U.S. patent application number 12/639674 was filed with the patent office on 2010-11-04 for methods and compositions for detection and diagnosis of infectious diseases.
Invention is credited to Reiko M. Nakamura.
Application Number | 20100278742 12/639674 |
Document ID | / |
Family ID | 26755032 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100278742 |
Kind Code |
A1 |
Nakamura; Reiko M. |
November 4, 2010 |
METHODS AND COMPOSITIONS FOR DETECTION AND DIAGNOSIS OF INFECTIOUS
DISEASES
Abstract
Methods and compositions for the detection and diagnosis of
infectious diseases are provided. In particular, efficient and
sensitive methods and compositions for the detection of active
mycobacterial disease are provided for distinguishing between
individuals having active disease, and individuals who have been
immunologically exposed, such as those infected with a
mycobacterium but are without active disease, or those who have
been vaccinated with BCG. The methods comprise topical application
of antigen compositions for transdermal delivery.
Inventors: |
Nakamura; Reiko M.; (Tokyo,
JP) |
Correspondence
Address: |
Johnson & Associates
317A E. Liberty Street
Savannah
GA
31401
US
|
Family ID: |
26755032 |
Appl. No.: |
12/639674 |
Filed: |
December 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11020974 |
Dec 22, 2004 |
7655218 |
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12639674 |
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09877802 |
Jun 6, 2001 |
6979450 |
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11020974 |
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09244701 |
Feb 4, 1999 |
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09877802 |
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60073911 |
Feb 6, 1998 |
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60096140 |
Aug 11, 1998 |
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Current U.S.
Class: |
424/9.2 ;
424/9.81 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 49/0006 20130101; A61K 9/7023 20130101; Y10S 424/81
20130101 |
Class at
Publication: |
424/9.2 ;
424/9.81 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61P 31/00 20060101 A61P031/00 |
Claims
1-20. (canceled)
21. A method of diagnosing infection with Mycobacterium
tuberculosis in a patient with immune deficiency disease (AIDS):
exposing the patient to a transdermal delivery device with an
antigen composition applied to or impregnated on the transdermal
delivery device wherein the antigen composition is an antigen
derived from MPB64.
22. The method of claim 21, wherein the antigen composition further
comprises a surfactant.
23. The method of claim 22, wherein the surfactant is a non-ionic
surfactant.
24. The method of claim 23, wherein the surfactant is
polyoxyethylene sorbitan derivative.
25. The method of claim 24, wherein the polyoxyethylene sorbitan
derivative is polyoxyethylene sorbitan monooleate.
26. The method of claim 21, wherein the transdermal device is
medical tape, medical plaster, gauze, patch, adhesive solution, or
a patch band.
27. A method of monitoring effect of drug therapy in a patient
infected with Mycobacterium tuberculosis comprising: exposing the
patient to a transdermal delivery device with an antigen
composition applied to or impregnated on the transdermal delivery
device wherein the antigen composition is an antigen derived from
MPB64.
28. The method of claim 27, wherein the antigen composition further
comprises a surfactant.
29. The method of claim 28, wherein the surfactant is a non-ionic
surfactant.
30. The method of claim 29, wherein the surfactant is a
polyoxyethylene derivative.
31. The method of claim 30, wherein the polyoxyethylene sorbitan
derivative is polyoxyethylene sorbitan monooleate.
32. The method of claim 27, wherein the transdermal device is
medical tape, medical plaster, gauze, patch, adhesive solution, or
a patch band.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/020,974, filed Dec. 22, 2004, now U.S. Pat.
No. 7,655,218, which is a divisional of U.S. patent application
Ser. No. 09/877,802, filed Jun. 6, 2001, now U.S. Pat. No.
6,979,701, which is a continuation of U.S. patent application Ser.
No. 09/244,701, filed Feb. 4, 1999, now abandoned. U.S. patent
application Ser. No. 09/244,701 claims priority to U.S. provisional
application Ser. No. 60/073,911 filed Feb. 6, 1998 and to U.S.
provisional application Ser. No. 60/096,140 filed Aug. 11, 1998
FIELD OF INVENTION
[0002] The present invention relates to methods and compositions
for detecting and diagnosing infectious diseases. In particular,
the invention relates to transdermal delivery systems or devices
comprising mycobacterial antigens, wherein the application of such
systems or devices stimulates an immunological response sufficient
for detection and diagnosis of active mycobacterial infection.
BACKGROUND OF THE INVENTION
[0003] The detection of infectious diseases is often accomplished
by use of tests that monitor immunological responses. Many times,
however such tests are cumbersome and frequently yield inconsistent
results. In addition, the absence of sophisticated laboratory
equipment often reduces the availability of testing to individuals
living in underdeveloped areas where the occurrence of infectious
disease may be disproportionately high. Accurate diagnosis and
detection of infectious disease is important not only for treatment
purposes, but also for the prevention of occurrence and
dissemination of disease. The need for sensitive and accurate
detection methods has become particularly pronounced recently
especially in light of the increase in infections such as those
caused by mycobacteria.
[0004] Mycobacterial infections often manifest as diseases such as
tuberculosis. Human infections caused by mycobacteria have been
widespread since ancient times. and tuberculosis remains a leading
cause of death today. Although the incidence of the disease
declined in parallel with advancing standards of living since at
least the mid-nineteenth century, mycobacterial diseases still
constitute a leading cause of morbidity and mortality in countries
with limited medical resources and can cause overwhelming,
disseminated disease in immunocompromised patients. In spite of the
efforts of numerous health organizations worldwide, the eradication
of mycobacterial diseases has never been achieved, nor is
eradication imminent. Nearly one third of the world's population is
infected with M. tuberculosis complex, commonly referred to as
tuberculosis (TB), with approximately 8 million new cases and 3
million deaths attributable to TB yearly. After decades of decline,
TB is on the rise. In the United States, up to 10 million
individuals are believed to be infected. Almost 28,000 new cases
were reported in 1990, a 9.4 percent increase over 1989. A sixteen
percent increase was observed from 1985 to 1990. Overcrowded living
conditions and shared air spaces are especially conducive to the
spread of TB, contributing to the increase in instances that have
been observed in the U.S. in prison inmates and among the homeless
in larger cities. Approximately half of all patients with acquired
immune deficiency syndrome (AIDS) will acquire a mycobacterial
infection, with TB being an especially devastating complication.
