Identification of specific differentially expressed antigens
United States Patent: 7,772,386
Issued: August 10, 2010
Inventors: Jungblut; Peter
(Berlin, DE), Kaufmann; Stefan H. E. (Berlin, DE), Schaible; Ulrich
(Berlin, DE), Mollenkopf; Hans (Berlin, DE), Raupach; Barbel (Berlin, DE),
Zimny-Arndt; Ursula (Berlin, DE), Lamer; Stephanie (Berlin, DE), Mattow;
Jens (Berlin, DE)
zur Forderung der Wissenschaften E.V. (Berlin, DE)
Appl. No.: 09/890,339
Filed: January 28, 2000
PCT Filed: January 28, 2000
PCT No.: PCT/EP00/00690
371(c)(1),(2),(4) Date: March
PCT Pub. No.: WO00/44392
PCT Pub. Date: August 03,
Training Courses -- Pharm/Biotech/etc.
The present invention relates to
compositions useful in immunization against pathogenic organisms of the
genus Mycobacterium and for diagnostic purposes. In particular, the
present invention relates to a composition comprising at least one protein
which is differentially expressed in a virulent strain as compared to an
avirulent strain of Mycobacteria. Furthermore, the invention relates to
compositions comprising fusion proteins, antigenic fragments, nucleic acid
molecules encoding the aforementioned proteinaceous compounds and/or
antibodies thereto. Additionally, the invention relates to pharmaceutical
and diagnostic compositions comprising or employing compounds of the
invention. In addition, the present invention relates to the use of the
compounds of the invention for the treatment of Mycobacterium induced
diseases and/or for the preparation of a vaccine for vaccination against
Mycobacterium induced diseases.
Description of the
This application is a continuation-of PCT
Application No. PCT/EP00/00690, filed Jan. 28, 2000, which claims the
benefit of EP99/101590.0, filed Jan. 29, 1999.
The instant application contains a Sequence Listing which has been
submitted in both a paper copy and a computer readable copy, and which
Sequence Listing is hereby incorporated by reference in its entirety.
The present invention relates to compositions useful in immunization
against pathogenic organisms of the genus Mycobacterium and for diagnostic
purposes. In particular, the present invention relates to a composition
comprising at least one protein which is differentially expressed in a
virulent strain as compared to an avirulent strain of a pathogenic
Mycobacterium. Furthermore, the invention relates to compositions
comprising fusion proteins, antigenic fragments, nucleic acid molecules
encoding the aforementioned proteinaceous compounds and/or antibodies
thereto. Additionally, the invention relates to pharmaceutical and
diagnostic compositions comprising or employing compounds of the
invention. In addition, the present invention relates to the use of the
compounds of the invention for the treatment of Mycobacterium induced
diseases and/or for the preparation of a vaccine for vaccination against
Mycobacterium induced diseases.
Several documents are cited throughout the text of this specification.
Each of the documents cited herein (including any manufacturer's
specifications, instructions, etc.) are hereby incorporated by reference;
however, there is no admission that any document cited is indeed prior art
of the present invention.
Since the beginning of the 1980s, a new trend has been observed in the
industrialized countries. On the one hand, resistances to antibiotics have
increased, which make it difficult or even impossible to treat many of the
disease-causing agents. On the other hand, new infectious diseases, which
had been unknown up to now, arise, and old diseases return. For example,
malaria and tuberculosis are old epidemics and increasingly surmounting in
many different parts of the world. Especially tuberculosis (TB), a chronic
infectious disease that is generally caused by infection with
Mycobacterium tuberculosis, is a disease of major concern. Each year, 8 to
10 million new cases of TB are described, and, causing more than three
million deaths per year, TB is a major disease in developing countries as
well as an increasing problem in developed areas of the world due to, for
example, antibiotic resistance.
Inhibiting the spread of TB will require effective vaccination and
accurate, early diagnosis of the disease. Currently, vaccination with live
bacteria is the most efficient method for inducing protective immunity.
The most common Mycobacterium for this purpose is Bacillus Calmette-Guerin
(BCG), an avirulent strain of Mycobacterium bovis.
However, the safety and efficacy of BCG is a source of controversy, and
some countries, such as the United States and the Netherlands, do not
vaccinate the general public.
