherein the antigen is administered alone or in combination with another mycobacterial antigen.

It is another object of the present invention to provide methods and compositions for the detection of active disease caused by mycobacteria, wherein the topically applied mycobacterial antigen composition comprises MPB 64.

It is yet another object of the present invention to provide methods and compositions for infectious disease diagnosis that are easy to perform.

It is a further object of the present invention to provide methods and compositions for active tuberculosis detection for monitoring the efficacy of treatment.

It is another object of the present invention to provide a kit for the diagnosis and detection of active diseases caused by mycobacteria.

It is also an object of the present invention to provide methods and compositions for the detection of active disease caused by mycobacteria in household TB or mycobacterial disease contacts.

It is another object of the present invention to provide methods and compositions for clinical status monitoring of mycobacterially infected patients following chemotherapy.

It is another object of the present invention to provide sensitive diagnostic methods and compositions for children with active tuberculosis.

It is a further object of the present invention to provide sensitive methods and compositions for the detection of active disease caused by mycobacteria, wherein the methods involve the use of a transdermal patch.

These and other objects, features and advantages of the present invention will become apparent after review of the following detailed description of the disclosed embodiments and the appended claims.

Brief description of the drawings

FIG. 1 shows a graph of the dose response of different amounts of MPB64 antigen by guinea pig skin reactions.

Fig. 2 is a graph showing the time course of delayed-type hypersensitivity reaction to the patch of MPB64 antigen in guinea pigs after inoculation with BCG.

Detailed Description

The invention may be understood more readily by reference to the following detailed description of specific embodiments included herein. Although the present invention has been described with reference to specific details of certain embodiments, these details should not be construed as limitations on the scope of the invention. The references mentioned herein are incorporated by reference in their entirety, including U.S. provisional application 60/073911 filed on 6/2/1998 and U.S. provisional application 60/096140 filed on 11/8/1998.

Mycobacterial infections that cause tuberculosis and the like, once considered to have a reduced incidence, rebound and again pose a serious health threat. People are crowded together or living environments are under-standard areas that are increasingly finding persons infected with mycobacteria. Persons without an immune response are at great risk of being infected with mycobacteria and dying from the infection. In addition, the emergence of drug resistant strains of mycobacteria has increased the difficulty of treating infected individuals.

Many people infected with mycobacteria are poor or live in areas where health care facilities are incomplete. These people are not readily tested for mycobacterial infections and they require inexpensive and non-invasive methods of infection detection. Furthermore, the persons in prisons or homeless persons generally do not have adequate health care conditions, are in poor physical condition, and often do not receive adequate or successful health care intervention.

The present invention provides methods and compositions for topical administration comprising antigenic compositions for transdermal delivery of antigens, particularly mycobacterial antigens. More specifically, the present invention provides methods and compositions for detecting diseases such as active tuberculosis, for distinguishing between individuals with active disease and individuals who have only been immunologically exposed to infectious agents such as mycobacteria.

The methods and compositions of the invention are useful for testing humans and other animals for the presence of mycobacterial infections. For example, the invention is particularly useful for disease detection in cows infected with mycobacterium bovis.

Medically, "active tuberculosis" is diagnosed by well-known medical procedures such as chest X-ray (CXR), saliva tests, or other symptoms. Because the precise identification of the presence of mycobacterial infectious agents is expensive and requires a long time, the diagnosis of active disease does not necessarily include the identification of the presence of mycobacteria. Thus, the diagnosis of active diseases such as tuberculosis may rely on the detection of other aspects of the mycobacterial infection, such as the generation of specific immune responses or the manifestation of certain symptoms. The term "tuberculosis" as used herein includes disease states commonly associated with infections caused by mycobacterial species including Mycobacterium tuberculosis complex. Mycobacterial infections caused by mycobacteria other than mycobacterium tuberculosis (MOTT) are typically caused by mycobacterial species including mycobacterium intracellulare, mycobacterium kansasii, mycobacterium fortuitum, mycobacterium cheloniae, mycobacterium leprae, mycobacterium africanum and mycobacterium microti.