AIDS patients are at higher risks of developing clinical TB and
anti-TB treatment seems to be less effective than in non-AIDS
patients. Consequently, the infection often progresses to a fatal
disseminated disease.
[0005] Mycobacteria other than M. tuberculosis are increasingly
found in opportunistic infections that plague the AIDS patient.
Organisms from the M. avium-intracellulare complex (MAC),
especially serotypes four and eight, account for 68% of the
mycobacterial isolates from AIDS patients. Enormous numbers of MAC
are found (up to 10.sup.10 acid-fast bacilli per gram of tissue)
and, consequently the prognosis for the infected AIDS patient is
poor.
[0006] The World Health Organization (WHO) continues to encourage
the battle against TB, recommending prevention initiatives such as
the "Expanded Program on Immunization" (EPI), and therapeutic
compliance initiatives such as "Directly Observed Treatment
Short-Course" (DOTS). For the eradication of TB, diagnosis,
treatment, and prevention are equally important. Rapid detection of
active TB patients will lead to early treatment by which about 90%
cure is expected. Therefore, early diagnosis is critical for the
battle against TB. In addition, therapeutic compliance will ensure
not only elimination of infection, but also reduction in the
emergence of drug-resistance strains.
[0007] The emergence of drug-resistant M. tuberculosis is an
extremely disturbing phenomenon. The rate of new TB cases proven
resistant to at least one standard drug increased from 10 percent
in the early 1980's to 23 percent in 1991. Compliance with
therapeutic regimens, therefore, is also a crucial component in
efforts to eliminate TB and prevent the emergence of drug-resistant
strains.
[0008] Although over 37 species of mycobacteria have been
identified, more than 95% of all human infections are caused by six
species of mycobacteria: M. tuberculosis, M. avium-intracellulare,
M. kansasii, M. fortuitum, M. chelonae, and M. leprae. The most
prevalent mycobacterial disease in humans is tuberculosis (TB)
which is caused by mycobacterial species comprising M.
tuberculosis, M. bovis, or M. africanum (Merck 35 Manual 1992).
Infection is typically initiated by the inhalation of infectious
particles which are able to reach the terminal pathways in lungs.
Following engulfment by alveolar macrophages, the bacilli are able
to replicate freely, with eventual destruction of the phagocytic
cells. A cascade effect ensues wherein destruction of the
phagocytic cells causes additional macrophages and lymphocytes to
migrate to the site of infection, where they too are ultimately
eliminated. The disease is further disseminated during the initial
stages by the infected macrophages which travel to local lymph
nodes, as well as into the blood stream and other tissues such as
the bone marrow, spleen, kidneys, bone and central nervous system.
(See Murray et al. Medical Microbiology, The C.V. Mosby Company
219-230 (1990)).
[0009] There is still no clear understanding of the factors which
contribute to the virulence of mycobacteria. Many investigators
have implicated lipids of the cell wall and bacterial surface as
contributors to colony morphology and virulence. Evidence suggests
that C-mycosides, on the surface of certain mycobacterial cells,
are important in facilitating survival of the organism within
macrophages. Trehalose 6,6' dimycolate, a cord factor, has been
implicated for other mycobacteria.
[0010] The interrelationship of colony morphology and virulence is
particularly pronounced in M. avium. M. avium bacilli occur in
several distinct colony forms. Bacilli which grow as transparent or
rough colonies on conventional laboratory media are able to
multiply within macrophages in tissue culture, are virulent when
injected to susceptible mice, and are resistant to antibiotics.
Rough or transparent bacilli which are maintained on laboratory
culture media often spontaneously assume an opaque colony
morphology at which time they fail to grow in macrophages, are
avirulent in mice, and are highly susceptible to antibiotics. The
differences in colony morphology between the transparent, rough and
opaque strains of M. avium are almost certainly due to the presence
of a glycolipid coating on the surface of transparent and rough
organisms which acts as a protective capsule. This capsule, or
coating, is comprised primarily of C-mycosides which apparently
shield the virulent M. avium organisms from lysosomal enzymes and
antibiotics. By contrast, the non-virulent opaque forms of M. avium
have very little C-mycoside on their surface. Both resistance to
antibiotics and resistance to killing by macrophages have been
attributed to the glycolipid barrier on the surface of M.
avium.
[0011] Diagnosis of mycobacterial infection is confirmed by the
isolation and identification of the pathogen, although conventional
diagnosis is based on a sputum smears, chest X-ray examination
(CXR), and clinical symptoms. Isolation of mycobacteria on a medium
takes as long a time as four to eight weeks. Species identification
takes a further two weeks. There are several other techniques for
detecting mycobacteria such as the polymerase chain reaction (PCR),
mycobacterium tuberculosis direct tests, or amplified mycobacterium
tuberculosis direct test (MTD), and detection assays that utilize
radioactive labels.
[0012] One diagnostic test that is widely used for detecting
infections caused by M. tuberculosis is the tuberculin skin test.
Although numerous versions of the skin test are available,
typically one of two preparations of tuberculin antigens are used:
old tuberculin (OT), or purified protein derivative (PPD). The
antigen preparation is either injected into the skin intradermally,
or is topically applied and is then invasively transported into the
skin with the use of a multiprong inoculator (Tine test). Several
problems exist with the skin test diagnosis method. For example,
the Tine test is not generally recommended because the amount of
antigen injected into the intradermal layer cannot be accurately
controlled. (See Murry et al. Medical Microbiology, The C.V. Mosby
Company 219-230 (1990)).
