U.S. patent application number 09/847365 was filed with the patent office on 2002-06-27 for composition comprising a carrier and a purified mycobacterial lipid cell-wall component and its use in the prevention, treatment and diagnosis of disease.
This patent application is currently assigned to ADCOCK INGRAM LIMITED. Invention is credited to Johannsen, Elzbieta, Lenaerts, Anne, Verschoor, Jan Adrianus.
Application Number | 20020082297 09/847365 |
Document ID | / |
Family ID | 27143737 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082297 |
Kind Code |
A1 |
Verschoor, Jan Adrianus ; et
al. |
June 27, 2002 |
Composition comprising a carrier and a purified mycobacterial lipid
cell-wall component and its use in the prevention, treatment and
diagnosis of disease
Abstract
A composition including a purified lipid cell-wall component or
analog or derivative thereof and a suitable pharmaceutical carrier,
medium, excipient or adjuvant is described. The composition is
useful in prophylactic and therapeutic methods of treating a
microbial infection in a subject, typically a mycobacterial
infection such as tuberculosis, and immune disorders, inflammatory
conditions and allergies in a subject, typically autoimmune
diseases. It is also useful in diagnostic methods. The purified
lipid cell-wall component is typically a purified mycolic acid or a
mixture of purified mycolic acids form a bacterium which produces
mycolic acids. The bacterium if from Mycobacterium, Corynbacterium,
Nocardia or Rhodococcus.
Inventors: |
Verschoor, Jan Adrianus;
(The Willows, ZA) ; Lenaerts, Anne; (Genk, BE)
; Johannsen, Elzbieta; (Lynwood Glen, ZA) |
Correspondence
Address: |
Ladas & Parry
26 West 61st Street
New York
NY
10023
US
|
Assignee: |
ADCOCK INGRAM LIMITED
|
Family ID: |
27143737 |
Appl. No.: |
09/847365 |
Filed: |
May 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09847365 |
May 2, 2001 |
|
|
|
09388725 |
Sep 2, 1999 |
|
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Current U.S.
Class: |
514/557 ;
514/552; 554/175 |
Current CPC
Class: |
G01N 33/5308 20130101;
Y10S 514/924 20130101; A61K 39/04 20130101; A61K 2039/6043
20130101; G01N 33/5695 20130101; A61K 2039/544 20130101 |
Class at
Publication: |
514/557 ;
514/552; 554/175 |
International
Class: |
A61K 031/23; A61K
031/20; C07C 051/43 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 3, 1997 |
ZA |
97/1817 |
Nov 14, 1997 |
ZA |
97/10300 |
Claims
1. A composition comprising a purified mycobacterial mycolic acid
or mixture of purified mycobacterial mycolic acids or ester form
thereof or synthetic form thereof and a pharmaceutically acceptable
carrier or adjuvant, wherein the pharmaceutically acceptable
carrier is suitable for direct administration to the lungs of a
subject.
2. A composition according to claim 1, wherein the purified
mycobacterial mycolic acid or mixture of purified mycobacterial
mycolic acids is from Mycobacterium tuberculosis.
3. A composition according to claim 1, wherein the pharmaceutically
acceptable carrier is a vaporisable liquid.
4. A composition according to claim 3, wherein the vaporisable
liquid is a saline solution.
5. A composition comprising a purified mycobacterial mycolic acid
or mixture of purified mycobacterial mycolic acids or ester form
thereof or synthetic form thereof and a pharmaceutically acceptable
carrier or adjuvant, wherein the composition comprises a
tolerogenic amount of the purified mycobacterial mycolic acid or
mixture of purified mycobacterial mycolic acids or ester form
thereof or synthetic form thereof.
6. A composition according to claim 5, wherein the purified
mycobacterial mycolic acid or mixture of purified mycobacterial
mycolic acids is from Mycobacterium tuberculosis.
7. A prophylactic pharmaceutical composition for an immune
disorder, comprising a purified mycobacterial mycolic acid or
mixture of purified mycobacterial mycolic acids or ester form
thereof or synthetic form thereof and a pharmaceutically acceptable
carrier or adjuvant.
8. A vaccine according to claim 7, wherein the immune disorder is
arthritis.
9. A method of decreasing the pathogenic effects of a mycobacterial
infection in the lungs of a subject by administering to the subject
a purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids or ester form thereof or synthetic form
thereof to stimulate the innate immunity of the subject.
10. A method according to claim 9, which is a prophylactic method
or a therapeutic method.
11. A method according to claim 10, which is an immunoregulatory
method.
12. A method according to claim 10 or 11, which is a prophylactic
method which enhances resistance or reduces susceptibility to a
mycobacterial infection in a subject.
13. A method according to claim 12, wherein the prophylactic method
promotes a pro-inflammatory response in the lungs of the
subject.
14. A method according to claim 10 or 11, wherein the prophylactic
method or the therapeutic method modulates or manipulates the
humoral immune system or cellular immune system or both in a
subject.
15. A method according to claim 12 or 13, wherein the prophylactic
method stimulates the expression of IL12 IFN-.gamma. in the lungs
of the subject.
16. A method according to claim 10, wherein the therapeutic method
stimulates the expression of TGF-.beta. in the lungs of the
subject.
17. A method according to claim 10, wherein the mycobacterial
infection is tuberculosis.
18. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids or ester form thereof or synthetic form
thereof for use in a method of decreasing the pathogenic effects of
a mycobacterial infection in the lungs of a subject by stimulating
the innate immunity of the subject.
19. Use of a purified mycobacterial mycolic acid or mixture of
purified mycobacterial mycolic acids or ester form thereof or
synthetic form thereof in a method of making a medicament for use
in a method of decreasing the pathogenic effects of a mycobacterial
infection in the lungs of a subject by stimulating the innate
immunity of the subject.
20. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 18 or 19, wherein
the method of treatment is a prophylactic method or a therapeutic
method.
21. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 20, wherein the
method of treatment is an immunoregulatory method.
22. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids or ester form thereof or synthetic form
thereof according to claim 18 or 19, wherein the method of
treatment is a prophylactic method which enhances resistance or
reduces susceptibility to a mycobacterial infection in a
subject.
23. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 22, wherein the
prophylactic method promotes a pro-inflammatory response in the
lungs of the subject.
24. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 20 or 21, wherein
the prophylactic method or the therapeutic method modulates or
manipulates the humoral immune system or cellular immune system or
both in a subject.
25. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 22, wherein the
mycobacterial infection is tuberculosis.
26. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 20, wherein the
method of treatment is a prophylactic method which stimulates the
expression of IL12 and IFN-.gamma. in the lungs of the subject.
27. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids according to claim 20, wherein the
method of treatment is a therapeutic method which stimulates the
expression of TGF-.beta. in the lungs of the subject.
28. A method of diagnosing an immune disorder in a subject
comprising the steps of: contacting a sample from the subject with
a purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids or ester form thereof or synthetic form
thereof; and detecting any immunological reaction between the
purified mycobacterial mycolic acid or mixture of purified mycolic
acids and the sample.
29. A method according to claim 28, wherein the step of detecting
any reaction between the purified mycobacterial mycolic acid or
mixture of purified mycobacterial mycolic acids or ester form
thereof or synthetic form thereof and the sample comprises
detecting the binding of an antibody present in the sample to the
purified mycobacterial mycolic acid component.
30. A method of treatment of an immune disorder in a subject
comprising the step of administering to the subject a purified
mycobacterial mycolic acid or mixture of purified mycobacterial
mycolic acids or ester form thereof or synthetic form thereof.
31. A method according to claim 30, which is a prophylactic method
or a therapeutic method.
32. A method according to claim 31, which is an immunoregulatory
method.
33. A method according to claim 31 or 32, which is a prophylactic
method which enhances resistance or reduces susceptibility to an
immune disorder in a subject.
34. A method according to claim 33, wherein the prophylactic method
suppresses inflammation of the joints in the subject.
35. A method according to claim 31 or 32, wherein the prophylactic
method or the therapeutic method modulates or manipulates the
humoral immune system or cellular immune system or both in a
subject.
36. A method according to any one of claims 30 to 35, wherein the
immune disorder is an inflammatory condition or allergy.
37. A method according to claim 36, wherein the inflammatory
condition is an autoimmune disease.
38. A method according to claim 37, wherein the autoimmune disease
is arthritis.
39. A purified mycobacterial mycolic acid or mixture of purified
mycobacterial mycolic acids or ester form thereof or synthetic form
thereof for use in a method of treatment of an immune disorder in a
subject.
40. Use of a purified mycobacterial mycolic acid or mixture of
purified mycobacterial mycolic acids or ester form thereof or
synthetic form thereof in a method of making a medicament for use
in the treatment of an immune disorder in a subject.
41. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 39 or 40, wherein the method of
treatment is a prophylactic method or a therapeutic method.
42. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 41, wherein the method of
treatment is an immunoregulatory method.
43. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 41 or 42, wherein the method of
treatment is a prophylactic method which enhances resistance or
reduces susceptibility to an immune disorder in a subject.
44. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 43, wherein the prophylactic
method suppresses inflammation of the joints in the subject.
45. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 41 or 42, wherein the
prophylactic method or the therapeutic method modulates or
manipulates the humoral immune system or cellular immune system or
both in a subject.
46. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to any one of claims 39 to 45, wherein the
immune disorder is an inflammatory condition or allergy.
47. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 46, wherein the inflammatory
condition is an autoimmune disease.
48. A purified mycobacterial mycolic acid, mixture, ester or
synthetic form according to claim 47, wherein the autoimmune
disease is arthritis.
49. A method of separating and purifying a specific microbial
cell-wall component of a lipid or carbohydrate nature or a
derivative or analog thereof from an extracted mixture of the
cell-wall component or derivative or analog thereof and
contaminants or from a synthetic mixture of the cell-wall component
or derivative or analog thereof and contaminants, comprising the
steps of: dissolving the extracted mixture or synthetic mixture in
a bi-phasic solvent containing sodium chloride to form a solution;
allowing the solution to separate to from an upper phase and a
lower phase; subjecting the phases to countercurrent
distribution/separation comprising a required number of cycles to
separate the microbial cell-wall component or analog or derivative
thereof in the upper phase or the lower phase; and removing the
separated microbial cell-wall component or derivative or analog
thereof from the upper or lower phase.
50. A method according to claim 49, which also comprises the
additional pre-purification steps of: dissolving the extracted
mixture of cell-wall component or derivative or analog thereof and
contaminants or the synthetic mixture of the cell-wall component or
derivative or analog thereof and contaminants in a first solvent
without sodium chloride; adding thereto a second solvent without
sodium chloride; mixing and allowing the solution to separate to
form a first upper phase (second solvent) and first lower phase
(first solvent); and removing the first upper phase and/or the
first lower phase for further processing.
51. A method according to claim 49 or 50, wherein the lower phase
containing the extracted mixture is removed and subjected to
countercurrent distribution/separation comprising a required number
of cycles to separate the microbial cell-wall component or analog
or derivative thereof in a second upper phase or lower phase and
the separated microbial cell-wall component or derivative or analog
thereof is removed from the second upper or lower phase.
52. A method according to any one of claims 49 to 51, which also
comprises the additional post-purification steps of: dissolving the
extracted microbial cell-wall component or derivative or analog
thereof in a suitable solvent; and adding a precipitant to the
solution to precipitate out the dissolved further purified
microbial cell-wall component or derivative or analog thereof.
53. A method according to claim 52, wherein the solvent is
chloroform.
54. A method according to claim 52 or 53, wherein the precipitant
is acetone.
55. A method according to any one of claims 49 to 54, which also
comprises the steps of: saponifying a microbial culture prior to
preparing therefrom an extracted mixture of a cell-wall component
or derivative or analog thereof on which to perform the method; and
resaponifying the separate and purified microbial cell-wall
component or derivative or analog thereof.
56. Detection means for detecting the presence of antibodies to a
purified mycolic acid or mixture of purified mycolic acids
comprising a solid phase and an unconjugated purified mycolic acid
or a mixture of unconjugated purified mycolic acids in a methyester
form or in a freshly resaponified form associated therewith.
57. Detection means according to claim 56, wherein the solid phase
is an ELISA plate.
58. A prophylactic pharmaceutical composition according to claim 7,
wherein the immune disorder is an autoimmune disorder.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to a composition comprising a
purified mycobacterial lipid cell-wall component or analog or
derivative thereof and a pharmaceutically acceptable excipient,
medium, carrier or adjuvant and the use of the purified
mycobacterial lipid cell-wall component or analog or derivative
thereof and the composition containing it in the prevention,
treatment and diagnosis of disease.
[0002] It has been known for a long time that BCG*) vaccination
leads to the induction of a positive tuberculin skin test,
resulting in delayed type hypersensitivity (DTH). This delayed
hypersensitivity in turn has been considered to be indicative of
the successful induction of protective immunity against
tuberculosis and has led to the almost world-wide BCG immunization
in the 1950s-1970s. This convenient test is in fact the only
immunological criterion/parameter on which epidemiological
assessments of the effectiveness of the immunization have been
based. *) BCG: (Bacillus of Calmette and Guerin) Calmette and
Guerin attenuated a strain of M. bovis by passaging it 231 times
over a period of 13 years through a medium containing glycerine and
ox-bile.
[0003] This view is no longer generally accepted and many
immunologists are of the opinion that
[0004] i) the induction of DTH is not directly related to the
degree of protective immunity;
[0005] ii) the protective efficacy obtained in vaccination with BCG
varies between 0 to 80% (Snider, 1994)
[0006] and in addition
[0007] iii) BCG vaccination has detrimental side-effects being
partially responsible for tissue destruction in patients, without
offering sufficient protection (Fine, 1994).
[0008] The unsatisfactory results observed and reported in a number
of countries with the BCG vaccine currently used for the prevention
of the spread of tuberculosis (Dolin, Raviglione and Koch, 1994;
Snider, 1994) could be explained by:
[0009] i) variations between BCG vaccines, which could be caused by
strain variation or by differences between manufacturing
processes;
[0010] ii) differences in pathogenesis of Mycobacterium
tuberculosis;
[0011] iii) differences in the exposure to the environmental
mycobacteria. The environmental mycobacteria may act
antagonistically or synergistically with BCG;
[0012] iv) genetic differences between population groups subjected
to vaccination with BCG;
[0013] v) differences in nutrition and exposure to sunlight between
various population groups;
[0014] vi) differences between designs of various studies;
[0015] vii) inadequacies of the criteria used for the evaluation of
protective effects of vaccination with BCG.
[0016] Efforts directed at finding an effective vaccine capable of
inducing long-lasting immunity have centered over the last decade
on three main approaches:
[0017] i) identifying "protective" antigens and epitopes of M.
tuberculosis presented by macrophages and recognized by human
lymphocytes;
[0018] ii) developing a DNA-based vaccine with protective antigen
and interleukin genes (Lowrie et al., 1994);
[0019] iii) identifying which types of cells of the immune system
and which types of cytokines are involved in tuberculosis in order
to manipulate their activity towards offering a cure or protection
against tuberculosis.
SUMMARY OF THE INVENTION
[0020] According to one aspect of the invention there is, provided
a conjugate comprising an organic carrier and a purified lipid
cell-wall component associated therewith, provided that if the
organic carrier is a protein, it is not bovine serum albumin (BSA),
gelatin, keyhole limpets haemocyanin or the CD.sub.1 molecule.
[0021] The organic carrier may be a protein excluding bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the
CD.sub.1 molecule.
[0022] The protein may be a microbial protein. More specifically,
it may be a modified bacterial protein and may be derived from a
bacterium from the genus Mycobacterium, Corynebacterium, Nocardia
or Rhodococcus. It may be a heat-shock protein, such as heat-shock
protein 60 (HSP60) or heat-shock protein 65 (HSP65), or it may be a
serum protein from an animal. The animal may have a mycobacterial
infection, such as a Mycobacterium tuberculosis infection. The
animal may be a mammal, typically a human.
[0023] Alternatively, the protein may be derived from a mammal,
particularly a human, and is preferably a protein which mimics the
structure of collagen or a collagen-derived protein or a plasma
protein, such as the collagen-like segment of human serum component
C1.sub.q.
[0024] Alternatively, the carrier may be a carbohydrate such as
galactomannan or arabinogalactan or a lipopolysaccharide.
[0025] Further alternatively, the organic carrier may be a micelle,
such as a liposome.
[0026] According to another aspect of the invention there is
provided a diagnostic kit comprising a support containing a
conjugate as described above immobilised thereon.
[0027] According to another aspect of the invention there is
provided a pharmaceutical composition which comprises a
therapeutic, prophylactic or tolerogenic amount of a conjugate as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component or analog or derivative thereof
associated therewith or a biologically active purified lipid
cell-wall component or analog or derivative thereof and a
pharmaceutically acceptable or compatible pharmaceutical excipient,
medium, carrier or adjuvant.
[0028] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0029] The pharmaceutical composition may also contain at least one
immunomodulator. The immunomodulator may be a cytokine. The
cytokine may be an interleukin, such as interleukin 4 (IL4),
interleukin 10 (IL10) or interleukin 12 (IL12), or may be an
interferon.
[0030] The suitable pharmaceutical carrier or adjuvant which may
also be suitable for veterinary applications may be a solid, such
as polymer dust, a liquid, such as an oil, typically Marcol 52, or
a water-in-oil emulsion, typically Freund's Incomplete Adjuvant
(FIA), or a solution, typically a saline solution or PBS, in which
case the composition may be in the form of a suspension or a
vapourised liquid, typically a neibulisable physiological saline
solution, or a gas, or a transdermal delivery system.
[0031] The composition may comprise a therapeutic, prophylactic or
tolerogenic amount of the purified lipid cell-wall component.
[0032] The pharmaceutical composition may comprise about 5 .mu.g or
less, typically 1 .mu.g, of the purified lipid cell-wall component
per ml of the composition.
[0033] A unit dose of the pharmaceutical composition for
administration to a human subject preferably comprises from about 5
to 10 mg of the purified lipid cell-wall component.
[0034] According to another aspect of the invention there is
provided a vaccine containing a purified lipid cell-wall component
or analog or derivative thereof or a conjugate or a pharmaceutical
composition as described above or a conjugate comprising any
organic carrier and a purified lipid cell-wall component associated
therewith for use in preventing an immune disorder or an
inflammatory condition in a subject.
[0035] According to another aspect of the invention there is
provided a vaccine containing a purified lipid cell-wall component
or analog or derivative thereof or a conjugate or a pharmaceutical
composition as described above or a conjugate comprising any
organic carrier, except bovine serum albumin (BSA) and a purified
lipid cell-wall component associated therewith, for use in
preventing a microbial infection in a subject.
[0036] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0037] According to another aspect of the invention there is
provided an isolated antibody which is capable of forming,
separately, an antigen/antibody complex with any two or more of the
following antigens: a purified lipid cell-wall component derived
from a microorganism: a protein derived from a bacterial species or
from a mammal; a conjugate as described above; and a conjugate
comprising any organic carrier and a purified mycobacterial lipid
cell-wall component associated therewith.
[0038] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0039] According to another aspect of the invention there is
provided a method of diagnosing a microbial infection in a subject
comprising the step of contacting a sample from the subject with a
conjugate as described above or with a support containing a
conjugate as described above; and detecting any reaction between
the conjugate and the sample.
[0040] More preferably, the method of diagnosis may comprise the
step of detecting the binding of an antibody present in the sample
to the conjugate.
[0041] According to another aspect of the invention there is
provided a conjugate or a pharmaceutical composition as described
above for use in a method of diagnosis of a microbial infection in
a subject.
[0042] According to another aspect of the invention there is
provided the use of a conjugate or a pharmaceutical composition as
described above in a method of making a medicament for use in a
method of diagnosis of a microbial infection in a subject.
[0043] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or a pharmaceutical composition
as described above or a conjugate comprising any organic carrier
and a purified lipid cell-wall component associated therewith for
use as a tolerogen to enhance resistance and/or reduce
susceptibility to a microbial infection in a subject.
[0044] According to another aspect of the invention there is
provided the use of a purified lipid cell-wall component or analog
or derivative thereof or a conjugate or a pharmaceutical
composition as described above or a conjugate comprising any
organic carrier and purified lipid cell-wall component associated
therewith in a method of making a medicament for use as a tolerogen
to enhance resistance and/or reduce susceptibility to a microbial
infection in a subject.
[0045] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or a pharmaceutical composition
as described above or a conjugate comprising any organic carrier
and a purified lipid cell-wall component associated therewith for
use in a method of enhancing resistance or lowering susceptibility
to microbial infections in a subject.
[0046] According to another aspect of the invention there is
provided use of a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith in a method
of making a medicament for use in enhancing resistance or lowering
susceptibility to microbial infections in a subject.
[0047] According to another aspect of the invention there is
provided a method of treatment of a microbial infection in a
subject comprising the step of administering to the subject a
purified bacterial lipid cell-wall component or analog or
derivative thereof or a pharmaceutical composition of the invention
to the subject.
[0048] According to another aspect of the invention there is
provided a conjugate or a pharmaceutical composition as described
above or a conjugate comprising any organic carrier and a purified
lipid cell-wall component associated therewith or a purified lipid
cell-wall component or analog or derivative thereof for use in a
method of treatment of a microbial infection in a subject.
[0049] According to another aspect of the invention there is
provided the use of a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith or a
purified lipid cell-wall component or analog or derivative thereof
in a method of making a medicament for use in a method of treatment
of a microbial infection in a subject.
[0050] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0051] The method of treatment may be a prophylactic and/or
therapeutic method and may be a high zone tolerance treatment or
may be a low zone tolerance treatment or a treatment aiming at an
idiotypic regulation involving the conjugate or the antibody.
[0052] The method may be an immunoregulatory method.
[0053] The method of treatment may be a prophylactic method which
enhances resistance or reduces susceptibility to a microbial
infection in a subject. The prophylactic method may promote an
inflammatory response in an infected organ, typically the lungs,
kidney and/or liver of the subject. The infected organ is usually
the lungs.
[0054] According to another aspect of the invention there is
provided a method of treatment of an immune disorder in a subject
comprising the step of administering to the subject a purified
bacterial lipid cell-wall component or analog or derivative thereof
or a pharmaceutical composition as described above.
[0055] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith for use in
a method of treatment and/or diagnosis of an immune disorder in a
subject.
[0056] According to another aspect of the invention there is
provided the use of purified lipid cell-wall component or analog or
derivative thereof or a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith in a method
of making a medicament for use in a method of treatment and/or
diagnosis of an autoimmune disease in a subject.
[0057] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0058] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith for use in
a method of treatment and/or diagnosis of an inflammatory condition
in a subject.
[0059] According to another aspect of the invention there is
provided the use of a purified lipid cell-wall component or analog
or derivative thereof or a conjugate or pharmaceutical composition
as described above or a conjugate comprising any organic carrier
and a purified lipid cell-wall component associated therewith in a
method of making a medicament for use in a method of treatment
and/or diagnosis of an inflammatory condition in a subject.
[0060] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0061] According to another aspect of the invention there is
provided a method of diagnosing an immune disorder in a subject
comprising the step of contacting a sample from the subject with a
purified lipid cell-wall component or analog or derivative thereof
or with a pharmaceutical composition as described above or with a
conjugate as described above or with a conjugate comprising any
organic carrier and a purified lipid cell-wall component associated
therewith or with a support containing either conjugate; and
detecting any reaction between the purified lipid cell-wall
component or conjugate and the sample.
[0062] More preferably, the method of diagnosis may comprise the
step of detecting the binding of an antibody present in the sample
to the purified lipid cell-wall component or the conjugate.
[0063] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0064] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or a pharmaceutical composition
as described above or a conjugate comprising any organic carrier
and a purified lipid cell-wall component associated therewith for
use as a toleragen to enhance resistance and/or reduce
susceptibility to an immune disorder or an inflammatory condition
in a subject.
[0065] According to another aspect of the invention there is
provided the use of a purified lipid cell-wall component or analog
or derivative thereof or a conjugate or a pharmaceutical
composition as described above or a conjugate comprising any
organic carrier and a purified lipid cell-wall component associated
therewith for use in a method of making a medicament as a tolerogen
to enhance resistance and/or reduce susceptibility to an immune
disorder or an inflammatory condition in a subject.
[0066] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0067] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or a pharmaceutical composition
as described above or a conjugate comprising any organic carrier,
except bovine serum albumin (BSA), and a purified lipid cell-wall
component associated therewith for use in a method of modulating or
manipulating the humoral immune system in a subject for the
treatment or prophylaxis of a microbial infection, an immune
disorder or an inflammatory condition in a subject.
[0068] According to another aspect of the invention there is
provided the use of a conjugate or a pharmaceutical composition as
described above or a conjugate comprising any organic carrier,
except bovine serum albumin (BSA), and a purified lipid cell-wall
component associated therewith in a method of making a medicament
for use in a method of modulating or manipulating the humoral
immune system in a subject for the treatment or prophylaxis of a
microbial infection, an immune disorder or an inflammatory
condition in a subject.
[0069] The organic carrier may be a protein including gelatin,
keyhole limpets haemocyanin and the CD1 molecule, or may be a
carbohydrate or may be a micelle.
[0070] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith for use in
a method of enhancing resistance or lowering susceptibility to
inflammatory conditions or allergies in a subject.
[0071] According to another aspect of the invention there is
provided use of a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or pharmaceutical composition as
described above or a conjugate comprising any organic carrier and a
purified lipid cell-wall component associated therewith in a method
of making a medicament for use in enhancing resistance or lowering
susceptibility to inflammatory conditions and/or allergies in a
subject.
[0072] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin, the CD1
molecule and a serum protein, or may be a carbohydrate or may be a
micelle.
[0073] The method of treatment may be a prophylactic and/or
therapeutic method and may be a high zone tolerance treatment or
may be a low zone tolerance treatment or a treatment aiming at an
idiotypic regulation involving the conjugate or the antibody.
[0074] The method may be an immunoregulatory method.
[0075] The method of treatment may be a prophylactic method which
enhances resistance or reduces susceptibility to an immune
disorder, inflammatory condition or allergy in a subject. The
prophylactic method may suppress inflammation in the Joints of the
subject.
[0076] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or a pharmaceutical composition
as described above or a conjugate comprising any organic carrier
and a purified lipid cell-wall component associated therewith for
use in the method of modulating or manipulating the cellular immune
system in a subject for the treatment or prophylaxis of a microbial
infection, an immune disorder or an inflammatory condition in a
subject.
[0077] According to another aspect of the invention there is
provided a purified lipid cell-wall component or analog or
derivative thereof or a conjugate or a pharmaceutical composition
as described above or a conjugate comprising any organic carrier
and a purified lipid cell-wall component associated therewith in a
method of making a medicament for use in a method of modulating or
manipulating the cellular immune system in a subject for the
treatment or prophylaxis of a microbial infection, an immune
disorder or an inflammatory condition in a subject.
[0078] The organic carrier may be a protein including bovine serum
albumin (BSA), gelatin, keyhole limpets haemocyanin and the CD1
molecule, or may be a carbohydrate or may be a micelle.
[0079] More particularly, the modulation or manipulation of the
immune system may be the modulation or manipulation of T-cell
effects, such as CD4.sup.+, Th0, Th1 and Th2, CD8.sup.+ or
CD4.sup.- CD8.sup.- (double negative (DN)) T-cells, natural killer
(NK) cells, and/or of macrophages.
[0080] According to another aspect of the invention there is
provided use of a purified lipid cell-wall component in a method of
forming a conjugate comprising any organic carrier and the purified
lipid cell-wall component, whether the conjugate is produced in
vitro or in vivo.
[0081] The purified lipid cell-wall component as referred to herein
is preferably a bacterial cell-wall component from a bacterium
which produces mycolic acids and it may be derived from the genus
Mycobacterium, Corynebacterium, Nocardia or Rhodococcus.
[0082] The bacterium is preferably Mycobacterium tuberculosis.
[0083] More preferably, the purified lipid cell-wall component is a
purified mycolic acid, a mixture of purified mycolic acids or a
mycolic acids fraction or derivative originating from a single or
different species or a synthetic source. Other lipid cell-wall
components may be high molecular weight lipids such as cord
factors.
[0084] The purified lipid cell-wall component may be a biologically
active purified mycolic acid, a mixture of biologically active
purified mycolic acids or a biologically active purified mycolic
acids fraction or derivative originating from a single or different
species or a synthetic source.
[0085] The purified lipid cell-wall component may be a resaponified
biologically active mycolic acid, a mixture of resaponified
biologically active mycolic acids or a biologically active mycolic
acids fraction or derivative of resaponified mycolic acids.
[0086] The derivative of the purified lipid cell-wall component as
referred to herein may be an ester of a mycolic acid, a mixture of
esters of mycolic acids or a derivative or fraction thereof.
[0087] The microbial infection as referred to herein may be a
mycobacterial infection, typically tuberculosis or leprosy.
[0088] The immune disorder as referred to herein may be an
inflammatory condition. It may be an autoimmune disorder, typically
arthritis.
[0089] The autoimmune disorder may be of mycobacterial origin.
[0090] According to yet another aspect of the invention there is
provided a method of separating and purifying a specific microbial
cell-wall component of a lipid or carbohydrate nature or a
derivative or analog thereof from an extracted mixture of the
cell-wall component or derivative or analog thereof and
contaminants or from a synthetic mixture of the cell-wall component
or derivative or analog thereof and contaminants comprising the
steps of:
[0091] dissolving the extracted mixture or synthetic mixture in a
bi-phasic solvent containing sodium chloride to form a
solution;
[0092] allowing the solution to separate to form an upper phase and
a lower phase;
[0093] subjecting the phases to countercurrent
distribution/separation comprising a required number of cycles to
separate the microbial cell-wall component or analog or derivative
thereof in the upper phase or the lower phase; and
[0094] removing the separated microbial cell-wall component or
derivative or analog thereof from the upper or lower phase.
[0095] The method may also comprise the additional pre-purification
steps of:
[0096] dissolving the extracted mixture of cell-wall component or
derivative or analog thereof and contaminants or the synthetic
mixture of the cell-wall component or derivative or analog thereof
and contaminants in a first solvent without sodium chloride;
[0097] adding thereto a second solvent without sodium chloride;
[0098] mixing and allowing the solution to separate to form a first
upper phase (second solvent) and a first lower phase (first
solvent); and
[0099] removing the first upper phase and/or the first lower phase
for further processing.
[0100] Preferably, the lower phase or first lower phase, containing
the extracted mixture, is removed and subjected to countercurrent
distribution purification, as described above.
[0101] The method may also comprise the additional
post-purification steps of:
[0102] dissolving the extracted microbial cell-wall component or
derivative or analog thereof in a suitable solvent; and
[0103] adding a precipitant to the solution to precipitate out the
dissolved further purified microbial cell-wall component or
derivative or analog thereof.
[0104] The solvent is preferably chloroform.
[0105] The precipitant is preferably acetone.
[0106] The method may comprise the steps of:
[0107] saponifying a microbial culture prior to preparing therefrom
an extracted mixture of a cell-wall component or derivative or
analog thereof on which to perform the method of the invention;
and
[0108] resaponifying the separated and purified microbial cell-wall
component or derivative or analog thereof.
[0109] According to yet another aspect of the invention a purified
mycolic acid or mixture of purified mycolic acids or a fraction
thereof is provided in a particular conformation that renders it
biologically active.
[0110] The purified mycolic acid or mixture of purified mycolic
acids may be in a conformation produced by the purified mycolic
acid or mixture of purified mycolic acids being in a methyl ester
form or a freshly resaponified form.
[0111] According to yet another aspect of the invention there is
provided a method of preparing detection means for detecting the
presence of anti-mycolic acids antibodies, the detection means
comprising a solid phase and mycolic acids in a methyl ester form
or in a freshly resaponified form associated therewith.