The present invention includes methods and compositions for topical administration that enable transdermal delivery of antigens that elicit an immune response, such as delayed-type hypersensitivity, in active disease subjects such as tuberculosis caused by mycobacterial infection. The antigen is derived from a mycobacterium or is cross-reactive with mycobacterial protein or carbohydrate moieties. Preferred antigens include, but are not limited to, MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80, or MPB83 derived from mycobacteria. Particularly preferred is antigen MPB64 (see Kawajiri et al, Japanese patent application publication 09206092, the entire disclosure of which is incorporated herein by reference).

Another preferred combination of antigens includes the use of MPB64 in combination with MPB 59. For example, a combination of MPB59 and MPB64 may indicate an infection with atypical mycobacteria, as subjects infected with other species of mycobacteria that do not secrete MPB64 will show a positive response to MPB 59.

MPB64 is a mycobacterial antigen that is frequently associated with Mycobacterium tuberculosis complex. Harboe et al first described as MPT64 (infection and immunity 1986; 52: 293-902, incorporated herein by reference in its entirety), and thereafter have been fully characterized and used in various laboratories (see, e.g., Yamaguchi et al, infection and immunity 1989; 57: 283-288, also incorporated herein by reference in its entirety). "MPB 64" and "MPT 64" refer to the same antigen: MPT64 was isolated from the culture filtrate of M.tuberculosis and was therefore designated M.tuberculosis protein, and MPB64 was later isolated from the culture filtrate of M.bovis (or BCG) and was therefore designated M.bovis protein. The two proteins were subsequently found to be identical. MPB64 and MPT64 refer to antigens selected from mycobacterial species including, but not limited to, certain strains of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG. The antigen is secreted during bacterial growth and is immunogenic, causing Delayed Type Hypersensitivity (DTH) in guinea pigs and humans.

Recombinant antigens may also be used in diagnostic methods and compositions contemplated by the present invention. See, e.g., Haga et al, J leukocyte Biol 1995; 57: 221-225; roche et al, clinical and Experimental immunology 1996; 103(2) 226-232; roche et al, journal of infectious diseases 1994; 107(5): 1326-30, each of which is incorporated herein in its entirety.

The antigens of the invention are typically for transdermal delivery into the skin of a subject. The antigen is used by keeping a composition comprising the antigen in close contact with the skin. The concentration of antigen in the composition is in the range of about 1 to 150 micrograms per dose administered, more specifically 10 to 100 micrograms per dose administered, and most specifically 30 to 75 micrograms per dose administered. The antigenic composition can comprise a physiologically effective solution comprising surfactants, buffers and solvents, which enable transdermal delivery of the antigenic composition. Preferably, surfactants, buffers and solvents are used which increase the penetration and transport of the antigen without itself triggering a reaction or interfering with the immunogenicity of the antigen. Preferred surfactants for use in the antigen composition include tween 20, tween 40, tween 60 and tween 80; the concentration of each in phosphate buffered saline may be 0.001-10%, 0.001-1%, preferably 0.005%. One preferred embodiment for administration comprises 30 to 75 micrograms of antigen in 100 microliters of phosphate buffered saline, further comprising the preferred surfactant tween 80. The most preferred embodiment for administration is 75 micrograms of antigen and 0.005% tween 80 in 100 microliters of Phosphate Buffered Saline (PBS). The prepared antigen composition can be stored in appropriate sterile glass or plastic containers in batches or in portions according to the required amount.

In particular, the invention relates to methods for transdermal delivery of antigen compositions to skin cells for the detection of active disease. All contemplated solvent and antigen compositions that are capable of effecting the release of infectious agent antigens to skin cells for active disease detection are included as such.

The term "transdermal delivery" as used herein refers to the delivery of a composition to various layers of the skin, including but not limited to the epidermis (stratum corneum, stratum lucidum, stratum granulosum, stratum germinatum, stratum basale), dermis, and subcutaneous layers. The term "topical application" as used herein means that the composition is applied or placed on the skin without piercing or invasive entry into the skin with a needle or the like.