[0013] Although tuberculin skin tests are widely used, they
typically require 2-3 days to generate results, and many times, the
results are inaccurate as false positives are sometimes seen in
subjects who have been exposed to mycobacterium but are healthy. In
addition, instances of mis-diagnosis are frequent since a positive
result is not observed only in active TB patients, but also in
BCG-vaccinated persons and those who had been infected with
mycobacteria but have not developed the disease. It is hard
therefore, to distinguish active TB patients from the others, such
as household TB contacts, by the tuberculin skin test.
Additionally, the tuberculin test often produces a cross-reaction
in those individuals who were infected with mycobacteria other than
M. tuberculosis (MOTT). Diagnosis using the skin tests currently
available is frequently subject to error and inaccuracies.
[0014] What is needed are effective tests for detecting the
presence of mycobacterial infection. In particular a test that does
not require the invasion of the skin surface of the tested person
would minimize the exposure of the health care professional
administering the test to the bodily fluids of the tested person
and lessen the risk of transmission of other infectious agents that
may be present in the tested person. In addition, a test that is
easily administered and has an easily determined positive or
negative outcome is essential when monitoring compliance with a
therapeutic regimen for highly infectious diseases such as
tuberculosis, particularly in individuals such as homeless persons,
prison inmates, schoolchildren and senior citizens.
[0015] What is also needed are inexpensive and accurate methods for
distinguishing between persons who have active disease states and
those persons who have only been immunologically exposed to
infectious agents, (such as those persons previously infected with
a mycobacterium) but are without active disease, or those persons
who have been vaccinated with BCG. Additionally, there is no known
method for monitoring the effects of drug therapy in persons
infected with a mycobacterium, such as tests that can distinguish
between active tuberculosis and other stages of healing or prior
exposure. Furthermore, what is also needed is a test that can be
easily administered to children, who are especially afraid of
currently used skin tests that involve needles or puncturing the
skin. Such tests are particularly desirable for monitoring patients
particularly AIDS patients who are highly susceptible to
mycobacterial infection. In addition, tests that are easily
administered and have an easily determined positive or negative
outcome are essential when monitoring a disease such as
tuberculosis in homeless persons or prison inmates.
SUMMARY OF THE INVENTION
[0016] The present invention comprises methods and compositions for
the detection of infectious diseases. In accordance with a
preferred embodiment of the present invention, transdermal delivery
systems or devices, such as patches containing mycobacterial
antigen compositions, are provided. Such patches are worn on the
skin and removed after a predetermined amount of time. The skin is
then examined for an immunogenic response to the presence of the
antigen in the patch
[0017] 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 especially effective in detecting M.
tuberculosis infection in active tuberculosis patients thereby
eliminating the possibility of misdiagnosing individuals who have
received vaccines or have been otherwise exposed to the organism
without disease manifestation.
[0018] The diagnostic methods described herein include the topical
application of compositions comprising mycobacterial antigens
including, but not limited to, MPB44, MPB45, MPB51, MPB59, MPB64,
MPB70, MPB80 or MPB83, for transdermal delivery to skin and for
subsequent detection of an immunogenic response. The antigens may
be applied individually or in combination. Particularly preferred
is the topical application of an antigen composition comprising
MPB64. The present invention contemplates any antigen that has the
characteristics of MPB64, in that there is a delayed-type
hypersensitivity reaction to the antigen in the presence of active
tuberculosis disease, and no reaction where there has been no
exposure to mycobacteria, or in exposure via vaccine or other
non-active tuberculosis state.
[0019] Accordingly, it is an object of the present invention to
provide methods and compositions for the detection of infectious
diseases.
[0020] Another object of the present invention to provide methods
and compositions for the detection of active tuberculosis.
[0021] It is another object of the present invention to provide
methods and compositions for the detection of active tuberculosis
using topical application of antigen compositions for transdermal
delivery to the skin.
[0022] Another object of the present invention to provide methods
and compositions for the detection of mycobacterial infections.
[0023] Yet another object of the present invention to provide
methods and compositions for the detection of active disease caused
by mycobacterial species comprising M. tuberculosis complex, M.
avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae, M.
leprae, M. africanum, and M. microti.
[0024] Another object of the present invention to provide methods
and compositions for the detection of active disease caused by M.
tuberculosis.
[0025] It is yet another object of the present invention to provide
methods and compositions for the detection of active disease caused
by M. bovis.
[0026] Another object of the present invention is to provide
methods and compositions for the immunological detection of
mycobacterial infection, that utilize topical application without
requiring invasive procedures.
[0027] Yet another object of the present invention is to provide
sensitive diagnostic methods and compositions for the detection of
active disease caused by mycobacteria wherein antigen compositions
are topically applied and transdermally delivered and skin is
subsequently examined for an immunogenic response.
[0028] Another object of the present invention is to provide
methods and compositions for the detection of active disease caused
by mycobacteria wherein the topically applied mycobacterial antigen
composition comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70,
MPB80 or MPB83.
[0029] Yet another object of the present invention is to provide
methods and compositions for the detection of active disease caused
by mycobacteria wherein the topically applied mycobacterial antigen
composition comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70,
MPB80 or MPB83, wherein the antigen is applied either individually
or in combination with another mycobacterial antigen.
[0030] 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 MPB64.
[0031] Yet another object of the present invention is to provide
methods and compositions for diagnosis of infectious disease that
is easy to administer.
[0032] An additional object of the invention is to provide methods
and compositions for detection of active tuberculosis for
monitoring the effectiveness of therapeutic treatments.
[0033] Another object of the present invention is to provide a kit
for diagnosis and detection of active disease caused by
mycobacteria.
[0034] Yet another object of the present invention is to provide
methods and compositions for detection of active disease caused by
mycobacteria in household TB or mycobacterial disease contacts.