[0112] The solid phase may be an ELISA plate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0113] FIG. 1 is a schematic presentation of the activation process
of naive T-cells;
[0114] FIG. 2a is an HPLC profile of mycolic acids originating from
M. tuberculosis, purified using the improved method of
countercurrent distribution in a sodium-chloride containing phase
system;
[0115] FIG. 2b is an HPLC profile of mycolic acids originating from
M. vaccae, purified using the improved method of countercurrent
distribution in a sodium-chloride containing phase system;
[0116] FIG. 3 is an infra-red spectrum of countercurrent purified
mycolic acids, originating from M. tuberculosis;
[0117] FIG. 4 is an infra-red spectrum of the resaponified
countercurrent purified mycolic acids, originating from M.
tuberculosis;
[0118] FIG. 5 is an infra-red spectrum of countercurrent-purified
and resaponified mycolic acids, originating from M. tuberculosis,
frozen at -70.degree. C.;
[0119] FIG. 6 is an infra-red spectrum of countercurrent-purified
and resaponified mycolic acids, originating from M. tuberculosis,
and maintained at 10.degree. C.;
[0120] FIG. 7 shows the stability of mycolic acids originating from
M. tuberculosis (resaponified and methylester form) upon dry
storage;
[0121] FIG. 8 shows cytokine profiles of IL-12, IFN-.tau.,
TNF-.alpha. and TGF-.beta. in the lungs of Balb/c mice treated with
250 .mu.g and boosted with 25 .mu.g mycolic acids from M.
tuberculosis. The lungs were removed 24 and 48 hours after the
boost;
[0122] FIG. 9 shows the expression of IL-12 in the spleen, liver,
kidney, lung and heart of Balb/c and C57BI/6 mice two weeks after
the infection with M. tuberculosis;
[0123] FIG. 10 shows the survival of M. tuberculosis-infected
Balb/c mice, pre-treated with mycolic acids (from M. tuberculosis)
one week before the infection, at the indicated doses;
[0124] FIG. 11 shows the survival of M. tuberculosis-infected
C57BI/6 mice, pre-treated with mycolic acids (from M. tuberculosis)
one week before the infection, at the indicated doses;
[0125] FIG. 12 shows the survival of M. tuberculosis-infected
Balb/c mice post-treated with mycolic acids (from M. tuberculosis)
three weeks after the infection, at the indicated doses, delivered
in three daily injections of equal dose;
[0126] FIG. 13 shows the survival of M. tuberculosis-infected C57B
I/6 mice post-treated with mycolic acids (from M. tuberculosis)
three weeks after the infection, at the indicated doses, delivered
in three daily injections of equal dose;
[0127] FIG. 14 shows the expression of IL-12 in M.
tuberculosis-infected Balb/c mice, pre-treated with mycolic acids
(from M. tuberculosis) one week before the infection, at the
indicated doses. Lungs were removed five weeks after the
infection;
[0128] FIG. 15 shows the correlation between IL-12 expression in
the lungs of Balb/c mice, pre-treated with mycolic acids (from M.
tuberculosis), at five weeks after the infection and their
survival;
[0129] FIG. 16 shows the expression of IFN-.gamma. in M.
tuberculosis-infected Balb/c mice pre-treated with mycolic acids
(from M. tuberculosis) one week before the infection, at the
indicated doses. Lungs were removed five weeks after the
infection;
[0130] FIG. 17 is a repeat experiment and shows the expression of
IFN-.gamma. in M. tuberculosis-infected Balb/c mice pre-treated
with mycolic acids (from M. tuberculosis) one week before the
infection, at the indicated doses. Lungs were removed five weeks
after the infection;
[0131] FIG. 18 shows the expression of TGF-.beta. in M.
tuberculosis-infected Balb/c mice pre-treated with mycolic acids
(from M. tuberculosis) one week before the infection, at the
indicated doses. Lungs were removed five weeks after the
infection;
[0132] FIG. 19 shows the correlation between TGF-.beta. expression
in the lungs of Balb/c mice, pre-treated with mycolic acids (from
M. tuberculosis) and their survival. The lungs were removed five
weeks after the infection;
[0133] FIG. 20 shows the expression of IL-12 in M.
tuberculosis-infected Balb/c mice, post-treated with mycolic acids
(from M. tuberculosis) three weeks after the infection, at the
indicated doses. Lungs were removed five weeks after the
infection;
[0134] FIG. 21 shows the expression of IFN-.gamma. in M.
tuberculosis-infected Balb/c mice post-treated with mycolic acids
(from M. tuberculosis) three weeks after the infection, at the
indicated doses. Lungs were removed five weeks after the
infection;
[0135] FIG. 22 shows the expression of TGF-.beta. in M.
tuberculosis-infected Balb/c mice post-treated with mycolic acids
(from M. tuberculosis) three weeks after the infection, at the
indicated doses. Lungs were removed five weeks after the
infection;
[0136] FIG. 23 shows cytokine profiles of IFN-.gamma. and IL-4 in
the spleen of Balb/c mice pre- and post-treated with mycolic acids
(from M. tuberculosis) at the indicated doses;
[0137] FIG. 24 shows the expression of IL-12 in the lungs of M.
tuberculosis-infected Balb/c mice, pre- and post-treated with
mycolic acids (from M. vaccae) at the indicated doses. Lungs were
removed five weeks after the infection;
[0138] FIG. 25a is a photograph of two rats, the rat on the left
being a control and having received only FIA, the rat on the right
having received a reagent for the induction of adjuvant arthritis
and showing a bleeding nose and arthritic nodules visible on the
front paws;
[0139] FIG. 25b is a photograph of two rats, the rat on the right
having been pre-treated with 1 mg of mycolic acids/serum in FIA
before having received a reagent for the induction of adjuvant
arthritis and showing minimal signs of arthritis, the rat on the
left not having been pre-treated before having received a reagent
for the induction of adjuvant arthritis and showing swollen and
inflamed arthritic hind legs;
[0140] FIG. 25c is a photograph of the rat showing the typical
deformation of the joints in the hind legs, the so-called "swimming
position" and a necrosis developing at the site of the
injection;
[0141] FIG. 26a shows X-ray photographs of the hind limbs of rats
used in the arthritis experiments. Group 5 of Tables 8a and 8b--a
"negative" control treated with Freund's Adjuvant only;
[0142] FIG. 26b shows X-ray photographs of the hind limbs of rats
used in the arthritis experiments. Group 1 of Tables 8a and 8b--a
"positive" control treated with M. tuberculosis H37Rv suspended in
Freund's Adjuvant;
[0143] FIG. 26c shows X-ray photographs of the hind limbs of the
rats pre-treated with 1 mg mycolic acids (from M. tuberculosis)
prior to the induction of arthritis. Group 4 of Tables 8a and
8b;
[0144] FIG. 27 shows the emaciation of a rat with typical induced
adjuvant arthritis;
[0145] FIG. 28 shows rat antibody response to mycolic acids
suspended in oil after three months' treatment using 1,0, 0,3 and
0,1 mg mycolic acids per immunization, after three months
treatment;
[0146] FIG. 29 shows ELISA results of human tuberculosis patients'
sera in comparison to healthy control on the plates coated with
mycolic acids;
[0147] FIG. 30 is a specificity assay of antibodies of human
tuberculosis patient No. 38 assessed by inhibition of ELISA;
[0148] FIG. 31 is a Western blot of mouse serum with and without
exposure to mycolic acids, probed with human tuberculosis patients'
sera and a healthy control serum;
[0149] FIG. 32a shows the stimulation by mycolic acids of human T
cells by CD1 presenting cells;
[0150] FIG. 32b shows the stimulation of human T cells by mycolic
acids.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0151] This invention is based on the involvement of isolated,
purified, biologically active mycobacterial lipid cell-wall
components, and more particularly purified, biologically active
mycolic acids or analogs or derivatives thereof, in the regulation
of the immune response in a subject or host to infection or
abnormal activation. These purified mycobacterial lipid cell-wall
components originate from bacteria assigned to the genus M.
mycobacterium and to the genera Corynebacterium, Nocardia and
Rhodococcus. In particular, the present invention teaches the
application of isolated and purified mycolic acids in:
[0152] 1. the modulation and immunoregulation of an immune response
towards infection with mycobacteria in humans and animals;
[0153] 2. the development of anti-tuberculosis and other
anti-mycobacterial treatment for human and veterinary use. It is
envisaged that tolerogenic doses of the purified lipid cell-wall
components of M. tuberculosis and other Mycobacteria, on their own
or presented on carriers such as proteins, liposomes or
carbohydrates or in a medium or pharmaceutical carrier or excipient
such as liquid (oil, saline, water) gas or vapour, could be used in
the modulation of immune responses to mycobacterial infections;
[0154] 3. the development and production of diagnostic tests (in
vitro and/or in vivo tests) for the confirmation of the presence of
mycobacterial cells in samples such as blood, cerebrospinal fluid,
tears and saliva or tests that would be based on the identification
and detection of auto-reactive antibodies and/or T-cells, and
antibodies against the purified lipid cell-wall components with or
without host carriers;
[0155] 4. the prevention and immunotherapy and regulation of
autoimmune illnesses of mycobacterial and other origin and
particularly those associated with infection with mycobacteria.
Such a therapy could be achieved by modulating the immune response
directed against mycobacterial lipid cell-wall components. It is
envisaged that the tolerogenic doses of the purified lipid
cell-wall components of M. tuberculosis and other Mycobacteria
presented in a supportive medium or in a pharmaceutically
acceptable carrier or excipient or on carriers such as liposomes or
proteins or suspended in an adjuvant, typically a water-in-oil
emulsion such as FIA, can be used for the regulation of immune
responses to mycobacterial antigens leading to the prevention of
tuberculosis and auto-immune diseases such as arthritis, often
associated with tuberculosis.
[0156] The invention utilises the method of isolation and
purification of a mycobacterial lipid cell-wall component described
in South African Patent No. 96/1412 and is based on the
demonstration of the immunogenicity of mycolic acids described in
South African Patent No. 95/3077.
[0157] The invention also provides an improvement on the method of
isolation and purification of mycobacterial lipid cell-wall
components described in South African Patent No. 96/1412. An
improvement involves a preliminary purification of crude
mycobacterial extract to remove redundant or unnecessary cellular
components prior to the countercurrent purification and to reduce
the soap content in the crude extract. A further improvement to the
method involves the addition of sodium chloride to the solvent.
This allows larger amounts of the extracted mycolic acids to be
purified per run of the countercurrent separation.
[0158] The availability of the isolated and purified required
components, particularly mycolic acids, is essential for the
development and assessment of the potential applications listed
above.
[0159] As indicated above, the conventional approaches to the
prevention of infection with M. tuberculosis by attempting to
induce humoral and/or cellular memory leading to a long-term
protection against tuberculosis have been unsuccessful. The present
invention offers another approach. It is based in part on the use
of appropriate tolerogenic doses of mycolic acids in a supportive
medium or pharmaceutically acceptable carriers or excipients or on
appropriate carriers, with or without appropriate interleukine(s)
(Heath and Playfair, 1992), to modulate/regulate the immune
response(s) in the human body. Such a treatment would help to
prevent or decrease mortality due to tuberculosis and could be used
as a potential treatment for the disease.
[0160] The presentation of purified mycolic acids in supportive
media, conjugates, pharmaceutical compositions comprising
pharmaceutically acceptable excipients, carriers and adjuvants,
uses and methods of the invention should be considered in the light
of what is known about the pathology of tuberculosis as described
below.
[0161] The encounter between the bacterium Mycobacterium
tuberculosis and its human host is exceptionally complex and
multifaceted and leads to a number of intertwined, interdependent
and interconnected processes, in which mycolic acids appear to be
playing a significant role. For the sake of clarity these processes
will be discussed under the following headings:
OUTLINE: CLINICAL STAGES OF TUBERCULOSIS
[0162] 1. Primary pulmonary tuberculosis
[0163] 2. Post-primary pulmonary/reactivation tuberculosis
[0164] 3. Immunology/immunopathology in tuberculosis
[0165] 3.1 Overview
[0166] 3.2 Synthesis of cytokines
[0167] 3.3 Activation of macrophages and the intracellular fate of
M. tuberculosis
[0168] 3.4 Delayed-Type Hypersensitivity (DHT)
[0169] 3.5 Activation of T cells and their functions
[0170] 3.6 Cytokine circuits in tuberculosis
[0171] 3.7 Cytokine induction by M. tuberculosis
[0172] 3.8 Cytokine profiles in tuberculosis patients
[0173] 3.9 Involvement of other T cells in tuberculosis
[0174] 4. Proposed approach
[0175] 5. Immunogenicity of mycolic acids
OUTLINE: CLINICAL STAGES OF TUBERCULOSIS
1. Primary Pulmonary Tuberculosis
[0176] It is generally accepted that the primary infection with M.
tuberculosis occurs by inhalation of a very small number of bacilli
into the respiratory tract. Usually fewer than 10% of the inhaled
microorganisms reach the respiratory bronchioles and alveoli, the
rest being successfully removed from the upper respiratory
epithelium by the nonspecific, external innate immunity, i.e., by
mucus and the ciliated epithelium of the upper respiratory tract
acting as filters. Mycobacteria that reach alveoli are dealt with
by the internal innate immunity mechanisms. In the case of the
mycobacterial infection, phagocytosis (engulfment and destruction
of the pathogens by mononuclear leucocytes) and inflammatory
reaction play the decisive part. The age of an individual, general
state of his/her health, conditions of living, nutrition as well as
ethnic and geographic differences affect the susceptibility to the
disease and its severity (Fine, 1994).
[0177] The inhaled cells of M. tuberculosis are ingested by
alveolar macrophages (see section 3.3 on the activation of
macrophages), which have a predilection for this function. Some of
the bacilli are destroyed within the phago-lysosomes of the
macrophages and some remain in the macrophagal vacuoles for
undetermined periods of time. In some cases, even replication of
the bacilli may occur in the macrophage's vacuoles. Within the
period of 2 to 3 weeks some of the surviving bacteria destroy their
host macrophages and, after being released, can infect additional
macrophages (Fenton and Vermeulen, 1996).
[0178] The chemotactic factors released by the destroyed
macrophages attract other leucocytes: monocytes, lymphocytes and
neutrophils, which are not capable of destroying the released
mycobacteria. The accumulation of lymphocytes and the formation of
macrophage-derived epitheloid giant cells constitute the beginning
of the inflammation process (see section 3.1 and 3.3) and lead to
the formation of nodules called granulomas, which in the case of
tuberculosis are referred to as tubercles. The degree of success of
granuloma formation depends on the initial number of mycobacteria
present in the tissue and on the number of macrophages present at
the site of infection (Fenton and Vermeulen, 1996).
[0179] The formation of granulomas is a result of an interaction
between the macrophages and T lymphocytes and the secretion of
proteins stimulating cells of both types. It constitutes the
beginning of the development of cell-mediated immunity (for a
detailed description see section 3.4). Although the cells of
ingested M. tuberculosis are not always completely destroyed, the
formation of granulomas is an effective defense mechanism of
"containing"/walling-off the pathogens, which stops the infection
from spreading further and retains it in a subclinical state. If,
however, the resistance of the infected person is low, the
formation of granulomas may not stop the growth and spread of the
mycobacteria and the disease progresses further to primary clinical
tuberculosis.
[0180] During s stage of the disease more tubercles are formed and
they become larger. A hypersensitivity reaction (see section 3.4)
accompanied by tissue necrosis and fusion of the dead macrophages
constitute the characteristic response. The fusion of macrophages
leads to the formation of caseation necrosis within the granulomas.
In the majority of cases, in order to seal off the necrotic site,
lymphocytes and other cells collect at the site and form a fibrous
tissue. The walled-off caseated granulomas frequently heal over
time, shrink and calcify. The disease frequently stops at this
stage and the damaged lung tissue, detected by X-ray, remains the
proof of the successfully combated disease. In the majority of
cases the encounter between mycobacteria and the human immune
system stops here and as a result of this exposure, most
individuals successfully control the focus of infection and develop
some degree of immunity.
[0181] If, however, the healing process is impaired, the lesions do
not calcify but expand, eroding adjacent bronchi and leading to the
formation of cavities, in which live cells of M. tuberculosis can
multiply freely, sometimes reaching numbers exceeding 10.sup.8 per
cavity (Nardell, 1993). Living mycobacteria can leak from such open
cavities directly into bronchia leading to even further spread of
the disease and a continuous discharge of these bacilli into the
sputum. The leaked bacilli can be inhaled into other parts of the
host's lungs resulting in tuberculous bronchopneumonia. As the
caseation necrosis develops further and spreads, the patient starts
to display the clinical symptoms of tuberculosis such as loss of
weight, night sweats, persistent cough, loss of appetite and
fatigue. At this stage an intensive therapy is required (Fenton and
Vermeulen, 1996).
[0182] If mycobacteria spread to the blood, they can localise in
any organ forming new tubercles and the disease becomes a
generalised form of tuberculosis, referred to as miliary
tuberculosis.
[0183] 2. Post-primary pulmonary/reactivation tuberculosis
[0184] Post-primary pulmonary tuberculosis or reactivation
tuberculosis can develop following either inhalation of additional
mycobacteria or by reactivation of a dormant primary lesion, in
which bacilli can survive in a dormant state for decades. The
post-primary tuberculosis progresses despite the existing immunity
developed during the primary exposure (Fenton and Vermeulen, 1996)
and is considered to be the most prevalent form of the disease
(Elgert, 1996a). It usually occurs concomitantly with a period of
excessive environmental stress, malnutrition or lowering of immune
competence of the body.
[0185] The pathogen, no longer held in check within the tubercles,
multiplies vigorously and leads to the development of symptoms
observed in the primary clinical tuberculosis. Generalized clinical
tuberculosis causes damage to various vital organs and can result
in death of the patient.
[0186] 3. Immunology/immunopathology in tuberculosis
[0187] 3.1 Overview
[0188] Although the breaking down and digestion of mycobacteria by
macrophages is considered to be essentially a non-antigen-specific
process, it initiates the development of the adaptive, i.e.,
antigen-specific immune responses, which develop after about 3
weeks since the initial exposure to M. tuberculosis and
significantly contribute to the combating of the infection. Certain
components of the macrophage-digested bacilli, after being released
from the phago-lysosomes, are transported by specialised proteins
referred to as major histocompatibility molecules (MHC molecules)
to the surface of the macrophages, where they can be recognised by
T lymphocytes (for details see section 3.3). If any of the
presented components of M. tuberculosis is recognised by a T cell,
certain proteins called cytokines*) (see section 3.2) capable of
initiating the proliferation of the T cell into a clone of cells
recognising this particular antigen, are synthesized and secreted.
*) Small protein molecules via the communication between various
parts and various cells of the immune system is accomplished.
[0189] The mycobacterial antigens presented by the infected
macrophages attract specific T cells and the process of
inflammation leading to the formation of granulomas is initiated.
The antigen recognition which takes place is due to the
complementarity of the combining sites between the {MHC-M.
tuberculosis component}-complex presented on the surface of the
macrophage and the receptors present on the T cell. The interaction
between the T cells and macrophages leads to the
stimulation/activation of macrophages (for details see section 3.3)
which start secreting various cytokines. One of the cytokines
produced by the activated macrophage, interleukin 1 (IL-1), induces
the proliferation of neighbouring T cells (inflammatory CD4 T
cells), which in turn start secreting interferon .gamma.
(IFN-.gamma.). IFN-.gamma. acting as a chemotaxin, attracts
monocytes to the site of infection and converts resting monocytes
into activated macrophages (Fenton and Vermeulen, 1996; Elgert,
1996b).
[0190] The further course of the disease depends to a large degree
upon the type of the activated lymphocyte, i.e., whether it is a
cytotoxic (CD8) or a helper (CD4) T cell, or whether T-helper cell
is a T-helper 1 (Th1) or a T-helper-2 (Th2) type. The stimulation
of CD8 or CD4 T cells depends on whether an antigen presenting cell
(APC) presented the antigen in association with a major
histocompatibility complex (MHC) molecule of class I or MHC class
II, respectively. The mechanism of the selection between Th1 or Th2
type T-cell response is less clearly understood.
[0191] On the basis of the accumulated evidence it appears that the
activated macrophages and Th1 CD4 cells play the most important
role in the immune response to tuberculosis, leading to the
development of acute phase inflammatory response i.e., the
development of delayed-type hypersensitivity (DTH) (see section
3.4) and the formation of granulomas. The Th1 cells are known to
secrete, apart from IL-2, interferon .gamma., in response to IL-12
secretion by activated macrophages. INF-.gamma. as the principal
cytokine activating macrophages is of critical importance in
combating intracellular mycobacterial infection (Toossi, 1996;
Kaufmann, 1995a and 1995b).
[0192] The involvement of CD4 cells of the Th2 type, which activate
B cells and induce the production of antibodies, has also been
confirmed in tuberculosis (Grange, 1984; Fine, 1994). Although a
number of various antibodies reactive with different types of
mycobacterial antigens have been detected in the sera of
tuberculosis patients (Grange, 1984; Dolin, Raviglione and Koch,
1994; Snider, 1994), their protective role has not been
demonstrated.
[0193] For a long time the activities of Th1 cells in tuberculosis
have been associated with the "good" immunity resulting in the
"containment" of the mycobacterial infection and leading to an
optimistic prognosis, whereas the Th2 cells activities have been
held responsible for the development of allergic reactions and
consequently were considered to be a "bad" type of immunity in this
disease. The current approach considers the activities of Th2 cells
as more positive and assigns to them a regulating function, which
allows them to terminate the defence reactions initiated by Th1
cells which, if left unchecked, could cause serious tissue damage
(Kaufmann, 1995a).
[0194] Furthermore, the importance of the initial innate immunity
and the decisive role it plays in the differentiation of T helper
cells (Th0) to Th1 and Th2 cells has become better understood. The
balance between macrophage-activating and -deactivating cytokines
determines the outcome of the infection (Toossi, 1996; Kaufmann,
1995a and 1995b). Secretion of IL-12 by the infected macrophages
and the production of IFN-.gamma. by NK cells leads to the
development of Th1 cells which initially offer protection against
the pathogen. On the other hand, mycobacteria are potent inducers
of IL-10 which inhibits the Th1 response to this pathogen, probably
by inhibiting the synthesis of IFN-.gamma. by NK cells, and has a
wide range of effects on antigen-presenting cells. IL-10 has
suppressive function, downregulates the major histocompatibility
complex molecules and inhibits the production of monokines (Gong et
al., 1996). The intracellular bacteria within the macrophage also
appear to suppress the early production of IL-4 by CD4.sup.+
NK1.sup.+ cells. Therefore if, at the early stage of the infection
when innate immunity plays a critical part, the production of IL-10
by macrophages and of IL-4 by CD4.sup.+ NK1.sup.+ takes place, the
differentiation of T cells will be biased towards the premature
development of Th2 cells with detrimental effects on the prognosis
of the illness (Kaufmann, 1995a).
[0195] 3.2 Synthesis of cytokines
[0196] Cytokines--general
[0197] Cytokines are low molecular weight, soluble proteins which
are transiently produced by some cells upon their activation and
which specifically affect the behaviour of other cells. They act at
picomolar to nanomolar concentrations on cytokine receptors
inducing significant effects on the proliferation and function of a
variety of cells involved in eliciting an adaptive immune response
(Elgert, 1996b). Cytokines are regulatory and effector molecules
which induce signal transduction, activation of genes responsible
for growth, differentiation and cell activity.
[0198] Cytokines can act as regulators of cell functions by binding
to specific, high-affinity receptors on the cells they affect.
Cytokines are produced by T and B lymphocytes, NK cells,
macrophages and granulocytes. The term interleukins is used for the
molecules secreted by lymphoid cells which allow them to
communicate with each other. Currently some 60 cytokines are known.
A list of the more important of them, i.e., those involved in the
"cytokine cascades" and in the "cytokine regulatory network" in
tuberculosis, is given in Table 1.
[0199] A large panel of monoclonal antibodies specific for human
and mouse cytokines, cytokine receptors and cell-surface
differentiation antigens has been established. These antibodies
constitute powerful probes for analyzing the roles played by
different cytokines as intercellular regulators and effector
molecules used by lymphocyte populations to mediate protective
natural and specific immune responses to foreign antigens, e.g.
bacteria, viruses and neoplasms.
[0200] Cytokine production by lymphocytes is restricted due to the
instability of their respective mRNA. The mRNA instability is
caused by the presence of an "instability sequence" in their 3'
untranslated region (Janeway and Travers, 1994a). This allows the
strict control of cytokine production and release. The
stabilization of mRNA increases the synthesis of the cytokines by
20 to 30-fold.
[0201] The production of IL-2 is a decisive factor determining
whether a T cell will proliferate and become a functional effector
cell. The interaction of IL-2 with its receptor enhances clonal
expansion of CD4 memory T cells at the site of active infections.
These cells, in contrast to naive CD4 T cells are capable of
producing and secreting IFN-.gamma.. The production of IL-2 and
other cytokines depends on the induction of several transcription
factors (Janeway and Travers, 1994a). The induction of these
factors is in turn initiated by the recognition of a specific
antigen by the T cell. One of these factors, nuclear factor of
activation (NF-AT), activates IL-2 transcription by binding to the
promoter region of the IL-2 gene. However, this event ion its own
will not result in the production of IL-2, for which an additional
binding of CD28 (a receptor molecule of Th cells) by B7 protein or
APC is required. A signal which stabilizes IL-2 mRNA is passed
through CD28. The stabilization of IL-2 mRNA together with the
ligand binding of CD28 leads to the increase in the production of
IL-2 by 100-fold (Janeway and Travers, 1994a). However, if the
recognition of a specific antigen by a T cell is not accompanied by
co-stimulation through CD28 molecule, the amount of IL-2 produced
is minimal and T cells are not stimulated.
1TABLE 1 Major cytokines (based on Elgert, 1996b) Abbre- viation
Aliases Functions IL-1 Interleukin-1 Induces thymocytes, T-cell and
B-cell (.alpha. + .beta.) (.alpha. + .beta.) proliferation; Acts as
a co-factor during antigenic and mitogenic stimulation; Increases
secretion of other cytokines, e.g.IL-2, Il-4, CSF; Increases
expression of IL-2 receptors; Induces maturation of pre-B-cells
IL-2 Interleukin-2 Stimulates T-cell growth; Co-stimulates B-cell
differentiation IL-3 Interleukin-3 Stimulates multipotential
haemopoietic cell growth; Stimulates mast cell growth IL-4
Interleukin-4 Co-stimulates B-cell differentiation; Stimulates
class II MHC molecule expression on B cells and macrophages;
Synergizes (with IL-3) in mast cell growth; Enhances IgG, and IgE
production; Co-stimulates prolifera- tion of several haemopoietic
progenitors IL-8 Interleukin-8 Stimulates chemotaxis of neutrophils
and T cells; Stimulates granulocyte activity IL-10 Interleukin-10
Inhibits cytokine synthesis by Th1 cells and activated macrophages;
T-cell growth factor; Cytotoxic T cells differentiation factor
IL-12 Interleukin-12 Induces IFN-.gamma. production by T and NK
cells; Augments the cytotoxic activity of NK cells; Stimulates
differentiation of CD.sub.4 T cells to Th1 cells IFN-.gamma.
Interferon-.gamma. A potent immunoregulatory molecule;
Cross-regulating factor between Th1 and Th2 T cells; Downregulating
factor of Th2 T cells activity; A strong activating factor for
macrophages; Increase of MHC I and II class molecule expression;
Inhibition of intracellular bacterial and protozoan growth;
Activation of neutrophils, NK cells and vascular endothelial cells;
Promotion of B- and T-cell differentiation; Stimulation of antibody
production, particularly IgG2a; Stimulation of IL-1 and IL-2
synthesis TNF-.alpha. Tumour necrosis Causes lysis of target cells;
Activation of factor-.alpha. endothelial cells GM- Granulocyte-
Induces localized haematopoiesis of CSF macrophage monocy tes and
neutrophils; colony stimulating factor TGF-.beta. Transforming
Regulates the formation of extracellular growth factor .beta.
matrix; Inhibits B-, T- and NK- cell activity CSF Colony
stimulating Induces stimulation of the growth of factor colonies of
granulocytes and macrophages; Activation of mature macrophages
[0202] The central role played by IL-2 in the adaptive immunity is
reflected by the number of drugs designed to suppress undesirable
immune responses and interfere with the synthesis of this
interleukin. The examples of such drugs are: cyclosporin and
rapamycin, administered in order to prevent tissue grafts
rejection.
[0203] Another interleukin having a profound influence on the
development of adaptive immunity is interleukin 12 (IL-12). It is a
product of mononuclear phagocytes (macrophages) and plays a crucial
role in the differentiation of T-helper cells, inducing the
development of Th1 cells in vitro and in vivo (Flynn et al., 1995;
Toossi, 1996), thus increasing the resistance to several
intracellular pathogens in experimental mice (Flynn et al., 1995).
IL-12 also enhances the production of IFN-.gamma. from T and NK
cells (D'Andrea et al., 1992; Kobayashi et al., 1989, Toossi,
1996), increases their proliferation (Flynn et al., 1995) and
enhances cytolytic activity of CD8 T and NK cells (Flynn et al.,
1995; Kobayashi et al., 1989; Gately et al., 1994). On the other
hand, cytokines such as TGF-.beta. and IL-10 inhibit the production
and activity of IL-12, suppress the Th1 response and stimulate the
development of intracellular infections (D'Andrea et al.,
1992).
[0204] 3.3 Activation of macrophages and the intracellular fate of
M. tuberculosis
[0205] Following phagocytosis the intracellular growth of
mycobacteria depends on their ability to avoid destruction by
lysosomal enzymes and reactive nitrogen intermediates. Sufficient
evidence has been accumulated to support the observation that M.
tuberculosis bacilli have the ability to block the fusion of
mycobacterium-containing phagosomes (acidic phagocytic vacuoles)
with lysosomes (organelles containing hydrolytic enzymes)
(McDonough, Kress and Bloom, 1993) and the ability to disrupt the
normal functioning of phagosomes (Rastogi, Bachelet and Carvalho de
Sousa, 1992). It is generally accepted that the ability of M.
tuberculosis to survive and multiply within the macrophages is
linked to the unusual physicochemical properties of the
mycobacterial cell wall, attributed mainly to the lipid and
lipid-associated components.
[0206] Normally, activated macrophages have the ability to process
the mycobacterial antigens and to transport them to the cell
surface where they are presented in association with major
histocompatibility complex (MHC) proteins. In this process,
macrophages become antigen presenting cells (APCs).
[0207] Macrophage activation i.e., the induction of antibacterial
mechanisms in macrophages, is initiated by the contact of the
macrophage containing ingested M. tuberculosis with an inflammatory
T cell.
[0208] Macrophages require two signals before becoming
activated:
[0209] the macrophage-activating cytokine, interferon-.gamma.
(IFN-.gamma.) (interferons are cytokines that can induce cells to
resist viral replication) and
[0210] membrane-bound form of tissue necrotic factor .alpha.
(TNF-.alpha.) or a small amount of bacterial lipopolysacharide
(Janeway and Travers, 1994a).
[0211] The first signal, which sensitizes the macrophage to the
second signal, is delivered by inflammatory CD4 T cells. The second
signal is delivered by membrane-bound molecules induced on effector
CD4 T cells. As the process of cytokine synthesis and of the
synthesis of cell-surface molecules mediating their effects
requires several hours, the inflammatory CD4 T cells must adhere to
their target macrophages for this period. The newly synthesised
cytokines are transferred through the microvesicles to the site of
contact between the CD4 T cell and the macrophage.
[0212] The activation of a macrophage through the stimulation with
IFN-.gamma. and the contact with CD4 T cell results in a series of
biochemical responses, which enable the macrophage to become highly
bactericidal. The activated macrophages:
[0213] i) fuse their lysosomes more efficiently to phagosomes,
which leads to the exposure of the intracellular bacteria to a
variety of destructive/bactericidal enzymes;
[0214] ii) secrete IL-12 (activating Th1 cells), IL-6, Il-8 and
TNF-.alpha.;
[0215] iii) produce oxygen radicals--antibacterial agents;
[0216] iv) produce nitric oxide--an antibacterial agent;
[0217] v) produce antibacterial peptides;
[0218] vi) amplify the immune response by increasing the number of
major histocompatibility molecules class II (MCH class II) and of
TNF-.alpha. receptors on their surface;
[0219] vii) recruit other immune cells to the site of infection
(Janeway and Travers, 1994a and 1994b; Tizard, 1995a; Fenton and
Vermeulen, 1996).
[0220] The TNF-.alpha. further contributes to the
INF.gamma.-activation of the macrophage particularly in the
induction of nitric oxide. These functions aim at the destruction
of the mycobacteria. However, if antigenic stimulation persists,
the macrophages become chronically activated and produce additional
cytokines, growth factors and lysosomal enzymes. The latter can
attack and destroy the surrounding tissue (Janeway and Travers,
1994a) leading to the formation of pulmonary cavities.
[0221] The enhancement of the macrophage microbicidal activities
can be brought about by T cells, especially CD4 .alpha..beta. T
cells which secrete IFN-.gamma. and IL-2 (Orme, 1993). As the
activities of Th1 CD4 cells are antigen specific, their involvement
in the activation of macrophages serves as one of the numerous
examples of the intertwining between the innate and specific
adaptive immunity.
[0222] In addition, the activated macrophages produce and secrete
other interleukins such as IL-6, Il-8, and tumour necrosis factor
.alpha. (TNF-.alpha.). Il-8 attracts T cells and neutrophils, while
IL-6 and IL-8 initiate the acute phase inflammatory response.
TNF-.alpha. plays a particularly prominent part in the immune
response to mycobacteria. The production of TNF-.alpha. is induced
by the presence of lipo-arabinomannan, a constituent of the
mycobacterial cell wall (Toossi, 1996). By attracting monocytes to
the site of infection, TNF-.alpha. is aiding the formation of
granulomas, in which, owing to the anaerobic environment, the
bacilli eventually die. Therefore, IL-8, IL-6 and TNF-.alpha.
participate in the protection of the patient's tissues against the
disease and lead to the "containment" of the disease.
[0223] 3.4 Delayed-Type Hypersensitivity (DTH)
[0224] The cell-mediated immune reaction occurring in M.
tuberculosis infection, in which the main effector cells are
activated macrophages, is called delayed-type hypersensitivity
(DTH) or type IV hypersensitivity. The tuberculin skin reaction
(subcutaneous or intradermal contact with the concentrated
derivatives of M. tuberculosis) is a classic example of the DTH
reaction mediated through T cells (and cytokines produced by them)
via activated macrophages (Elgert, 1996a).
[0225] The conversion from tuberculin-negative to
tuberculin-positive skin reaction develops within six to eight
weeks of infection (Boom, 1996) and frequently constitutes the
first sign of the infection with M. tuberculosis. The second
example of the DTH is the systemic granuloma formation resulting
from the inflammatory reaction in tuberculosis.