Additionally, it has been shown that BCG vaccination affords greater
protection against leprosy than against tuberculosis (Ponninghaus, Lancet
339 (1992), 639). Furthermore, M. bovis BCG has failed to protect against
TB in several trials (WHO, Tech. Rep. Ser. (1980), 651, 1-15) for reasons
that are not entirely clear (Fine, Tubercle 65 (1984), 137-153).
Additionally, it has been shown that the vaccine strain of M. bovis BCG
only confers protection against the severe form of miliary tuberculosis in
children (Fine, Lancet 346 (1995), 1339-1345). In contrast, its protective
capacity against the most common form, pulmonary tuberculosis in adults,
is low and highly variable (Colditz (1994), JAMA 271, 698).
Diagnosis of TB is commonly achieved using a skin test, which involves
intradermal exposure to tuberculin PPD (protein-purified derivative).
Antigen-specific T cell responses result in measurable induration at the
injection site by 48-72 hours after injection, which indicates exposure to
Mycobacterial antigens. Sensitivity and specificity have, however, been a
problem with this test, and individuals vaccinated with BCG cannot be
distinguished from infected individuals.
Therefore, it is of major concern that effective and safe vaccines and
therapies for the immunization and the treatment of TB as well as useful,
reliable diagnostics be developed.
The technical problem of the present invention was thus to provide
compositions useful for effective immunization against pathogenic
organisms, for effective therapy of infected humans and animals that can
be reliably used in low doses and with substantially no side effects
and/or for detection/diagnosis of pathogenic organisms in
The solution to this technical problem is achieved by providing the
embodiments characterized in the claims.
Accordingly, the present invention relates to a composition comprising at
least one protein which is differentially expressed in a virulent strain
as compared to an avirulent strain of the genus Mycobacterium.
The term "composition", as used in accordance with the present invention,
comprises at least one protein, an antigenic fragment of said protein, a
fusion protein, a nucleic acid molecule and/or an antibody of this
invention and, optionally, further molecules, either alone or in
combination, like e.g. molecules which are capable of optimizing antigen
processing, cytokines, immunoglobulins, lymphokines or CpG-containing DNA
stretches or, optionally, adjuvants. The composition may be in solid,
liquid or gaseous form and may be, inter alia, in form of (a) powder(s),
(a) tablet(s), (a) solution(s) or (an) aerosol(s). In a preferred
embodiment, said composition comprises at least two, preferably three,
more preferably four, most preferably five differentially expressed
The term "protein" means, in accordance with the present invention, a
peptide(s) or (a) (poly)peptide(s) which encompass amino acid chains of
any length, wherein the amino acid residues are linked by covalent peptide
bonds. However, peptidomimetics of such proteins wherein amino acid(s)
and/or peptide bond(s) have been replaced by functional analogs are also
encompassed by the invention. In accordance with this invention, a protein
may comprise different protein species. A protein species is defined by
its chemical composition and modifications of said peptide(s)/(poly)peptide(s)
by, inter alia, glycosylations, acetylations, phosphorylations,
lipidations or by amino acid exchanges, the term describes a chemically
clearly-defined molecule and corresponds, inter alia, to one spot on a
high-performace 2-DE pattern (Jungblut, Electorphoresis 17 (1996),
839-847). The term protein species is therefore defined as the smallest
unit of a protein classification, defined by its chemical structure.
The term "differentially expressed" denotes in the context of the present
invention proteins/protein species which are distinctly expressed,
regulated and/or modified. Therefore, the term "differentially expressed"
includes protein(s)/protein species that are absent in, that occur in
different amounts in and/or that comprise different post-translating
modifications in a "virulent" strain compared to an "avirulent" strain of
a pathogenic organism. The term "differentially expressed" as used in
accordance with the invention denotes therefore not only proteins/protein
species which are missing in one strain as compared to another (+/-
variants), but also comprises mobility variants and/or intensity variants.
Intensity variants are protein species occurring in comperative protein
2DE-patterns which differ in amount. A +/- variant can be considered as an
extreme intensity variant, where the protein species occurs in one pattern
and is absent in the other. If the protein occurs in two different
compared patterns at different positions, these two positions can be
considered as indication for two different protein species of this protein
(inter alia, due to secondary modifications as explained herein above)
which are defined as mobility variants. These variants (+/-, intensity or
mobility) can be detected by proteome analysis.