[0035] It is another object of the present invention to provide
methods and compositions for the monitoring of the clinical status
of a mycobacteria-infected patient following chemotherapy.
[0036] Another object of the present invention is to provide
sensitive diagnostic methods and compositions for children with
active tuberculosis.
[0037] 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 method involves the use
of a skin patch.
[0038] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 shows photographs of the dose response of varying
amounts of MPB64 antigen by skin reactions in guinea pigs.
[0040] FIG. 2 is a graph showing the time course of delayed-type
hypersensitivity response to a patch with MPB64 antigen following
inoculation of guinea pigs with BCG.
DETAILED DESCRIPTION
[0041] The present 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 thereof, it is not intended that such details should be
regarded as limitations upon the scope of the invention. The entire
text of the references mentioned herein are hereby incorporated in
their entireties by reference, including U.S. provisional
application Ser. No. 60/073,911 filed Feb. 6, 1998, and U.S.
provisional application Ser. No. 60/096,140 filed Aug. 11,
1998.
[0042] Mycobacterial infections such as those causing tuberculosis,
once thought to be declining in occurrence, have rebounded and
again constitute a serious health threat. Areas where humans are
crowded together or living in substandard housing are increasingly
found to have persons infected with mycobacteria. Persons who are
immunocompromised are at great risk of being infected with
mycobacteria and dying from such infection. In addition, the
emergence of drug-resistant strains of mycobacteria has added to
the treatment problems of such infected persons.
[0043] Many people who are infected with mycobacteria are poor or
live in areas with inadequate health care facilities. Such people
are not easily tested for mycobacterial infection and need
inexpensive and noninvasive methods for detection of infections.
Additionally, persons who are in prison or are homeless, generally
have inadequate healthcare, poor physical condition and adequate or
successful health care intervention is typically unavailable.
[0044] The present invention provides methods and compositions
comprising topical applications of antigen compositions for
transdermal delivery of antigens, particularly mycobacterial
antigens. More particularly, the present invention provides methods
and compositions for detecting disease such active tuberculosis,
and distinguishing persons with active disease from persons who
have only been exposed immunologically to infectious agents such as
mycobacteria.
[0045] The methods and compositions of the present invention may be
used for testing the presence of mycobacteria infection in humans
as well as other animals. For example, the present invention may be
particularly useful for the detection of disease in cows infected
with M. bovis.
[0046] Medically "active tuberculosis" is diagnosed by the well
known medical procedures of chest X-ray (CXR), sputum tests, or
other symptoms. Because precise identification of the presence of
mycobacterial infectious agents is expensive and takes a long time,
diagnosis of active disease does not necessarily include the
identification of the presence of mycobacteria. Accordingly,
diagnosis of active disease such as tuberculosis may be dependent
upon detecting other aspects of mycobacterial infection such as the
generation of particular immune responses or the manifestation of
certain symptoms. As used herein the term "tuberculosis" comprises
disease states usually associated with infections caused by
mycobacteria species comprising M. tuberculosis complex.
Mycobacterial infections caused by mycobacteria other than M.
tuberculosis (MOTT) are usually caused by mycobacterial species
comprising M. avium-intracellulare, M. kansasii, M. fortuitum, M.
chelonae, M. leprae, M. africanum, and M. microti.
[0047] The present invention includes methods and compositions for
topical application enabling transdermal delivery of antigens that
elicit an immune response, such as a delayed-type hypersensitivity
response in persons who have active disease such as tuberculosis
caused by mycobacterial infections. Such antigens are derived from
mycobacteria, or are cross-reactive with mycobacterial proteins or
carbohydrate moieties. Preferred antigens comprise those that are
derived from mycobacteria including, but not limited to, MPB44,
MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83. A particularly
preferred antigen is MPB64. (See Kawajiri et al. Japanese Patent
Application, Pub. No. 09206092 which is incorporated by reference
herein in its entirety)
[0048] Another preferred combination of antigens comprises the
combined use of MPB64 and MPB59. For example, combination of MPB59
and MPB64 may indicate the infection of atypical mycobacteria since
a subject infected by other kinds of mycobacteria that do not
secrete MPB64 will show a positive response to MPB59.
[0049] MPB64 is a mycobacterial antigen frequently associated with
the M. tuberculosis complex. It was first described as MPT64 by
Harboe et al. (Infect. Immun. 1986; 52:293-902, which is herein
incorporated in its entirety), and has been well characterized and
used in various laboratories since then. (See for example Yamaguchi
et ale Infect. and Immu. 1989; 57:283-288, which is also
incorporated herein in its entirety.) "MPB64" and "MPT64" refer to
the same antigen: MPT64 was isolated from the culture filtrate of
M. tuberculosis, and was therefore named as mycobacterial protein
of tuberculosis, and MPB64 was later isolated from the culture
filtrate of M. bovis (or BCG) and was therefore named as
mycobacterial protein of bovis. It was subsequently discovered that
both proteins are the same. MPB64 and MPT64 refer to antigens
secreted from mycobacteria species including, but not limited to,
M. tuberculosis, M. bovis, and some strains of M. bovis BCG. The
antigen is secreted during bacterial growth and is immunogenic,
eliciting delayed type hypersensitivity (DTH) in guinea pigs and
humans.
[0050] Recombinant antigens may also be used in the diagnostic
methods and compositions contemplated by the present invention. See
for example Haga et al., Journ. of Leukocyte Biology 1995;
57:221-225; Roche et al., Clin. Exp. Immunol. 1996; 103(2)226-232;
and Roche et al., J. Infect. Dis. 1994; 107(5): 1326-30, each of
which is incorporated herein in its entirety.
[0051] The antigens of the present invention are topically applied
for transdermal delivery into the skin of the person to be tested.