A preferred embodiment of the present invention comprises a transdermal delivery system or device for maintaining the above-described composition in intimate contact with human skin. A highly preferred embodiment comprises a patch tape, such as a skin patch tape, for maintaining the composition in intimate contact with the skin. Materials suitable for use in patches for delivering antigenic compositions of the present invention include the TORII patch bandage "Torniban^TM"(available from Torri and Co., Ltd., Tokyo), Finn-chamber and Perme-aid S. (Nitto-Denkou Co., Japan). Alternatively, a material such as medical adhesive plaster or tape, in which a part of plaster or tape includes a part of the material impregnated with the antigen composition, may be used, and the material may be fixed so as to be in direct contact with the skin. Suitable medical plasters, tapes and fabrics are manufactured by several manufacturers, such as Nichiban (Japan), Kimberly-Clark (Neenah, Wisconsin) and 3M (St. Paul, Minnesota). The device may be of any of a variety of types well known to those skilled in the artAnd fixing by a fixing method. For example, the device may be tethered to the subject's arm using a string, or the device may be attached using a plaster. Preferably, the material used for the securing means, such as plaster, should be breathable and waterproof so that it does not fall off by perspiration or bathing.

One particularly advantageous aspect of the present invention relates to the ease of use and implementation of the present invention. For example, the antigen composition as a whole may be preserved by shipping or holding the composition in a sterile container under appropriate temperature conditions. The antigen composition may be stored in aliquots of the required amount, for example 100 or 200. mu.l, and then applied to gauze, plaster or tape, etc., as necessary for the test patient. Furthermore, the essential components of the composition and the method of the invention may be provided together in a kit to facilitate use.

In addition to patch-type embodiments, the present invention may also take the form of other transdermal drug delivery vehicles known to those skilled in the art, including, but not limited to, gels, creams, liquid sprays, and the like.

Preferred methods contemplated by the present invention include topical administration of a composition comprising a mycobacterial antigen for transdermal delivery to a human. For example, a patch containing the antigen composition is applied to a person's forearm and is maintained in intimate contact with the skin. The patch is left for a predetermined period of time to allow sufficient transdermal delivery of the antigen. Such a time period may range from 1 to 7 days, preferably from 2 to 5 days, most preferably 3 days. After a specified period of time, the patch was removed and the skin examined for immunogenic response.

It is known in the art that a delayed-type hypersensitivity reaction is observed on the skin in response to the presence of certain antigens. Typically, this response is observed following invasion of the antigenic composition and is characterized by redness, erythema, induration (thickening of the skin), appearance of red blisters or ulceration. The response sought in the method of the invention is similar to that seen with intradermal injection of mycobacterial antigen.

The present inventors have surprisingly found that transdermal delivery of the antigens of the invention by topical application results in an immunogenic response (particularly a delayed-type hypersensitivity (DTH) response) in persons suffering from active mycobacterial diseases such as tuberculosis. As shown in the examples herein, TB-infected guinea pigs exhibit a delayed-type hypersensitivity skin response to the antigen MPB64 as long as the bacteria continue to grow; in contrast, guinea pigs immunized with BCG-Tokyo lost delayed-type hypersensitivity to MPB64 at some time post-vaccination. As disclosed by the inventors, the methods and compositions of the invention comprising mycobacterial antigens, particularly MPB64, are useful in the diagnosis of active mycobacterial disease, such as active TB. Surprisingly, individuals infected with mycobacterium tuberculosis but who have not developed tuberculosis and individuals who have previously received BCG vaccination did not show a positive skin response in response to MPB 64.

The discovery that transdermal delivery of antigens, particularly MPB64, by topical administration for the diagnosis of active mycobacterial disease such as tuberculosis is particularly desirable because invasive procedures are not required. Indeed, intradermal release of MPB64 has recently been shown to be unsuccessful in distinguishing TB patients from healthy controls (Wilcke et al tuberculosis and Lung disease (1996) 77: 250-. Effective transdermal delivery of mycobacterial antigens by topical application has heretofore been unknown and the present inventors have successfully investigated diagnostic procedures for differentiating active TB patients from healthy PPD-positive controls using the methods and compositions of the present invention.