Previously, the determination of immunogenic antigenic and/or pathogenic
determinants of pathogenic organisms had been hampered by the fact that it
was not possible to analyze the whole proteome of such organisms, like
Mycobacteria, by conventional means. However, the previously employed
analysis of cellular fractions and/or fragments (like bacterial membranes)
can only reflect a limited number of differentially expressed protein(s)/protein
species, if any, due to the loss of proteinaceous material during
fractionation and isolation of such fragments. In accordance with the
present invention, a new method (as examplified in the examples) has been
employed that allows the analysis of whole pathogenic organisms and it was
surprisingly found, that a great number of differentially expressed
proteins in a virulent strain as compared to an avirulent strain of
Mycobacteria can be identified.
Differentially expressed proteins (protein species) may be identified,
detected and/or brought into a biological correlation, inter alia, by
proteome analysis of whole organisms (like mycobacteria) or, less
preferred, of biochemically defined fractions (like, inter alia,
lipoproteins, glycoproteins, phosphoproteins) or of biologically defined
fractions (like, inter alia, membranes, cytosol, structural elements of a
pathogenic organism); see, e.g. Wilkins (1997), "Proteome Research: New
Frontiers in Functional Genomics, Springer-Publishers Berlin; Kahn,
Science 270 (1995), 369-370; Jungblut, J. Biotech. 41 (1995), 111-120;
Bluggel, Biospektrum 5 (1998), 39-44; Lohaus, Biospekturm 5 (1998), 32-39;
Jungblut Electrophoresis 17 (1996), 839-847; Scheler, Electrophoresis 19
As known to the person skilled in the art, analysis of proteomes of lower
complexity, e.g. ribosomes with 60 protein species, can be performed,
inter alia, by protein/protein species separation and identification
strategies, comprising, for example, 2-dimensional gel electrophoresis
(2-DE; Kaltschmidt, Anal. Biochem. 36 (1970), 401) or HPLC (Kamp, J.
Chromatogr. 317 (1984), 181). However, analysis of proteomes of higher
complexity can be carried out, inter alia, by a combination of isoelectric
focusing and SDS-PAGE (Vesterburg, Acta Chem. Scand. 20 (1966), 820;
Laemmli, Nature 227 (1970), 680) and the use of large-sized gels (Jungblut,
Electrophoresis 15 (1994), 685; Klose, Electrophoresis 16 (1995), 1034).
Comparison of individual, specific 2-DE gels allows for the identification
of differentially expressed proteins and the identification of proteins
separated by 2-DE is known to the skilled artisan (see, e.g. Patterson,
Electrophoresis 16 (1995), 1791; Jungblut, Electrophoresis 17 (1996), 839;
Jungblut, Mass Spectrometry Reviews 16 (1997), 145; Kaufmann, Jahrbuch der
MPG (1998), 42-57; Bluggel (1998), loc. cit., Schaible, DGHM-Kongress
(1998), Einhoon-Resse Verlag (ISSN 1433-3988), 20).
In order to further identify differentially expressed proteins, several
techniques which are known in the art can be used. These techniques
comprise, but are not limited to, in-gel digestions, electroelution
procedures, microsequencing, amino acid analysis, Edman-sequencing or mass
spectroscopy. For example, some techniques start directly from gel(s),
others need a transfer to membranes by blotting. To the first group
belong, inter alia, coelectrophoresis, internet comparison of position,
peptide mapping by SDS-PAGE (Cleveland, J. Biol. Chem. 252 (1977), 1102),
protein elution and MALDI-MS or N-terminal sequencing by Edman degradation
(Edman, Acta Chem. Scand. 4 (1950), 283), enzymatic in-gel digestion,
analysis of peptides directly in the mixture by mass spectrometry, peptide
mass fingerprinting (Pappin, Curr. Biol. 3, (1993), 327), PSD-MALDI-MS
(Spengler, Rapid Commun. Mass Spectrom. 6, (1992), 105), ESI-MS (electrospray-ionization-MS)
and/or (after separation) by micro-HPLC. HPLC separated peptides may be
further analysed, inter alia, by Edman degradation, PSD-MALDI-MS, MS/MS (Wilm,
Nature 379, (1996), 466) or ladder sequencing (Thiede, FEBS Lett. 357,
(1995), 65) in order to obtain a peptide sequence. Proteins immobilized on
membranes allow the identification by immunostaining (Towbin, Proc. Natl.