The antigen is applied by maintaining a composition comprising the
antigen in close contact with. the skin. The concentration of the
antigen in the composition is in a range of approximately 1 to 150
micrograms/dosage applied, more particularly 10 to 100
micrograms/dosage applied, most particularly 30 to 75
micrograms/dosage applied. The antigen composition may comprise a
physiologically effective solution comprising surfactants, buffers
and solvents that enable transdermal delivery of the antigen
composition. Preferably surfactants, buffers and solvents that
improve permeation and transport of the antigen, and that do not
themselves trigger a reaction or interfere with the immunogenicity
of the antigen, are used. Preferred surfactants for the antigen
composition comprise, TWEEN.RTM. 20, TWEEN.RTM. (polysorbate
surfactant) 40, TWEEN.RTM. 60, and TWEEN.RTM. 80; each of which may
be used at concentrations ranging from 0.001-10%, 0.001-1% and
preferably 0.005%, in phosphate buffered saline. A preferred
embodiment for the application comprises 30 to 75 micrograms of
antigen in 100 microliters of phosphate buffered saline further
comprising the preferred surfactant TWEEN.RTM. 80. A most preferred
embodiment for application is 75 micrograms of antigen in 100
microliters of phosphate buffered saline (PBS) with 0.005%
TWEEN.RTM. 80. Prepared antigen compositions may be stored in
suitable aseptic glass or plastic containers, in batches or
aliquoted according to desired quantities.
[0052] The present invention is particularly directed to methods of
transdermal delivery of antigen compositions to skin cells for the
detection of active disease. Accordingly, all contemplated solvent
and antigen combinations that enable the delivery of infectious
agent antigens to skin cells, and result in the detection of active
disease, are included herewith.
[0053] As used herein, the term "transdermal delivery" refers to
the delivery of a composition to all layers of the skin, including
but not limited to, the epidermis (stratum corneum, stratum
lucidum, stratum granulosum, stratum spinosum, stratum basale), the
dermis, and the subcutaneous layer. As used herein, the term
"topical application" refers to the application or placement of a
composition on skin without puncturing or otherwise invasively
entering the skin by use of needles and the like.
[0054] A preferred embodiment of the present invention comprises a
transdermal delivery system or device for holding the compositions
described above in close contact with the skin of a person. A
highly preferred embodiment comprises a patch band, such as skin
patch band, for holding the composition in close contact with the
skin. Materials that are suitable for use in the patch for
delivering the antigen composition of the present invention include
TORII's patch band TORIIBAN.TM. (obtained from Torii and Co., Ltd.,
Tokyo), FINN-CHAMBER.RTM., and Perme-aid S. (Nitto-Denkou Co.
Japan). In addition, materials such as medical adhesive plaster or
tape may also be used wherein a portion of the plaster or tape
includes a portion of a material impregnated with the antigen
composition, and wherein the material is located so that it is in
direct contact with the skin. Suitable medical adhesive plasters,
tapes and fabrics are made by numerous manufacturers such as
Nichiban (Japan), Kimberly-Clark (Neenah, Wis.), and 3M (St. Paul,
Minn.). The device may be held in place by various fastening means
well known to those skilled in the art. For example, the device may
be tied to the subject's arm by use of a string, or it may be
attached by use of adhesive. Preferably, material such as the
adhesive used for holding the device in place should be gas
permeable and water-resistant so that it does not fall off as a
result of becoming wet due to perspiration or bathing.
[0055] One particularly advantageous aspect of the present
invention relates to the ease with which the invention may be used
and executed. For example, integrity of the antigen compositions
may be preserved by shipping or maintaining compositions in aseptic
containers under appropriate temperature conditions. The antigen
compositions may be stored in aliquots of desired amounts, for
example, 100 or 200 .mu.l, and then applied to gauze plaster or
tape or the like, as necessary for testing patients. Furthermore,
each of the components necessary for the compositions and methods
of the present invention may be provided together in a kit to
facilitate use.
[0056] In addition to a patch-type embodiment, 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.
[0057] A preferred method contemplated by the present invention
comprises the topical administration of a composition comprising
mycobacterial antigen for transdermal delivery to the skin of a
human. For example, a patch containing the antigen composition is
applied to the forearm of a person and held in close contact with
the skin. The patch is left in place for a predetermined amount of
time so as to enable sufficient transdermal administration of the
antigen. Such an amount of time may range from 1 to 7 days,
preferably 2 to 5 days, most preferably 3 days. After the specified
time has passed, the patch is removed and the skin is examined for
an immunogenic response.
[0058] It is known in the art that a delayed-type hypersensitivity
reaction is observed in skin in response to the presence of some
antigens. Typically such reactions are observed following invasive
introduction of an antigen composition, and usually such reactions
are characterized by redness, erythema, induration (raised
thickening of the skin), presence of red vesicles or ulcers. The
response looked for in the present method is similar to the
response seen with the intradermal injection of mycobacterial
antigen.
[0059] The inventors of the present invention have surprisingly
found that transdermal delivery by topical application the antigens
of the present invention, cause an immunogenic response
(specifically a delayed-type hypersensitivity (DTH) reaction) in
persons who have active mycobacterial disease such as tuberculosis.
As shown in the examples herein, TB-infected guinea pigs express a
delayed-type hypersensitivity skin reaction 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 some time after vaccination. As discovered by the inventors,
the methods and compositions of present invention, comprising
mycobacterial antigens, particularly MPB64, can be used for the
diagnosis of active mycobacterial disease such as active TB.
Surprisingly, individuals who were infected with M. tuberculosis
but had not developed tuberculosis, and individuals who had
previously received BCG vaccinations, do not show a positive skin
reaction in response to MPB64.
[0060] The novel discovery of transdermal delivery of antigens by
topical application, particularly MPB64, for diagnosing active
mycobacteria disease such as tuberculosis, is especially desirable
because no invasive procedures are required. In fact, intradermal
delivery of MPB64 has been recently shown to be unsuccessful for
distinguishing between TB patients and healthy controls (Wi/eke et
al. Tubercle and Lung Disease (1996) 77:250-256). Effective
transdermal delivery of mycobacterial antigens by topical
application was heretofore unknown, and using the methods and
compositions of the present invention, the inventors have
successfully developed diagnostic procedures for distinguishing
between active TB patients and healthy PPD positive controls.