One reason why intradermal injection of antigens may not be as effective as transdermal administration is believed to be related to inadequate solubility of the antigen and/or opportunity for antigen presentation, although not wishing to be bound by this theory. For example, with intradermal injection, the antigen may dissolve rapidly in the body fluid and the immune system does not have sufficient time to recognize and/or respond to it. In contrast, the novel methods of the present invention enable the gradual introduction of antigen into the immune system of a subject, which has sufficient time to mount a response, usually in the form of a delayed-type hypersensitivity reaction, due to the slow penetration of antigen through the skin pores and sweat glands to the local part of the skin. Intradermal and transdermal introduction of antigens may also elicit significant immune responses.

Hypersensitivity reactions (also known as type IV or cell-mediated immune reactions) are believed to be mediated in large part by T cells, followed by the involvement of monocytes, although without wishing to be bound by this theory. This reaction usually has a response that is not observed until a period of time has elapsed, i.e., 18-24 hours, and is therefore referred to as delayed-type hypersensitivity. Antigen presenting cells are thought to present antigen to T cells, which upon activation release lymphokines, leading to accumulation and activation of macrophages, monocytes and non-immune lymphocytes.

The invention can also be used to detect active tuberculosis in family contacts of previously infected TB patients. This allows monitoring of the spread of the disease to others who have had close contact with the person initially infected. Because the public may have tuberculosis and be transmitted by coughing, the present invention can be used to monitor exposure to personnel working with the public, such as aircraft crews or health care personnel. Persons who work for homeless or prison populations are also readily able to monitor for the presence of active tuberculosis.

A particularly preferred use of the invention is to monitor the efficacy of treatment for persons suffering from mycobacterial infections. For example, with the increase of drug resistant mycobacteria, the ability to detect that active tuberculosis patients no longer have active TB, i.e. the treatment is effective, is highly desirable. A preferred method comprises topically administering the antigen of the invention to a person with active TB prior to treatment, and after a sufficient period of time, observing skin reactions. In a later step of the treatment regimen, the antigen is reapplied and the skin reaction is observed. The absence of skin reactions indicates that the treatment has effectively altered the active tuberculosis state. The continuous response of the skin indicates that the treatment is not effective, or that there has not been sufficient time for the treatment to take effect, and that the person still has active TB. At this point, the treatment may be altered, the drug sensitivity of the infected mycobacteria may be determined, or the same treatment may be continued for a longer period of time.

One use of the invention would be for rapid screening of populations, such as to investigate infection rates in adjacent or deceased areas, for testing prisoners, hospices or homeless on the street. For example, incoming prisoners are issued patches containing the antigens of the invention upon entering the prison. After wearing the patch for a given period of time, the skin reaction of the prisoner is examined. Positive responders will be placed individually. Because the present invention detects active TB, these prisoners can be isolated from other prisoners and treatment started immediately. Other TB tests can detect prisoners who have been exposed to TB at some time during their life and who may not be able to transmit TB to others. Prisoners who are unable to transmit TB to others do not need isolation and treatment.

In many parts of the world, people are vaccinated against TB. It is extremely difficult to detect active TB in these persons, since all persons exposed to mycobacteria are positive under the conditions of the standard TB skin test, regardless of the presence of active TB or the outcome of vaccination. The invention is used in this case to detect the presence of active TB in individuals, distinguishing them from those who have been previously vaccinated.

The ease of administration is a particularly advantageous aspect of the invention. For example, children are not afraid of topical devices such as patches to administer drugs, and wear such devices without hesitation for a period of time sufficient to produce a skin reaction. Topical devices such as patches are easy to store and transport to occluded locations where refrigerated conditions and clean water may be lacking. The invention can be made of cheap materials, has low production cost and can be used by health care organizations worldwide.

The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. Rather, various other embodiments, modifications, and equivalents may be resorted to by those skilled in the art after reading the description without departing from the spirit of the invention.

Example 1

To test a novel, simple and rapid method of active tuberculosis diagnosis, subjects were tested for skin reactions to MPB64 using a removable patch for topical application of the antigenic composition followed by transdermal delivery.

Skin patch

Although antigens such as MPB59, MPB70, MPB44, MPB45, MPB51 or MPB64 can be used in the patch test of the present invention, the antigen used in the following examples is MPB64 at a concentration of about 75 μ g per patch. It is expected that the use of about 50 to 100 μ g of antigen per patch will be successful. A preferred antigen solution comprises approximately 750 μ g antigen per ml Phosphate Buffered Saline (PBS), wherein the PBS consists of 0.005% Tween 80.