[0226] The DTH reaction constitutes a part of the immune response
to many intracellular infectious microorganisms, particularly those
causing chronic diseases such as tuberculosis or leprosy. Although
the development of DTH in tuberculosis involves sensitization
rather than immunization of the infected person, nevertheless it
results in a certain degree of protection.
[0227] With effective cellular immunity, the infection should be
arrested permanently at this stage, with healed granulomas leaving
small fibrous and calcified lesions and memory CD4 cells. If the
cellular immune response is insufficient, macrophages containing
viable cells of M. tuberculosis may escape via the intrapulmonary
lymphatic vessels which may lead to the rapid spread of infection
(Elgert, 1996a; Fenton and Vermeulen, 1996).
[0228] Although the precise mechanism for the development of the
initiated DTH response remains not fully understood, the following
has been established:
[0229] i) the interaction between the macrophage-processed and
macrophage-presented antigen and the specific receptors present on
CD4 T cells leads to the formation of sensitized inflammatory T
helper cells, referred to as Th1 cells;
[0230] ii) a portion of Th1 cells become memory cells;
[0231] iii) on reactivation/restimulation Th1 cells produce a
number of cytokines, the most important being:
[0232] 1. IL-2, which causes the proliferation of antigen-primed
Th1 cells;
[0233] 2. GM-CSF, which induces localized haematopoiesis of
monocytes and neutrophils;
[0234] 3. TNF-.alpha. and -.beta. which participate in endothelial
cell binding and activation of leucocytes;
[0235] 4. IFN-.gamma. which is responsible for the enhanced
expression of MHC proteins class II molecules on macrophages and
endothelial cells as well as for the activation of macrophages
(Elgert, 1996a; Fenton and Vermeulen, 1996).
[0236] In addition, chemotaxic factors (chemokines) such as
interleukins of the IL-8 group may attract monocytes to the sites
of antigen deposition and the TNF-.beta. together with the
macrophage-secreted TNF-.alpha. and IL-1 as well as IFN-.gamma.
induce and control the movement of leucocytes within the area of
inflammation.
[0237] 3.5 Activation of T cells and their functions
[0238] Lymphocytes which have left the primary lymphoid organs,
e.g. the thymus, and have never encountered their specific antigen
and therefore have not responded to it, are referred to as naive T
cells. The critical point in their development and in the
development of the adaptive immune response is their activation.
The activation is brought about by their contact with
"professional" antigen-presenting cells (APC: macrophages,
dendritic cells and B cells) and requires the concomitant presence
of two independent signals (Janeway and Travers, 1994a and 1994b).
FIG. 1 presents a graphic illustration of this process.
[0239] The first signal is delivered by the binding of MHC-antigen
complex to the T-cell antigen specific receptor and its co-receptor
(either CD4 or CD8). This signal is transmitted by the T-cell
co-receptor, indicating that the antigen has been recognised. The
second co-stimulatory signal is delivered to the T cell by the same
antigen-presenting cell, APC. The best characterised co-stimulatory
molecules on APC are molecules called B7 and B7.2. The molecule on
the surface of the T cell acting as a receptor for B7 is CD28 and
the ligation of these two molecules will stimulate the
proliferation of the particular clone of T cells. Subsequently, an
additional receptor called CTLA-4 is expressed on the surface of
the activated T cell and binds the B7 molecule with a higher
affinity.
[0240] Naive T cells will respond to a particular antigen only when
one APC cell presents both stimulatory signals: antigen specific to
the T-cell receptor and a co-stimulating signal (B7, B7.2). Only
APC possess the ability to express both classes of MHC molecules as
well as to deliver a co-stimulatory signal and therefore to perform
the so called "priming" of the naive T cells.
[0241] It is important to know that the T cells recognising the
specific antigen in the absence of co-stimulatory signal, fail to
produce IL-2, do not multiply and become anergic, i.e., unable to
respond to a given antigen. This dual requirement for the
proliferation of T cells is a preventative measure aimed at
inhibiting the response of T cells to self tissues, which would be
detrimental to the host. The phenomenon of anergy is frequently
encountered in tuberculosis.
[0242] The interaction of T cells with APCs is influenced to a
varying degree by a range of adhesion molecules such as selecting,
integrins, some mucin-like molecules and CD44 molecule.
[0243] The activation of T cells by APCs results in their clonal
proliferation leading to the production of large numbers of
antigen-specific lymphocytes and the differentiation of their
progeny into armed effector cells. The production of IL-2 is a
decisive factor which determines whether a T cell will proliferate
and differentiate into effector cells.
[0244] The antigen presentation takes place with the help of two
classes of major histocompatibility molecules (MHC). MHC class I
molecules present to CD8 T-cells antigens originating from the
pathogens multiplying in the cytosol of the macrophages, thus
initiating cellular or cell-mediated immune response. MHC molecules
class II present antigens derived from ingested extracellular
bacteria and toxins. These antigens are presented to CD4
inflammatory cells referred to as Th1 cells and to CD4 helper
cells, referred to as Th2 cells. The Th2 cells activate the
specific B cells, thus initiating the humoral immune response.
[0245] Once the T cells are activated and start the clonal
expansion, they can act on any target cell which displays the
specific antigen on its surface. Effector T cells can perform a
number of functions such as:
[0246] i) killing of infected cells by CD8 cytotoxic T cells;
[0247] ii) activation of macrophages and peripheral mononuclear
cells by CD4 inflammatory cells (an essential activity in
tuberculosis leading to the destruction of the phagocytosed
mycobacterial cells);
[0248] iii) activation of B cells to produce antibodies.
[0249] The first two activities constitute elements of
cell-mediated immunity, whereas the third one represents the
humoral immunity (Janeway and Travers, 1994a, 1994b and 1994c;
Elgert, 1996c).
[0250] 3.6 Cytokine circuits in tuberculosis
[0251] Experimental evidence accumulated so far indicates that the
protective immune response against M. tuberculosis in man is
mediated primarily by CD4 Th1 cells and mononuclear phagocytes
(Kaufmann, 1995a and 1995b, Fenton and Vermeulen, 1996, Boom,
1996). Among the cytokines secreted in the process of mounting host
anti-mycobacterial responses, IL-12, IL-2 and IFN.gamma. appear to
be playing the most prominent parts. The treatment of the M.
tuberculosis-infected Balb/c mice with IL-12 effectively increased
their survival and reduced 10 to 50-fold the number of viable
bacilli in their organs (Flynn et al., 1995).
[0252] As there exists a distinct possibility of T-cell
hyper-responsiveness at sites of active infection, the production
of other cytokines which can inhibit the anti-mycobacterial immune
responses such as transforming growth factor .beta. (TGF-.beta.)
also takes place. The intricate interplay between various cytokine
circuits which are activated by M. tuberculosis and its
constituents may be amplified and contribute to pathology at the
site of the infection (Toossi, 1996).
[0253] The overall outcome of the host immune response/host defence
and the course of the disease will be determined by the balance
between macrophage-activating and -deactivating cytokines. Some
cytokines e.g. tumour necrosis factor .alpha. (TNF-.alpha.) and
transforming growth factor .beta. (TGF-.beta.) may contribute to
symptoms of tuberculosis such as tissue destruction, fibrosis
formation, fever and weight loss. In addition, in the HIV-infected
tuberculosis patients, cytokines may promote viral replication and
in this way contribute to the progression of the disease.
[0254] The well documented role of IFN-.gamma. in the infection
with M. tuberculosis, becomes closer to be understood. The
administration of IFN-.gamma. to mice deficient in this cytokine
prolongs their survival after the challenge with M. tuberculosis
(Cooper et al., 1993; Flynn et al., 1993). The protective effects
of IFN-.gamma. appear to be due to the enhancement of macrophage
activity towards M. tuberculosis, namely due to the upregulation of
TNF-.alpha. and 1,25-hydroxy vitamin D (Bermudez and Young, 1988;
Rook et al., 1987).
[0255] Both, IFN-.gamma. and TNF-.alpha. increase
anti-mycobacterial activity of human (Denis, 1991; Hirsch et al.,
1994) and murine macrophages (Bermudez and Young, 1988). Both,
IFN-.gamma. and TNF-.alpha. counteract the effects of TGF-.beta..
These effects interfere with the production of anti-mycobacterial
nitrogen intermediaries within the infected macrophage and
down-regulate IFN-.gamma. and TNF-.alpha.. The balance between
these three macrophage-activating and -deactivating cytokines,
i.e., IFN-.gamma., TNF-.alpha. and TGF-.beta. influences the final
outcome of the infection (Chantry et al., 1989; Ding, Nathan and
Srimal, 1990; Espevik et al., 1987; Tsunawki, Sporn and Nathan,
1988; Toossi, 1996).
[0256] 3.7 Induction of cytokines by M. tuberculosis' antigens or
components
[0257] The currently available information concerning the
involvement of M. tuberculosis' antigens or components on the
induction of cytokines secretion (Toossi, 1996), can be summarized
as follows:
[0258] i) both, partially purified and completely purified
mycobacterial proteins were found to induce the production of Il-1,
IL-2, IL-12, TGF-.beta. and of TNF-.alpha.;
[0259] ii) the 38 kDa antigen was reported to induce the secretion
of IL-12 and IFN-.gamma. (Agrewala and Mishra, 1995) whereas the 58
kD antigen (present in the culture filtrate) was found to induce
the production of TNF-.alpha. (Wallis, Paranjape and Phillips,
1993);
[0260] iii) two purified protein derivatives PPD1 and PPD2 were
found to induce the production of IL-5 and IFN-.gamma.,
respectively (Ebtekar and Khanasri, 1996);
[0261] iv) the 30 kD antigen (a major secretory protein and
cell-wall component) was found to induce cytokines in monocytes as
well as the production of TGF-.beta. (Toossi, 1996);
[0262] v) lipo-arabinomannan was found to be associated with the
induction of TGF-.beta. but not TNF-.alpha., IL-1 or IL-10 (Toossi,
1996);
[0263] vi) heat-shock proteins studied, i.e., 10 kD, 65 kD and 71
kD molecules were found to induce proliferative responses in human
CD4.sup.+ T cells, with the 65-kDa molecule apparently playing part
in human autoimmune responses (Boom, 1996; Beagly et al.,
1993);
[0264] vii) some components of M. tuberculosis' cell walls were
reported to induce potent immunosuppressive agents such as
TGF-.beta. (Toossi, 1996).
[0265] 3.8 Cytokine profiles in tuberculosis patients
[0266] Whereas in mice there exists a sequential production of Th1
and Th2 cytokines in response to live mycobacteria (Sander et al.,
1995), in human tuberculosis there is an absence of a prominent Th2
cytokine response (Lin et al., 1996). Depressed Th1 cytokines
responses, however, are characteristic of the advanced human
tuberculosis, as illustrated by the following observations:
[0267] 1. Several studies confirmed that tuberculosis sufferers
display a defect in the production of IFN-.gamma. when challenged
with various mycobacterial antigens (Vilcek et al., 1986; Hirsch et
al., 1996; Huygen et al., 1988; Onwubalili, Scott and Robinson,
1985);
[0268] 2. The results observed in in vitro experiments indicate
that CD4 lymphocytes originating from tuberculosis patients have a
limited capacity to synthesise IL-2 and IFN-.gamma., when
challenged with M. tuberculosis antigens (Toossi, 1996);
[0269] 3. Lower peripheral blood mononuclear cell responses were
reported and are thought to be caused by a functional suppression
of T-cell production of IL-2 and expression of IL-2 receptors. The
relative numbers of the two main populations/groups of T cells,
namely CD4 and CD8, remain unchanged (Toossi, Kleinhenz and Ellner,
1986; Kleinhenz and Ellner, 1987; Vanham et al., 1996);
[0270] 4. A dominant role played by monocytes in suppression of
T-cell responses resulting in the lowering of the synthesis of IL-2
is well established (Ellner, 1978; Kleinhenz and Ellner, 1987;
Toossi et al., 1989) and high numbers of these cells are frequently
encountered in cases of active tuberculosis (Toossi, 1996). The
molecular mechanisms by which monocytes suppress T-cell responses
in patients with tuberculosis have been to a large degree
elucidated and were discussed in a recent review by Toossi
(1996);
[0271] 5. Cytokines produced by CD4 cells belonging to Th2
subpopulations such as IL-4 and IL-10 are reported by some groups
of researchers to be higher in patients with tuberculosis than in a
control group (Sucrel et al., 1994; Toossi, 1996). However, there
exists a certain degree of controversy on this subject (Hirsch et
al., 1996, Toossi, 1996). As both of these cytokines have been
associated with the ability to de-activate macrophages, their
presence could adversely affect the course of the disease;
[0272] 6. Transforming growth factor .beta. (TGF-.beta.) is a
potent immunosuppressor that inhibits the clonal expansion of T
cells by interfering with the proliferative signal of IL-2 and
suppresses the production of INF-.gamma. and IL-2 (even at
femtomolar concentrations). TGF-.beta. is known for its
auto-induction, through which it can significantly increase its
levels at sites of active tuberculosis infection. Through this
action TGF-.beta. can have a direct influence on several cytokines
and seriously interfere with the host immune defense
mechanisms.
[0273] 7. Granulocyte-macrophage colony stimulating factor (GM-CSF)
is secreted by macrophages and some T cells. GM-CSF, by acting
directly on bone marrow cells, stimulates the expansion of
granulocytes and macrophages. It influences, therefore, both
humoral and cell-mediated immune responses (Boom, 1996).
[0274] 3.9 Involvement of other T cells in tuberculosis
[0275] Apart from the immune processes dependent on CD4 cells, CD8
T cells are also involved in the immune response to the infection
with M. tuberculosis. As CD8 cells can be directly cytotoxic and
have the ability to kill the macrophages harbouring mycobacteria,
they play a part in the destruction of M. tuberculosis, either by
lysis of the macrophages or by releasing of M. tuberculosis to the
extracellular environment where they can be phagocytosed by other
activated macrophages.
[0276] Recently carried out investigations indicate that additional
subsets of T cells are involved in the immune reaction to
tuberculosis. These T cells produced large amounts of IFN-.gamma.
and varying amounts of IL-2, IL4, IL-5 and IL-10, and cannot be
assigned to the clearly defined Th1 or Th2 groups. They are
classified as Th0 subset of CD4 cells (Boom, 1996).
[0277] Another subset of T cells which may have an important role
in the cellular response to the infection with M. tuberculosis are
the T cells expressing As T-cell receptor (Haanen et al., 1991;
Boom, 1996; Kaufmann, 1995a). They have been reported to recognise
phosphate-containing non-proteinaceous components of mycobacteria
and, on stimulation with these components, have been shown to
display a Th1 cytokine pattern (Kaufmann, 1995a). It has been
postulated (Kaufmann, 1995a) that the rapid activation of
.gamma./.delta. T cells, preceding that of .alpha./.beta. T cells
could attribute to them a function of a link between innate
immunity by NK cells and the specific adaptive immunity effected by
.alpha./.beta. T cells. The .gamma./.delta. T cells appear to
control the local tissue response at the site of bacterial
replication and the TCR.delta. (T-cell receptor .delta.) gene
deletion mice mutants were found to be more susceptible to death
when challenged by M. tuberculosis inocula tolerated by
immunocompetent mice (Kaufmann, 1995a).
[0278] The CD1b-restricted .alpha./.beta. T cells produce
IFN-.gamma. and express cytolytic activity, in which they resemble
Th1 cells (Kaufmann, 1995a).
[0279] 4. Proposed approach to the prevention of infection with M.
tuberculosis and proposed approach to the prevention of rheumatoid
arthritis associated with tuberculosis
[0280] Accepting that mycolic acids possess immunoregulatory
properties in spite of their simple, long chain fatty acid
structure, it is proposed that the prevention of infection with M.
tuberculosis could be achieved by a successful induction of humoral
and/or cellular memory against mycolic acids leading to a long-term
protection against the disease. Another approach is based on the
assumption that by using appropriate tolerogenic doses of purified,
biologically active mycolic acids used alone, in a supportive
medium or pharmaceutical carrier or excipient or on appropriate
carriers, with or without the simultaneous introduction of
appropriate cytokines (Heath and Playfair, 1992), it should be
possible to successfully modulate the immune response(s) in the
human body. Such a treatment could potentially help to prevent or
decrease mortality due to tuberculosis and be used as a potential
treatment of the disease.
[0281] On the other hand, vaccination with mycolic acids used with
or without the appropriate carriers, may regulate the immune system
upon the infection with M. tuberculosis, by modulating the induced
response of the recipient.
[0282] Prevention of rheumatoid arthritis associated with
tuberculosis could be achieved by preventing the generation of
auto-immune antibodies directed against collagen. Recently
published results of the immunomodulatory properties of a synthetic
10-kD heat shock protein (hsp10) from M. tuberculosis, in relation
to adjuvant-induced arthritis in rats, indicate that the
administration of this compound could indeed lead to the delayed
onset of the disease and the development of less severe symptoms
(Ragno et al., 1996).
[0283] Accepting once again that mycolic acids possess
immunoregulatory properties, the proposed approach is based on the
assumption that by using appropriate tolerogenic doses of mycolic
acids, possibly on suitable carriers, either on their own or,
simultaneously with appropriate interleukin(s) (Heath and Playfair,
1992), it should be possible to successfully manipulate or regulate
the immune response(s) in the human body. Such a treatment could
potentially help to prevent or delay the onset of rheumatoid
arthritis associated with tuberculosis, or decrease the severity of
this disease.
[0284] In order to investigate this approach, adjuvant arthritis
was induced in rats, which were treated with mycolic acids either
prior to or after the induction of the disease. The induction of
this form of arthritis, using a suspension of heat-killed and
freeze-dried cells of an avirulent strain of M. tuberculosis H37 Ra
was achieved following the method of Wauben, Wagenaar-Hilber and
Van Eden (1994). Investigations into this approach are set out
below in Example 2.
[0285] 5. Immunogenicity of mycolic acids
[0286] Immunogenicity of a molecule, i.e., its ability to induce an
immune reaction depends on the chemical structure and properties of
the molecule and on the ability of a particular immune system to
recognise it. Many compounds such as proteins, peptides, nucleic
acids and polysaccharides are naturally highly immunogenic and
capable of eliciting strong immune reactions when recognised as
"foreign" by the immune system. On the other hand, the majority of
lipid compounds, with the exception of some glycolipids, has until
recently not been considered to be immunogenic.
[0287] Mycolic acids are the major lipids of the cell wall of
Mycobacteria and constitute approximately 40% of the dry weight of
these bacteria. Mycolic acids are high molecular weight
.beta.-hydroxy fatty acids (C.sub.60 to C.sub.90), which have
moderately long aliphatic chains in the .alpha.-position and are
characterised by a highly restricted solubility. Their aliphatic
structure and absence of aromaticity suggest that, similarly to
other lipid compounds, they should have very weak/limited
immunogenic properties (Savelkoul, Claassen and Benner, 1997). In
addition, the mechanism by which lipids could elicit immune
responses in a host and the manner in which they could be presented
to the immune system, have, until recently, been unknown.
[0288] However, the evidence for immunogenicity of mycolic acids,
i.e., for their ability to induce an immune reaction, has been
accumulated over the last three/four years on the basis of the
results reported by various research centers.
[0289] It was observed by the present inventors in 1994 that
mycolic acids adsorbed to proteins and administered to mice elicit
an antibody response (South African Patent Application No. 95/3077
and International Patent Application No. 95/00856 relating to the
induction of antibodies to mycolic acids upon immunization of mice
with mycolic acids adsorbed to proteins). The response appears to
be specific for mycolic acids on two accounts. It was elicited by
bovine serum albumin conjugates but could be detected on ELISA
wells coated with mycolic acids-gelatin conjugate in the
immunoassay and the response measured on mycolic acids-gelatin
conjugate as antigen could be partly inhibited by co-incubating the
antisera with bovine serum albumin-mycolic acids conjugate.
[0290] The immunogenicity of mycolic acids and their
immunoregulatory properties have been supported by evidence which
has recently became available from other sources:
[0291] i) The discovery of Beckman et al., (1994) that mycolic
acids activate DN T-cells upon presentation on antigen presenting
cells
[0292] While investigating presentation of non-peptide microbial
antigens, Beckman et al., (1994) discovered that mycolic acids
originating from M. tuberculosis stimulated the proliferation of a
rare subset of human T cells. The group of stimulated human T cells
was identified as double negative, i.e., neither CD4.sup.+ (helper
function) nor CD8.sup.+ (cytotoxic function), T-cell clones.
[0293] Similarly, Rosat et al., (1995) reported stimulation and
subsequent expansion of a human T-cell line (OGD1) upon exposure to
unique cell-wall lipids isolated by organic extraction from M.
tuberculosis and presented on CD1b molecules.
[0294] Additional observations concerning the presentation of
lipo-arabinomannan originating from the cell-wall fractions of M.
leprae by CD1 molecules were reported by Sieling et al., (1995),
who isolated two T-cell lines responding to the stimulation with
lipoarabinomannan.
[0295] On the basis of these reports and results it appears
that:
[0296] i) mycolic acids are in fact immunogenic at least in terms
of eliciting some kind of cellular and humoral immune response;
[0297] ii) mycolic acids are presented by one of a group of five
CD1 glycoproteins in humans, of which CD1b plays the major role in
presentation of mycolic acids.
[0298] ii) The role of CD1 presented molecules in the presentation
of mycolic acids and other mycobacterial lipids
[0299] CD1 molecules constitute a group of glycoproteins occurring
on antigen presenting cells. They appear to perform a novel and
unique function, by presenting mycobacterial antigens originating
from the lipid fraction of bacterial lysates to the immune system.
CD1 molecules appear to be homologous in their function to
peptide-presenting MHC (Major Histocompatibility Complex) proteins
(Beckman et al., 1994; 1995) but specialized in presenting antigens
of lipid or hydrophobic nature.
[0300] The compounds presented by CD1 molecules are recognised by
human T cells displaying on their surface receptor chains .alpha.
and .beta.*) (Beckman et al., 1994, 1995) as well as .gamma. and
.delta. chains **) (Rosat et al., 1995). The subset of
.gamma..delta. T cells was found to express only receptors coded by
V.delta.1.sup.+ gene and to proliferate on exposure to unique
cell-wall lipids isolated by organic extraction from M.
tuberculosis, presented on CD1b molecules. *) .alpha. and .beta.
chains--transmembrane glycoprotein receptor chains are responsible
for the recognition of antigens on antigen presenting cells (APC)
after processing and presentation on MHC or other professional
antigen presenting molecules such as CD1. **) .gamma. and .delta.
chains--polypeptide receptor chains are homologous to .alpha. and
.beta., appearing at an early stage of thymocytes differentiation,
with a very limited variability and poorly understood functions.
They can recognise antigens without the requirement of prior
processing and presentation by APC, even recognising antigen in
solution as long as it is multivalent (Schild et al., 1994).
[0301] Both subsets of these T cells were activated on exposure to
the antigens of mycobacterial origin and showed enhanced
proliferation (Beckman et al., 1994, 1995; Rosat et al., 1995).
[0302] Experiments described by Tangri et al., (1995), carried out
with the mouse CD1 molecules, established peptide sequences binding
to this type of antigen-presenting molecules and found such
peptides to be highly hydrophobic. This observation confirms the
distinct role which CD1 molecules appear to be playing in the
presentation of lipid or other hydrophobic compounds in a non MHC
restricted manner.
[0303] iii) The anticipated role of double negative (DN) T cells in
auto-immunity and immunoregulation
[0304] Double negative (DN) T cells form a small, highly
heterogenous group of cells residing in the thymus, comprising
several early stages in T-cell development. A small percentage of
these cells expresses genes coding for .gamma..delta. while the
remaining DN express genes for .alpha..beta. receptor chains. They
precede the appearance of the functional receptor chains and the
expression of CD4 and CD8 markers (Janeway and Travers, 1994d).
[0305] DN T cells constitute less than 2% of human lymphocytes
present in peripheral blood (Niehues et al., 1994). They were found
to differ from single positive (SP) subsets of T cells in:
[0306] i) proliferating in response to the presence of interleukin
3 (IL-3);
[0307] ii) becoming activated on exposure to non-peptide antigens
presented by CD1 molecules;
[0308] iii) not responding to the stimulation with antigens
presented by MHC class I and II molecules (Niehues et al.,
1994).
[0309] Although the main function of this small and poorly defined
T cell subset group remains unknown, there is some evidence
indicating their involvement in auto-immune reactions and in the
regulation of auto-immunity. The studies of von Boehmer, Kirberg
and Rocha (1991) and Kisielow et al., (1988) demonstrated that in
transgenic mice, the receptors for self antigens were predominantly
expressed on DN cells. In humans, levels of DN T-cell populations
were found to be elevated in patients with auto-immune disorders
such as systemic lupus erythematosus (Shivakumar, Tsokos and Datta,
1989) and systemic sclerosis (Sakamoto et al., 1992).
[0310] The involvement of DN T cells in immunoregulation was
demonstrated by Niehues et al., (1995a), who reported that the
stimulated DN T cells secreted interleukin 10 (IL-10). As IL-10 can
downregulate the expression of MHC proteins class II by the antigen
presenting cells and, at certain concentrations, can suppress the
expression of CD1 molecules (Chomssen, Kahnn and Londei, 1995) and
inhibit the functions of inflammatory Th1 cells (Janeway and
Travers, 1994a), an important role played by DN T cells in
regulating and probably suppressing immune functions and in
auto-immunity is anticipated.
[0311] If it is accepted that mycolic acids are immunogenic and,
when presented on CD1 molecules can activate human DN T cells, it
can be postulated that two immunological phenomena observed in
tuberculosis, namely the occurrence of anergy and the induction of
post-tuberculosis rheumatoid arthritis could be associated with
mycolic acids.
[0312] Anergy, i.e., the inability of the infected person to mount
an immune response despite the presence of antigen, is commonly
observed during the initial stage of infection with M. tuberculosis
and M. leprae. It is believed that the specific immunocompetent
lymphocytes are suppressed as a consequence of either the way in
which the molecules are presented to them (Schwartz, 1993) or as
the result of the absence of co-stimulatory signals (Janeway and
Travers, 1994a).
[0313] If the presentation of lipid antigens on CD1 molecules
creates the conditions necessary for the initiation of anergy in
tuberculosis, it can be hypothesised that mycolic acids could play
a direct role in this phenomenon, probably by the activated DN T
cells secreting IL-10 (Niehues, et al., 1995a).
[0314] Auto-immunity or auto-reactivity is a pathological condition
caused by the adaptive immune response directed at self antigens.
Such responses can be generally produced by:
[0315] i) a sudden exposure of normally hidden self antigens (as in
the case of sympathetic ophthalmia);
[0316] ii) self antigens becoming immunogenic due to chemical,
physical or biological changes (as in the case of contact
dermatitis);
[0317] iii) coincidental similarity between a foreign antigen
(pathogen) and the self tissue antigen, referred to as molecular
mimicry (as in the case of streptococcal protein M and human heart
muscle) (Merck Manual, 1987; Janeway and Travers, 1994e).
[0318] Rheumatoid arthritis, is an auto-immune disease associated
with the presence of auto-antibodies and auto-reactive T cells
damaging the joints' cartilage (Laycock et al, 1995). The
mycobacterial antigens could be implicated in this pathological
state in two ways. Either heat-shock proteins of M. tuberculosis
(HSP 60, HSP 65) elicit the production of auto-antibodies due to
the genuine molecular mimicry between them and the aggrecan i.e.,
the core protein of host cartilage (Roitt, 1994; Tizard, 1995b;
Voet and Voet, 1995) or the production of such auto-immune
antibodies is caused by a contamination of heat-shock proteins
secreted by mycobacteria with mycolic acids. The latter possibility
finds some support in the observation reported by Buzas et al.,
(1995) who could not detect any cross-reactivity between aggrecan
and the mycobacterial HSP 65 produced by genetically manipulated E.
coli, therefore not contaminated by mycolic acids. Additional
evidence that the HSP 65 may not be directly involved in the
induction of adjuvant arthritis, came from Moudgil et al., (1995).
These authors worked with two groups of rats, resistant and
susceptible to adjuvant arthritis and found that they shared
identical MHC. As only peptides can be presented by MHC, this
finding implies that compounds other than proteins (e.g. lipids)
are probably involved in inducing this form of arthritis.
[0319] Further evidence of the lipid nature of the molecules
potentially involved in the induction of rheumatoid arthritis comes
from the studies of Beech et al., (1995) and Lemonidis et al.,
(1995) who studied pristane*)-induced arthritis in mice. In both
instances the response was characterized by the presence of
T-lymphocytes and antibodies strongly cross-reacting with
mycobacterial HSP 65. *) A mineral oil of defined structure,
including several methyl branches.
[0320] The evidence presented above concerning the role which
lipids/oils could play in inducing rheumatoid arthritis supports
the hypothesis that in the case of tuberculosis this role could be
performed by mycolic acids forming natural conjugates with proteins
present in the infected host.
[0321] iv) Our observations concerning cross-reactivity of mice
antisera against mycolic acids-BSA with gelatin
[0322] During our experimental work substantiating Patent No
94/2575, it was observed that the antibodies produced during
immunization with mycolic acids-protein conjugates cross-reacted
with gelatin, the denatured form of collagen. Without wishing to be
bound by theory, we propose a hypothesis to explain this
observation, namely that the murine immune system probably does not
recognize mycolic acids as such, but rather as modified epitopes on
protein molecules.
[0323] Similarly, during infection with M. tuberculosis, mycolic
acids originating from the pathogen may become attached to some
proteins present in the host's body, such as heat-shock proteins
known to be expressed on the surface of infected macrophages
(Grange, Stanford and Rook, 1995) or host protein to create
"foreign" epitopes. The presence of such epitopes can lead to the
production of auto-antibodies and auto-reactive T cells against
collagen (gelatin), which could attack host collagen leading to the
development of an auto immune reaction. The auto-antibodies and
auto-reactive T cells thus generated, may significantly influence
the degree of severity of the disease and may play a crucial role
in TB patient survival.
[0324] If it is remembered that:
[0325] i) collagen is the native form of gelatin;
[0326] ii) the mice antibodies generated against mycolic
acids-protein conjugates were observed to recognise gelatin;
[0327] iii) arthritis can be produced in experimental animals by
injection of collagen, collagen reactive T cells or anti-collagen
antibodies (Brand et al., 1995); and
[0328] iv) collagen is the most abundant protein in higher
vertebrates (Sakai, 1995) and that lungs, where the original
contact with M. tuberculosis usually takes place, comprise large
numbers of collagen fibrils (Leeson and Leeson, 1981)
[0329] the potential of mycolic acids' attachment to the host's
major class of fibrous protein and their involvement in the
generation of auto-immune antibodies directed against collagen and
probably leading to the development of rheumatoid arthritis
associated with tuberculosis, becomes evident.
[0330] Furthermore, the presence of antibodies recognising
collagen/gelatin in a tuberculosis patient could be responsible for
impaired resistance against bacterial infection (Bras and Aguas,
1995). Such antibodies could react with the collagen-like region of
human serum (segment C1q) thus impairing this serum protein's
crucial role in the cytotoxic reaction towards bacteria and
infected host cells. The presence of anti-C1q antibodies in
patients with systemic lupus erythematosus was associated with
persistent hypo-complementaemia and defective ability to opsonise
bacteria, which led to the patients' inability to dispose of
life-threatening infections (Davies, Norsworthy and Walport,
1995).
IMMUNOREGULATORY AND IMMUNOGENIC PROPERTIES OF
COUNTERCURRENT-PURIFIED MYCOLIC ACIDS
[0331] The substantiation/evidence for the immunogenic and
immunoregulatory properties of countercurrent-purified mycolic
acids is illustrated by the three examples described below.
EXAMPLE 1
[0332] Protection against tuberculosis in mice provided by the
administration of purified mycolic acids. Experiments involving
animals injected with M. tuberculosis by intravenous
administration.
1.1 Materials
[0333] 1.1.1 Cultures
[0334] Mycobacterium tuberculosis H37Rv ATCC 27294--a virulent
strain, originally isolated from an infected human lung. Type
strain of the species.
[0335] Mycobacterium vaccae ATCC 15483--a strain originally
isolated from cow's milk. Type strain of the species.
[0336] The cultures were purchased in lyophilized form from the
American Type Culture Collection (ATCC), Maryland, USA.
[0337] 1.1.2 Media
[0338] 1.1.2.1 Growth media
[0339] The following media were used for the cultivation of M.
tuberculosis:
[0340] Lowenstein-Jensen-(LJ) medium (slants) and
[0341] Middlebrook 7H-10 agar medium (plates).
[0342] A detailed composition of the ingredients necessary for the
preparation of these media as well as the conditions recommended
for their sterilization, are given in the Laboratory Manual of
Tuberculosis Methods, Tuberculosis Research Institute of the SA
Medical Research Council (1980, Chapter 6, pp 83-105; Second
Edition, revised by E E Nel, H H Kleeberg and E M S Gatner).
[0343] The media were prepared by the National Tuberculosis
Institute of the Medical Research Council of South Africa, in
Pretoria.
[0344] 1.1.2.2 Media used for washing and diluting of
Mycobacteria
[0345] The harvested bacteria were washed in sterile 0,9% m/v NaCl
(Saarchem, Chemically Pure, RSA).
[0346] Medium used for the preparation of serial dilutions,
preceding the determination of viable counts of M. tuberculosis,
was prepared by dissolving Tween 80 (Merck, Chemically Pure) in
0,9% m/v NaCl (Saarchem, Chemically Pure) to a concentration of
0,01% v/v and distributing it in 9,0 ml aliquots into test-tubes.