[0061] Although not wishing to be bound by the following theory, it
is thought that one reason intradermal injection of antigen may be
less effective than transdermal application is related to
solubility aspects of the antigen and/or inadequate opportunity for
antigen presentation. For example, it is possible that with
intradermal injection the antigen is quickly dissolved in body
fluids with insufficient time for the immune system to recognize
and/or respond to it. In contrast, the novel methods of the present
invention enable the antigen to be gradually introduced to the
subject's immune system, and as the antigen slowly permeates
through the skin pores and sweat glands into the intradermal local
portion, there is enough time for the immune system to mount a
response, typically in the form of a delayed hypersensitivity
reaction. It is also possible that intradermal and transdermal
introduction of antigen elicit distinct immune responses.
[0062] Although not wishing to be bound by the following theory, it
is thought that hypersensitivity reactions (also known as Type IV
or cell mediated immunity reactions) are mediated largely by T
cells with consequent involvement of monocytes. Such reactions
typically have responses that are observed after a passage of time,
i.e. 18-24 hours, and are therefore referred to as delayed-type
hypersensitivity. It is thought that an antigen presenting cell
presents the antigen to a T cell and following activation, the T
cells release lymphokines that cause accumulation and activation of
macrophages, monocytes and nonimmune lymphocytes.
[0063] The present invention may also be used to detect active
tuberculosis in household contacts of previously infected TB
patients. This allows for the monitoring of the spread of the
disease to others in close contact with the originally infected
persons. Because members of the public may have tuberculosis and
spread it by coughing, the present invention can be used to monitor
the exposure of persons who work with the public, such as airline
stewards or health care professionals. Persons who work with the
homeless or prison populations are also easily monitored for the
presence of active tuberculosis.
[0064] An especially desirable use of the present invention is the
monitoring of the effectiveness of treatment of persons with
mycobacterial infections. For example, the ability to detect when
the active tuberculosis patient no longer has active TB thus, the
treatment is effective, is highly desirable with the rise in drug
resistant mycobacteria. A preferred method includes topically
applying the antigens of the present invention to the person with
active TB prior to treatment, and after a sufficient time,
observing the skin reaction. At a later point in the treatment
regimen, the antigens are again applied and the skin reaction is
observed. Lack of response by the skin indicates that the treatment
has been effective in changing the active tuberculosis state. A
continued response by the skin indicates that the treatment either
is not effective, or that there has not been enough time for the
treatment to be effective, and the person still has active TB. At
this time, the treatment could be changed, the drug sensitivity of
the infecting mycobacterium could be determined, or the same
treatment could be continued for a longer amount of time.
[0065] One use of the present invention would be for rapid
screening of a population such as when investigating a neighborhood
or slum for infection rates, for testing incoming prisoners, or a
group of homeless persons in a shelter or on the street. For
example, incoming prisoners would have patches containing the
antigens of the present invention issued to them upon entry to the
prison. After the specified time of wearing the patch, the skin
reactions of the prisoners are examined. Those with positive
reactions would be housed separately. Because the present invention
detects active TB, those prisoners could be isolated from the other
prisoners and immediately begin treatment. Other tests for TB would
enable detection of prisoners who have had exposure to TB at some
time in their lives, and who may not be capable of transmitting TB
to others. There is no need to isolate and treat persons who are
not capable of transmitting TB to others.
[0066] In many parts of the world, persons are vaccinated against
TB. It is extremely difficult to detect active TB in these persons
because with standard TB skin tests, all persons exposed to
mycobacteria test positive, whether there is active TB or the
effects from vaccination. The present invention is used in such
areas to detect the presence of active TB in individuals and
distinguish such individuals from those who were previously
vaccinated.
[0067] The ease of administration is a particularly beneficial
aspect of the present invention. For example, children are not
frightened by the application of a topical device, such as a patch,
and are not hesitant to wear such a device for a time sufficient to
create a skin response. Such topical devices as patches are easily
stored and transported to isolated places that may lack
refrigeration and clean water. The present invention can be made
from inexpensive materials that can be produced at low cost and
used by health care organizations worldwide.
[0068] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had: to various other
embodiments, modifications, and equivalents thereof, which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention.
Example 1
[0069] To test the development of a new, simple and rapid
diagnostic method for active tuberculosis, subjects were tested for
skin reaction to the antigen MPB64 by a transdermal delivery method
following topical application of an antigen composition using
removable patches.
Skin Patch Preparation
[0070] Although antigens such as MPB59, MPB70, MPB44, MPB45 or
MPB51 or MPB64 may be used in the patch test of the present
invention, in the following example the antigen used was MPB64 in a
concentration of approximately 75 .mu.g per patch. It is
contemplated that approximately 50 to 100 .mu.g of antigen may
successfully be used per patch. A preferred antigen solution
comprises approximately 750 .mu.g antigen per ml of phosphate
buffered solution (PBS), wherein the PBS consists of 0.005%
TWEEN.RTM. 80.
[0071] The patches were applied on the skin of the test subjects,
left on for three days, and the results were interpreted by
observing the site following removal of the patch. The presence of
an immunological response such as a delayed-type hypersensitivity
(DTH) reaction (redness, induration, or small red vesicles
indicated a positive result), i.e. the presence of active
tuberculosis. No change at the site was concluded as a negative
response.
Test Subjects
[0072] 53 patients with active tuberculosis, and 43 healthy
purified protein derivative (PPD) positive controls were tested to
determine whether or not the reaction to MPB64 was positive only in
active tuberculosis patients. Tuberculosis patients from four
clinics, in the vicinity of Manila, Philippines, Our Lady of Grace
Parish, St. Nino de Tondo Parish, Canossa Health and Social Center,
and Health Care Development Center, were examined.