The patch was applied to the skin of the subject, left for three days, and the site of application was observed after removing the patch. The presence of an immune response indicates a positive result, such as a Delayed Type Hypersensitivity (DTH) reaction (redness, induration, or small red vesicles), i.e. the presence of active tuberculosis. No change in the site of application indicates a negative reaction.

Test subject

53 active tuberculosis patients and 43 healthy Purified Protein Derivative (PPD) positive controls were tested to determine whether the response to MPB64 was positive only in active tuberculosis patients. Tuberculosis patients from four clinics, Our Lady of Grace Parish, Sto. Nino de Tondo Parish, Canossa Health and Social Center, and Health Care Development Center, near Philippine Maryla were examined.

Of 53 active tuberculosis patients, 52 showed a positive response to MPB64, while none of the 43 PPD-positive controls had a positive response to MPB 64. The specificity of MPB64 to active tuberculosis was 100%, and the sensitivity was 98.1%. The efficacy of the test was 98.9%.

The MPB64 patch test is an effective and accurate method for the diagnosis of active tuberculosis, distinguishing patients from BCG-vaccinated individuals and individuals who are naturally infected but do not develop tuberculosis.

The experimental design used for this finding is detailed in example 2.

Example 2

To determine the reliability of MPB64 as a specific antigen for the transdermal patch method for the diagnosis of active TB, comparative tests were carried out between three different groups of individuals:

(1) active TB patients;

(2) healthy tuberculin-positive persons;

(3) family TB contacts people.

A correlation between the skin response of humans to MPB64 and the clinical status of TB was observed.

Since the purpose of this study was to determine MPB64 as a specific diagnostic antibody for active TB

Original reliability, so the choice of active TB patients is of paramount importance.

Clinical records of outpatients were examined. Patients who were sputum smear positive, had an abnormal CXR and had clinical symptoms of active TB were divided into active TB patients group 1. Culture results are not available in most cases. Patients who have just begun chemotherapy shortly before are preferred because the effect of long-term chemotherapy on the MPB64 skin response is unknown. However, some patients in group 1 have been treated for 6 months. They were considered as active TB patients based on smear positive in the most recent examination and symptom manifestation.

Patients live near clinics where socio-economic conditions are very poor. The geographical location of their residence is important as they are scheduled to return to the clinic 3 days later so the results are read. Only 12 of the 105 patients tested did not return. Of the returned patients, 53 were available for final analysis. The analysis results are shown in table 1. The remaining patients were excluded because their patches had been peeled or dropped off prior to the day of reading.

Patients were screened according to clinical records and only patients with smear positive, CXR abnormalities and other symptoms with cough, fever or weight loss indicative of TB were selected as active TB patients. Patients who are culture positive are preferred, but only seven cases of culture results are available. Most active TB patients have been chemotherapy for 1-4 months. Some had been treated for 6 months at the time of the study. Healthy tuberculin-positive volunteers were filipinos and japanese who did not show any indication of TB. Some family members also come to the clinic with TB patients at the time of the trial. They were tested as "family TB contacts". All subjects were informed of the general condition of the study and agreed to conduct the study. The number of three groups of subjects was as follows: group 1 active TB patients, 53; group 2 healthy controls, 43; group 3 family TB contacts, 41.

MPB64 was isolated from a 8-day culture filtrate of Mycobacterium bovis BCG (obtained from the Japanese BCG laboratory in Tokyo, Japan). The purified protein was suspended in PBS and stored at-20 ℃. The amount of protein was measured by the Lowry method. The whole protein precipitated from 8-day culture filtrate of Mycobacterium bovis BCG with ammonium sulfate was named PPD-eT to distinguish purified protein derivative tuberculin (PPD), which was used as a control for the patch test. PPD prepared from Mycobacterium tuberculosis AoyamaB was obtained from the Japanese BCG laboratory (Tokyo, Japan). Five Tuberculin Units (TU) of PPD suspended in 0.1ml reconstitution buffer were used for Mantoux intradermal injection experiments.