The autoclaved media were stored at 4.degree. C.
[0347] 1.1.2.3 Media used for the amplification of the competitive
plasmid pGEM-3Z
[0348] Dulbecco's Modified Eagle's Medium (DMEM)--manufactured by
Life Technologies Inc., Country
[0349] LB agar comprising:
[0350] 10% m/v Tryptone (Biolab Diacgnostics, RSA)
[0351] 5% m/v Yeast extract (Difco Laboratories, Michigan, USA)
[0352] 10% m/v NaCl (Saarchem, Chemically Pure, RSA) and
[0353] 1,5% m/v agar (Saarchem, Chemically Pure, RSA).
[0354] LB broth comprising:
[0355] 10% m/v Tryptone (Biolab Diagnostics, RSA)
[0356] 5% m/v Yeast extract (Difco Laboratories, Michigan, USA)
and
[0357] 10% m/v NaCl (Saarchem, Chemically Pure, RSA).
[0358] Ampicillin and streptomycin (Life Technologies Inc.,
Scotland)
[0359] Sodium pyruvate (Life Technologies Inc)
[0360] Agarose (Promega Corporation, Madison, USA)
[0361] 1.1.3 Reagents
[0362] 1.1.3.1 For the preparation of the reagents used for the
extraction, derivatization and High-Performance Liquid
Chromatography (HPLC) analysis of mycolic acids, HPLC Grade
methanol (BDH) and double-distilled deionized water were used.
[0363] Reagent A: 25% potassium hydroxide (Saarchem, Analytical
Grade) dissolved in methanol-water (1:1), i.e., 62,5 g potassium
hydroxide was dissolved in 125 ml water and 125 ml methanol (BDH,
HPLC Grade) was added.
[0364] Reagent B: Concentrated hydrochloric acid (Saarchem,
Analytical Grade) diluted 1:1 with water.
[0365] Reagent C: 2% potassium bicarbonate (BDH. Analytical Grade)
dissolved in methanol-water (1:1), 10 g potassium bicarbonate was
dissolved in 250 ml water and 250 ml methanol was added.
[0366] Reagent D: para-bromophenacylbromide dissolved in
acetonitrile and crown ether (Pierce Chemical Co, Cat. No 48891)
was dispensed in 500 .mu.l quantities into small amber-coloured
screw cap vials with Teflon-coated septa. The caps were tightened
and the vials were wrapped with Parafilm. Reagent D was stored at
4.degree. C.
[0367] Reagent E: Reagent E was prepared by mixing reagent B 1:1
with methanol.
[0368] HPLC Standard: High Molecular Weight Internal Standard
(C-100) from Ribi ImmunoChem Research Company, Cat No R-50. The
standard, 1 mg, was dissolved in 20 ml chloroform (BDH, HPLC Grade)
at 4.degree. C. and aliquots of 100 .mu.l were dispensed into 4 ml
amber WISP vials, dried, capped with Teflon-coated septa and stored
at 4.degree. C.
[0369] Chloroform (Saarchem, Analytical Grade, RSA)
[0370] Methylene chloride (BDH, UK, HPLC-Grade)
[0371] Reagents A, B, C and E were prepared fresh prior to
experiments, taking all the necessary safety precautions.
[0372] 1.1.3.2 The following reagents were used for the preliminary
purification of crude bacterial extracts ("funnel extraction") and
for the countercurrent purification of the extracted mycolic
acids:
[0373] Chloroform (Saarchem, Chemically Pure)
[0374] Methanol (Saarchem, Chemically Pure)
[0375] Acetone (Saarchem, Chemically Pure)
[0376] Sodium chloride (Saarchem, Analytical Grade)
[0377] Double-distilled deionized water was used for the
preparation of the required reagent concentrations. i.e.,:
[0378] 39% v/v methanol
[0379] 42% v/v chloroform
[0380] 0,2 M NaCl
[0381] 1.1.3.3 Reagents used in the Semi-Quantitative Competitive
Reverse Transcriptase Polymerase Chain Reaction (QC-RT-PCR):
[0382] The following reagents were used:
[0383] Ethidium bromide (Boehringer Mannheim, Germany)
[0384] Formamide and formaldehyde (BDH, UK)
[0385] Tris (Hydroxymethyl)-aminomethane (Merck, Germany)
[0386] EDTA (Ethylenediaminetetra-acetic acid) (Merck)
[0387] Sodium acetate (Merck)
[0388] TRI-reagent (Molecular Research Centre Inc, USA)
[0389] Formazol (Molecular Research Centre Inc)
[0390] MOPS (3-(N-morpholino) propanesulfonic acid) (Sigma
Chemicals, USA)
[0391] Diethyl pyrocarbonate (DEPC) (Sigma)
[0392] Oligo dT primers (Life Technologies Inc., Scotland)
[0393] Superscript RNase H Reverse Transcriptase (Life Technologies
Inc.)
[0394] Recombinant Taq Polymerase Dynazyme (Finnzmes OY)
[0395] Amplitaq Gold (Roche Molecular Systems, USA)
[0396] Qiagen mini preparatory column Kit (Qiagen)
[0397] Tris EDTA buffer: Tris base 10 mM disodium ethylene diamine
tetraacetate.2H.sub.2O, pH adjusted to pH 8,3.
[0398] 1.1.3.4 Reagents used in the purification of
.alpha..beta.TCR.sup.+ CD4.sup.+, .alpha..beta.TCR.sup.+ CD8.sup.+
single positive (SP) and .alpha..beta.TCR.sup.+, CD4.sup.- and
CD8.sup.- double negative (DN) T cells from the human peripheral
blood
[0399] The reagents used in this part of the experimental work were
described by Niehues et al., (1994, 1995b).
[0400] 1.1.4 Experimental Animals
[0401] Eight to twelve weeks old female Balb/c (a
tuberculosis-susceptible strain) and C57/bIJ6 mice (a
tuberculosis-resistant strain) were used in the "immunoregulatory"
experiments. The mice were inbred for 11 and 9 generations,
respectively, by the Animal Centre at the South African Institute
for Medical Research in Johannesburg. Male mice of corresponding
age were used for the collection of serum necessary for the
preparation of mycolic acids/mouse serum conjugates.
[0402] Seventeen weeks old Sprague-Dawley female rats were used for
the induction of anti-mycolic acids antibodies.
[0403] Feed and Water
[0404] Mice cubes, manufactured by EPOL and tap, autoclaved water
were provided ad libitum.
[0405] Sanitation:
[0406] Bronocide, manufactured by Essential Medicines (Pty) Ltd,
was used for sanitation purposes.
[0407] 1.1.5 Plasticware
[0408] The following plasticware was used:
[0409] Disposable Petri's dishes (Promex, RSA)
[0410] ELISA plates (Sterin, UK)
[0411] Sterile, disposable 50 ml centrifuge tubes (Corning,
USA)
[0412] Disposable tips (Elkay, Denark)
[0413] 96-well round bottom microplates (Nunc, Denmark)
1.2 Methods
[0414] The following methods were used in the experimental
work:
[0415] 1.2.1 Cultivation of the Bacterial Strains
[0416] The bacteria were cultivated at 37.degree. C. using
Lowenstein-Jensen (LJ) medium slants and Middlebrook 7H-10 agar
medium plates.
[0417] The sterility of all the media was confirmed, before they
were used in the experiments by incubating them at 37.degree. C.
for 24 h.
[0418] For routine extraction of mycolic acids approximately 4-week
old M. tuberculosis and 2-week old cultures of M. vaccae, grown on
LJ slants, were used. When Middlebrook 7H-10 agar medium plates
were used, 2-week old cultures of M. tuberculosis were harvested
for the extraction of mycolic acids. For the preparation of
bacterial suspensions used for the experimental induction of
tuberculosis, approximately 2-week old cultures of M. tuberculosis,
grown on LJ slants were used.
[0419] 1.2.2 Viable and Total Bacterial Counts
[0420] For the viable count determination, serial suspensions of
the harvested bacteria were prepared in the diluent medium (as
specified under 1.1.2.2) to a density corresponding to a McFarland
standard 4 (approximately OD of 1,0; using a Beckman DU 65
spectrophotometer, at 486 nm). Tenfold serial dilutions were
prepared using 9 ml aliquots of the diluent medium. From the last
three dilutions corresponding to 10.sup.-3, 10.sup.-4 and 10.sup.-5
of the original suspension, aliquots of 0,1 ml (100 .mu.l) were
withdrawn and spread over the surface of Middlebrook 7H-10 plates.
The plates were incubated at 37.degree. C. and the developed
colonies counted after two to three weeks for M. tuberculosis and
after one week for the plates seeded with M. vaccae.
[0421] The direct total count was performed using a Neubauer
counting chamber and the autoclaved cultures of M. tuberculosis and
M. vaccae, originally adjusted to a density corresponding to a
McFarland standard 4 and suitably diluted with the diluent
medium.
[0422] Statistical analysis of the bacterial counts included the
mean values of bacterial counts and standard deviations.
[0423] 1.2.3 Preparation of Mycolic Acids from Bacterial
Samples
[0424] The preparation of bacterial samples comprised three
steps:
[0425] harvesting of the Mycobacteria cells;
[0426] saponification and
[0427] extraction of mycolic acids.
[0428] Glassware used for the harvesting, extraction,
derivatization and HPLC analyses of mycolic acids was washed in 2%
(v/v) Contrad (Merck), rinsed in water, followed by rinsing in
chloroform, water, Technical Grade methanol, water and finally
rinsed in double distilled deionized water. The washed glassware
was dried in a warm air oven.
[0429] Harvesting was done by scraping the bacterial growth from
the surface of media slants or agar plates (using sterile plastic
loops) and by suspending them in Reagent A. Initial bacterial
suspensions were prepared in Reagent A, by vortexing the harvested
cells with sterile glass beads. Homogenous bacterial suspensions
were prepared using sterile tissue homogenizers. Prior to the
saponification, the density of the bacterial suspensions was
adjusted to a density corresponding to a McFarland standard 4.
[0430] The saponification, extraction and derivatization of mycolic
acids were carried out as described by Butler, lost and Kilburn
(1991), with minor modifications and are described under the
relevant headings.
[0431] Saponification of the Mycobacteria in Reagent A was carried
out in an autoclave at 121.degree. C., for 30 min.
[0432] 1.2.4 Extraction of Mycolic Acids
[0433] The saponified samples were allowed to cool after
autoclaving. Into 2 ml samples containing crude extract, 1,5 ml
Reagent B was introduced. After vortexing, the pH of each sample
was checked and if necessary, adjusted to pH 1 with Reagent B.
[0434] Subsequently, 2,0 ml chloroform was added to each sample and
vortexed for 30 seconds. The layers were allowed to separate. The
bottom layers were removed with Pasteur pipettes, transferred to
amber WISP vials and evaporated to dryness at 85.degree. C. in a
heat block-evaporator under a stream of nitrogen. To neutralize
traces of acid carried over, 100 .mu.l of reagent C was added to
each sample and the fluid evaporated to dryness at 85.degree. C. in
a heat block-evaporator under a stream of nitrogen.
[0435] 1.2.5 Storage of the Crude Extracted Mycolic Acids
[0436] The material obtained from the large-scale extraction of
mycolic acids originating from M. tuberculosis and M. vaccae, i.e.,
the crude bacterial extracts, was stored under acetone, at
4.degree. C. in 4 ml amber WISP vials. To prevent
evaporation/drying and the exposure to light, the caps of the WISP
vials were covered with Parafilm.
[0437] 1.2.6 Determination of Mycolic Acids Contents in Crude
Extracts
[0438] Extracted mycolic acids were derivatized as follows:
[0439] To a cooled sample of crude extract (approximately 10 .mu.g
in 2,0 ml Reagent A), an aliquot of 1,0 ml chloroform was
introduced, followed by the addition of 100 .mu.l of Reagent D
(derivatization reagent). The capped samples were vortexed for 30
seconds and heated for 20 minutes at 85.degree. C. in a heat
block-evaporator. Subsequently, the samples were cooled and 1,0 ml
of Reagent E added. The samples were vortexed for 30 seconds and
the layers allowed to separate. The bottom layers were removed with
Pasteur pipettes and transferred to WISP-vials. The vials were
placed in a heat block-evaporator and their contents evaporated to
dryness at 85.degree. C. using a stream of nitrogen.
[0440] The residues were resuspended in 0,212 g (which corresponds
to 160 .mu.l) methylene chloride, capped and vortexed. Each
reconstituted sample was introduced into a WISP vial containing 5
.mu.g of the HPLC internal standard (prepared as described under
1.1.3.1), filtered through a 0,22 .mu.m Millex GV4 filter with a
polyethylene housing into another amber-coloured WISP-vial. The
recapped vials were stored at 4.degree. C. until ready for HPLC
analysis.
[0441] 1.2.7 HPLC Analysis and Quantification of Mycolic Acids
[0442] Repeatability and accuracy of the pipette used for the
distribution of the HPLC standard was determined. The precision was
established to be +/-1% and was confirmed prior to each aliquoting
of the internal standard.
[0443] For the HPLC analysis 10 .mu.l from each sample (maintained
on ice during handling), was analyzed. Control samples, i.e., 10
.mu.l of filtered methylene chloride, were run prior to each set of
samples analyzed. If a large number of samples was analyzed, in
order to validate the reliability of the HPLC apparatus, control
samples were run after every three or four test samples.
[0444] The reverse-phase HPLC analyses were carried out using a
Waters 600 E System Controller High Performance Liquid
Chromatography apparatus consisting of:
[0445] Microsep M741 Data Module;
[0446] Waters 712 WISP Autosampler;
[0447] Detector (Waters 486 Tunable Absorbance Detector);
[0448] Column: Nova-Pak C18 4 .mu.m 3,9.times.150 mm and an end
connector set for steel cartridge columns.
[0449] RKC Rex-C 4 Column Temperature regulator.
[0450] Running conditions were:
[0451] Mobile phase:
[0452] Solvent A: HPLC Grade methanol
[0453] Solvent B: HPLC Grade methylene chloride
[0454] Flow Rate: 2,5 ml/min
[0455] Column temperature: 30.degree. C.
[0456] The detector was set at 260 nm.
[0457] Prior to use, the solvents were sparged with Instrument
Grade helium. High Purity Nitrogen was used to control hydraulics
of the WISP vials autosampler.
[0458] The HPLC gradient initially comprised 98% (v/v) methanol
(Solvent A) and 2% (v/v) methylene chloride (Solvent B). The
gradient was increased linearly to 80% A and 20% B at one minute;
35% A and 65% B at ten minutes, held for 30 seconds and then
decreased over 10 seconds back to 98% A and 2% B. This ratio was
maintained for 4 minutes to allow for stabilization of the system
prior to injection of the next sample.
[0459] Mathematical quantification of mycolic acids was carried out
by comparing the combined peak areas of the tested samples to the
peak area of the introduced quantity of the High Molecular Weight
Internal HPLC Standard.
[0460] 1.2.8 Preliminary Purification of Crude Mycobacterial
Extracts
[0461] In order to shorten the time required for the countercurrent
purification of the crude mycobacterial extracts, an additional
preliminary extraction step was introduced. This step had a dual
purpose:
[0462] i) to remove unnecessary cellular components from the crude
extract prior to the countercurrent purification and
[0463] ii) to reduce soap fraction in the crude bacterial
extracts.
[0464] A portion of the crude extracted material (approximately 3-4
g) was suspended in a minimum volume of the lower phase solvent
(usually 100 ml), transferred into a separation funnel and mixed
with an equal volume of the upper phase solvent. The phases were
allowed to separate and the upper phase was removed and stored at
4.degree. C. Into the remaining lower phase an equal volume of the
upper phase solvent was again introduced and the process of the
phase separation was repeated.
[0465] The second upper phase was removed and stored at 4.degree.
C. and the second lower phase was dried in a Buchi Rotoevaporator
RE 120, at 75.degree. C. and its mass recorded.
[0466] 1.2.9 Countercurrent Purification of Mycolic Acids
Originating from M. tuberculosis and M. vaccae
[0467] Countercurrent Apparatus
[0468] A countercurrent apparatus produced by H O POST, Instrument
Company Inc., Middle Village, N.Y. was used during the
investigations. The "trains" in this model consisted of 2.times.250
inter-connected tubes.
[0469] Solvent System Used in the Countercurrent Apparatus
[0470] The solvent system used for the countercurrent separation
consisted of:
[0471] 42% v/v chloroform (Saarchem, Chemically Pure Reagent)
[0472] 39% v/v methanol (Saarchem, Chemically Pure)
[0473] 19% v/v 0,2 M NaCl (Saarchem, Chemically Pure).
[0474] Double-distilled deionized water was used for the
preparation of the solvent system.
[0475] The components were mixed, equilibrated and the upper and
lower phases were collected using a separation funnel.
[0476] The composition of the upper phase was established to
be:
[0477] 15% v/v chloroform, 52% v/v methanol and 33% v/v 0,2 M
NaCl.
[0478] The composition of the lower phase was established to
be:
[0479] 68% v/v chloroform, 27% v/v methanol and 5% v/v 0,2 M
NaCl.
[0480] The countercurrent purification process was carried out
under the following conditions:
[0481] A countercurrent distribution train comprising 55 tubes,
numbered 0-54, was used in the experiments. The upper phase
solvent, a volume of 600 ml, was introduced into a buffer
reservoir. A sample of 125 mg of mycolic acids after the
preliminary purification was dissolved in 50 ml of the lower phase
solvent, divided into five aliquots and introduced into first five
tubes, numbered 0-4. Subsequently, 10 ml of the upper phase solvent
was introduced into each of the first five countercurrent tubes.
Into the remaining 50 tubes aliquots of 10 ml of the lower phase
were introduced. Upper phase, in volumes of 10 ml per cycle, was
automatically dispensed into tube number 0, repeatedly over 55
cycles resulting in approximately 5 hour operations Thus, fifty
five countercurrent cycles were performed, with each cycle
consisting of 10 mixing pendula and 3 minutes phase separation
time.
2 Initial load of crude extract after the funnel extraction: 125 mg
Number of cycles: 55 Equilibration time: 3 min
[0482] 1.2.11 Removal of Malachite Green from the
Countercurrent-Purified Mycolic Acids
[0483] To remove traces of malachite green derived from bacterial
growth media (when M. tuberculosis was grown on LJ slants), the
countercurrent-purified material was selectively precipitated in
the following manner. Countercurrent-purified mycolic acids (92 mg)
were placed in a WISP vial into which 1,0 ml chloroform was
introduced. The dissolved mycolic acids were transferred into a
pre-weighed round-bottom flask. The vial was rinsed twice with 1,0
ml chloroform and the two aliquots of chloroform were added to that
already present in the round-bottom flask. Subsequently, acetone
was introduced drop-wise in 500 .mu.l aliquots. In total, 26 ml of
acetone was introduced and the white flakes of the precipitated-out
mycolic acids were washed twice with 20 ml acetone. The acetone
supernatant, with the dissolved malachite green, was removed and
the mycolic acids dried by evaporation.
[0484] The procedure was carried out at room temperature.
[0485] 1.2.12 Determination of Mycolic Acids after Countercurrent
Purification
[0486] In order to increase the accuracy of the HPLC determination
of mycolic acids, the High Molecular Weight Internal Standard
(C-100) was introduced into the countercurrent-purified mycolic
acids before the saponification.
[0487] A sample of 0,5 mg of the countercurrent-purified mycolic
acids was introduced into a WISP vial containing 5 .mu.g of the
High Molecular Weight Internal Standard (C-100). Saponification of
mycolic acids was carried out with 2 ml of Reagent A at room
temperature. The WISP vial was vortexed for 30 seconds. The
extraction was carried out with 1,5 ml of Reagent B. After
vortexing, the pH of the sample was checked and if necessary,
adjusted to pH 1 with Reagent B.
[0488] Subsequently, 2,0 ml chloroform was added to each sample and
vortexed for 30 seconds. The layers were allowed to separate. The
bottom layers were removed with Pasteur pipettes, transferred to
amber WISP vials and evaporated to dryness at 85.degree. C. in a
heat block-evaporator under a stream of nitrogen. To neutralize
traces of acid carried over, 100 .mu.l of reagent C was added to
each sample and the fluid evaporated to dryness at 85.degree. C. in
the heat block-evaporator under a stream of nitrogen.
[0489] Therefore, the main difference between the determination of
mycolic acids after countercurrent purification and in the crude
extract was the time of introduction of the Internal Standard.
[0490] 1.2.13 Determination of Yield of the Countercurrent
Separation
[0491] In order to calculate the approximate yield of
purification/separation, the amount of the mycolic acids present in
the samples obtained after the countercurrent
separation/purification was compared to the amount of these
compounds present in the crude cellular extract introduced into the
countercurrent apparatus. The calculations were based on the
results obtained by the HPLC analysis.
[0492] It should be stressed, that it is essential for the
calculation of the yield of the countercurrent separation, that the
mycolic acids determined by HPLC should be within the tested linear
range of the HPLC UV detector.
[0493] 1.2.14 Infra-Red Spectroscopy
[0494] Samples of mycolic acids to be analyzed by infra-red
spectroscopy were prepared in the following manner.
Countercurrent-purified mycolic acids, 1 mg, were dissolved in 1 ml
chloroform, introduced into 200 mg KBr and thoroughly mixed. After
the evaporation of chloroform, a pellet of mycolic acids in KBr was
prepared by using a Shimadzu tablet die and applying a force of
approximately 100 kilonewtons on the sample for 10 minutes. A
control pellet was prepared using only chloroform, without mycolic
acids added to the preparation. The control pellet was used to
determine the background infra-red spectrum. The spectra were
analyzed on a Perkin Elmer 1600 series FT-IR system and plotted on
a Roland Digital Group X-Y Plotter DXY-1200.
[0495] 1.2.15 Determination of the Stability of the
Countercurrent-Purified Mycolic Acids
[0496] A pooled sample of the countercurrent-purified mycolic acids
was prepared by introducing five batches of countercurrent-purified
mycolic acids into a container, dissolving them in chloroform and
mixing the contents very well. The chloroform was evaporated using
a Buchi Rotoevaporator RE 120, at 75.degree. C. and the sample
dried under a stream of nitrogen. The pooled sample was divided
into two parts which constituted two stock samples. The first stock
sample was re-saponified and the second was left as a
non-saponified stock sample. From both stock samples individual
aliquots were withdrawn and placed at -20.degree. C., 4.degree. C.
and 25.degree. C. Three samples were prepared per each time point
and HPLC analyses were carried out after 6 weeks, 3, 6, 9 and 12
months of storage.
[0497] 1.2.16 Methods Used in Handling Experimental Animals in the
Immunoregulatory Experiments
[0498] 1.2.16.1 Environmental conditions under which the
experimental animal were maintained
[0499] Experimental Animals
[0500] Eight to twelve weeks old female Balb/c (a
tuberculosis-susceptible strain) and C57/bIJ6 mice (a
tuberculosis-resistant strain) were used in the "immunoregulatory"
experiments.
[0501] Experimental animals were accommodated in cages with a floor
area of 450 cm.sup.2, with 8 mice per cage.
[0502] Environmental conditions: Temperature and humidity in the
animal facility were set at 20.degree. C. (+/-1.degree. C.) and 40%
(+/-10%), respectively. Lighting was provided by means of
fluorescent tubes. A light-darkness cycle of alternating 12 hour
periods was set up.
[0503] Cages
[0504] Mice were housed in transparent polypropylene cages with
tight fitting stainless steel lids. Wooden shavings, after
autoclaving, were provided as nestling material.
[0505] Sanitation
[0506] Animal rooms, mice cages and glass bottles were cleaned and
decontaminated once a week using Bronocide. Water bottles after
washing were autoclaved once a week.
[0507] Glove Isolator
[0508] Mice infected with M. tuberculosis H37Rv were maintained in
a glove isolator manufactured by Labotec, South Africa. The
isolator was inflated by a positive pressure of 4 atm. It was
equipped with an air inlet pre-filter (with the pore size of 0,6
.mu.m) through which the incoming air was filtered and an outlet
HEPA (High Efficiency Particulate Air) filter (with a pore size of
0,22 .mu.m) through which the outgoing air was filtered before
leaving the isolator. The air-flow rate was regulated at 7
exchanges per hour.
[0509] Sanitation
[0510] Animal rooms, mouse cages, the glove isolator and water
bottles were cleaned and decontaminated once a week using
Bronocide. Water bottles, after washing, were autoclaved once a
week.
[0511] 1.2.16.2 Identification of the experimental animals
[0512] Individual identification of mice was accomplished by making
ear marks.
[0513] 1.2.16.3 Collection of blood samples and preparation of
mouse serum
[0514] Mice were bled from the tail vein and the blood collected
into sterile Eppendorf's tubes. The collected blood was incubated
at 37.degree. C. for one hour and then left at 4.degree. C.
overnight for the clot to retract. The serum was recovered by
centrifugation (in a Beckman J-6 centrifuge, at 1000 g for 15 min),
aliquoted in volumes of 1,0 ml and stored frozen at -70.degree.
C.
[0515] 1.2.16.4 Preparation of mycolic acids--mouse serum
conjugates
[0516] The required mass of mycolic acids (2,5 mg) was dissolved in
200 .mu.l chloroform and added to 10,0 ml of mouse serum (see
1.2.19.3), previously filtered through a 0,22 .mu.m filter. Thus,
the volume of dissolved mycolic acids constituted 2% of the volume
of mouse serum.
[0517] The sample was sonicated using a Branson Sonifier B 30 Cell
Disruptor, (at 20% duty cycle, output control of 2, for 50 pulses,
at room temperature). The sample was maintained for 1 hour at room
temperature, to allow air bubbles formed during sonication to
escape. In order to remove chloroform, nitrogen was bubbled through
the conjugate until the chloroform odour was removed. The conjugate
was prepared immediately before administration to the experimental
animals.
[0518] 1.2.16.5 Preparation of bacterial suspensions for the
induction of tuberculosis in mice
[0519] The cells of M. tuberculosis H37 Rv, harvested from LJ
slants were suspended in the diluting buffer (0,01% v/v Tween 80 in
0,9% m/v NaCl) and homogenized. After centrifugation in a Beckman
J-6 centrifuge for 20 min at 1580 g, the cells were washed with a
sterile solution of 0,9% m/v of NaCl and adjusted to a
concentration corresponding to a McFarland standard No. 4. After
the confirmation of the total direct bacterial count, carried out
on an autoclaved suspension in a Neubauer counting chamber, the
suspension was further diluted in the sterile solution of 0,9% NaCl
to obtain concentrations of M. tuberculosis corresponding to
10.sup.3, 10.sup.4 and 10.sup.5 cells/ml.
[0520] The viable counts of the mycobacteria in the suspensions
were confirmed by plating 100 .mu.l aliquotes of the relevant
dilutions onto Middlebrook 7H-10 agar medium, incubating the plates
at 37.degree. C. for two weeks and counting the number of colony
forming units (CFU).
[0521] The suspensions were introduced into the experimental
animals in aliquots of 100 .mu.l per animal.
[0522] 1.2.16.6 Introduction of the M. tuberculosis H37 Rv
suspensions, mycolic acids-mouse serum conjugate and mouse
serum
[0523] The introduction of the bacterial suspensions and of the
mycolic acids conjugates was carried out via the intravenous route.
Prior to injections, mice were heated for 5 min in a heating box
until vasodilation of the tail veins could be observed.
[0524] The respective bacterial suspensions were introduced in
aliquots of 100 .mu.l per mouse. The mycolic acids-mouse serum
conjugate was administered by introducing 25 .mu.g mycolic
acids/100 .mu.l mouse serum per mouse.
[0525] Control animals received 100 .mu.l of mouse serum introduced
in the same manner.
[0526] 1.2.16.7 Experimental set-up
[0527] The experimental set-up is presented in Tables 2a -2d.
3TABLE 2a Experimental set-up for the immunoregulatory experiment
IR-III Experimental set-up Number of Number of Time schedule
Description of experiment Group mice Set-up injections Pretreatment
Infection Treatment Controls Controls were performed to test: group
1 9 -- -- -- -- M tb infection group 2 9 M tb M tb effect of the
serum carrier group 3 3 serum one serum effect of the way of
administration of MA group 4 3 MA one MA 5 .mu.g as single or
multiple injection group 5 3 serum three serum group 6 3 MA three
MA 5 .mu.g Pre-treatment Pretreatment of mice with serum alone or
group 7 10 M tb + serum one serum M tb MA adsorbed on mouse serum
one week group 8 10 M tb + MA one MA 5 .mu.g M tb prior to
infection group 9 10 M tb + MA one MA 25 .mu.g M tb Treatment
Treatment of mice with serum alone or MA group 10 10 M tb + serum
one M tb serum adsorbed on mouse serum two weeks after group 11 10
M tb + MA one M tb MA 5 .mu.g infection in a single or multiple
injection group 12 10 M tb + MA one M tb MA 25 .mu.g group 13 10 M
tb + serum three M tb serum group 14 10 M tb + MA three M tb MA 5
.mu.g group 15 10 M tb + MA three M tb MA 25 .mu.g Abbreviations: M
tb--Mycobacterium tuberculosis MA--Mycolic acids
[0528]
4TABLE 2b Experimental set-up for the immunoregulatory experiment
IR-IV (Balb/c mice) Experimental set-up Number of Time schedule
Description of experiment Group mice Set-up Pre-treatment Infection
Treatment Controls Controls were performed to test: group 1 16 --
-- -- M tb infection group 2 16 M tb M tb Pre-treatment (single)
effect of the serum carrier group 3 16 MA + M tb 12,5 .mu.g MA M tb
effect of the way of administration of group 4 16 MA + M tb 25,0
.mu.g MA M tb MA as single or multiple injection group 5 16 MA + M
tb 50,0 .mu.g MA M tb group 6 16 serum + M tb serum M tb Treatment
(multiple) Treatment of mice with serum alone or group 7 16 M tb +
MA M tb 3 .times. 8,0 .mu.g MA MA adsorbed on mouse serum two weeks
group 8 16 M tb + MA M tb 3 .times. 16,0 .mu.g MA after infection
in multiple injections group 9 16 M tb + serum M tb 3 .times. serum
Abbreviations: M tb--Mycobacterium tuberculosis MA--Mycolic
acids
[0529]
5TABLE 2c Experimental set-up for the immunoregulatory experiment
IR-IV (C57/bl6 mice) Experimental set-up Number of Time schedule
Description of experiment Group mice Set-up Pre-treatment Infection
Treatment Controls Controls were performed to test: group 1 14 --
-- -- M tb infection group 2 14 M tb M tb Pre-treatment (single)
effect of the serum carrier group 3 14 MA + M tb 12,5 .mu.g MA M tb
effect of the way of administration of group 4 14 MA + M tb 25,0
.mu.g MA M tb MA as single or multiple injection group 5 13 MA + M
tb 50,0 .mu.g MA M tb group 6 13 serum + M tb serum M tb Treatment
(multiple) Treatment of mice with serum alone or group 7 14 M tb +
MA M tb 3 .times. 8,0 .mu.g MA MA adsorbed on mouse serum two weeks
group 8 13 M tb + MA M tb 3 .times. 16,0 .mu.g MA after infection
in multiple injections group 9 13 M tb + serum M tb 3 .times. serum
Abbreviations: M tb--Mycobacterium tuberculosis MA--Mycolic
acids
[0530]
6TABLE 2d Experimental set-up for the immunoregulatory experiment
No V Experimental set-up Number Time schedule Description of
experiment Group of mice Set-up Pre-treatment Infection Treatment
Controls Controls were performed to test the effect of: Untreated,
uninfected group 1 25 -- -- -- M tb infection group 2 25 M tb M tb
effect of the serum pretreatment group 3 25 serum pre-treat. serum
M tb effect of the serum treatment 21, 24 and group 4 25 serum
treatment M tb serum 27 days after infection Pretreatment
Pretreatment of mice with MA from M tb group 5 25 MA (M tb) + M tb
MA 25 .mu.g M tb adsorbed on mouse serum one week prior to
infection Pretreatment of mice with MA from M vac group 6 25 MA (M
vac) + M tb MA 25 .mu.g M tb adsorbed on mouse serum one week prior
to infection Treatment Treatment of mice with MA from M tb group 7
25 MA (M tb) + M tb .times. 3 M tb 3 .times. 8 .mu.g MA (M tb)
adsorbed on mouse serum 21, 24 and 27 days after infection
Treatment of mice with MA from group 8 25 MA (M vac) + M tb .times.
3 M tb 3 .times. 8 .mu.g MA (M vac) M vac adsorbed on mouse serum
21, 24 and 27 days after infection Abbreviations: M
tb--Mycobacterium tuberculosis; MA--Mycolic acids; M
vac--Mycobacterium vaccae;
[0531] 1.2.16.8 Assessment of pathology of the experimental
animals
[0532] Individual mass measurements of all the experimental animals
were carried out at seven-day intervals, at the same time of a
particular day. These measurements were carried out using a
Sartorius electronic scale (with a range of 0,00-200,00 g and
accuracy of 0,01 g) and a plastic beaker to contain the mice.
[0533] Post mortem analyses were performed on control and infected
mice. Dissection of the diseased mice and histological examination
of the appropriate organs were carried out by Dr J H Vorster of the
Section of Pathology of the Veterinary Research Institute,
Onderstepoort, 0110.