[0073] Of the 53 active tuberculosis patients, 52 showed positive
reaction to MPB64, while none of the 43 PPD-positive controls had a
positive reaction to MPB64. The specificity of MPB64 for active
tuberculosis was 100% and the sensitivity was 98.1%. Efficacy of
the test was 98.9%.
[0074] The patch test with MPB64 is an effective and accurate
method for the diagnosis of active tuberculosis, distinguishing
such patients from BCG-vaccinated individuals and those naturally
infected, but not developing tuberculosis. The experimental design
for this finding is more fully described in Example 2.
Example 2
[0075] To determine the reliability of MPB64 as a specific antigen
for diagnosing active TB using the skin patch method, comparative
tests were conducted among three different classes of individuals:
[0076] (1) active TB patients [0077] (2) healthy
tuberculin-positive persons [0078] (3) household TB-contacts
[0079] Correlation between skin reaction to MPB64 and the clinical
status of TB in humans was observed. Because the purpose of this
study was to determine the reliability of MPB64 as the specific
diagnosing antigen for active TB, the selection of active TB
patients was most important.
[0080] Clinical records of the outpatients coming to the clinics
were checked. Those patients who were sputum-smear positive, had an
abnormal CXR, and had clinical symptoms indicating active TB were
classified as active TB patients, Group 1. Culture results were not
available in most cases. The patients who had started chemotherapy
shortly before were preferable because the effect of long-term
chemotherapy on the MPB64 skin reaction was not known. However,
there were some patients in Group 1 who had been treated for 6
months. They were considered as active TB patients because of
positive smears in recent examinations and because of their
symptoms.
[0081] The patients were living near the clinics where the
socioeconomic conditions were very poor. The geographical situation
of their residences was important because they were scheduled to
return to the clinic 3 days later for the results to be read. Only
12 out of 105 patients tested did not return. Among the patients
who returned, 53 were available for the final analysis. The
analysis is shown in Example 1. The rest of the subjects were
excluded because their patches had been removed or fallen off
before the reading day.
[0082] The patients were screened according to their clinical
records and only those who were smear positive, had an abnormal CXR
and had other symptoms such as cough, fever, or weight loss to
indicate TB were selected as active TB patients. Culture positive
patients were preferable, but the results of culture were available
in only seven cases. Most of the active TB patients had been on
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 sign of TB. At the time
of the test, some family members came to the clinic along with TB
patients. They were tested as "household TB-contacts". All the
subjects were informed of the outline of the study and gave consent
for the test. The number of subjects in the three groups were as
follows; Group 1, active TB patients, 53; Group 2, healthy
controls, 43; and Group 3, household TB contacts 41.
[0083] MPB64 was isolated from an 8-day culture filtrate of M.
bovis BCG Tokyo (obtained from Japan BCG Laboratory, Tokyo, Japan).
The purified protein was suspended in PBS and stored at -200 C. The
amount of protein was measured by Lowry's method. Ammonium
sulfate-precipitated whole protein from the 8-day culture filtrate
of M. bovis BeG was named PPD-eT to distinguish it from purified
protein derivative tuberculin (PPD), and used as the control for
the patch test. PPDs prepared from M. tuberculosis Aoyama B was
obtained from Japan BCG Laboratory (Tokyo, Japan). Five tuberculin
units (TU) of PPDs suspended in 0.1 ml of reconstitution buffer
were used for the Mantoux test with an intradermal injection.
Materials for the Patch Test
[0084] TORII's patch band TORIIBAN.TM. (obtained from Torii and
Co., Ltd., Tokyo) of 15-mm gauze size was used. The antigen
solution (75 .mu.g of the antigen in 100 .mu.l of PBS containing
0.005% TWEEN.RTM. 80) was applied on the gauze and the patch was
attached to a forearm of a human subject after cleaning with
alcohol the skin area to which the patch was to be applied. The
patch was placed on the subject's skin such that the gauze
impregnated with the antigen solution contacted the skin directly.
The patch was left on for 72 hours.
Patch Test Schedule
[0085] Active TB patients and tuberculin-positive healthy controls
were tested for a skin reaction to the MPB64 patch on the left arm,
and to the PPD-eT patch on the right arm. Each patch contained 75
.mu.g of antigen. A PPD (5 TU/100 ml) dose was injected
intradermally into the right forearm in a place separate from the
patch. A PPD-eT patch was used to confirm that the protein antigens
did get into the body transdermally. If the PPD test was positive
and the PPD-eT patch test was negative, transdermal administration
was incomplete. Such cases were excluded from the test results. The
attached patch was removed 3 days later (72 hr) and the reaction
was read as positive or negative. No change in the skin at the site
was "negative", while erythema, induration, or a few small red
vesicles at the site were recorded as a "positive" response to the
antigen.
[0086] We have compared applying the patch on the forearm and the
upper arm. Patches detached more easily from the upper arm (41.2%)
than the forearm (17.6%) before the reading day. Therefore, the
forearm is recommended for the test in adults.
Statistics
[0087] The two-by-two contingency test was used to evaluate the
results of the MPB64 patch tests in humans.
Active TB Patients and Tuberculin Positive Healthy Controls
[0088] Table 1 shows the actual numbers in Groups 1 and 2 with
positive or negative reaction to MPB64. All the subjects were
positive to the PPDs Mantoux test and the PPD-eT patch test. From
these results, the following values were calculated: Sensitivity,
98.1%; Specificity, 100%; False positive rate, 0%; False negative
rate, 1.9%: Positive predictive value, 100%; Negative predictive
value, 97.7%; Efficacy of the test, 98.9%. The results indicated
that the MPB64 patch test is an effective method to distinguish
active TB from healthy tuberculin-positive persons.