Acad. Sci. USA 76, (1979), 4350), N-terminal sequencing (either directly
or after deblocking) (Hirano, Electrophoresis 14, (1993), 839),
determination of the protein mass (Eckerskorn, Electrophoresis 13, (1992),
664), amino acid analysis (Jungblut, J. Prot. Chem. 11, (1992), 603)
and/or enzymatic digestion with the same proteinchemical techniques as
described for in-gel digestions. Results of such analysis are mass
The resulting peptide masses are searched by search programs (e.g.
in sequence databases (EMBL, PIR, NCBI, MIPS, Swiss-Prot, OWL). By use of
such mass fingerprints amino acid sequences can be deduced and sequenced.
From these sequenced amino acid fragments degenerative oligonucleotides
may be deduced and synthesized that may be used to screen, for example,
genomic or cDNA libraries to identify and clone the corresponding GENE/cDNA.
Identified proteins may be produced by, for example, recombinant
techniques or by biochemical or synthetic techniques which are known to
the skilled artisan (Sambrook et al., "Molecular Cloning, A Laboratory
Manual", Cold Spring Harbor Laboratory, N.Y. (1989); Ausubel, "Current
Protocols in Molecular Biology", Green Publishing Associates and Wiley
Interscience, N.Y. (1989)).
Other methods for the elucidation of differentially expressed proteins
include, but are not limited to, enzyme activity, receptor activity
measurements, immunostainings, immunohistochemical methods.
As shown in the appended examples, differential protein expression can be
detected by preparation of microorganisms or, less preferred,
compartment/fragments thereof, 2-DE, subtractive analysis and
identification of proteins by peptide mass fingerprinting (PMF) with or
without confirmation by further methods.
Identification of protein species from 2-DE patterns by only one of the
above-described methods, peptide mass fingerprinting or amino acid
analysis, was described to lead to false identification (Cordwell,
Electrophoresis 16 (1995), 438; Mortz, Biol. Mass. Spec. 23 (1993), 249).
However, the present invention, surprisingly showed that differentially
expressed proteins may be identified by peptide mass fingerprinting
without confirmation by a further method. As examplified in the appended
examples, improvements in the sample preparation, e.g. reduction of
volumes and surface contacts, use of volatile buffers and improvements in
mass spectrometry, introduction of delayed extraction, results in improved
mass accuracy, resolution, and sensitivity, leading to high sequence
coverage of at least 30%. This sequence coverage is sufficient for
identification and needs no further confirmation. Thus, the present
invention also concerns a method for identification of differentially
expressed proteins as discussed above and illustrated in examples 2, 4 and
The term "virulent strain", in accordance with the present invention,
denotes the capacity of a pathogenic strain of the genus Mycobacterium to
infect a host and/or to cause disease--defined broadly in terms of
severity of symptoms in a host. Thus, a "virulent strain" might cause
symptoms in a susceptible host, whereas another host might be unaffected
by this strain, which can be therefore considered as being an "avirulent
strain" in this second host. As used in accordance with the present
invention, the term "avirulent strain" denotes strains of a Mycobacteria
which is not capable of inducing infection and/or causing disease in a
specific host or in a host species. The term "avirulent strains" denotes
furthermore attenuated strains of microorganisms.
The terms "virulent" and "avirulent" strains not only relate to laboratory
strains but also comprise wildtype strains. The virulency of a strain is
known in the art and described, inter alia, in Brandis et al., "Lehrbuch
der medizinischen Mikrobiologie", Gustav Fischer Verlag, 7. Auflage
(1994), Zinsser Microbiology, ed Joklik, Willett, Amos, Wilten 20.sup.th
edition, Appleton & Lange, 1992.
In a preferred embodiment of the composition of the present invention said
strains are selected from the group consisting of M. tuberculosis, M.
bovis, M. avium, M. africanum, M. kanasasii, M. intracellulare, M.
ulcerans, M. paratuberculosis, M. simiae, M. scrofulaceam, M. szulgai, M.
xenopi, M. fortuitum, M. chelonei M. leprae and M. marinum.