[0534] Methods Used in Histopathological Assessments
[0535] After dissecting of various organs, i.e., the lungs, spleens
and livers, from mice cadavers, they were individually weighed and
photographed.
[0536] Macroscopic Assessment
[0537] Macroscopic assessment of the degree of infection in various
organs was carried out by comparing individual organs originating
from various groups of experimental mice to the control organs. The
evaluators were not aware of the treatment to which individual
animals were subjected.
[0538] Microscopic Assessment
[0539] Fixation of the organs was carried out by submerging them in
10% v/v formaldehyde solution in PBS buffer. The organs/tissues
were subsequently embedded in paraffin-wax and sections of 5 .mu.m
thickness prepared by cutting with microtome.
[0540] For granuloma counts, organ sections were stained in
haematoxylin/eosin solution according to Luna (1968). Lesions
observed in the tissues were graded by counting the number of
granulomas per field, using 10 fold magnification for the liver and
lung tissues and 40 fold magnification for the spleen tissue.
[0541] A qualitative grading system for the assessment of the
severity of lung lesions was devised as follows:
[0542] 1-small, well defined granulomas in the lungs;
[0543] 2-larger, more diffused granulomas which sometimes formed
extended focal areas of granulomatous pneumonia, occupying less
than a third of the lung tissue. Interstitial pneumonia was
slightly more pronounced;
[0544] 3-mostly fused granulomas which were extensive and affected
more than one third of the lung tissue.
[0545] For counts of add-fast microorganisms, the sections were
stained by Ziehl-Neelsen technique (Heifets and Good, 1994). (Using
this technique, acid-fast bacteria stain red, nuclei stain dark
blue and other tissue constituents are pale blue).
[0546] Ziehl-Neelsen staining was used for qualitative assessment,
which was made by comparing the number of organisms and their
density within the stained tissue. The level of infection in the
lungs, livers and spleens originating from various groups of mice
was compared for each specific organ, but could not be compared
among different organs, due to the difference in appearance and
size of lesions characteristic for individual organ types.
[0547] Biochemical Assessment
[0548] Five to seven weeks after the infection with M.
tuberculosis, the mice were sacrificed for cytokine profiling, the
required organs removed aseptically and snap-frozen in liquid
nitrogen. The frozen organs were maintained at -70.degree. C. and
analyzed for the expression of various cytokines.
[0549] 1.2.17 Methods Used in the Semi-Quantitative Competitive
Reverse Transcriptase Polymerase Chain Reaction (SQC-RT-PCR)
Determination
[0550] Background Information:
[0551] RT-PCR--Principle
[0552] The Polymerase Chain Reaction (PCR) is a technique used for
the amplification of DNA and the complementary DNA (cDNA) of
specific mRNAs, which was invented by Mullis in the late 1980's
(Mullis and Faloona 1987; Saiki et al., 1988) for the amplification
of DNA sequences in vitro.
[0553] PCR is based on a series of incubation steps carried out at
different temperatures. The template DNA (or cDNA) is denatured at
a temperature above 90.degree. C. (denaturing step). The
oligonucleotide primers are then annealed to the single stranded
DNA (ssDNA) at a temperature varying between 50.degree. C. and
60.degree. C., depending on the type of primers used (annealing
step). This process is followed by an extension of the primers by
incorporating dNTPs, using a heat-resistant DNA polymerase and an
incubation temperature of 70-72.degree. C. (extension step). The
extension products of one primer provide templates for the other
primers in subsequent cycles, so that each successive cycle
essentially doubles the amount of DNA synthesised in the previous
cycle. The result is the exponential amplification of the target
DNA to approximately 2.sup.n (n=number of cycles) (Zubay, 1993;
Tamarin, 1996).
[0554] Taking into consideration a number of variables*) which may
interfere with the quantification of the exact amount of mRNA
originally present in the analyzed sample, an improved technique,
that of Semi-Quantitative Competitive Reverse Transcriptase
Polymerase Chain Reaction, was employed. *) The variables include:
differences in the stability and purity of polymerase enzyme, dNTP
and in buffer preparation in various batches; Mg.sup.2+
concentration; DNA (template) concentration; primer concentration;
annealing, extension and denaturing temperatures; length and number
of cycles; rate of primer-dimer formation; presence of
contaminating DNA.
[0555] Scanning of gels was done with a densitometer (Apple Mac).
The density of the unknown cDNA band was compared to the density of
that of .beta.-actin and the concentration of the unknown cDNA
could thus be estimated. The relative densitometric measurements
were done using a Macintosh NIH Image Program.
[0556] This technique can be used to accurately quantify less than
1 fg (femtogram, 10.sup.-15 g) of target cDNA obtained from total
RNA after the RT reaction. The accuracy of this method can be
improved by using the same master mix for all the samples. The
master mix should contain the appropriate primers, PCR buffer,
dNTPs, MgCl.sub.2 and the polymerase enzyme. The mix should be
divided equally between all the tubes used for the PCR.
[0557] The competitive plasmid for murine IL-12 p40 (obtained as a
gift from K. L. Bost, University of Tulane, New Orleans, La., USA)
was a pGEM-3Z derivative with a IL-12 fragment (334 base pairs)
cloned into the Xba I site from the multicloning site of the vector
(Bost and Clements, 1995).
[0558] The construction of the plasmid was described by Bost and
Clements (1995) and Chong, Bost and Clements (1996).
[0559] 1.2.17.1 Preparation of the organs used for RNA
extraction
[0560] The organs originating from both infected and uninfected
mice, used for the RNA extraction experiments were lungs, spleens
and kidney. Mice were sacrificed by rapid cervical dislocation. The
organs were removed from each mouse aseptically, and kept at
-70.degree. C. after snap-freezing in liquid nitrogen.
[0561] A single-cell suspension of the spleen was made by cutting
the spleen into small pieces on a nylon sieve (70 .mu.m mesh) in
the presence of ice cold medium (DMEM containing streptomycin and
sodium pyruvate). The spleen cells were concentrated by
centrifugation and the excess of medium was removed. The
erythrocytes present in the preparation were lysed by hypotonic
shock, i.e., by treating the cells with a 1/10 dilution of DMEM in
sterile distilled water for 15 seconds. The lysis of the cells was
stopped by adding excess medium. After centrifugation, the excess
medium was removed and the cells were snap-frozen in liquid
nitrogen (McCarron, et al., 1984). The cells were maintained as a
dry pellet at -70.degree. C.
[0562] 1.2.17.2 RNA Extraction from control and infected organs
[0563] RNA was isolated from all the organs using the TRI-Reagent
protocol based on an acid guanidium thiocynate-phenol-chloroform
extraction, a method first developed by Chomczynski and Sacchi
(1987). The isolated RNA was quantified by a Shimadzu UV-Visible
Recording Spectrophotometer model UV-160, at wave-lengths of 260 nm
and 280 nm. Pure RNA (absorption ratio at 260/280 nm>=2,0) was
used for PCR.
[0564] Integrity of the isolated RNA was determined using a
denaturing formaldehyde gel (Maniatis, 1982). These denaturing
conditions prevent degradation of the RNA by RNases. The water used
in these experiments was diethyl pyro-carbonate (DEPC)-treated.
[0565] Ethidium bromide was added to the RNA sample, before it was
loaded on the gel, at a concentration of 0,5 ng/nl to enabler
visualization of the DNA with UV light. Pure, undegraded RNA giving
the three rRNA bands (the 26S rRNA, 18S rRNA and the 5S rRNA) on
agarose gel electrophoresis, was used for the reverse transcriptase
reaction (Maniatis, 1982).
[0566] 1.2.17.3 The optimization of the different cytokine PCRs and
the 6-actin PCR
[0567] Cytokine PCRs were optimised by using different plasmids
containing DNA sequence fragments of the various cytokines to be
evaluated/to be tested. These fragments of DNA are deletion
mutations of fragments of the wild type cDNA for the individual
cytokine. Both the mutated and the wild type cDNA can be amplified
by using the same primers.
[0568] Three different plasmids were used for this purpose:
[0569] i) a plasmid used for the determination of IL-12 was
obtained from K Bost (University of Tulane, USA);
[0570] ii) a plasmid used for the determination of TNF-.alpha. was
obtained from R L Tarleton (University of Georgia, USA);
[0571] ii) a plasmid used for the determination of IL4, IL-10,
IFN-.gamma. and TGF-.beta. was obtained from R M Locksley
(University of California, San Franciso USA).
[0572] 1.2.17.4 The amplification of the competitor plasmids
[0573] The competitive plasmids for the determination of IL-12,
TNF-.alpha., TGF-.beta. were amplified after transformation in the
SURE E. coli strain and isolated with the Qiagen mini preparatory
column kit. The recovered plasmids were resuspended in TE buffer
(10 mM Tris pH 8, 1 mM EDTA) and stored at -20.degree. C.
[0574] A list of the sequence of sense and anti-sense primers,
their annealing temperatures and the wild type fragment size is
given below in Table 3.
7TABLE 3 A list of the sequences of the sense and antisense
primers, their annealing temperature and the wild type fragment
size Type of Fragment Annealing PCR Sequence of the primers Size
Temperature 1. .beta.-actin.sup.1 S:.sup.5'CTC CAT CGT GGG CCG CTC
TAG.sup.3' 133 bp 59.degree. C. AS:.sup.5'GTA ACA ATG CCA TGT TCA
AT.sup.3' 2. IL-12.sup.2 S:.sup.5CCA CTC ACA TCT GCT GCT CCA CAA
G.sup.3' 266 bp 60.degree. C. AS:.sup.5ACT TCT CAT AGT CCC TTT GGT
CCA G.sup.3' 3. TNF-.alpha..sup.3 S:.sup.5' GTC TAC TTT AGA GTC ATT
GC.sup.3' 275 bp 48.degree. C. AS:.sup.5' GAC ATT CGA GGC TCC AGT
G.sup.3' 4. TGF-.beta..sup.4 S:.sup.5' ACA GGG CTT TCG ATT CAG
CGC.sup.3' 306 bp 60.degree. C. AS:.sup.5' CAC CTA GGT GCT CGG CTT
CCC.sup.3' 5. IFN-.gamma..sup.4 S:.sup.5' CAT TGA AAG CCT AGA AAG
TCT G.sup.3' 267 bp 60.degree. C. AS:.sup.5' GCT TTT TCC TAC GTA
AGT ACT C.sup.3' 6. IL-10.sup.4 S:.sup.5' CCA GTT TTA CCT GGT AGA
AGT GAT G.sup.3' 324 pb 60.degree. C. AS:.sup.5'AAC TCA GAC CTG AGG
TCC TGG ATC TGT.sup.3' 1. Ma et al 1994 2. Chong et al, 1996. 3.
Benavides et al, 1995. 4. Reiner et al, 1994.
[0575] 1.2.17.5 Semi-Quantitative Competitive Reverse Transcriptase
Polymerase Chain Reaction (SQC-RT-PCR) determination of IL-12
[0576] The primers were individually diluted in TE to a
concentration equal to 132-133 pmol). PCR reactions, carried out
using the MJ Research Peltier Thermal Cycler (PTC-200), were
performed based on the protocol developed by Bost and Clements
(1995). The protocol was adjusted due to a different type of PCR
apparatus and to a different enzyme used (see Table 3).
[0577] The enzymes used in the SQC-RT-PCR during the exploratory
phase were Dynazyme (Recombinant Taq Polymerase). Amplitaq Gold
(Taq polymerase) was used in the final PCR experiments. Amplitaq
Gold is a more sensitive and heat-stable enzyme-antibody complex,
which is activated at temperatures above 90.degree. C. The PCR
conditions for using these two enzymes differ.
[0578] For Dynazyme, a 3 min Hot Start (3 min at 96.degree. C.
followed by 1 min at 80.degree. C.) was required before the enzyme
was introduced into the reaction mixture. After the enzyme was
added, three cycles consisting of:
[0579] 45 sec at 94.degree. C.
[0580] followed by 75 sec at 58.degree. C. and
[0581] 105 sec at 72.degree. C.
[0582] were run to initiate the synthesis of the second cDNA
strands.
[0583] The subsequent amplification cycle consisted of the
following shorter steps:
[0584] 35 sec at 94.degree. C.,
[0585] 45 sec at 58.degree. C. and
[0586] 75 sec at 72.degree. C.
[0587] This cycle was repeated 29 times.
[0588] Because Amplitaq Gold enzyme is heat stable, the enzyme was
added before the PCR cycling was initiated by an incubation step
for 10 min at 94.degree. C. At this temperature, the enzyme became
activated. The rest of the cycling profile remained the same as for
the Dynazyme enzyme.
[0589] The optimum concentration of MgCl.sub.2 and primer was
determined for each enzyme in a series of experiments.
[0590] Each PCR reaction nix consisted of 2 mM MgCl.sub.2 (for
Dynazyme) or 1,5 mM MgCl.sub.2 (for Amplitaq Gold), 0,2 mM dNTP,
PCR buffer supplied with the enzyme and 2U polymerase enzyme, 500
ng of each primer and plasmid DNA was added to the mixture. The
final reaction mixture volume was made up to 50 .mu.l with sterile
deionised water.
[0591] A first QC-RT-PCR approach protocol was described by Bost
and Clements (1995) for interleukin 12. For the QC-RT-PCR both the
RT mix and the IL-12 p40 plasmid DNA were added to the reaction
mixture. The RT-mix (for the first strand cDNA) had a total volume
of 20 .mu.l. This volume was divided into different percentages eg.
60%; 20%; 6%; 3% etc, according to the protocol of Bost and
Clements (1995). A constant concentration of plasmid DNA was added
to the PCR reaction mix.
[0592] A different approach described by Chong, Bost and Clements
(1996)was subsequently applied for optimization of PCR conditions.
According to this protocol, the RT-mix was constant at 20% (4 .mu.l
of RT-mix were used), and different concentrations of the plasmid
DNA were added. Plasmid was added in the following dilution range:
0.5, 0.25, 0.125, 0.062, 0.032, 0.016 and 0.008 pg.
[0593] The final PCR product was visualised on a 2% agarose gel
containing 0.5 .mu.g/ml ethidium bromide. It was found that the
digested plasmid gave better results than the undigested plasmid
due to better denaturation of the plasmid material. The digestion
was performed with the enzyme Xba I at 37.degree. C. for 3
hours.
[0594] 1.2.17.6 .beta.-actin PCR:
[0595] The .beta.-actin PCR was performed as an indication of the
amount of intact mRNA. Amplitaq Gold was used as DNA polymerase.
The same PCR cycling protocol as that of IL-12 p40 was used, except
for the annealing temperature for the .beta.-actin primers which
was 59.degree. C. instead of 58.degree. C. The elongation and
amplification cycles were the same as that of IL-12 PCR.
[0596] The sequences of the primers used are given in Table 3.
1.3 Results and Discussion
[0597] 1.3.1 The Influence of the Modified Method of Purification
on Yield and Purity of Mycolic Acids
[0598] By applying modifications to the previously patented
purification procedure (SA Patent Applications No 9511464 and
96/1412), i.e., by using NaCl as described under 1.2.8 and 1.2.9,
larger amounts of the extracted mycolic acids could be purified in
a single run of countercurrent separation, without impairing the
degree of their purity. This is illustrated by the results
summarized in Table 4 as well as in FIGS. 2a and 2b, for mycolic
acids originating from M. tuberculosis and M. vaccae, respectively.
Although a yield of approximately 10% m/m of the purified mycolic
acids was previously reported, it was subsequently established that
yields of pure mycolic acids using either method, varied between 3
and 10% m/m depending on the particular batch of bacteria used for
extraction.
8TABLE 4 Yield and purity of the mycolic acids originating from M.
tuberculosis, purified using the improved method Parameter Original
method Method with NaCl Loaded mass of mycolic acids-crude 31,1 mg
3 760 mg extract Mass of countercurrent-purified mycolic 3,5 mg 218
mg acids Equilibration time 40 min 5 min Number of cycles 24 30
Duration of the run 18 hours 3,5 hours Yield 5,3%-10% 5,8%-7,8%
[0599] 1.3.2 Structural Analysis of Mycolic Acids Originating from
M. tuberculosis, Using Infra-Red Spectroscopy
[0600] In order to evaluate the influence of the process of
saponification, freezing and storage on the conformation of
purified mycolic acids, the infra-red spectra of a number of
samples were analyzed.
[0601] The infra-red spectrum of countercurrent-purified mycolic
acids, originating from M. tuberculosis, prior to saponification is
presented in FIG. 3.
[0602] The spectrum in FIG. 3 provides evidence that mycolic acids
after countercurrent purification exist in the methylester form.
The absence of a broad absorption band spanning the 3000-2000
cm.sup.-1 frequency range indicates that there are no free
carboxylic acids. The intense narrow band at 2800-2950 cm.sup.-1
indicates aliphatic nature of the compound. In addition, the narrow
band of absorption at 1750 cm.sup.-1 indicates the presence of an
ester form.
[0603] The infra-red spectrum in FIG. 4 shows the pattern observed
after resaponification of the countercurrent-purified mycolic
acids. The anticipated presence of free carboxylic acid groups
could not be confirmed, which suggests that inter- or
intra-molecular interaction of the carboxylic acids with other
functional groups took place.
[0604] FIG. 4 also shows that this inter- or intra-molecular
rearrangement of the carboxylic acids restricts the degree of
aliphatic breathing (a decreased absorption in the 1720-1500
cm.sup.-1 range) in comparison to that of the methyl ester form
(FIG. 3), indicating a conformationally rigid structure. This would
imply a significant structural alteration caused by the process of
resaponification.
[0605] The carboxylic acids rearrangement over time did not
manifest as changes observed in the infra-red spectra. In addition,
the influence of freezing at -70.degree. C. and storage at
10.degree. C. on the configuration of mycolic acids was also
investigated. The respective infra-red spectra are presented in
FIGS. 5 and 6.
[0606] Prior to freezing at -70.degree. C. and storage at
10.degree. C., the samples of mycolic acids were freshly
resaponified. Freezing at -70.degree. C. appears to loosen up the
structure of mycolic acids and leads to the increase of aliphatic
breathing and "out of plane" bending within the molecules. This is
probably due to the decrease in the strength of van der Waals'
forces caused by the withdrawal of water as ice crystals.
[0607] 1.3.3 Stability of Mycolic Acids
[0608] Stability of mycolic acids was investigated by maintaining
equal aliquots of samples of countercurrent-purified mycolic acids
in the methylester form as well as saponified mycolic acids,
originating from the same batch, at -20.degree. C., 4.degree. C.
and 25.degree. C. for 12 months in either the dry form,
precipitated under acetone, or dissolved in chloroform.
[0609] The results obtained after 12 months are summarized in FIG.
7 for mycolic acids stored in the dry state. Similar results were
obtained for mycolic acids stored in the acetone-precipitated and
chloroform-dissolved state, but with higher variance.
[0610] On the basis of the HPLC analysis of the various samples of
mycolic acids stored at -20.degree. C., 4.degree. C. and 25.degree.
C., it was concluded that mycolic acids in either methylester or
saponified form were stable for at least 12 months. The apparent
gradual increase in absorptivity could be an artifact of
calibrating the new internal standard with old samples of mycolic
acids but is not due to a chemical process, which would have
manifested itself by different values for different temperatures of
storage.
[0611] 1.3.4 Immunoregulatory Properties of Countercurrent-Purified
Mycolic Acids
[0612] Investigations of the immunoregulatory properties of
countercurrent-purified mycolic acids were centred on two main
aspects, i.e.:
[0613] i) their ability to extend the survival of the M.
tuberculosis-infected mice; and
[0614] ii) their cytokine profile in various organs over short and
long term with or without concomitant infection with M.
tuberculosis.
[0615] These investigations were based on the following
experiments:
[0616] i) The investigation into the influence of pre- and
post-treatment of the experimental mice with resaponified mycolic
acids on their survival after the infection with M.
tuberculosis;
[0617] ii) The immunoregulatory effect of mycolic acids on the
expression of interleukin 4 (IL-4), interleukin 10 (IL-10),
interleukin 12 (IL-12), interferon .gamma. (IFN-.gamma.), tumour
necrosis factor .alpha. (TNF-.alpha.) and transforming growth
factor .beta. (TGF-.beta.) in the lungs of M. tuberculosis-infected
and non-infected experimental animals.
[0618] Before the results obtained in the course of this
investigation are presented and discussed, a number of technical
aspects having a direct influence on the outcome of the experiments
as well as on the repeatability of various immunoregulatory
experiments is listed and briefly discussed below:
[0619] 1. The infection with M. tuberculosis was introduced not by
inhalation but by intravenous injections. Although the inhalation
is a more natural method of infection, it is very difficult to
control and consequently to quantify the number of mycobacterial
cells introduced. The intravenous injections permitted a more
accurate introduction of the intended dose of M. tuberculosis.
[0620] 2. The introduction of the mycolic acids-mouse serum
conjugates to the experimental mice was likewise done by
intravenous injections. This method is however restricted by
procedural difficulties. The difficulties are associated with the
introduction of a thick suspension in relatively large volumes into
a vein of the mouse tail. Not infrequently this procedure leads to
a leakage of the introduced material.
[0621] 3. The preparation of mycolic acids-mouse serum conjugate
involves a random adsorption process which normally does not result
in a uniform distribution of mycolic acids over the surface of
mouse serum protein.
[0622] 4. The actual number of live cells of Mycobacterium in the
introduced dose is very difficult to establish and can differ from
the intended number of bacterial cells. The number of viable
bacteria is usually determined on the basis of the number of
colonies formed when a suspension of the bacteria to be enumerated,
appropriately diluted, is spread on the surface of a solid growth
medium and incubated. Ideally, such a suspension should consist of
single bacterial cells. The living bacteria, in the presence of
required nutrients will multiply, and, within a short period of
time (24-48 hours), each living cell should give rise to a single
colony. However, in the case of bacteria which form long chains or
clusters of cells, or which branch and do not separate easily
during the preparation of the suspension, a single colony will
frequently originate from a cluster of living cells. In such cases,
the number of colonies formed is lower than the actual number of
living cells in the enumerated suspension, and is denoted by the
term "colony forming units" (cfu)" rather than the viable number of
cells.
[0623] Mycobacterium cells during multiplication tend to branch and
this in turn leads to the formation of cell clusters. Such clusters
are difficult to disrupt and convert to a unicellular suspension,
despite attempts aimed at homogenising the suspensions. When
introduced on to the surface of solid media such clusters,
comprising a number of cells will, form colonies. Therefore, an
underestimation of the number of viable cells is usually
obtained.
[0624] 5. Variations in the stability of individual interleukin
mRNA in various organs (discussed in the Background to the
Invention, section 3.2) constitute another source of inaccuracies
in the determination of cytokine profiles.
[0625] 6. The mouse serum, used as a carrier of mycolic acids in
some of the experiments, was found not to be a neutral molecule,
but to possess non-specific immunoregulatory activity. Therefore,
the protective properties of the purified mycolic acids, reported
in these investigations, might have been distorted, and to a
certain degree even decreased, by the immunological activity of
mouse serum.
[0626] 7. Taking also into account low accuracy, typical of
densitometric quantification of electrophoresis bands, the cytokine
profiling reported in the work was undertaken as a tentative,
qualitative screening only.
[0627] Preliminary Experiments
[0628] The aim of these experiments was to determine the effects of
resaponified mycolic acids on healthy, uninfected mice and to
confirm the difference in the resistance of Balb/c and C57BI/6 mice
to the infection with M. tuberculosis. These experiments are
discussed in sections 1.3.4.1 and 1.3.4.2.
[0629] 1.3.4.1 The influence of treatment of the experimental mice
with resaponified mycolic acids originating from M.
tuberculosis
[0630] In order to investigate the effects of mycolic acids on the
experimental animals, Balb/c mice were injected with 250 .mu.g
mycolic acids/mouse serum conjugate. After a boost with 25 .mu.g
mycolic acids/mouse serum conjugate two weeks later, short (24 and
48 hours) and long term (14 days ) effects of this treatment were
measured. The cytokine profiles of IL-12, IFN-.gamma., TNF-.alpha.
and TGF-.beta. in the lungs of the mycolic acids-treated mice and
the control, serum-treated mice, were determined. No significant
response to the mycolic acids/mouse serum conjugate was observed in
the spleens of the experimental animals. The cytokine profiles of
IL-12, IFN-.gamma., TNF-.alpha. and TGF-.beta. in the lunas are
presented in FIG. 8.
[0631] As becomes apparent from FIG. 8, levels of the
pro-inflammatory cytokines, IL-12 and of IFN-.gamma., in the lungs
of mice treated with mycolic acids/mouse serum conjugate were
higher than those observed for the mice treated with serum only.
For the anti-inflammatory TGF-.beta., there was no significant
difference between the levels observed in the lungs of the mice
treated with mycolic acids/mouse serum conjugate and those treated
with mouse serum only. The results obtained for the
pro-inflammatory TNF-.alpha. show that this cytokine appears
responsive towards the treatment with mouse serum only.
[0632] 1.3.4.2 The resistance of Balb/c and C57BI/6 mice to the
infection with M. tuberculosis
[0633] The variable resistance of different mouse strains towards
the infection with M. tuberculosis has been reported not to be due
to genetic differences of the major histocompatibility (MHC) gene
complex. Rather, the genetic difference responsible for the extent
to which interleukin 12 (IL-12) is synthesized and secreted by
various strains of mice appeared to be one of the responsible
factors (Flynn et al., 1995). Expression of the inducible IL-12 is
lower in spleens from mice susceptible to infection with M.
tuberculosis compared to the more resistant mouse strains
(Kobayashi et al., 1996; Yoshida, Koide and Uchijima, 1995).
[0634] This conclusion was corroborated by our experiments using
inbred Balb/c susceptible to infection with M. tuberculosis and
C57BI/6, a more resistant mouse strain. Firstly, the survival
studies were carried out to establish the degree of resistance of
these mice. The results are presented in Table 5.
[0635] Secondly, IL-12 mRNA expression was compared in these two
strains of mice. Balb/c and C57BI/6 mice were infected with equal
doses of M. tuberculosis (approximately 10.sup.5 cells/mouse) and
sacrificed after 14 days. The results presented in FIG. 9 indicate
that the organs that responded in their IL-12 expression towards
the infection with M. tuberculosis were the liver and kidneys in
both strains and the lungs in only the susceptible Balb/c strain.
In the lungs of the more resistant C57BI/6 strain, the uninfected
organs already expressed a high level of IL-12, which did not
change upon infection.
[0636] By treatment with mycolic acids, IL-12 expression in the
lungs was enhanced (see FIG. 8) thus providing the protection
typical of the more resistant strain. It is important to realise
that the serum carrier on its own appeared to suppress the IL-12
levels in the animal organs (see point 6 in section 1.3.4).
9TABLE 5 Survival of Balb/c and C57BI/6 mice upon infection with M.
tuberculosis Survival (weeks) Bacterial dose Balb/c C57B1/6
10.sup.6 3.box-solid. 1.box-solid. 3.box-solid. 3.box-solid.
3.box-solid. 3.box-solid. 3.box-solid. 3.box-solid. 3.box-solid.
10.box-solid. 10.sup.5 3.box-solid. 15.box-solid. 3.box-solid.
18.box-solid. 11.smallcircle. 19.smallcircle. 11.smallcircle.
20.smallcircle. 11.smallcircle. 20.smallcircle. 10.sup.4
6.smallcircle. alive 7.box-solid. alive 8.box-solid. alive
19.box-solid. alive 21.box-solid. 22.smallcircle. .box-solid. =
Natural death due to tuberculosis .smallcircle. = Death by
euthanasia after severe TB symptoms have developed
[0637] Immunoregulatory Properties of Mycolic Acids Originating
from M. tuberculosis and M. vaccae
[0638] The aim of these experiments was to establish whether
mycolic acids originating from M. tuberculosis and M. vaccae could
offer a degree of protection against tuberculosis to the
experimental animals and whether such a protection was reflected in
the profiles of the selected cytokines.
[0639] The results obtained in the treatment with mycolic acids
preceding (pre-treatment) and following (post-treatment) the
infection with M. tuberculosis are presented in sections 1.3.4.3,
1.3.4.4 and 1.3.4.5.
[0640] 1.3.4.3 The influence of pre-treatment and post-treatment of
the experimental mice with resaponified mycolic acids originating
from M. tuberculosis on their survival after the infection with M.
tuberculosis
[0641] In order to establish whether the immunoregulatory
properties of mycolic acids could protect against or enhance the
susceptibility to the infection with M. tuberculosis, an experiment
described in section 1.2.16 was carried out. The expression of IL4,
IL-10, IL-12, IFN-.gamma., TNF-.alpha. and TGF-.beta. was measured
by PCR in the tissues originating from the lungs and spleens of the
experimental animals.
[0642] The results obtained in IR IV provide evidence that
pre-treatment of Balb/c mice with 25 .mu.g mycolic acids, one week
prior to the infection with M. tuberculosis, enhanced the survival
of the infected mice (FIG. 10). This protective effect was also
seen in C57BI/6 mice but was less pronounced (FIG. 11). The
introduction of mycolic acids to Balb/c mice three weeks after
infection with M. tuberculosis, did not lead to protection (FIG.
12). At double the dose, however, a degree of protection was
observed illustrating the potential of mycolic acids also as an
anti-tuberculosis agent (FIG. 12). Protection was also observed in
M. tuberculosis-infected C57BI/6 mice upon post-treatment with
mycolic acids three weeks after the infection (FIG. 13).
[0643] In two more experiments (Nos IR III and IR VII), mycolic
acids induced protection, while in another experiment (IR V) less
significant differences in survival were observed between treated
and non-treated animals (results not shown). In the latter case,
the duration of survival before the first death occurred was
considerably longer, suggesting that protection was provided
especially when relatively large doses of pathogenic bacteria were
administered. In IR V however, macroscopic observation of the
degree of tubercle formation in the infected lungs showed a degree
of protection in the mycolic acids-treated animals seven weeks
after the infection, with no evidence for protection in other
organs (Table 6). It therefore appears probable that at subacute
doses of the pathogen, a short term protection of the lung by
mycolic acids pre-treatment could be rendered redundant due to the
progression of the infection in the other organs.
[0644] The assessment of pathological changes in the organs
dissected from the mice is presented in Table 6.
10TABLE 6 Assessment of pathological changes in the organs
dissected from mice of immunoregulatory experiment No V (IR V) 1st
Organ extraction 2nd Organ extraction 3rd Organ extraction (5
weeks) (7 weeks) (11 weeks) Group M Spleen Lungs Liver M Spleen
Lungs Liver M Spleen Lungs Liver -TB infection 1 norm norm norm 1
norm norm norm 1 -- -- -- 2 norm norm norm 2 norm norm norm 2 -- --
-- 3 norm norm norm 3 norm norm norm 3 -- -- -- -TB infection 1
> ++ norm 1 >> + red 1 >> +++ norm 2 > ++ norm 2
>>> ++ red 2 > +++ red 3 > ++ red 3 >>> +++
red 3 >> +++ norm MA tb pre-treated 1 > ++ red 1 norm +
norm 1 > ++ norm 2 > +++ red 2 > + norm 2 > ++ norm 3
> +++ red 3 > + norm 3 > ++ norm MA vaccae pre-treated 1
> ++ norm 1 > + red 1 > +++ norm 2 > ++ red 2 > +
red 2 > +++ norm 3 > +++ norm 3 >> + norm 3 >> ++
red Serum pre-treated 1 > ++ norm 1 >> +++ norm 1 > ++
norm 2 > +++ red 2 >>> +++ norm 2 >> ++ norm 3
> ++ red 3 > ++ norm 3 > ++ red MA tb post-treated 1 >
+++ norm 1 > + norm 1 > ++ norm 2 > ++ norm 2 >> ++
norm 2 > +++ norm 3 > ++ norm 3 >>> +++ norm 3 >
++ norm MA vaccae post-treated 1 > ++ norm 1 >> ++ norm 1
>> ++ red 2 > +++ norm 2 > +++ norm 2 >> +++ red
3 > ++ norm 3 > ++ norm 3 >> ++ norm Serum post-treated
1 > ++ norm 1 norm +++ norm 1 > +++ red 2 > + norm 2 >
++ red 2 > +++ norm 3 > ++ norm 3 > +++ norm 3 > +++
norm Key: Spleen: the degree of enlargement was compared to the
negative control mice (group 1): (>)--moderate (1.times. bigger)
(>>)--much (2.times. bigger) (>>>)--extreme
(3.times. bigger) Lungs: lesions started with grey spots. The next
stage is characterised by the formation of white nodules around the
grey spots. The different stages are indicated by "+", "++" and
"+++" or "normal" if no lesions were visible. Liver: colour of
liver was noted "normal" if light brown, or "red". MA tb--mycolic
acids originating from M. tuberculosis MA vaccae--mycolic acids
originating from M. vaccae.
[0645] 1.3.4.4 The influence of pre-treatment and post-treatment of
the experimental mice with resaponified mycolic acids originating
from M. tuberculosis on cytokine profiles in the lungs
[0646] Lungs used in the cytokine determinations were removed from
mice five weeks after the infection with M. tuberculosis.
[0647] 1.3.4.4.1 Pre-treatment with mycolic acids and its effect on
IL-12 in the lungs
[0648] Interleukin 12 was determined in the lungs because:
[0649] i) IL-12 is mainly expressed by macrophages, which are
abundant in the lungs;
[0650] ii) it is known to play a role as a pro-inflammatory
cytokine in protection against tuberculosis;
[0651] iii) its expression in mice has been found to be enhanced by
the introduction of mycolic acids (see section 1.3.4.1).