Medicine plaster test material

Torriban, using a Torrii patch bandage with a 15mm gauze size^TM"obtained from Torii and dc, ltd, Tokyo). The antigen solution (75. mu.g of antigen in 100. mu.l PBS containing 0.005% Tween 80) was poured onto gauze and the patch was applied to the forearm of the subject, the skin area on which the patch was applied having been cleaned with alcohol. The patch was placed on the skin of the subject with the gauze impregnated with the antigen solution in direct contact with the skin. The patch was left for 72 hours.

Patch test arrangement

Active TB patients and tuberculin-positive healthy controls were tested for skin reactions to the MPB64 patch on the left arm and the PPD-eT patch on the right arm. Each patch contained 75 μ g of antigen. A PPD was injected intradermally (5 TU/100. mu.l) into the right forearm at a location different from the patch. The PPD-eT patch was used to confirm that the protein antigen did not enter the body transdermally. Transdermal administration is incomplete if the PPD test is positive and the PPD-eT patch test is negative. This is excluded from the test results.

After 3 days (72 hours), the patch was removed and the reaction was judged to be positive or negative. No change in the skin at the site of application was "negative", while the appearance of erythema, induration or several small red blisters at the site of application was recorded as a "positive" response to the antigen.

We compared the application of the patch to the forearm and upper arm. The patch fell off the upper arm (41.2%) more easily than the front arm (17.6%) before the day of reading. Therefore, the forearm is recommended for adult testing.

Statistics of

A two-by-two contingency test was used to evaluate the results of the patch test on human MPB 64.

Active TB patients and tuberculin Positive healthy controls

Table 1 shows the actual number of positive or negative responses to MPB64 in groups 1 and 2. All subjects were positive to the PPD Mantoux test and the PPD-eT patch test. From these results the following values were calculated: the sensitivity is 98.1%; the specificity is 100%; false positive rate is 0%; false negative rate is 1.9%; positive predictive value 100%; negative predictive value 97.7%; the efficacy of the test is 98.9%. The results indicate that the MPB64 patch test is an effective method for distinguishing active TB from healthy tuberculin-positive persons.

TABLE 1

MPB64 Patch test

Two-by-two contingency test between group 1 and group 2

Group of	Positive for	Negative of	Total of
				1: TB patients	52	1	53
2: healthy controls	0	43	43
				Total of	52	44	96

Household TB contacter

The number of family TB contacts (group 3) was 41, including 12 males and 29 females. The patch test results are shown in Table 2. 26 subjects showed positive reactions to the PPD-eT and MPB64 patches (63.4%), and none were positive to PPD-eT and negative to MPB64 (22.0%). Six people (14.6%) were negative for PPD-eT and MPB 64. Of these double negative persons, three were negative to the PPD Mantoux test.

Group 3 subjects were not clinically recorded as TB patients. The clinical status of each individual is unknown, although some symptoms suggestive of TB are observed.

TABLE 2

MPB64 and PPD-eT patch test for household TB-contactants

This study strongly suggested that the MPB64 patch test is a promising tool for the rapid diagnosis of active TB. It can distinguish active TB patients from individuals vaccinated with BCG or individuals infected with TB but not yet developed the disease with a sensitivity of 98.1% and a specificity of 100%. The patch test also has advantages over intradermal injection, namely simple technique and safe administration. The patch test is believed to be able to provide a continuous supply of antigen in response to a patient, although not wishing to be bound by any theory.

Example 3

To determine the reliability of MPB64 as a specific diagnostic antigen for active TB using the transdermal patch method, comparative tests were performed on guinea pigs.

Female albino Hartley guinea pigs were purchased from Japan Laboratory Animals, inc., Tokyo and weighed 300 to 400g at the start of the experiment. Animals were maintained in the japanese BCG laboratory under specific pathogen free conditions.

Antigens

Antigens were prepared according to the methods and materials described in example 2.

Immunization of guinea pigs

Live BCG vaccine (Japan BCG laboratory, Tokyo, Japan) was reconstituted according to the manufacturer's instructions and injected subcutaneously into guinea pigs without adjuvant at a dose of 0.5mg per animal. Animals were tested between 4 and 25 weeks after BCG injection.