In a more preferred embodiment of the composition of the present invention
said protein is differentially expressed in M. tuberculosis and in M.
In a particularly preferred embodiment the present invention relates to a
composition wherein said virulent strain is M. tuberculosis H37Rv or M.
tuberculosis Erdman and said avirulent strain is M. bovis BCG.
Furthermore, the present invention relates to a composition wherein said
protein is differentially expressed in M. tuberculosis H37Rv and M.
tuberculosis Erdman as compared to M. bovis BCG.
In an even more preferred embodiment of the composition of the present
invention said differentially expressed protein is 2-isopropyl malate
synthase (Rv3710), s-adenosylmethionine synthase (metK, RV1392),
succinyl-CoA synthase a-chain (sucD, RV0952), oxidoreductase of aldo/keto
reductase family (Rv2971), oxidoreductase (Rv0068), elongation factor G
(FusA2, Rv0120c), uridylate kinase (PyrH, Rv2883c), ABC-type transporter
(Rv1463), short chain dehydrogenase/reductase family (RV1856C), hydrolase
(LinB, Rv2579), phosphoribosylamino-imidazole carboxylase catalytic
subunit (PurE, Rv3275c), hypothetical protein (Rv2557), hypothetical
protein (Rv3407), hypothetical protein (Rv3881c), hypothetical protein
(Rv2449c), hypothetical protein (Rv0036c), hypothetical protein (Rv2005c)
or transcriptional regulator (Crp/Fr family) (Rv 3676). As shown in the
appended examples, whereas 2-isopropyl malate synthase (Rv3710) is
expressed in M. tuberculosis H37Rv, it is not detected and identified in
M. bovis BCG. Furthermore, s-adenosylmethionine synthase (metK, RV1392),
succinyl-CoA synthase a-chain (SUCD, Rv0952), oxidoreductase of aldo/keto
reductase family (Rv2971) or oxidoreductase (Rv0068), represent protein
species which are differentially expressed in M. tuberculosis H37Rv and M.
bovis BCG and represent mobility variants. As intensity variants may be
considered proteins corresponding to the Rv numbers Rv0652, Rv2429,
Rv2428, RV0569, Rv0475, Rv3463, Rv3054c. As +/--variants may be considered
Rv2883c, Rv0120c, Rv1463, Rv2579, Rv3275c, Rv3407, Rv3881c, Rv2449c,
Rv0036c, Rv2005c or Rv3676. As shown in the appended examples, whereas
elongation factor G (Rv0120c), uridylate kinase (Rv2883c), ABC-type
transporter (Rv1463), short chain dehydrogenase/reductase family protein (Rv
1856c), 1,3,4,6-tetracholoro-1,4,-cyclohexadiene hydrolase (Rv2579),
phosphoribosylaminoimidazole carboxylase catalytic subunit (Rv3275c),
hypothetical protein (Rv2557), and hypothetical protein (Rv3407) are
expressed in M. tuberculosis H37Rv and M. tuberculosis Erdman, they are
not detected in M. bovis BCG Chicago and M. bovis BCG Copenhagen.
Furthermore, protein spot A607 in M. tuberculosis H37Rv and the
corresponding spot A148 in M. tuberculosis Erdman have no counterparts in
M. bovis BCG Chicago and M. bovis BCG Copenhagen. This protein was
identified herein as the hypothetical protein Rv3881c. Furthermore, spots
C434 from M. tuberculosis H37Rv and the corresponding spot C508 from M.
tuberculosis Erdman have no counterparts in M. bovis BCG Chicago and M.
bovis Copenhagen. They were identified as a hypothetical protein
(Rv2005c). Rv2005c occurs at the 2-DE pattern in another form at a
different position in all four strains. Additionally, the spots B69, C176,
D12 and D115 of M. tuberculosis H37Rv with their counterparts in M.