[0652] Three doses of mycolic acids, i.e., 12,5 .mu.g, 25 .mu.g and
50 .mu.g were used in IR IV for pre-treatment. The results
presented in FIG. 14 indicate that mycolic acids enhanced IL-12
expression in the lungs up to an optimum dose (25 .mu.g), after
which expression was suppressed. This correlated with the
protection that was induced by mycolic acids pre-treatment (FIG.
15). Protection by mycolic acids as well as concomitantly enhanced
expression of IL-12 was confirmed in IR V (Table 6).
[0653] 1.3.4.4.2 Pre-treatment with mycolic acids and its effect on
IFN-.gamma. in the lungs
[0654] Interleukin 12 is known to exert some of its
immunoregulatory properties through the stimulation of IFN-.gamma.,
which then provides protection against tuberculosis infection. In
order to determine whether this correlation held true for the
protection provided by mycolic acids, the degree of expression of
IFN-.gamma. was determined in the lungs, five weeks after the
infection with M. tuberculosis.
[0655] The results presented in FIG. 16 do not clearly support a
model in which IFN-.gamma. is the cytokine stimulated by IL-12 to
exert a protective effect in animals against tuberculosis. This
also applies to the measurements of TNF-.alpha. (results not
shown). In a repeat experiment (IR V, as shown in FIG. 17) the
results presented in FIG. 16 could not be confirmed for both
IFN-.gamma. and TNF-.alpha..
[0656] It was concluded that the semi-quantitative PCR is not
sufficiently reliable in providing quantitative data on subtle
differences between IFN-.gamma. and TFN-.alpha. expressions of
mycolic acids in treated and untreated mice. However, it is
adequate to show qualitatively that IFN-.gamma. and TNF-.alpha. are
expressed upon infection with M. tuberculosis. The data do not
exclude the possibility that IFN-.gamma. and possibly TNF-.alpha.
may play effector roles in response to the increased IL-12
expression induced by mycolic acids.
[0657] 1.3.4.4.3 Pre-treatment with mycolic acids and its effect on
TGF-.beta. in the lungs
[0658] The correlation that was found between the protective effect
of mycolic acids and its influence on IL-12 expression indicated
that protection was brought about by a pro-inflammatory mechanism.
The expression in response to pre-treatment with mycolic acids of
two other pro-inflammatory cytokines, IFN-.gamma. and TNF-.alpha.
(results not shown), did not yield satisfactory quantitative
results, but at least did not argue against a pro-inflammatory
effect induced by mycolic acids. TGF-.beta. is an anti-inflammatory
cytokine expressed in macrophages which might respond to mycolic
acids.
[0659] The results in FIG. 18 indicate that the levels of
expression of the anti-inflammatory cytokine TGF-.beta. were
already high in the non-infected control mice. This is the only one
of the four cytokines investigated that gave this result. It would
not be unusual for an anti-inflammatory cytokine in the lungs to
maintain a high level of expression under normal conditions when
the inflammatory response could do harm to normal, uninfected lung
tissue. After the infection with M. tuberculosis, the levels of
TGF-.beta. expression were not significantly altered, although a
broad spread of measured values was obtained, indicating the
possibility of unstable mRNA structure. Introduction of homologous
serum into the animals significantly lowered the expression of
TGF-.beta., while there was a tendency to restore this level by
addition of mycolic acids adsorbed onto the serum. Due to the
spread of the measurements for every point of data, a definite
conclusion based on a fine resolution between experimental groups
of animals could not be made.
[0660] These measurements of expression of TGF-.beta. illustrate
the complicating role that homologous serum played in defining the
immunoregulatory role of mycolic acids. Ignoring the effect of the
serum carrier, the data might have suggested a reduced expression
of TGF-.beta. under the influence of mycolic acids, as FIG. 19
illustrates.
[0661] 1.3.4.4.4 Post-infection treatment with mycolic acids and
its effect on IL-12, IFN-.gamma. and TGF-.beta. in the lungs
[0662] Balb/c mice infected with M. tuberculosis and treated with
mycolic acids three weeks after the infection, showed protection
against tuberculosis (FIG. 12). This was also observed in C57BI/6
mice (FIG. 13). The measured values for expression of IL-12 in the
lungs of Balb/c mice post-treated with mycolic acids (FIG. 20) do
not support a model in which this cytokine mediates the protection,
as was found in pre-treatment. Although the IFN-.gamma. expression
levels correlated positively with IL-12 expression levels (FIG.
21), they did not correlate with increased survival. FIG. 22
indicates a decreased TGF-.beta. expression in the lungs of Balb/c
mice that were treated with 48 .mu.g of mycolic acids after the
infection with M. tuberculosis. This decreased TGF-.beta.
expression correlated with an increased survival.
[0663] These observations indicate a different model of protection
provided by mycolic aids as a potential therapeutic agent against
infection with M. tuberculosis, i.e., the down-regulation of the
anti-inflammatory process in the lungs.
[0664] 1.3.4.5 The influence of pre-treatment and post-treatment of
the experimental mice with resaponified mycolic acids originating
from M. tuberculosis on cytokine profiles in the spleens
[0665] Spleens used in the cytokine determinations were removed
from mice five weeks after the infection with M. tuberculosis.
[0666] The Th1 response has been previously indicated as the
protective mode of Th cells to provide protection against
tuberculosis. Because infection with M. tuberculosis was, in our
experiments, routed systemically through the tail vein, the spleen
was regarded as a prime lymphoid organ from which the Th1/Th2 bias
could be determined, using IFN-.gamma. as the indicator for a Th1
and IL-4 as the indicator for a Th2 response. These cytokines would
have acted antagonistically, if the spleen became biased in one of
the two modes upon immune challenge by M. tuberculosis. However,
from the results presented in FIG. 23 it is evident that the
infection with M. tuberculosis induced the expression of both
cytokines and neither the expression of IFN-.gamma. nor of IL-4 was
significantly altered by the pre-treatment or post-treatment with
mycolic acids.
[0667] These results suggest that:
[0668] i) the spleen does not appear to be the organ where the role
of mycolic acids as a protector against tuberculosis is
decided;
[0669] ii) the T cells do not appear to play the decisive role in
providing protection against tuberculosis upon the administration
of mycolic acids.
[0670] 1.3.4.6 The influence of pre-treatment and post-treatment of
the experimental mice with resaponified mycolic acids originating
from M. vaccae after the infection with M. tuberculosis
[0671] The immunoregulatory effects of mycolic acids were found to
be present irrespective of the source species of Mycobacterium from
which they were obtained. Mycolic acids extracted from M. vaccae,
after the countercurrent purification, stimulated the expression of
interleukin 12 in Balb/c mice (FIG. 24) and induced protection
against tuberculosis in the lungs of the experimental animals
(Table 6). These results confirm the effects observed in the mice
pre- and post-treated with mycolic acids originating from M.
tuberculosis, described in sections 1.3.4.3 and 1.3.4.5.
[0672] 1.3.5 Assessment of the Significance of the Results
[0673] The results obtained in the above experiments indicate that
mycolic acids can protect mice against infection with M.
tuberculosis, particularly when administered before the
infection/onset of the disease. The kidneys, liver and lungs in
mice were found to respond to mycolic acids even without the
infection with M. tuberculosis (FIG. 8).
[0674] It should be stressed that the lungs from the mycolic
acids-pre-treated animals were the only organ in which a
significantly reduced tubercle formation could be observed upon
macroscopic post-mortem assessment (Table 6) and were also the only
organ that responded differently to the infection with M.
tuberculosis in respect of IL-12 expression. It was, therefore
concluded, that the protection provided by mycolic acids manifested
itself mainly in the lungs. Preliminary evidence from IR V
indicated that mycolic acids from other species of Mycobacterium
(e.g. M. vaccae) could also induce immune effects in the lungs of
mice infected with tuberculosis (FIG. 24).
[0675] The cytokine profiling of the lungs and spleen five weeks
after the infection with M. tuberculosis, with or without pre- or
post-treatment with mycolic acids, confirmed the observation that
the lungs were the responsive organs to the antigenic challenge
with mycolic acids. Although cytokine expression in the spleen was
induced upon the infection with M. tuberculosis, it was not
significantly altered by the pre- or post-treatment with mycolic
acids. No bias towards either Th1 or Th2 mode of immune response
was evident from the measurements of the IFN-.gamma. and IL-4
expression levels in mice infected with tuberculosis, treated or
untreated with mycolic acids (FIG. 23).
[0676] The results of IL-12, IFN-.gamma. and TGF-.beta.
measurements in the lungs of the experimental animals support the
view that a pro-inflammatory mechanism of protection against
tuberculosis was elicited by pretreatment with mycolic acids.
Interleukin 12 expression correlated positively with the survival
of the M. tuberculosis-infected mice, while IFN-.gamma. (although
induced) and TNF-.alpha., could not be determined with sufficient
accuracy to clearly demonstrate its role in the protection. The
TGF-.beta. levels in the organs of the experimental animals
remained largely unaltered.
[0677] During the post-infection treatment with mycolic acids, the
levels of expression of IL-12 positively correlated with those of
IFN-.gamma.. No correlation with the protection against
tuberculosis by mycolic acids was observed. Rather, a
down-regulation of TGF-.beta. appeared to be a possible mechanism
of protective inflammation in the lungs. This mechanism might
explain the lower efficiency of the post-infection therapy with
mycolic acids.
[0678] Overall, the mechanistic model of mycolic acids-induced
protection against tuberculosis, supported by the experimental
evidence provided above, allows for the prediction of a much
enhanced protection offered by mycolic acids against the disease,
when the infection with M. tuberculosis occurs by the normal route,
i.e., by inhalation into the lungs. It appears possible that the
administration of mycolic acids directly into the lungs of
tuberculosis-infected individuals, via inhaled vapours generated by
a nebuliser could greatly reduce the incidence of infection with M.
tuberculosis and also aid towards recovery from the disease.
[0679] The above findings can be extrapolated to human beings, and
it can be expected that the response in humans towards the
treatment with mycolic acids should be better. This assumption is
based on the fact that humans possess mycolic acids presenting
molecules, CD1b, expressed on their antigen presenting cells, the
homologue of which has not yet been identified in mice.
[0680] Positive evidence was found for short term prevention or
therapy of tuberculosis by mycolic acids in rats and mice. The
results obtained would suggest that Th cells did not participate in
the immune response upon challenge with mycolic acids either before
or after the infection with M. tuberculosis. However, as humans
possess CD1b molecules mycolic acids may have a role in human
vaccines, i.e., for using them to provide long term immune memory
against infection with M. tuberculosis.
[0681] The experimental evidence supports a role of cells of innate
immunity, such as macrophages and natural killer cells, in
responding towards mycolic acids administration in a manner that
decreases the pathogenic effects induced in the lungs by M.
tuberculosis.
EXAMPLE 1a
[0682] Protection against tuberculosis in mice provided by the
administration of purified mycolic acids. Experiments involving
animals infected with M. tuberculosis by intra-nasal
administration
[0683] 1a. 1 Rationale:
[0684] Dissemination of M. tuberculosis occurs in nature by
inhalation of airborne droplets containing a few bacilli,
expectorated by infected individuals with "open" pulmonary disease
i.e., by smear-positive patients. Such patients have very high
numbers of bacilli in their sputum (at least 5 000 bacilli/ml)
which are visible on standard microscopical examination. As the
lungs are the most frequent portal of entry, they are the most
frequently affected organ in man, although virtually any organ or
system of the body may be invaded by M. tuberculosis.
[0685] In order to imitate the natural way of entry of mycobacteria
into the experimental animals, an additional experiment was carried
out in which the animals were infected by the intra-nasal
administration of mycobacteria, while the route of administration
of mycobacterial mycolic acids remained intravenous.
[0686] The results obtained from the treatment of mice with mycolic
acids in the absence of M. tuberculosis infection (Example 1),
indicated the lungs as the site of mycolic acids-induced IL-12
expression. This suggested that mycolic acids could be especially
effective as prophylactant if tuberculosis infection was given
through the intra-nasal route, targeting the lungs directly.
[0687] The survival of mycolic acids pre-treated mice Balb/c,
infected with M. tuberculosis either intravenously or
intra-nasally, was therefore tested.
[0688] 1a. 2 Materials
[0689] The materials used in the Example 1a, i.e. the bacterial
culture, media, reagents, the experimental animals and plasticware
were identical to those described in Example 1.
[0690] Methods
[0691] The methods used in the experimental work carried out to
perform Example 1a i.e., the cultivation of M. tuberculosis, viable
and total bacterial counts, the preparation, extraction,
purification and analysis of mycolic acids, the preparation of
mycolic acids-mouse serum conjugates, the preparation of bacterial
suspensions for the induction of tuberculosis in mice as well as
the methods used in handling experimental animals and the
conditions of their maintenance, were identical to the
corresponding methods described in Example 1.
[0692] However, the manner in which the suspensions of M.
tuberculosis cells were introduced into Balb/c mice was different
and is described in detail below.
[0693] 1a 2.1 Introduction of the M. tuberculosis H37 Rv
Suspensions
[0694] The cells of M. tuberculosis H37 Rv, harvested from LJ
slants, were suspended in the diluting buffer (0.01% v/v Tween 80
in 0,9% m/v NaCl) and homogenized. After centrifugation in a
Beckman J-6 centrifuge for 20 min at 1 580 g, the cells were washed
with a sterile solution of 0,9% m/v of NaCl and adjusted to a
concentration corresponding to a McFarland standard No. 4. After
the confirmation of the total direct bacterial count, carried out
on an autoclaved suspension in a Neubauer counting chamber, the
suspension was further diluted in the sterile solution of 0,9% NaCl
to obtain concentrations of M. tuberculosis corresponding to
10.sup.3, 10.sup.4 and 10.sup.5 cells/ml.
[0695] The viable counts of the mycobacteria in the suspensions
were confirmed by plating 100 .mu.l aliquots of the relevant
dilutions onto Middlebrook 7H-10 agar medium, incubating the plates
at 37.degree. C. for two weeks and counting the number of colony
forming units (CFU).
[0696] The introduction of the M. tuberculosis's supensions was
performed in a biosafety cabinet class III in the PIII facilities
at the Tuberculosis Institute of Medical Research Council in
Pretoria.
[0697] In the intra-nasally infected group of mice, the bacterial
suspensions prepared in sterile saline were introduced into the
nostrils of mice anaesthetized with 5% diethylether, in aliquots of
60 .mu.l per animal. The suspensions were released dropwise into
the nostrils using autoclaved pipette tips, while the animals were
in dorsal recumbence. The intravenous administration of
mycobacteria was performed as described in Example 1.
[0698] Control animals received an equivalent volume sterile
saline, i.e., 60 .mu.l introduced intra-nasally or 100 .mu.l
administered intravenously.
[0699] The administration of the mycolic acids conjugates was
carried out for both types of the infection with M. tuberculosis
via the intravenous route, after mice were heated for 5 min in a
heating box to effect vasodilation of the tail veins. The mycolic
acids-mouse serum conjugate was administered intravenously by
introducing 5 .mu.g or 25 .mu.g mycolic acids in 100 .mu.l mouse
serum per mouse.
[0700] Control animals received 100 .mu.l of mouse serum introduced
in the same manner.
[0701] Balb/c mice were divided into nine groups and treated as
indicated in Table 6a.
11TABLE 6a Experimental set-up for immunoregulatory experiment No
1a Number of mice Group No. Group No/treatment per group 1 1. Serum
10 Saline 2 2. Serum 15 M tb.sup.1) intra nasally 3 3. 5 .mu.g MA
iv.sup.2) 15 M tb intra-nasally 4 4. 25 .mu.g MA iv 15 M ib
intra-nasally 5 5. 25 .mu.g MA iv 15 Saline iv 6 6. Serum iv 10 M
tb iv 7 7. 5 .mu.g MA iv 10 M tb iv 8 8. 25 .mu.g MA iv 10 M tb iv
9 9. Control 15 No treatment .sup.1)M. tb - M. tuberculosis
.sup.2)iv - intravenous administration
[0702] 1a. 3 Results
[0703] All the mice in the intravenously infected pre-treatment and
control groups were dead by week 36, with a very small difference
in the average survival time (Table 6b). This corroborates the
previous results (Example 1) where an increase in time of survival
was seen in the mycolic acids-pre-treated group, but all the mice
eventually died.
[0704] In contrast, 8 out of 9 mice (which corresponds to a 89%
survival) of the intra-nasally infected mice pre-treated with
mycolic acids-serum conjugate survived for more than 36 weeks,
compared to the control mice where only 4 mice survived, which
constituted 44% survival (Table 6b). After 36 weeks, the,
intra-nasally infected, mycolic acids pre-treated mice appeared to
have the tuberculosis infection under control, while the control
animals that were not pre-treated with mycolic acids were either
dead, or sick at the end of week 36.
[0705] A single mouse in the mycolic acids pre-treatment group
died, but the cause of death could not be established, because it
was eaten by other mice in the cage. The remaining mice in the
mycolic acids pre-treated group appeared healthy at week 36. The
subsequent culturing of the spleens and lungs of the surviving
animals confirmed the presence of M. tuberculosis infection in both
organs in all the mice, including those of the mycolic acids
pre-treated group.
12TABLE 6b The survival of Balb/c mice pre-treated with 25 .mu.g
mycolic acids: intra- nasal versus intravenous route of infection
with M. tuberculosis. Observation carried out over a period of 36
weeks. Intravenous infection Intra-nasal infection with 4 .times.
10.sup.4 cfu with 1 .times. 10.sup.5 cfu MA -pre- MA -pre- treated
Control treated Control Type of treatment mice mice mice mice
Average survival time 25.56 24.48 35.64 31.3 (in weeks) Number of
mice surviving 0/7 0/7 8/9 4/9 0% 0% 88% 44% MA--mycolic acids
cfu--colony forming units
[0706] 1a. 4 Discussion:
[0707] The normal route of infection with M. tuberculosis is by
inhalation into the lungs. While mycolic acids induced the
protective IL-12 and IFN-.gamma. cytokines mainly in the lungs, it
was predicted that the protection provided by mycolic acids as
prophylactant against tuberculosis would be much more pronounced
when M. tuberculosis infection was given by the intra-nasal, rather
than the intravenous route. This was confirmed (Example 1a),
showing that mycolic acids administration could sufficiently
stimulate the immune system of the experimental animals to enable
them to bring the infection with M. tuberculosis under control,
although it was unable to prevent the infection from establishing
itself in the lungs and spleen.
EXAMPLE 2
[0708] Protection against tuberculosis-induced arthritis in
rats
2. Materials
[0709] 2.1 Culture
[0710] Mycobacterium tuberculosis H37Rv ATCC 27294--a virulent
strain, originally isolated from an infected human lung, was used
in the experiments.
[0711] The culture was purchased in lyophilized form from the
American Type Culture Collection (ATCC), Maryland, USA.
[0712] M. tuberculosis H37Ra ATCC 27294--an avirulent strain, was
purchased in lyophilized form from Difco (Cat No: 3114-33-8).
[0713] 2.1.2 Media
[0714] 2.1.2.1 Growth media
[0715] The following media were used for the cultivation of M.
tuberculosis:
[0716] Lowenstein-Jensen (LJ) medium (slants) and
[0717] Middlebrook 7H10 agar (plates).
[0718] A detailed composition of the ingredients necessary for the
preparation of these media as well as the conditions recommended
for their sterilization, are given in the Laboratory Manual of
Tuberculosis Methods, Tuberculosis Research Institute of the SA
Medical Research Council (1980, Chapter 6, pp 83-105; Second
Edition, revised by E E Nel, H H Kleeberg and E M S Gatner).
[0719] The media were prepared by the National Tuberculosis
Institute of the Medical Research Council of South Africa, in
Pretoria.
[0720] 2.1.2.2 Media used for washing and diluting of
Mycobacteria
[0721] The harvested bacteria were washed in sterile 0,9% m/v NaCl
(Saarchem, Chemically Pure, RSA).
[0722] Medium used for the preparation of serial dilutions
preceding the determination of viable counts of M. tuberculosis was
prepared by dissolving Tween 80 (Merck, Chemically Pure) in 0,9%
m/v NaCl (Saarchem, Chemically Pure) to a concentration of 0,01%
v/v and distributing it in 9,0 ml aliquots into test-tubes. The
autoclaved media were stored at 4.degree. C.
[0723] 2.1.3 Reagents
[0724] 2.1.3.1 For the preparation of the reagents used for the
extraction, derivatization and High-Performance Liquid
Chromatography (HPLC) analysis of mycolic acids, HPLC Grade
methanol (BDH) and double-distilled deionized water were used.
[0725] Reagent A: 25% potassium hydroxide (Saarchem, Analytical
Grade) dissolved in methanol-water (1:1), i.e., 62,5 g potassium
hydroxide was dissolved in 125 ml water and 125 ml methanol (BDH,
HPLC Grade) was added.
[0726] Reagent B: Concentrated hydrochloric acid (Saarchem,
Analytical Grade) diluted 1:1 with water.
[0727] Reagent C: 2% potassium bicarbonate (BDH, Analytical Grade)
dissolved in methanol-water (1:1), 10 g potassium bicarbonate was
dissolved in 250 ml water and 250 ml methanol was added.
[0728] Reagent D: para-bromophenacylbromide dissolved in
acetonitrile and crown ether (Pierce Chemical Co, Cat. No 48891)
was dispensed in 500 .mu.l quantities into small amber-coloured
screw cap vials with Teflon-coated septa. The caps were tightened
and the vials were wrapped with Parafilm. Reagent D was stored at
4.degree. C.
[0729] Reagent E: Reagent E was prepared by mixing reagent B 1:1
with methanol.
[0730] HPLC Standard: High Molecular Weight Internal Standard
(C-100) from Ribi ImmunoChem Research Company, Cat No R-50. The
standard, 1 mg, was dissolved in 20 ml chloroform (BDH, HPLC Grade)
at 4.degree. C. and aliquots of 100 .mu.l were dispensed into 4 ml
amber WISP vials, dried, capped with Teflon-coated septa and stored
at 4.degree. C.
[0731] Chloroform (Saarchem, Analytical Grade, RSA)
[0732] Methylene chloride (BDH, UK, HPLC-Grade)
[0733] Reagents A, B, C and E were prepared fresh prior to
experiments, taking all the necessary safety precautions.
[0734] 2.1.3.2 The following reagents were used for the preliminary
purification of crude bacterial extracts ("funnel extraction") and
for the countercurrent purification of the extracted mycolic
acids:
[0735] Chloroform (Saarchem, Chemically Pure)
[0736] Methanol (Saarchem, Chemically Pure)
[0737] Acetone (Saarchem, Chemically Pure)
[0738] Sodium chloride (Saarchem, Analytical Grade)
[0739] Double-distilled deionized water was used for the
preparation of the required reagent concentrations, i.e.:
[0740] 39% v/v methanol
[0741] 42% v/v chloroform
[0742] 0,2 M NaCl
[0743] 2.1.3.3 Reagents used for the induction of adjuvant
arthritis:
[0744] Heat-killed and freeze-dried cells of M. tuberculosis H37Ra
(Difco, Cat No 3114-33-8), 100 mg, suspended in 10 ml of Freund's
Incomplete Adjuvant (FIA, Difco Cat No 0639) were used for the
induction of adjuvant arthritis in Lewis rats.
[0745] 2.1.3.4 Reagents used for the prevention of adjuvant
arthritis:
[0746] Freshly saponified mycolic acids (MA) originating from M.
tuberculosis H37Rv were suspended in FIA (10 mg/ml FIA) and diluted
with FIA to required concentrations, i.e.:
[0747] 0,1 mg MA/100 .mu.l, 0.3 mg MA/100 .mu.l and 1,0 mg MA/100
.mu.l.
[0748] 2.1.3.5 Reagents used in monitoring the production of
anti-mycolic acids antibodies:
[0749] Glycerol (Merck, Analytical Grade) was used for diluting
sera of the experimental animals.
[0750] ELISA Reagents:
[0751] Basic buffer--PBS buffer: 8,0 g NaCl, 0,2 g KCl, 0,2 g
KH.sub.2PO.sub.4 and 1,05 g Na.sub.2HPO.sub.4 per 1 l distilled
water, adjusted to pH 7,4.
[0752] Diluting buffer: 0,5% (m/v) casein in PBS buffer adjusted to
pH 7,4 was used for diluting of the experimental animals' sera
(mixed with glycerol 1:1) and for the preparation of suitable
dilutions of immunoreagents.
[0753] Blocking buffer: 0,5% (m/v) casein in PBS buffer adjusted to
pH 7,4 was used for blocking of ELISA plates.
[0754] Washing buffer: 0,5% (m/v) casein in PBS buffer adjusted to
pH 7,4 was used for washing of ELISA plates.
[0755] Coating antigen: unsaponified mycolic acids originating from
M. tuberculosis H37Rv, at a final concentration of 0,067
.mu.g/.mu.l. To prepare the coating antigen, 1 mg mycolic acids was
dissolved in 100 .mu.l chloroform and the solution introduced into
15,0 ml PBS buffer adjusted to pH 7,4. The solution was autoclaved
at 121.degree. C. for one hour.
[0756] Conjugates: Goat anti-rat antibody conjugated to peroxidase
(H+L chains), Cappel (Cat No 55770). Rabbit anti-human gamma chain
specific peroxidase conjugate (Sigma; A 8419).
[0757] Substrate: O-Phenylenediamine (Sigma; Cat No P-1526) and
hydrogen peroxide (BDH).
[0758] Substrate buffer: 0,1 M citrate buffer (0,1 M citric acids
and 0,1 M Tri sodium citrate), adjusted to pH 4,5.
[0759] 2.1.4 Experimental Animals
[0760] Six weeks old, female Lewis rats were purchased from Shaw's
farm, Blackthorn, Bicester, Oxon, England. This strain of rats is
susceptible to the induction of arthritis.
[0761] The animals were maintained at the Animal Facilities of the
Medical Research Council in Pretoria.
[0762] Feed and Water
[0763] Mice cubes, manufactured by Epol, South Africa and tap,
autoclaved water were provided ad libitum.
[0764] Sanitation:
[0765] Bronocide--manufactured by Essential Medicines (Pty) Ltd,
was used for sanitation purposes.
[0766] 2.1.5 Plasticware
[0767] The following plasticware was used:
[0768] Disposable Petri's dishes (Promex, RSA)
[0769] ELISA plates (Sterilin, UK)
[0770] Sterile, disposable 50 ml centrifuge tubes (Corning,
USA)
[0771] Disposable tips (Elkay, Denmark)
[0772] 96-well round bottom microplates (Nunc, Denmark)
2.2 Methods
[0773] The following methods were used in the experimental
work:
[0774] 2.2.1 Cultivation of the Bacterial Strains
[0775] The mycobacteria for the production of mycolic acids, i.e.,
M. tuberculosis H37Rv, were cultivated at 37.degree. C. using
Lowenstein-Jensen (LJ) medium slants and Middlebrook 7H-10 agar
medium plates.
[0776] The sterility of all the media was confirmed, before they
were used in the experiments by incubating them at 37.degree. C.
for 24 h.
[0777] For routine extraction of mycolic acids, approximately
4-week old cultures of M. tuberculosis grown on LJ slants or 2-week
old cultures of M. tuberculosis grown on Middlebrook 7H-10 agar
medium plates were used.
[0778] 2.2.2 Viable and Total Bacterial Counts
[0779] For the viable count determination, serial dilutions of the
harvested bacteria were suspended in the diluent medium (prepared
as specified under 2.1.2.2) to a density corresponding to a
McFarland standard 4 (approximately OD of 1,0; using a Beckman DU
65 spectrophotometer, at 486 nm). Tenfold serial dilutions were
prepared using 9 ml aliquots of the diluent medium. From the last
three dilutions corresponding to 10.sup.-3, 10.sup.-4 and 10.sup.-5
of the original suspension, aliquots of 0,1 ml (100 .mu.l) were
withdrawn and spread over the surface of Middlebrook 7H-10 plates.
The plates were incubated at 37.degree. C. and the developed
colonies counted after two to three weeks for M. tuberculosis and
after one week for the plates seeded with M. vaccae.
[0780] The direct total count was performed using a Neubauer
counting chamber and the autoclaved cultures of M. tuberculosis and
M. vaccae, originally adjusted to a density corresponding to a
McFarland standard 4 and suitably diluted with the diluent
medium.
[0781] Statistical analysis of the bacterial counts included the
mean values of bacterial counts and standard deviations.
[0782] 2.2.3 Preparation of Mycolic Acids from Bacterial
Samples
[0783] The preparation of bacterial samples comprised three
steps:
[0784] harvesting of the Mycobacterial cells;
[0785] saponification and
[0786] extraction of mycolic acids.
[0787] Glassware used for the harvesting, extraction,
derivatization and HPLC analyses of mycolic acids was washed in 2%
(v/v) Contrad (Merck), rinsed in water, followed by rinsing in
chloroform, water, technical Grade methanol, water and finally
rinsed in double distilled deionized water. The washed glassware
was dried in a warm air oven.
[0788] Harvesting was done by scraping the bacterial growth from
the surface of media slants or agar medium plates (using sterile
plastic loops) and by suspending them in Reagent A. Initial
bacterial suspensions were prepared in Reagent A, by vortexing the
harvested cells with sterile glass beads. Homogenous bacterial
suspensions were prepared using sterile tissue homogenizers. Prior
to saponification, the density of the bacterial suspensions was
adjusted to a density corresponding to a McFarland standard 4. This
density of bacteria corresponds to approximately
10-12.times.10.sup.8 colony forming units/ml.
[0789] The saponification, extraction and derivatization of mycolic
acids were carried out as described by Butler, Jost and Kilburn
(1991), with minor modifications and are described under the
relevant headings.
[0790] Saponification of the Mycobacteria in Reagent A was carried
out in an autoclave at 121.degree. C., for 30 min.
[0791] 2.2.4 Extraction of Mycolic Acids
[0792] The saponified samples were allowed to cool after
autoclaving. Into 2 ml samples containing crude extract, 1,5 ml
Reagent B was introduced. After vortexing, the pH of each sample
was checked and if necessary, adjusted to pH 1 with Reagent B.
[0793] Subsequently, 2,0 ml chloroform was added to each sample and
vortexed for 30 seconds. The layers were allowed to separate. The
bottom layers were removed with Pasteur pipettes, transferred to
amber WISP vials and evaporated to dryness at 85.degree. C. in a
heat block-evaporator under a stream of nitrogen. To neutralize
traces of acid carried over, 100 .mu.l of reagent C was added to
each sample and the fluid evaporated to dryness at 85.degree. C. in
the heat block-evaporator under a stream of nitrogen.
[0794] 2.2.5 Storage of the Crude Extracted Mycolic Acids
[0795] The material obtained from the large-scale extraction of
mycolic acids originating from M. tuberculosis H37Rv, the crude
bacterial extracts, was stored under acetone, at 4.degree. C. in 4
ml amber WISP vials. To prevent evaporation/drying and the exposure
to light, the caps of the WISP vials were covered with
Parafilm.
[0796] 2.2.6 Determination of Mycolic Acids Contents in Crude
Extracts
[0797] Extracted mycolic acids were derivatized as follows:
[0798] To a cooled sample of crude extract (approximately 10 .mu.g
in 2,0 ml Reagent A), an aliquot of 1,0 ml chloroform was
introduced, followed by the addition of 100 .mu.l of Reagent D
(derivatization reagent). The capped samples were vortexed for 30
seconds and heated for 20 minutes at 85.degree. C. in a heat
block-evaporator. Subsequently, the samples were cooled and 1,0 ml
of Reagent E added, The samples were vortexed for 30 seconds and
the layers allowed to separate. The bottom layers were removed with
Pasteur pipettes and transferred to WISP-vials. The vials were
placed in the heat block-evaporator and their contents evaporated
to dryness at 85.degree. C. using a stream of nitrogen.
[0799] The residues were resuspended in 0,212 g (which corresponds
to 160 .mu.l) methylene chloride, capped and vortexed. Each
reconstituted sample was introduced into a WISP vial containing 5
.mu.g of the HPLC internal standard (prepared as described under
2.1.3.1), filtered through a 0,22 .mu.m Millex GV4 filter with a
polyethylene housing into another amber-coloured WISP-vial. The
recapped vials were stored at 4.degree. C. until ready for HPLC
analysis.
[0800] 2.2.7 HPLC Analysis and Quantification of Mycolic Acids
[0801] Repeatability and accuracy of the pipette used for the
distribution of the HPLC standard was determined. The precision was
established to be +/-1% and it was confirmed prior to each
aliquoting of the internal standard.
[0802] For the HPLC analysis 10 .mu.l from each sample (maintained
on ice during handling), was analyzed. Control samples, i.e., 10
.mu.l of filtered methylene chloride, were run prior to each set of
samples analyzed. If a large number of samples was analyzed, in
order to validate the reliability of the HPLC apparatus, control
samples were run after every three or four test samples.
[0803] The reverse-phase HPLC analyses were carried out using a
Waters 600 E System Controller High Performance Liquid
Chromatography apparatus consisting of:
[0804] Microsep M741 Data Module;
[0805] Waters 712 WISP Autosampler;
[0806] Detector (Waters 486 Tunable Absorbance Detector);
[0807] Column: Nova-Pak C18 4 .mu.m 3,9.times.150 mm and an end
connector set for steel cartridge columns.