Medicine plaster test material

A 7mm gauze sized TORII patch bandage (available from TORII and co., ltd., Tokyo, Japan) was used. The antigen solution (75. mu.g of antigen in 15. mu.l PBS containing 0.005% Tween 80) was poured onto gauze, and the patch was attached to the hair-removed part of each guinea pig.

MPB64 was applied to patches at the doses shown in FIG. 1, which were applied to the right and left flanks of BCG-immunized guinea pigs and had hair removed.

Patch test arrangement

After 24 hours the patch was removed and the reaction was read immediately. No change in the skin at the site of application was "negative", while the appearance of erythema, induration or several small red blisters at the site of application was recorded as a "positive" response to the antigen.

Dose response to MPB64 in a BCG-immunized guinea pig patch test

Guinea pigs previously immunized with BCG Tokyo for 4 weeks were used for the MPB64 patch test with varying antigen doses. The highest dose for the patch test was 75 μ g/patch. Animals were tested with patches containing 2.3 to 75 μ g/patch of MPB 64. After 24 hours the patch was removed and the reaction was read as positive or negative. To confirm the sensitivity of the animals to BCG, 0.05. mu.g of PPD in 0.1ml of buffer solution was injected intradermally and the skin response was measured after 24 hours. Figure 1 shows the results of a dose-response experiment. At a dose of 4.7 μ g/patch or higher, the response to MPB64 was positive. No positive reaction was observed at the dose of 2.3. mu.g MPB64 per patch. The negative control patch contained only PBS containing 0.005% Tween 80 and did not cause any skin reaction in BCG-immunized guinea pigs. The non-immunized guinea pigs did not show any response to PPD or MPB 64.

Time course of the cutaneous response of BCG-immunized guinea pigs to MPB64

It is known that BCG-immunized guinea pigs lose the skin response to MPB64 15 weeks after BCG immunization when tested with intradermal injection of MPB 64. To confirm whether this was also the case in the patch test, guinea pigs were immunized with BCG Tokyo and tested with MPB64 patches at various times after BCG injection. Each animal was tested only once to avoid booster effects. As a control, a PPD-eT patch test was applied to each animal simultaneously. The results are shown in FIG. 2. DTH is expressed as 3+, 2+, etc., since the reaction diameter is adjusted by the patch size, not by the antigen dose. All animals were positive for skin reactions to the MPB64 patch test up to 13 weeks after BCG injection. Later became barely detectable and was completely negative after 23 weeks. In contrast, the response to the PPD-eT patch test remained positive until the end of the experiment at 25 weeks after BCG injection.

Delayed Type Hypersensitivity (DTH) to MPB64 and PPD-eT was examined at various times after BCG injection. FIG. 2 is a bar chart representing delayed type hypersensitivity in 3 guinea pigs as follows: 3+: erythema and induration; 2+: erythema; 1+: a vesicle; +/-: color blur (suspicious response); -: there was no reaction.

The guinea pig patch test confirmed that 1/16, the dose of antigen used in humans, elicited a positive response.

It will of course be appreciated that the foregoing relates only to preferred embodiments of the invention and that numerous modifications or alterations may be made thereto without departing from the spirit and scope of the invention.

Claims (6)

1. Use of a composition comprising a mycobacterial antigen MPB64 and a physiologically effective solution for the manufacture of a test product for the local detection of an active disease, wherein the active disease is tuberculosis; the test product comprises a transdermal delivery device comprising the composition, and the topical test comprises:

topically applying a transdermal delivery device to the skin;

removing said transdermal delivery device after a predetermined period of time;

observing the immune response of the skin; and

correlating the presence of an immune response with active disease.

2. The use of claim 1, wherein the transdermal delivery device comprises a material selected from the group consisting of medical tape, medical plaster, and gauze.

3. The use of claim 1, wherein the transdermal delivery device comprises a patch material.

4. The use of claim 1, wherein the transdermal delivery device comprises a patch material.

5. The use of claim 1, wherein the physiologically effective solution comprises a surfactant, a buffer and a solvent.

6. The use of claim 1, wherein the physiologically effective solution comprises a phosphate buffered solution, and the phosphate buffered solution further comprises tween 20, tween 40, tween 60 or tween 80.