TABLE-US-00001 TABLE 1 MPB64 Patch Test Two-by-Two Contingency Test
Between Group 1 and Group 2 Group Positive Negative Total 1: TB
Patients 52 1 53 2: Healthy Controls 0 43 43 Total 52 44 96
Household TB-Contacts
[0089] The number of household TB-contacts (Group 3) were 41
including 12 males and 29 females. The results of the patch test
are shown in Table 2. There were 26 subjects showing a positive
reaction to both PPD-eT and MPB64 patches (63.4%), and nine
subjects positive to PPD-eT but negative to MPB64 (22.0%). Six
persons (14.6%) were negative to PPD-eT and MPB64. Among these
double-negative persons, three were negative in PPDs Mantoux
test.
[0090] The subjects in Group 3 were not, registered as TB patients
at the clinic. The clinical status of each person was not known
although some symptoms suggesting TB were observed.
TABLE-US-00002 TABLE 2 Patch Test with MPB64 and PPD-eT in
Household TB-contacts PDD-et/MPB64 Subjects +/+ +/- -/- Total Male
7 3 2 12 Female 19 6 4 29 Total 26 9 6 41
[0091] From this study it is strongly suggested that MPB64 patch
test is a promising tool for rapid diagnosis of active TB. It can
distinguish active TB patients from individuals who were vaccinated
with BCG or those who were TB-infected but had not developed the
disease with 98.1% sensitivity and 100% specificity. The patch test
also has advantages over an intradermal injection in the technical
ease and the safety of its application. Though no wishing to be
bound by any theory, it is thought that the patch test can supply
antigen continuously for response by the patient.
Example 3
[0092] In order to determine the reliability of MPB64 as a specific
diagnosing antigen for active TB using the skin patch method,
comparative tests were conducted on guinea pigs.
[0093] Female albino Hartley guinea pigs weighing 300 to 400 g at
the beginning of the experiments, were purchased from Japan
Laboratory Animals, Inc., Tokyo. Animals were maintained under
specific pathogen free conditions at the Japan BCG Laboratory.
Antigens
[0094] Antigens were prepared according to the methods and
materials set forth in Example 2.
Immunization of Guinea Pigs
[0095] Live BCG vaccine (Japan BCG Laboratory, Tokyo, Japan) was
reconstituted according to the manufacturer's instructions and
injected subcutaneously without adjuvant into guinea pigs at a dose
of 0.5 mg per animal. The animals were tested between 4 and 25
weeks after the BCG injection.
Materials of Patch Test
[0096] TORII's patch band (Torii and Co., Ltd., Tokyo, Japan) of
7-mm gauze size was used. An antigen solution (75 .mu.g of antigen
in 15 .mu.l of PBS containing 0.005% TWEEN.RTM. 80) was applied on
the gauze, and the patch was attached to a shaved area of each
guinea pig.
[0097] MPB64 was applied to a patch at various doses as indicated
in FIG. 1 and the patches were attached to the right and left
flanks of a BCG-immunized guinea pig, where the hairshad been
removed.
Patch Test Schedule
[0098] Patches were removed at 24 hours and the reaction was read
immediately. No change in the skin at the site was "negative",
while erythema, induration, or a few small red vesicles at the site
were recorded as a "positive" response to the antigen.
Dose Response to MPB64 in the Patch Test in BeG-Immunized Guinea
Pigs
[0099] Guinea pigs immunized with BCG Tokyo 4 weeks previously were
used for the MPB64 patch test with various doses of the antigen.
The highest dose for the patch test was 75 .mu.g/patch. The animals
were tested with the patches containing MPB64 in various doses
between 2.3 and 75 mg/patch. The patches were removed 24 hours
later and the reaction was read as positive or negative. To make
sure that the animals were sensitized to BCG, 0.05 .mu.g of PPDs in
0.1 ml of the buffer was injected intradermally and the skin
reaction was measured at 24 hours. FIG. 1 shows the results of the
dose-response experiment. The response to MPB64 was positive at a
dose of 4.7 .mu.g/patch or higher concentration. Positive reaction
was not observed at a dose of 2.3 .mu.g of MPB64 per patch.
Negative control patches which contained only PBS containing 0.005%
TWEEN.RTM. 80 did not elicit any skin reaction in the BCG-immunized
guinea pigs. Nonimmunized guinea pigs did not show any response to
either PPDs nor MPB64.
Time Course of the Skin Reaction to MPB64 in BCG-Immunized Guinea
Pigs
[0100] It is known that BCG-immunized guinea pigs lost skin
reaction to MPB64 15 weeks after BCG immunization when tested by
the intradermal injection of MPB64. To address the question of
whether this was true in the case of the patch test, guinea pigs
were immunized with BCG Tokyo and tested with MPB64 patches at
various times after the BCG injection. Individual animals were
tested only one time to avoid the booster effect. As the control, a
PPD-eT patch test was applied at the same time to each animal. The
results are shown in FIG. 2. DTH was expressed as 3+, 2+, etc.,
because the diameter of a reaction was regulated by the size of a
patch, not by the antigen dose. The skin reaction to the MPB64
patch test was positive in all the animals until 13 weeks after the
BCG injection. It became hardly detectable afterwards, and was
completely negative at 23 weeks. In contrast, the reaction to the
PPD-eT patch test remained positive until the end of the experiment
at 25 weeks after BCG injection.
[0101] Delayed-type hypersensitivity (DTH) to MPB64 and PPD-eT were
examined at various times after the BCG injection. Each point of
FIG. 2 represents the delay-type hypersensitivity of 3 guinea pigs
expressed as follows: 3+, erythema and induration; 2+, erythema;
1+, small vesicles; +/-, faint colored (questionable reaction); -,
no reaction.
[0102] Patch tests in guinea pigs confirmed that 1/16 of the
antigen dose used for humans elicited a positive reaction.
[0103] It should be understood, of course, that the foregoing
relates only to preferred embodiments of the present invention and
that numerous modifications or alterations may be made therein
without departing from the spirit and the scope of the
invention.
* * * * *