[0808] RKC Rex-C 4 Column Temperature regulator.
[0809] Running conditions were:
[0810] Mobile phase:
[0811] Solvent A: HPLC Grade methanol
[0812] Solvent B: HPLC Grade methylene chloride
[0813] Flow Rate: 2,5 ml/min
[0814] Column temperature: 30.degree. C.
[0815] The detector was set at 260 nm.
[0816] Prior to use, the solvents were sparged with Instrument
Grade helium. High Purity Nitrogen was used to control hydraulics
of the WISP vials autosampler.
[0817] The HPLC gradient initially comprised 98% (v/v) methanol
(Solvent A) and 2% (v/v) methylene chloride (Solvent B). The
gradient was increased linearly to 80% A and 20% B at one minute;
35% A and 65% B at ten minutes, held for 30 seconds and then
decreased over 10 seconds back to 98% A and 2% B. This ratio was
maintained for 4 minutes to allow for stabilization of the system
prior to injection of the next sample.
[0818] Mathematical quantification of mycolic acids was carried out
by comparing the combined peak areas of the tested samples to the
peak area of the introduced quantity of the High Molecular Weight
Internal HPLC Standard.
[0819] 2.2.8 Preliminary Purification of Crude Mycobacterial
Extracts
[0820] In order to shorten the time required for the countercurrent
purification of the crude mycobacterial extracts, an additional
preliminary extraction step was introduced. This step had a dual
purpose:
[0821] i) to remove unnecessary cellular components from the crude
extract prior to the countercurrent purification and
[0822] ii) to reduce soap fraction in the crude bacterial
extracts.
[0823] A portion of the crude extracted material (approximately 3
to 4 g) was suspended in a minimum volume of the lower phase
solvent (usually 100 ml), transferred into a separation funnel and
mixed with an equal volume of the upper phase solvent. The phases
were allowed to separate and the upper phase was removed and stored
at 4.degree. C. Into the remaining lower phase an equal volume of
the upper phase solvent was again introduced and the process of the
phase separation was repeated.
[0824] The second upper phase was removed and stored at 4.degree.
C. and the second lower phase was dried in a Buchi Rotoevaporator
RE 120, at 75.degree. C. and its mass recorded.
[0825] 2.2.9 Countercurrent Purification of Mycolic Acids
Originating from M. tuberculosis
[0826] Countercurrent Apparatus
[0827] A countercurrent apparatus produced by H O POST, Instrument
Company Inc., Middle Village, N.Y. was used during the
investigations. The "trains" in this model consisted of 2.times.250
inter-connected tubes.
[0828] Solvent System Used in the Countercurrent Apparatus
[0829] The solvent system used for the countercurrent separation
consisted of:
[0830] 42% v/v chloroform (Saarchem, Chemically Pure Reagent)
[0831] 39% v/v methanol (Saarchem, Chemically Pure)
[0832] 19% v/v 0,2 M NaCl (Saarchem, Chemically Pure).
[0833] Double distilled deionized water was used for the
preparation of the solvent system.
[0834] The components were mixed, equilibrated and the upper and
lower phases were collected using a separation funnel.
[0835] The composition of the upper phase was established to
be:
[0836] 15% v/v chloroform, 52% v/v methanol and 33% v/v 0,2 M
NaCl.
[0837] The composition of the lower phase was established to
be:
[0838] 68% v/v chloroform, 27% v/v methanol and 5% v/v 0,2 M
NaCl.
[0839] The countercurrent purification process was carried out
under the following conditions:
[0840] A countercurrent distribution train comprising 55 tubes,
numbered 0-54, was used in the experiments. The upper phase
solvent, a volume of 600 ml, was introduced into a buffer
reservoir. A sample of 125 mg of mycolic acids after the
preliminary purification was dissolved in 50 ml of the lower phase
solvent, divided into five aliquots and introduced into first five
tubes, numbered 04. Subsequently, 10 ml of the upper phase solvent
was introduced into each of the first five countercurrent tubes.
Into the remaining 50 tubes aliquots of 10 ml of the lower phase
were introduced. Upper phase, in volumes of 10 ml per cycle, was
automatically dispensed into tube number 0, repeatedly over 55
cycles resulting in approximately 5 hour operation. Thus, fifty
five countercurrent cycles were performed, with each cycle
consisting of 10 mixing pendula and 3 minutes phase separation
time.
13 Initial load of crude extract after the funnel extraction: 125
mg Number of cycles: 55 Equilibration time: 3 min
[0841] 2.2.11 Removal of Malachite Green from the
Countercurrent-Purified Mycolic Acids
[0842] To remove traces of malachite green derived from bacterial
growth media (when M. tuberculosis was grown on LJ slants), the
countercurrent-purified material was selectively precipitated in
the following manner. Countercurrent-purified mycolic acids (92 mg)
were placed in a WISP vial into which 1,0 ml chloroform was
introduced. The dissolved mycolic acids were transferred into a
pre-weighed round-bottom flask. The vial was rinsed twice with 1,0
ml chloroform and the two aliquots of chloroform were added to that
already present in the round-bottom flask. Subsequently, acetone
was introduced drop-wise in 500 .mu.l aliquots. In total, 26 ml of
acetone was introduced and the white flakes of the precipitated-out
mycolic acids were washed twice with 20 ml acetone. The acetone
supernatant, with the dissolved malachite green was removed and the
mycolic acids dried by evaporation.
[0843] The procedure was carried out at room temperature.
[0844] 2.2.12 Determination of Mycolic Acids after Countercurrent
Purification
[0845] In order to increase the accuracy of the HPLC determination
of mycolic acids, the High Molecular Weight Internal Standard
(C-100) was introduced into the countercurrent-purified mycolic
acids before the saponification.
[0846] A sample of 0,5 mg of the countercurrent-purified mycolic
acids was introduced into a WISP vial containing 5 .mu.g of the
High Molecular Weight Internal Standard (C-100). Saponification of
mycolic acids was carried out with 2 ml of Reagent A at room
temperature. The WISP vial was vortexed for 30 seconds. The
extraction was carried out with 1,5 ml of Reagent B. After
vortexing, the pH of the sample was checked and if necessary,
adjusted to pH 1 with Reagent B.
[0847] Subsequently, 2,0 ml chloroform was added to each sample and
vortexed for 30 seconds. The layers were allowed to separate. The
bottom layers were removed with Pasteur pipettes, transferred to
amber WISP vials and evaporated to dryness at 85.degree. C. in a
heat block-evaporator under a stream of nitrogen. To neutralize
traces of acid carried over, 100 .mu.l of reagent C was added to
each sample and the fluid evaporated to dryness at 85.degree. C. in
a heat block-evaporator under a stream of nitrogen.
[0848] 2.2.13 Determination of Yield of the Countercurrent
Separation
[0849] In order to calculate the approximate yield of
purification/separation, the amount of the mycolic acids present in
the samples obtained after the countercurrent
separation/purification was compared to the amount of these
compounds present in the crude cellular extract introduced into the
countercurrent apparatus. The calculations were based on the
results obtained by the HPLC analysis.
[0850] It should be stressed, that it is essential for the
calculation of the yield of the countercurrent separation, that the
mycolic acids determined by HPLC should be within the tested linear
range of the HPLC UV detector.
[0851] 2.2.14 Methods Used in Monitoring Levels of Anti-Mycolic
Acids Antibodies
[0852] Bleeding: the animals were bled from the sublingual vein 12
days after the induction of arthritis. The blood was collected into
sterile centrifuge tubes and allowed to clot for 16 hours at
4.degree. C. The collected serum was centrifuged at 700-750 g for
20 minutes, diluted 1:1 v/v in glycerol and stored at -20.degree.
C.
[0853] ELISA Protocol:
[0854] Coating of ELISA plates: The autoclaved coating antigen (in
PBS buffer. pH 7.4), still hot, was introduced into ELISA wells in
aliquots of 50 .mu.l/well, with the solution being continuously
stirred. Approximately 3 .mu.g mycolic acids per well were
introduced. The coated ELISA plates were incubated at room
temperature for 16 hours. Subsequently the antigen solution was
removed, the ELISA plates dried and the dry plates were stored at
4.degree. C.
[0855] Blocking of ELISA plates: The blocking buffer (0,5% (m/v)
casein in PBS pH 7,4) was introduced in aliquotes of 200
.mu.l/well. The ELISA plates were incubated at room temperature for
2 hours.
[0856] Binding of animal antibodies: Rat sera (mixed with glycerol
1:1 v/v) were diluted further in the diluting buffer 1:10 v/v. The
final dilution was therefore 1:20 v/v. Aliquotes of 50 .mu.L were
introduced into wells in duplicate. The plates were incubated at
room temperature for one hour. The sera were removed and the plates
washed three times with the washing buffer using an Anthos
Automatic Washer.
[0857] Quantification of the bound antibodies: Peroxidase anti-rat
antibody conjugate diluted 1:1000 was introduced in aliquotes of 50
.mu.l per well and incubated at room temperature for 30 minutes.
After the removal of the conjugate, the ELISA plates were washed
three times with the washing buffer.
[0858] The substrate solution comprising 10,0 mg O-phenylenediamine
and 8,0 mg hydrogen peroxide in 10 ml of 0,1 M citrate buffer pH
4,5, was prepared immediately before use and introduced in 50 .mu.l
aliquotes per well. The plates were placed in a dark place and the
colour development was monitored at 15, 30 and 60 minutes intervals
using a SLT 340 ATC photometer at a wavelength of 450 nm.
[0859] 2.2.15 Methods Used in Handling Experimental Animals in the
Adjuvant Arthritis Experiments
[0860] 2.2.15.1 Environmental conditions under which the
experimental animal were maintained
[0861] Experimental animals: Six weeks old, female Lewis rats were
accommodated in cages with a floor area of 864 cm.sup.2 and a
height of 12,5 cm. Four rats were maintained in each cage, except
for the animals of group 6, which were maintained three per
cage.
[0862] The animals were maintained at the Animal Facilities of the
Medical Research Council in Pretoria.
[0863] Environmental conditions: Temperature and humidity in the
animal facility were set at 20.degree. C. (+/-1.degree. C.) and 40%
(+/-10%), respectively. Lighting was provided by means of
fluorescent tubes. A light-darkness cycle of alternating 12 hour
periods was set up.
[0864] Rats were fed on nutritionally controlled pellets
manufactured by Epol, South Africa.
[0865] Cages: Rats were housed in transparent polypropylene cages
with tightly fitting stainless steel lids. Wooden shavings, after
autoclaving, were provided as nestling material.
[0866] Sanitation: Animal rooms, rat cages and glass bottles were
cleaned and decontaminated once a week using Bronocide. Water
bottles after washing were autoclaved once a week.
[0867] Identification of the experimental animals: Individual
identification was accomplished by making ear marks.
[0868] 2.2.15.2 Preparation of the reagent used for the induction
of adjuvant arthritis
[0869] The method used for the induction of adjuvant arthritis was
based on the method described by Wauben, Wagenaar-Hilber and Van
Eden (1994).
[0870] In order to obtain a coarse surface, the bottom of a mortar
bowl was ground with coarse grinding paper (100 grain). After the
dust particles were removed, freeze-dried cells of M. tuberculosis
H37 Ra, 100 mg, were transferred into the bowl. After the
introduction of 3 drops of Freund's Incomplete Adjuvant (FIA) the
bacteria were mixed very well with FIA for 2 minutes using a
pestle. Once a thick paste was obtained, a few additional drops of
FIA were introduced into the mortar and mixed with continuous
grinding for a further half a minute. The thick paste was
transferred into a 50 ml test tube using a glass Pasteur pipette.
The mortar bowl was "rinsed" several times with the remaining FIA
using a few drops at a time, until the entire volume of 10 ml was
used. The final suspension of the freeze-dried cells of M.
tuberculosis H37 Ra in FIA contained 100 mg of cells per 10 ml
FIA.
[0871] 2.2.15.3 Preparation of the reagent used for the prevention
of adjuvant arthritis
[0872] An accurately weighed-off sample (10 mg) of freshly
saponified mycolic acids, originating from M. tuberculosis H37 Rv,
was introduced into 1 ml of FIA in a glass vial and heated on a
heat-block evaporator at 80.degree. C. until completely dissolved.
After vortexing, the dissolved sample was removed from the heat
block-evaporator and left at room temperature to cool down. From
this stock solution the required concentrations of mycolic acids
were prepared by introducing additional aliquotes of FIA.
[0873] 2.2.15.4 Experimental set-up
[0874] The experimental set-up is presented in Table 7.
[0875] A day before the start of the experiment, the rats were
weighed and the thickness of the joints of their front and hind
limbs was measured with a micrometer. Individual identification of
rats was done by making ear marks.
[0876] Heat-killed and freeze-dried cells of M. tuberculosis H37Ra,
100 mg, were suspended in 10 ml of FIA by emulsifying the bacterial
cells in FIA using a mortar and pestle. (For details, see section
2.2.15.2.). The administration of killed cells of M. tuberculosis
H37Ra suspended in FIA, FIA alone and various doses of mycolic
acids in FIA, using aliquots of 100 .mu.l, was carried out in the
form of intradermal injections at the base of the rats' tail.
[0877] The rats were divided into 12 groups and treated as
illustrated in Table 7.
[0878] The rats were monitored daily for the appearance of any
symptoms of arthritis such as swollen limbs, necrosis of the tail
and nose bleeding. Two weeks into the experiment, the diameter of
the joints in the front and hind limbs was measured every second
day. Rats' mass was likewise monitored every second day.
[0879] To determine the level of anti-mycolic acids antibodies in
the sera of the experimental animals, the rats were bled from the
sublingual vein in the tongue on the 12-th day after the induction
of arthritis.
14TABLE 7 Experimental set-up for the induction of adjuvant
arthritis Day of the experiment Day 0 Day 7 Day 11 Number of rats
Induction of Group per adjuvant number group Pre-treatment
arthritis Post-treatment Group 1 4 FIA only, 100 .mu.l 1 mg H37Ra 0
in 100 .mu.l FIA Group 2 4 0,1 mg MA 1 mg H37Ra 0 in 100 .mu.l FIA
in 100 .mu.l FIA Group 3 4 0,3 mg MA 1 mg H37Ra 0 in 100 .mu.l FIA
in 100 .mu.l FIA Group 4 4 1,0 mg MA 1 mg H37Ra 0 in 100 .mu.l FIA
in 100 .mu.l FIA Group 5 4 0 100 .mu.l FIA 0 Group 6 6 0 1 mg H37Ra
0 in 100 .mu.l FIA Group 7 4 0 1 mg MA 0 in 100 .mu.l FIA Group 8 4
0 1 mg H37Ra 100 .mu.l FIA in 100 .mu.l FIA Group 9 4 0 1 mg H37Ra
0,1 mg MA in 100 .mu.l FIA in 100 .mu.l FIA Group 10 4 0 1 mg H37Ra
0,3 mg MA in 100 .mu.l FIA in 100 .mu.l FIA Group 11 4 0 1 mg H37Ra
1,0 mg MA in 100 .mu.l FIA in 100 .mu.l FIA Group 12 4 0 0 0
[0880] 2.2.15.5 Methods used in the radiological assessment of
arthritis
[0881] Radiographs of the cadaver limbs originating from the
control, arthritis and mycolic acids-treated rats were made using a
Siemens Polymat 50 diagnostic X-ray machine. Fuji HRF film and
Trimax T2 detail screens were used at a source-to-image distance of
109 cm. Exposure factors were 42 kVp and 4 mAs to optimise soft
tissue visibility and bony detail.
[0882] Radiological examinations were carried out by Prof. R M
Kirberger the Section of Pathology of the Veterinary Research
Institute, Onderstepoort, Pretoria.
2.3 Results and Discussion
[0883] The results obtained concerning:
[0884] i) the influence of the modified method of purification on
yield and purity of mycolic acids;
[0885] ii) the structural analysis of mycolic acids originating
from M. tuberculosis using infra-red spectroscopy; and
[0886] iii) the stability of mycolic acids
[0887] were presented and discussed in sections 1.3.1, 1.3.2 and
1.3.3.
[0888] 2.3.1 Monitoring of the Symptoms of Adjuvant Arthritis
[0889] Successful induction of adjuvant arthritis (experimental
details given 2.2.15.2, 2.2.15.3 and 2.2.15.4) was first observed
four days after the administration of an arthritis-inducing dose of
the suspension of M. tuberculosis H37Ra freeze-dried cells in
Freund's Incomplete Adjuvant (FIA). The first symptoms were those
of a necrosis developing at the site of the injection. Other
symptoms were observed at about 11 days after the administration of
the cells of M. tuberculosis H37Ra in FIA and included swelling of
the knuckles and joints as well as nose bleeding (FIGS. 25a, 25b
and FIG. 26b). These symptoms peaked after approximately 16 to 21
days and subsequently subsided, except for the necrosis. Complete
recovery was observed within the next two weeks.
[0890] The rats which were pre-treated with 0,1 mg and 0,3 mg
mycolic acids developed less severe symptoms than those treated
with FIA alone. Three rats pre-treated with 1 mg MA did not develop
any symptoms indicative of the presence of the disease. The fourth
rat in the same group (rat number 3 in FIG. 26c) did not receive
the full dose of mycolic acids during, pre-treatment, as part of
the dose leaked out after the injection. This rat showed only
moderate symptoms of arthritis in the hind limbs.
[0891] The rats which received injections of FIA only (FIG. 26a),
did not show any symptoms. No toxic effects were observed among the
control rats which were treated with mycolic acids suspended in FIA
but without challenge with an arthritis-inducing dose of M.
tuberculosis H37Ra in FIA.
[0892] The results obtained in the experiment are summarized in
Tables 8a and 8b and illustrated by photographs presented in FIGS.
25 to 27.
[0893] FIGS. 25a and 26b show the typical swelling of the joints
and knuckles in the front paws of the experimental rats. FIG. 25a
illustrates the bleeding from the nose caused by thrombocytopenia
induced by the presence of a high concentration of immune complexes
in the blood of the experimental animals.
15TABLE 8a Results obtained in rats treated with mycolic acids
prior to the induction of adjuvant arthritis Necro- Nose Mass Avg
Group Rat Days LF RF LB RB sis bleed (g) mass 1 1 16 2.1 1.6* 5.3
4.3 ++N + +12 13.4 FLA + 2 3.2 2.9 3.6 3 +++N + +7.8 H37Ra 3 * 2.3
3.8 2.7 +++N + +8.3 4 3 3.7 3.6 4.7 +++N + -2.6 2 1 16 * * 2 2.3
+++N + +26.2 24.5 0.1 mg 2 2.4 2.9 * 4.9 +++N + +22.6 MA + 3 2.2
0.7 1.2 2.7 +++N + +24.7 H37Ra 4 0.6 2.4 2.6 3.8 ++N + +24.5 3 1 16
-- -- -- -- +++N + +31.2 17.6 0.3 mg 2 0.5 2.5 2.9 3.6 ++N + +22.4
MA + 3 2.1 2.8 3.3 5.5 +++N + +12.2 H37Ra 4 1.1 1 2.9 3.5 ++++N +
+4.4 4 1 16 -- 0.9 -- -- ++N + +40.6 30.6 1 mg 2 -- -- -- 1 +++N +
+31.4 MA + 3 1.5 2 4.2 4.3 +++N + +4.9 H37Ra 4 -- -- -- -- +N +
+45.3 5 1 16 -- 1.1 -- -- -- + +35.2 29.6 FIA 2 -- 0.9 -- 0.7 -- -
+33.1 3 -- -- -- -- -- - +23.9 4 -- 0.9 -- 0.5 -- + +26.1 7 1 21
0.5 -- -- -- -- + +39.9 36 1 mg 2 0.6 0.9 -- 0.5 -- - +26.5 MA 3 --
0.8 -- -- -- - +37.7 4 -- 0.6 -- -- -- + +39.6 Abbreviations:
Increase in the joint diameter of: LF Left front paw RF Right front
paw LB Left back paw RB Right back paw Nose bleed Nose bleeding
Mass Increase or decrease in mass Avg mass Average increase in mass
per group Max sympt Day on which the most severe symptoms were
observed
[0894]
16TABLE 8b Results obtained in rats treated with mycolic acids
administered after the induction of adjuvant arthritis Nose Mass
Avg Group Rat Days LF RF LB RB Necrosis bleed (g) mass H37Ra 1 21
1.5 1.5 2.3 4.3 ++++N + +19.8 17.5 2 1.35 -- -- -- +++N + +29.5 3
-- -- -- 0.9 ++N + +14.5 4 -- * 1 -- ++N - +19.5 5 * 1.5* 5.7 4.2
+N + +5.3 6 * 0.6 1.6 0.5 +++N + +16.6 7 1 21 0.5 -- -- -- -- +
+39.9 36 1 mg 2 0.6 0.9 -- 0.5 -- - +26.5 MA 3 -- 0.8 -- -- -- -
+37.7 4 -- 0.6 -- -- -- + +39.6 8 1 21 * -- 4.3 3.1 +++N - +24 26.6
H37Ra 2 -- -- 0.9 0.5 +++N - +27.6 + FIA 3 1.9 * 2.7 3 +++N + +9.9
4 -- 0.5 -- -- +++N + +44.7 9 1 21 * * * 2.7 +++N + +6.4 14.8 H37Ra
2 2.2* * -- 3 ++++N + +14.6 + 0.1 mg 3 -- -- 0.7 0.9 +++++N + +20.9
MA 4 0.7 2.1 3.1 3 +++N + +17.3 10 1 21 -- -- -- -- -- - +32.5 27.2
H37Ra 2 -- -- -- -- +++N + +30.4 + 0.3 mg 3 -- -- 0.5 -- +++++N -
+32.9 MA 4 1.5 2.9* 3.1 2.2 ++++++N + +13.1 11 1 21 1.5 1.1 1.2 2.1
++++N + +25.2 23.1 H37Ra 2 2.45 1* 2.75 1.45 ++++N + +18.2 + 1.0 mg
3 -- 1.1 0.75 -- +++N + +25.7 MA 4 2.7 0.65 -- 2.25 +++N + +23.1 12
1 21 -- 0.5 -- -- -- - +34.5 37.3 Nothing 2 -- 0.5 -- -- -- - +29.1
3 -- -- -- -- -- - +46.5 4 -- -- -- -- -- - +37.2 Abbreviations:
Increase in the joint diameter of: LF Left front paw RF Right front
paw LB Left back paw RB Right back paw Nose bleed Nose bleeding
MASS Increase or decrease in mass Avg mass Average increase in mass
per group Max sympt Day on which the most severe symptoms were
observed
[0895] FIGS. 25b as well as FIGS. 26b and 26c show a comparison
between the rat pre-treated with 1 mg MA in FIA and the arthritic
rat. The pronounced swelling and inflammation of hind leg joints of
the rat in which adjuvant arthritis was successfully induced can be
clearly distinguished from those rats that were protected with
mycolic acids pre-treatment (FIGS. 25b and 26c).
[0896] FIG. 25c shows the typical deformation of the joints in the
hind legs, known as the "swimming position". The emaciation caused
by adjuvant arthritis is evident from FIG. 27.
[0897] The results obtained in the experiment indicate a protective
influence of mycolic acids at a dose of 1 mg administered before
priming with M. tuberculosis H37Ra in FIA.
EXAMPLE 3
[0898] Immunogenic properties of countercurrent-purified mycolic
acids
3.1 Materials
[0899] 3.1.1 Culture
[0900] Mycobacterium tuberculosis H37Rv ATCC 27294--a virulent
strain, originally isolated from an infected human lung. Type
strain of the species.
[0901] The culture was purchased in lyophilized form from the
American Type Culture Collection (ATCC), Maryland, USA.
[0902] 3.1.2 Media
[0903] 3.1.2.1 Growth media
[0904] The following media were used for the cultivation of M.
tuberculosis:
[0905] Lowenstein-Jensen (LJ) medium (slants) and
[0906] Middlebrook 7H-10 agar medium (plates).
[0907] A detailed composition of the ingredients necessary for the
preparation of these media as well as the conditions recommended
for their sterilization, are given in the Laboratory Manual of
Tuberculosis Methods, Tuberculosis Research Institute of the SA
Medical Research Council (1980, Chapter 6, pp 83-105; Second
Edition, revised by E B Nel, H H Kleeberg and E M S Gatner).
[0908] The media were prepared by the National Tuberculosis
Institute of the Medical Research Council of South Africa, in
Pretoria.
[0909] 3.1.2.2 Media used for washing and diluting of
Mycobacteria
[0910] The harvested bacteria were washed in sterile 0,9% m/v NaCl
(Saarchem, Chemically Pure, RSA).
[0911] Medium used for the preparation of serial dilutions,
preceding the determination of viable counts of M. tuberculosis,
was prepared by dissolving Tween 80 (Merck, Chemically Pure) in
0,9% m/v NaCl (Saarchem, Chemically Pure) to a concentration of
0,01% v/v and distributing it in 9,0 ml aliquots into test-tubes.
The autoclaved media were stored at 4.degree. C.
[0912] 3.1.3 Reagents
[0913] 3.1.3.1 For the preparation of the reagents used for the
extraction, derivatization and High-Performance Liquid
Chromatography (HPLC) analysis of mycolic acids, HPLC Grade
methanol (BDH) and double-distilled deionized water were used.
[0914] Reagent A: 25% potassium hydroxide (Saarchem, Analytical
Grade) dissolved in methanol-water (1:1), i.e., 62,5 g potassium
hydroxide was dissolved in 125 ml water and 125 ml methanol (BDH,
HPLC Grade) was added.
[0915] Reagent B: Concentrated hydrochloric acid (Saarchem,
Analytical Grade) diluted 1:1 with water.
[0916] Reagent C: 2% potassium bicarbonate (BDH, Analytical Grade)
dissolved in methanol-water (1:1), 10 g potassium bicarbonate was
dissolved in 250 ml water and 250 ml methanol was added.
[0917] Reagent D: para-bromophenacylbromide dissolved in
acetonitrile and crown ether (Pierce Chemical Co, Cat. No 48891)
was dispensed in 500 .mu.l quantities into small amber-coloured
screw cap vials with Teflon-coated septa. The caps were tightened
and the vials were wrapped with Parafilm. Reagent D was stored at
4.degree. C.
[0918] Reagent E: Reagent E was prepared by mixing reagent B 1:1
with methanol.
[0919] HPLC Standard: High Molecular Weight Internal Standard
(C-100) from Ribi ImmunoChem Research Company, Cat No R-50. The
standard, 1 mg, was dissolved in 20 ml chloroform (BDH, HPLC Grade)
at 4.degree. C. and aliquots of 100 .mu.l were dispensed into 4 ml
amber WISP vials, dried, capped with Teflon-coated septa and stored
at 4.degree. C.
[0920] Chloroform (Saarchem, Analytical Grade, RSA)
[0921] Methylene chloride (BDH, UK, HPLC-Grade)
[0922] Reagents A, B, C and E were prepared fresh prior to
experiments, taking all the necessary safety precautions.
[0923] 3.1.3.2 The following reagents were used for the preliminary
purification of crude bacterial extracts ("funnel extraction") and
for the countercurrent purification of the extracted mycolic
acids:
[0924] Chloroform (Saarchem, Chemically Pure)
[0925] Methanol (Saarchem, Chemically Pure)
[0926] Acetone (Saarchem, Chemically Pure)
[0927] Sodium chloride (Saarchem, Analytical Grade)
[0928] Double-distilled deionized water was used for the
preparation of the required reagent concentrations. i.e.,:
[0929] 39% v/v methanol
[0930] 42% v/v chloroform
[0931] 0,2 M NaCl
[0932] 3.1.3.3 Reagents used in the induction of anti-mycolic acids
antibodies
[0933] Unsaponified mycolic acids originating from M.
tuberculosis;
[0934] Marcol 52 immunization oil manufactured by Esso, RSA.
[0935] 3.1.3.4 Reagents used in monitoring the production of
anti-mycolic acids antibodies.
[0936] Glycerol (Merck, Analytical Grade) was used for diluting
sera of the experimental animals.
[0937] ELISA Reagents:
[0938] Basic buffer--PBS buffer: 8,0 g NaCl, 0,2 g KCl, 0.2 g
KH.sub.2PO.sub.4 and 1,05 g Na.sub.2HPO.sub.4 per 1 l distilled
water, adjusted to pH 7,4.
[0939] Diluting buffer: 0,5% (m/v) casein in PBS buffer adjusted to
pH 7,4 was used for diluting of the experimental animals' sera
(mixed with glycerol 1:1) and for the preparation of suitable
dilutions of immunoreagents.
[0940] Blocking buffer: 0,5% (m/v) casein in PBS buffer adjusted to
pH 7,4 was used for blocking of ELISA plates.
[0941] Washing buffer: 0,5% (m/v) casein in PBS buffer adjusted to
pH 7,4 was used for washing of ELISA plates.
[0942] Coating antigen: unsaponified mycolic acids originating from
M. tuberculosis, at a final concentration of 0,067 .mu.g/.mu.l. To
prepare the coating antigen, 1 mg mycolic acids was dissolved in
100 .mu.l chloroform and the solution introduced into 15,0 ml PBS
buffer adjusted to pH 7,4. The solution was autoclaved at
121.degree. C. for one hour.
[0943] Conjugates: Goat anti-rat antibody conjugated to peroxidase
(H+L chains), Cappel (Cat No 55770). Rabbit anti-human gamma chain
specific peroxidase conjugate (Sigma; A 8419).
[0944] Substrate: O-Phenylenediamine (Sigma; CatNo P-1526) and
hydrogen peroxide (BDH).
[0945] Substrate buffer: 0,1 M citrate buffer (0,1 M citric acids
and 0,1 M Tri sodium citrate), adjusted to pH 4,5.
[0946] ELISA plates were manufactured by Sterilin, UK.
[0947] 3.1.3.4 Reagents used in the preparation of SDS-PAGE
gels
[0948] Laemmli buffer: 0,5 M Tris-HCl pH 6, 8, 10% v/v glycerol,
10% m/v SDS and 0,05% m/v bromo phenol blue
[0949] CAPS buffer pH 9,0: 3-[Cyclohexylamino]-1 propane sulphonic
acid, (Sigma) buffer pH 9,0
[0950] TBS buffer
[0951] pH 7,4: 20 mM Tris and 55 mM NaCl, containing 1% m/v
fat-free milk powder and 0,05% v/v Tween 20
[0952] SDS-PAGE gels: Sodium dodecyl sulphate polyacrylamide slab
electrophoresis gel:
[0953] a 4% stacking gel and a 6% separating gel comprising 30 mM
Tris pH 8,0, 200 mM glycine and 17 mM SDS.
[0954] Substrate: 0,03 mM 4-chloronaphtol, 3% v/v hydrogen peroxide
in 20 ml methanol, made up to 100 ml with TBS buffer pH 7,4
[0955] Immobilon-P Transfer membranes
[0956] Coomassie blue
[0957] 3.1.4 Human Sera
[0958] Human sera used in the ELISA experiments originated from the
Serum Bank of the Medical Research Council Tuberculosis Institute,
Pretoria.
[0959] 3.1.5 Experimental Animals
[0960] Seventeen weeks old Sprague-Dawley female rats were used for
the induction of anti-mycolic acids antibodies. The animals were
purchased from the Animal Centre at the South African Institute for
Medical Research in Johannesburg.
[0961] Feed and Water
[0962] Mice cubes, manufactured by EPOL and tap, autoclaved water
were provided ad libitum.
[0963] Sanitation:
[0964] Bronocide, manufactured by Essential Medicines (Pty) Ltd,
was used for sanitation purposes.
[0965] 3.1.6 Plasticware
[0966] The following plasticware was used:
[0967] Disposable Petri's dishes (Promex, RSA)
[0968] ELISA plates (Stefilin, UK)
[0969] Sterile, disposable 50 ml centrifuge tubes (Corning,
USA)
[0970] Disposable tips (Elkay, Denmark)
[0971] 96-well round bottom microplates (Nunc, Denmark)
3.2 Methods
[0972] The following methods were used in the experimental
work:
[0973] 3.2.1 Cultivation of the Bacterial Strains
[0974] The bacteria were cultivated at 37.degree. C. using
Lowenstein-Jensen (LJ) medium slants and Middlebrook 7H-10 agar
medium plates.
[0975] The sterility of all the media was confirmed, before they
were used in the experiments, by incubating them at 37.degree. C.
for 24 h.
[0976] For routine extraction of mycolic acids approximately 4-week
old M. tuberculosis and 2-week old cultures of M. vaccae, grown on
LJ slants, were used. When Middlebrook 7H-10 agar medium plates
were used, 2-week old cultures of M. tuberculosis were harvested
for the extraction of mycolic acids. For the preparation of
bacterial suspensions used for the experimental induction of
tuberculosis, approximately 2-week old cultures of M. tuberculosis,
grown on LJ slants were used.
[0977] 3.2.2 Viable and Total Bacterial Counts
[0978] For the viable count determination, serial suspensions of
the harvested bacteria were prepared in the diluent medium (as
specified under 1.1.2.2) to a density corresponding to a McFarland
standard 4 (approximately OD of 1,0; using a Beckman DU 65
spectrophotometer, at 486 nm). Tenfold serial dilutions were
prepared using 9 ml aliquots of the diluent medium. From the last
three dilutions corresponding to 10.sup.-3, 10.sup.-4 and 10.sup.-5
of the original suspension, aliquots of 0,1 ml (100 .mu.l) were
withdrawn and spread over the surface of Middlebrook 7H-10 plates.
The plates were incubated at 37.degree. C. and the developed
colonies counted after two to three weeks for M. tuberculosis and
after one week for the plates seeded with M. vaccae.
[0979] The direct total count was performed using a Neubauer
counting chamber and the autoclaved cultures of M. tuberculosis and
M. vaccae, originally adjusted to a density corresponding to a
McFarland standard 4 and suitably diluted with the diluent
medium.
[0980] Statistical analysis of the bacterial counts included the
mean values of bacterial counts and standard deviations.
[0981] 3.2.3 Preparation of Mycolic Acids from Bacterial
Samples
[0982] The preparation of bacterial samples comprised three
steps:
[0983] harvesting of the Mycobacteria cells;
[0984] saponification and
[0985] extraction of mycolic acids.
[0986] Glassware used for the harvesting, extraction,
derivatization and HPLC analyses of mycolic acids was washed in 2%
(v/v) Contrad (Merck), rinsed in water, followed by rinsing in
chloroform, water, Technical Grade methanol, water and finally
rinsed in double distilled deionized water. The washed glassware
was dried in a warm air oven.
[0987] Harvesting was done by scraping the bacterial growth from
the surface of media slants or agar plates (using sterile plastic
loops) and by suspending them in Reagent A. Initial bacterial
suspensions were prepared in Reagent A, by vortexing the harvested
cells with sterile glass beads. Homogenous bacterial suspensions
were prepared using sterile tissue homogenizers. Prior to the
saponification, the density of the bacterial suspensions was
adjusted to a density corresponding to a McFarland standard 4.
[0988] The saponification, extraction and derivatizaion of mycolic
acids were carried out as described by Butler, Jost and Kilburn
(1991), with minor modifications and are described under the
relevant headings.
[0989] Saponification of the Mycobacteria in Reagent A was carried
out in an autoclave at 121.degree. C., for 30 min.
[0990] 3.2.4. Extraction of Mycolic Acids
[0991] The saponified samples were allowed to cool after
autoclaving. Into 2 ml samples containing crude extract, 1,5 ml
Reagent B was introduced. After vortexing, the pH of each sample
was checked and if necessary, adjusted to pH 1 with Reagent B.
[0992] Subsequently, 2,0 ml chloroform was added to each sample and
vortexed for 30 seconds. The layers were allowed to separate. The
bottom layers were removed with Pasteur pipettes, transferred to
amber WISP vials and evaporated to dryness at 85.degree. C. in a
heat block-evaporator under a stream of nitrogen. To neutralize
traces of acid carried over, 100 .mu.l of reagent C was added to
each sample and the fluid evaporated to dryness at 85.degree. C. in
a heat block-evaporator under a stream of nitrogen.
[0993] 3.2.5 Storage of the Crude Extracted Mycolic Acids
[0994] The material obtained from the large-scale extraction of
mycolic acids originating from M. tuberculosis and M. vaccae, i.e.,
the crude bacterial extracts, was stored under acetone, at
4.degree. C. in 4 ml amber WISP vials. To prevent
evaporation/drying and the exposure to light, the caps of the WISP
vials were covered with Parafilm.
[0995] 3.2.6 Determination of Mycolic Acids Contents in Crude
Extracts
[0996] Extracted mycolic acids were derivatized as follows:
[0997] To a cooled sample of crude extract (approximately 10 .mu.g
in 2,0 ml Reagent A), an aliquot of 1,0 ml chloroform was
introduced, followed by the addition of 100 .mu.l of Reagent D
(derivatization reagent). The capped samples were vortexed for 30
seconds and heated for 20 minutes at 85.degree. C. in a heat
block-evaporator. Subsequently, the samples were cooled and 1,0 ml
of Reagent E added. The samples were vortexed for 30 seconds and
the layers allowed to separate. The bottom layers were removed with
Pasteur pipettes and transferred to WISP-vials. The vials were
placed in a heat block-evaporator and their contents evaporated to
dryness at 85.degree. C. using a stream of nitrogen.
[0998] The residues were resuspended in 0,212 g (which corresponds
to 160 .mu.l) methylene chloride, capped and vortexed. Each
reconstituted sample was introduced into a WISP vial containing 5
.mu.g of the HPLC internal standard (prepared as described under
1.1.3.1), filtered through a 0,22 .mu.m Millex GV4 filter with a
polyethylene housing into another amber-coloured WISP-vial. The
recapped vials were stored at 4.degree. C. until ready for HPLC
analysis.
[0999] 3.2.7 HPLC Analysis and Quantification of Mycolic Acids
[1000] Repeatability and accuracy of the pipette used for the
distribution of the HPLC standard was determined. The precision was
established to be +/-1% and was confirmed prior to each aliquoting
of the internal standard.
[1001] For the HPLC analysis 10 .mu.l from each sample (maintained
on ice during handling), was analyzed. Control samples, i.e., 10
.mu.l of filtered methylene chloride, were run prior to each set of
samples analyzed. If a large number of samples was analyzed, in
order to validate the reliability of the HPLC apparatus, control
samples were run after every three or four test samples.
[1002] The reverse-phase HPLC analyses were carried out using a
Waters 600 E System Controller High Performance Liquid
Chromatography apparatus consisting of:
[1003] Microsep M741 Data Module;
[1004] Waters 712 WISP Autosampler;
[1005] Detector (Waters 486 Tunable Absorbance Detector);
[1006] Column: Nova-Pak C18 4 .mu.m 3,9.times.150 mm and an end
connector set for steel cartridge columns.
[1007] RKC Rex-C 4 Column Temperature regulator.
[1008] Running conditions were:
[1009] Mobile phase:
[1010] Solvent A: HPLC Grade methanol
[1011] Solvent B: HPLC Grade methylene chloride
[1012] Flow Rate: 2,5 ml/min
[1013] Column temperature: 30.degree. C.
[1014] The detector was set at 260 m.
[1015] Prior to use, the solvents were sparged with Instrument
Grade helium. High Purity Nitrogen was used to control hydraulics
of the WISP vials autosampler.
[1016] The HPLC gradient initially comprised 98% (v/v) methanol
(Solvent A) and 2% (v/v) methylene chloride (Solvent B). The
gradient was increased linearly to 80% A and 20% B at one minute;
35% A and 65% B at ten minutes, held for 30 second and then
decreased over 10 seconds back to 98% A and 2% B. This ratio was
maintained for 4 minutes to allow for stabilization of the system
prior to injection of the next sample.
[1017] Mathematical quantification of mycolic acids was carried out
by comparing the combined peak areas of the tested samples to the
peak area of the introduced quantity of the High Molecular Weight
Internal HPLC Standard.
[1018] 3.2.8 Preliminary Purification of Crude Mycobacterial
Extracts
[1019] In order to shorten the time required for the countercurrent
purification of the crude mycobacterial extracts, an additional
preliminary extraction step was introduced. This step had a dual
purpose:
[1020] i) to remove unnecessary cellular components from the crude
extract prior to the countercurrent purification and
[1021] ii) to reduce soap fraction in the crude bacterial
extracts.
[1022] A portion of the crude extracted material (approximately 3-4
g) was suspended in a minimum volume of the lower phase solvent
(usually 100 ml), transferred into a separation funnel and mixed
with an equal volume of the upper phase solvent. The phases were
allowed to separate and the upper phase was removed and stored at
4.degree. C. Into the remaining lower phase an equal volume of the
upper phase solvent was again introduced and the process of the
phase separation was repeated.
[1023] The second upper phase was removed and stored at 4.degree.
C. and the second lower-phase was dried in a Buchi Rotoevaporator
RE 120, at 75.degree. C. and its mass recorded.
[1024] 3.2.9 Countercurrent Purification of Mycolic Acids
Originating from M. tuberculosis and M. vaccae
[1025] Countercurrent Apparatus
[1026] A countercurrent apparatus produced by H O POST, Instrument
Company Inc., Middle Village, N.Y. was used during the
investigations. The "trains" in this model consisted of 2.times.250
inter-connected tubes.
[1027] Solvent System Used in the Countercurrent Apparatus
[1028] The solvent system used for the countercurrent separation
consisted of:
[1029] 42% v/v chloroform (Saarchem, Chemically Pure Reagent)
[1030] 39% v/v methanol (Saarchem, Chemically Pure)
[1031] 19% v/v 0,2 M NaCl (Saarchem, Chemically Pure).
[1032] Double-distilled deionized water was used for the
preparation of the solvent system.
[1033] The components were mixed, equilibrated and the upper and
lower phases were collected using a separation funnel.
[1034] The composition of the upper phase was established to
be:
[1035] 15% v/v chloroform 52% v/v methanol and 33% v/v 0,2 M
NaCl.
[1036] The composition of the lower phase was established to
be:
[1037] 68% v/v chloroform, 27% v/v methanol and 5% v/v 0,2 M
NaCl.
[1038] The countercurrent purification process was carried out
under the following conditions:
[1039] A countercurrent distribution train comprising 55 tubes,
numbered 0-54, was used in the experiments. The upper phase
solvent, a volume of 600 ml, was introduced into a buffer
reservoir. A sample of 125 mg of mycolic acids after the
preliminary purification was dissolved in 50 ml of the lower phase
solvent, divided into five aliquots and introduced into first five
tubes, numbered 0-4.
[1040] Subsequently, 10 ml of the upper phase solvent was
introduced into each of the first five countercurrent tubes. Into
the remaining 50 tubes aliquots of 10 ml of the lower phase were
introduced. Upper phase, in volumes of 10 ml per cycle, was
automatically dispensed into tube number 0, repeatedly over 55
cycles resulting in approximately 5 hour operation. Thus, fifty
five countercurrent cycles were performed, with each cycle
consisting of 10 mixing pendula and 3 minutes phase separation
time.
17 Initial load of crude extract after the funnel extraction: 125
mg Number of cycles: 55 Equilibration time: 3 min
[1041] 3.2.11 Removal of Malachite Green from the
Countercurrent-Purified Mycolic Acids
[1042] To remove traces of malachite green derived from bacterial
growth media (when M. tuberculosis was grown on LJ slants), the
countercurrent-purified material was selectively precipitated in
the following manner. Countercurrent-purified mycolic acids (92 mg)
were placed in a WISP vial into which 1,0 ml chloroform was
introduced. The dissolved mycolic acids were transferred into a
pre-weighed round-bottom flask. The vial was rinsed twice with 1,0
ml chloroform and the two aliquots of chloroform were added to that
already present in the round-bottom flask. Subsequently, acetone
was introduced drop-wise in 500 .mu.l aliquots. In total, 26 ml of
acetone was introduced and the white flakes of the precipitated-out
mycolic acids were washed twice with 20 ml acetone. The acetone
supernatant, with the dissolved malachite green, was removed and
the mycolic acids dried by evaporation.
[1043] The procedure was carried out at room temperature.
[1044] 3.2.12 Determination of Mycolic Acids After Countercurrent
Purification
[1045] In order to increase the accuracy of the HPLC determination
of mycolic acids, the High Molecular Weight Internal Standard
(C-100) was introduced into the countercurrent-purified mycolic
acids before the saponification.
[1046] A sample of 0,5 mg of the countercurrent-purified mycolic
acids was introduced into a WISP vial containing 5 .mu.g of the
High Molecular Weight Internal Standard (C-100). Saponification of
mycolic acids was carried out with 2 ml of Reagent A at room
temperature. The WISP vial was vortexed for 30 seconds. The
extraction was carried out with 1,5 ml of Reagent B. After
vortexing, the pH of the sample was checked and if necessary,
adjusted to pH 1 with Reagent B.
[1047] Subsequently, 2,0 ml chloroform was added to each sample and
vortexed for 30 seconds. The layers were allowed to separate. The
bottom layers were removed with Pasteur pipettes, transferred to
amber WISP vials and evaporated to dryness at 85.degree. C. in a
heat block-evaporator under a stream of nitrogen. To neutralize
traces of acid carried over, 100 .mu.l of reagent C was added to
each sample and the fluid evaporated to dryness at 85.degree. C. in
the heat block-evaporator under a stream of nitrogen.
[1048] Therefore, the main difference between the determination of
mycolic acids after countercurrent purification and in the crude
extract was the time of introduction of the Internal Standard.
[1049] 3.2.13 Determination of Yield of the Countercurrent
Separation
[1050] In order to calculate the approximate yield of
purification/separation, the amount of the mycolic acids present in
the samples obtained after the countercurrent
separation/purification was compared to the amount of these
compounds present in the crude cellular extract introduced into the
countercurrent apparatus. The calculations were based on the
results obtained by the HPLC analysis.
[1051] It should be stressed, that it is essential for the
calculation of the yield of the countercurrent separation, that the
mycolic acids determined by HPLC should be within the tested linear
range of the HPLC TV detector.
[1052] 3.2.14 Infra-Red Spectroscopy
[1053] Samples of mycolic acids to be analyzed by infra-red
spectroscopy were prepared in the following manner.
Countercurrent-purified mycolic acids, 1 mg, were dissolved in 1 ml
chloroform, introduced into 200 mg KBr and thoroughly mixed. After
the evaporation of chloroform, a pellet of mycolic acids in KBr was
prepared by using a Shimadzu tablet die and applying a force of
approximately 100 kilonewtons on the sample for 10 minutes. A
control pellet was prepared using only chloroform, without mycolic
acids added to the preparation. The control pellet was used to
determine the background infra-red spectrum. The spectra were
analyzed on a Perkin Elmer 1600 series FT-IR system and plotted on
a Roland Digital Group X-Y Plotter DXY-1200.
[1054] 3.2.15 Determination of the Stability of the
Countercurrent-Purified Mycolic Acids
[1055] A pooled sample of the countercurrent-purified mycolic acids
was prepared by introducing five batches of countercurrent-purified
mycolic acids into a container, dissolving them in chloroform and
mixing the contents very well. The chloroform was evaporated using
a Buchi Rotoevaporator RE 120, at 75.degree. C. and the sample
dried under a stream of nitrogen. The pooled sample was divided
into two parts which constituted two stock samples. The first stock
sample was re-saponified and the second was left as a
non-saponified stock sample. From both stock samples individual
aliquots were withdrawn and placed at -20.degree. C., 4.degree. C.
and 25.degree. C. Three samples were prepared per each time point
and HPLC analyses were carried out after 6 weeks, 3, 6, 9 and 12
months of storage.
[1056] 3.2.16 Methods Used in the Experimental Production of
Anti-Mycolic Acids Antibodies
[1057] Experimental animals: Sprague-Dawley female rats, 17 weeks
old were used. Three animals were used per each antigen dose.
[1058] The animals were maintained at the Animal Facilities of the
Medical Research Council in Pretoria.
[1059] Environmental conditions: Temperature and humidity in the
animal facility were set at 20.degree. C. (+/-1.degree. C.) and 40%
(+/-10%), respectively. Lighting was provided by means of
fluorescent tubes. A light-darkness cycle of alternating 12 hour
periods was set up.
[1060] Cages: Rats were housed in transparent polypropylene cages
with tight fitting stainless steel lids. Wooden shavings, after
autoclaving, were provided as nestling material.
[1061] Sanitation: Animal rooms, rat cages and glass bottles were
cleaned and decontaminated once a week using Bronocide. Water
bottles after washing were autoclaved once a week.
[1062] Identification of the experimental animals: Individual
identification was accomplished by making ear marks.
[1063] Antigen: Unsaponified mycolic acids originating from M.
tuberculosis, suspended in Marcol 52 oil.
[1064] Dose: Three doses of the antigen were used: 1,0, 0,1 and
0,01 mg mycolic acids in 100 .mu.l of Marcol 52 oil per rat per
immunization procedure. A control group received 100 .mu.l Marcol
52 oil, only.
[1065] Route of antigen introduction: The antigen was injected
subcutaneously at the underneath site of the rat's tail base.
[1066] Frequency of immunization: the animals were immunized at 14
days intervals.
[1067] Bleeding: the animals were bled from the tongue vein at 14
days intervals. The blood was collected into sterile centrifuge
tubes and allowed to clot for 16 hours at 4.degree. C. The
collected serum was centrifuged at 700-750 g for 20 minutes,
diluted 1:1 v/v in glycerol and stored at -20.degree. C.
[1068] 3.2.17 Methods Used in Monitoring Levels of Anti-Mycolic
Acids Antibodies
[1069] ELISA Protocol:
[1070] Coating of ELISA plates: The autoclaved coating antigen (in
PBS buffer. pH 7.4), still hot, was introduced into ELISA wells in
aliquots of 50 .mu.l/well, with the solution being continuously
stirred. Approximately 3 .mu.g mycolic acids per well were
introduced. The coated ELISA plates were incubated at room
temperature for 16 hours. Subsequently, the antigen solution was
removed, the ELISA plates dried and the dry plates were stored at
4.degree. C.
[1071] Blocking of ELISA plates: The blocking buffer (0,5% (m/v)
casein in PBS pH 7,4) was introduced in aliquotes of 200
.mu.l/well. The ELISA plates were incubated at room temperature for
2 hours.
[1072] Binding of animal and human antibodies: Rat or human sera
(mixed with glycerol 1:1 v/v) were diluted further in the diluting
buffer 1:10 v/v. The final dilution was therefore 1:20 v/v.
Aliquotes of 50 .mu.l were introduced into wells in duplicate. The
plates were incubated at room temperature for one hour. The sera
were removed and the plates washed three times with the washing
buffer using an Anthos Automatic Washer.
[1073] Quantification of the bound antibodies: Peroxidase anti-rat
antibody conjugate (or peroxidase anti-human conjugate) diluted
1:1000 was introduced in aliquotes of 50 .mu.l per well and
incubated at room temperature for 30 minutes. After the removal of
the conjugate, the ELISA plates were washed three times with the
washing buffer.
[1074] The substrate solution comprising 10,0 mg O-phenylenediamine
and 8,0 mg hydrogen peroxide in 10 ml of 0,1 M citrate buffer pH
4,5, was prepared immediately before use and introduced in 50 .mu.l
aliquotes per well. The plates were placed in a dark place and the
colour development was monitored at 15, 30 and 60 minutes intervals
using a SLT 340 ATC photometer at a wavelength of 450 n.
[1075] 3.2.18 Methods Used in Evaluating Specificity of Human
Anti-Mycolic Acids Antibodies
[1076] For the determination of the specificity of antibodies
recognising mycolic acids, the inhibition ELISA was used. Coating
of the ELISA plates with mycolic acids and blocking of the plates
were carried out as described under 3.2.17.
[1077] Competition step: Human patient's (patient No 38) serum, 75
.mu.l, was mixed with an equal volume of mycolic acids/mouse serum
conjugate (prepared as described in section 1.2.16.4), Human
control serum was likewise mixed with 75 .mu.l of the conjugate.
Two additional controls were prepared by mixing 75 .mu.l of control
mouse serum with 75 .mu.l of the human patient's and control human
sera. The samples were incubated for 1 hour at room
temperature.
[1078] Subsequently, 625 .mu.l of diluting buffer (section 3.1.3.4)
was introduced into the each mixture, resulting in a final volume
775 .mu.l (final dilution of the human sera was therefore 1:10).
The diluted samples were mixed and 50 .mu.l aliquots were loaded in
triplicate onto ELISA plates, coated with mycolic acids. The plates
were incubated on an ELISA shaker for 1 hour at room temperature.
After washing (three times with the washing buffer using an Anthos
Automatic Washer) the wells were aspirated and the anti-human
gamma-chain specific peroxidase conjugate diluted 1:1000 was
introduced into each well. The plates were incubated for 30 minutes
at room temperature and again washed three times. The preparation
of the substrate and quantification of the bound antibodies was
carried out in the same manner as described under 3.2.17.
[1079] 3.2.22 Preparation of Gel Electrophoresis
[1080] Preparation of Human Sera
[1081] Patients' sera were centrifuged at 3 000 g, for 10 min at
4.degree. C., using a BHG Hermle centrifuge model 2320. After
centrifugation and heat inactivation at 56.degree. C. for 30 min,
the sera were maintained at -70.degree. C.
[1082] Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE)
[1083] The mouse serum and mycolic acids-mouse serum conjugates
(prepared as described under 1.2.16.3 and 1.2.16.4, respectively)
were diluted with Laemmli buffer (Laemmli, 1970) comprising 0,5 M
Tris-HCl pH 6,8, 10% v/v glycerol, 10% m/v SDS and 0,05% m/v bromo
phenol blue, and separated on a vertical sodium dodecyl sulphate
polyacrylamide slab electrophoresis gel system (SDS-PAGE) (Owl
system 1,5 mm.times.160 mm.times.140 mm). The gel consisted of a 4%
stacking gel and a 6% separating gel in an electrode buffer
comprising 30 mM Tris pH 8,0, 200 mM glycine and 17 mM SDS.
[1084] The SDS-PAGE gels were initially run at a voltage of 60
V/sec for one hour, after which the voltage was turned up to 100
V/sec for additional two to three hours. The gels were run in an
electric field created by the Electrophoretic Constant Power Supply
(ECPS 2000/300) produced by Pharmacia Biotechnology.
[1085] Western Blot
[1086] After the separation of the mouse serum proteins, the gel
was equilibrated in CAPS buffer pH 9,0 (as specified under
3.1.3.4). An Immobilon-P Transfer membrane was equilibrated in
methanol for one minute and then washed with CAPS
(3-[Cyclohexylamino]-1 propane sulphonic acid, Sigma) buffer. The
separated mouse proteins were transferred from the SDS-PAGE gel to
the Immobilon membrane with a Biorad Transblot-SP semi-dry transfer
cell (power supply: ECPS 2000/300 from Pharmacia
Biotechnology).
[1087] The strips present on the Immobilon membrane were cut out
and blocked by incubation in TBS buffer pH 7,4 (20 mM Tris, 55 mM
NaCl) containing 1% m/v fat-free milk powder and 0,05% v/v Tween
20.
[1088] Each strip of the Immobilon membrane contained one lane of
the mouse serum and one lane of the mouse serum-mycolic acids
conjugate. The control strip comprised one lane of the standard Low
Molecular Weight Markers, one lane of the mouse serum and of the
mouse serum-mycolic acids conjugate. The control strip was stained
with Coomassie blue. The remaining strips were individually
incubated in either patient or control sera at 4.degree. C. for 16
hours. The sera of both types were diluted 1:6 v/v in the blocking
buffer (TBS pH 7,4, 1% m/v fat free milk powder and 0,05% v/v Tween
20).
[1089] The membrane strips were subsequently incubated with a
mixture of anti-human IgG+IgM peroxidase conjugate diluted 1:500
with the blocking buffer, at room temperature for three hours and
excess antibody was removed by three rinses in TBS buffer pH 7,4
containing 1% m/v fat free milk powder. The blots were developed by
adding the substrate, i.e.: 0,03 mM 4-chloronaphtol, 3% v/v
hydrogen peroxide in 20 ml methanol, made up to 100 ml with TBS
buffer pH 7,4.
[1090] 3.2.23 Methods Used in the Purification of CD4.sup.+ Single
Positive (SP), CD8.sup.+ Single Positive, CD4- and CD8-Double
Negative (DN) .alpha..beta. TCR Positive Cells from the Human
Peripheral Blood
[1091] Purification of these cells was performed according to the
procedure described by Niehues et al., (1994) with small
modifications.
[1092] Peripheral blood mononuclear cells (PBMC) were isolated from
100 to 200 ml of blood from healthy individuals, using density
gradient centrifugation over Ficoll-Hypaque. Red blood cells were
lysed with 0,015 M NH.sub.4Cl and the remaining cells were
resuspended in PBS buffer containing 1% v/v BSA and 0,01% m/v
sodium amide to a concentration of 10 to 20.times.10.sup.4
cells/ml. The cells were then incubated on ice with the specific
mouse monoclonal antibody recognising a framework determinant of
the .alpha..beta.TCR WT31, at 10 .mu.l/10.sup.4 cells.
[1093] After 30 min, the cells were washed and positively selected
with Dynal M-450 magnetic beads coated with a goat anti-mouse IgE
antibody. The volume ratio between target cells and the beads was
1:40. The cells and beads mixture was gently rotated for 30 min at
4.degree. C.
[1094] The cell suspensions were then exposed to a strong magnetic
field and the non-adherent cells were removed. Adherent cells were
allowed to detach by an overnight incubation at 37.degree. C. and
were then subjected to two rounds of immunodepletion with anti-CDS
mAb covalently coupled to magnetic beads and one round of depletion
with anti-CD4 mAb, both covalently coupled to magnetic beads (M-450
Dynal).
[1095] Immunomagnetic depletions were performed at bead to target
cell ratios of 40:1 with gentle rotation for 30 min at 4.degree.
C.
[1096] Cells bound to the anti-CD4 magnetic beads and to the
anti-CD8 magnetic beads were used directly as a source of T-cell
RNA.
[1097] The isolated CD4, CD8 and DN cells did not contain CD20,
CD13, CD14 or CD34.sup.+ cells and were >90% viable. The
assessment of the cell viability was carried out using propidium
iodide staining and Fluorescence Activated Cell Sorter (FACS).
[1098] Cells that did not bind to WT31 in the first selection step
were irradiated (30 Gy) and used as autologous total APCs (Antigen
Presenting Cells).
[1099] Induction of CD1 on autologous APC was carried out as
described by Porcelli, Morita and Brenner (1992). Human monocytes
were isolated from leucocyte concentrates originating from blood of
normal donors by plastic adherence and detached by incubation at
37.degree. C. in PBS with 0,53 mM EDTA. Adherent cells comprised
typically more than 90% CD 13.sup.+ and MHC class II.sup.+ but
tested negative for CD1a, -b and -c by surface staining. To induce
CD1 expression, monocytes were cultured in RPM1 1640 tissue culture
medium containing 10% fetal calf serum with 100 units/ml of GM-CSF
and IL-4 for 60 hours. The cells were collected by desorption using
PBS with 0,53 mM EDTA.
[1100] Proliferation assay: The cells were suspended in RPM1 1640
tissue culture medium supplemented with 2 mM glutamine, 0,25% m/v
refobacine and 5% v/v heat-inactivated, pooled human AB serum. SP
and DN cells were plated in triplicate into round-bottomed 96-well
tissue culture microplates, stimulated with mycolic acids (at
concentrations of 5, 25, and 50 .mu.g/ml and a mutagen,
phytohemagglutinin (PHA), at a concentration of 3.3 .mu.g/ml). The
cells were incubated for 72 hours at 37.degree. C. in a humidified
CO.sub.2 incubator (5% CO.sub.2).
[1101] During the final 16 hours of incubation, the cells in the
microplates were pulsed with .sup.3H-thymidine (0,5 .mu.Ci/well).
Proliferation of SP and DN cells was determined by incorporation of
.sup.3H-thymidine as measured by standard liquid scintillation
counting. Dose response curves were generated. Autologous,
irradiated (30 Gy) APC or CD1.sup.+ APC cells were added at a ratio
of 4:1 in all experiments. The results were expressed at mean cpm
+/- SEM, from which background values (medium alone) were
subtracted.
3.3 Results and Discussion
[1102] The results obtained concerning:
[1103] i) the influence of the modified method of purification on
yield and purity of mycolic acids;
[1104] ii) the structural analysis of mycolic acids originating
from M. tuberculosis using infra-red spectroscopy; and
[1105] iii) the stability of mycolic acids
[1106] were presented and discussed in sections 1.3.1, 1.3.2 and
1.3.3.
[1107] 3.3 Investigations of the Immunogenic Properties of
Countercurrent-Purified Mycolic Acids
[1108] These investigations were based on the following
experiments:
[1109] 3.3.1 The induction of antibodies against mycolic acids in
the experimental rats;
[1110] 3.3.2 The detection of anti-mycolic acids antibodies in
human tuberculosis patients;
[1111] 3.3.3 Response of human CD4 T cells to the in vitro
stimulation with mycolic acids.
[1112] The following results were obtained.
[1113] 3.3.1 The Induction of Antibodies Against Mycolic Acids in
the Experimental Animals
[1114] In order to determine the immunogenicity of mycolic acids,
suspensions of a methylester form of mycolic acids in oil were used
for the immunization of Sprague-Dawley rats, as described under
3.2.16. The antibody response was monitored and the ELISA results
obtained after a treatment period of 3 months, are presented in
FIG. 28. A dose related response was observed for the induction of
antibodies specific for mycolic acids, immobilized on the ELISA
plates as described under 3.2.17.
[1115] The results presented in FIG. 28 support the hypothesis that
mycolic acids are immunogenic in respect of being able to induce
anti-mycolic acids antibodies.
[1116] 3.3.2 The Detection of Anti-Mycolic Acids Antibodies in
Human Tuberculosis Patients
[1117] Two out of 58 human tuberculosis patients sera screened,
revealed the presence of antibodies recognising methyl-ester form
of mycolic acids (FIG. 29).
[1118] The specificity of these antibodies was confirmed by an
inhibition ELISA reaction carried out as described under 3.2.18.
The results obtained are presented in FIG. 30.
[1119] The results imply that mycolic acids shed from the cell
walls of M. tuberculosis infecting the human host may induce the
formation of anti-mycolic acids antibodies in patients. However,
the antibodies are produced at a low frequency, or there is a stage
of the infection during which the antibodies are not detected. The
antibodies are specific (FIG. 30), but their affinity is low due to
the high serum concentration required to give a detectable
signal.
[1120] Anti-mycolic acids antibodies from human tuberculosis
patients recognise a preferred serum protein of +/-80 kDa on
control mouse serum exposed to purified mycolic acids in
methyl-ester form. An illustration of this observation is given in
FIG. 31.
[1121] The result presented in FIG. 31 could not be easily
reproduced with the sera of other human tuberculosis patients,
probably due to the fact that these antibodies occur at low
frequencies. The +/-80 kDa mouse protein appears to enhance the
antigenicity of mycolic acids and its human homologue may therefore
also increase the immunogenicity of mycolic acids in blood of human
tuberculosis patients.
[1122] 3.3.3 Response of Human CD4 T Cells to the In Vitro
Stimulation with Mycolic Acids
[1123] In order to establish the extent of the human T-cell
response to mycolic acids stimulation, besides the known
stimulation of double negative (DN) T cells (Porcelli, Morita and
Brenner, 1992; Beckman et al., 1994), CD4 T cells and CD8 T cells
were exposed to CD1-expressing antigen presenting cells and the
cell proliferation was measured. The results are presented in FIGS.
32a and 32b.
[1124] The results in FIG. 32a indicate stronger stimulation of CD4
T cells by mycolic acids at a frequency higher than that of DN T
cells. This effect was not previously reported by Beckman et al.,
(1995) or other researchers.
[1125] On the other hand, upon exposure to non-CD1 expressing APCs,
CD4 T cells also showed a significant but weak cell proliferation
after mycolic acids treatment (FIG. 32b). No stimulation of DN T
cells was observed. This result may, therefore, suggest a non-CD1
dependent way of mycolic acids presentation by APC cells in order
to activate CD4 T cells.
[1126] On the basis of these results, immunoregulatory properties
of mycolic acids appear to be mediated by both DN and CD4 T cells,
but not by CD8 T cells.
CONCLUSIONS
[1127] 1. On the basis of the results reported above it can be
concluded that mycolic acids originating from M. tuberculosis H37Rv
purified using counter-current purification, possess
immunoregulatory and imunogenic properties and can be used in the
prophylaxis and/or therapy of diseases, particularly those induced
by or associated with Mycobacteria.
[1128] 2. Mycolic acids can protect mice against infection with M.
tuberculosis, especially when administered before the
infection/onset of the disease.
[1129] 3. The protection provided by mycolic acids manifested
itself mainly in the lungs. The lungs from the mycolic
acids-pre-treated animals were the only organ in which a
significantly reduced tubercle formation could be observed upon
macroscopic post-mortem assessment.
[1130] 4. On the basis of cytokine profiling of IL-12, IFN-.tau.,
TNF-.alpha. and TFG-.beta. in the organs of the experimental
animals, mycolic acids appear to induce upon pre-treatment a
pro-inflammatory mechanism of protection against tuberculosis.
[1131] 5. Preliminary evidence from IR V indicates that mycolic
acids from other species of Mycobacterium (e.g. M. vaccae) could
also induce the protective immune effects in the lungs.
[1132] 6. Mycolic acids impair the development of arthritic
symptoms when introduced before the administration of an
arthritis-inducing dose of heat-killed and freeze-dried cells of M.
tuberculosis H37Ra. This reveals an immunosuppressive regulatory
property of mycolic acids, which might be applied in the prevention
of auto-immune side-effects of bacterial infections, particularly
in the case of M. tuberculosis or mycobacteria infections.
[1133] 7. The results do not exclude a protective effect of mycolic
acids treatment after the administration of arthritis-inducing
doses of M. tuberculosis H37Ra.
[1134] 8. The immunogenic properties of mycolic acids were
confirmed in the experiments in which they induced the formation of
antibodies in the experimental animals upon immunization with these
compounds. Anti-mycolic acids antibodies occurring spontaneously
and detected in human serum were found to be specific but of low
affinity.
[1135] 9. Countercurrent-purified mycolic acids were also found to
stimulate human DN and CD4 T cells, but did not appear to have an
effect on CD8 T cells.
[1136] 10. No toxic effects of mycolic acids were detected in
control rats within the tested doses, i.e., between 8 and 50 .mu.g
for mice and 0,1 and 1 mg for rats.
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