tuberculosis Erdman, B54, C404, D115 and D130, respectively, have no
counterparts in M. bovis BCG Chicago and M. bovis BCG Copenhagen. B69 was
identified as a hypothetical protein (Rv2449c). C176 was identified as a
hypothetical protein (Rv0036c). D12 and D115 of M. tuberculosis H37Rv were
identified as transcriptional regulator (Crp/Fnr family) (Rv3676). As will
be described herein below these proteins/protein species might serve,
inter alia, in pharmaceutical and diagnostic compositions. Cole (Nature
393 (1998), 537) published the complete sequence of the M. tuberculosis
H37Rv genome and identified a total of 3924 individual genes which were
classified according to the classification of Riley (Microbiol. Rev. 57
(1993), 862). Identifications of this putative genes were performed by
homology searches of deduced open reading frames from other
microorganisms. Therefore, the term "Rv numbers" as employed herein
corresponds to clearly defined nucleic acid sequences (deduced open
reading frames) as describes in Cole et al., (loc. cit.). However, for
most of the identified putative genes of M. tuberculosis, it is not
clearly shown that they are actually expressed additional sequence
information on mycobacterial genes is also available from the Sanger
Centre, U. K. Under www.sanger.ac.uk/Projects/M_tuberculosis/ information
on the genomic sequence of M. tuberculosis is available. Therefore, the "Rv-numbers"
not only refer to nucleic acid sequences but also to protein sequences as
deposited in the Sanger database. Further information on M. tuberculosis
sequence is available from the Institut Pasteur, Paris under
The invention also relates to a composition comprising an antigenic
fragment of the protein as defined herein.
The term "antigenic fragment", as used herein, refers to the ability of
said fragment to elicit an immune response (e.g. humoral or cellular) in a
subject, such as a human, and/or in a biological sample. These fragments
may consist entirely of the antigenic and/or immunogenic portion of the
protein or may contain additional sequences. The additional sequences may
be derived from said protein or may be heterologous, and such additional
sequences may (but need not) be antigenic and/or immunogenic. The
antigenicity of an amino acid sequence can be deduced/predicted by methods
known to the person skilled in the art as for example described in Parker,
J. Immunol. 152 (1994), 163 (bimas.dcrt.nih.gov:80/molbio/hla_bind/),
Meister, Vaccine 13 (1995), 581-591 or Bull, Biochem. Biophys. 161 (1974),
665-670. Furthermore, computer predictions may be employed to elucidate
hydrophilicity and/or antigenicity of amino acid sequences and stretches.
Such computer programs may be Garnier analysis of the on the plot v. 2.5e
package, the GCG-software derived from HGMP resource Center Cambridge
(Rice (1995) Programme Manual for the EGCG package, Cambridge (B10 IKQ,
England) or the programme based on Kyte/Dolittle, J. Mol. Biol. 157
(1982), 105-132 (see also www.expasy.ch/cgi-bin/protscale.pl).
Antigenic fragment may be produced recombinantly using a polynucleotide
sequence that encodes the antigenic fragment or may be produced by
biochemical or synthetic techniques. Those methods are known to those of
ordinary skill in the art (see, e.g. Sambrook et al., loc. cit.; Harlow
and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor,
N.Y. (1988); Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2146; Stewart,
"Solid Phase Peptide Synthesis", WH Freeman Co, San Francisco (1969);
Scopes, "Protein Purification", Springer Verlag, New York, Heidelberg,
Berlin (1987); Janson, "Protein Purification, Principles, High Resolution
Methods and Applications", VCH Publishers, New York, Weinheim, Cambridge
(1989); Wrede, "Concepts in Protein Engineering and Design", Walter de
Gruyter, Berlin, N.Y. (1994); Wittmann-Liebold, Jungblut "Analysis and
Characterization of Proteins", 47-107).
Additionally, the invention relates to a fusion protein comprising a
protein and/or an antigenic fragment as defined in the above.
The protein and/or the antigenic fragment of the present invention can
comprise a further domain, said domain being linked by covalent or
non-covalent bonds. The linkage can be based on genetic fusion according
to the methods known in the art (Sambrook et al., loc. cit.; Ausubel, loc.
cit.) or can be performed by, e.g., chemical cross-linking as described
in, e.g., WO 94/04686. The additional domain present in the fusion protein
comprising the protein of the invention may be joined directly (i.e. with
no intervening amino acids) or may be linked by a flexible linker,
advantageously a polypeptide linker, wherein said polypeptide linker
comprises plural, hydrophilic, peptide-bonded amino acids of a length
sufficient to span the distance between the C-terminal end of said further
domain and the N-terminal end of the protein or vice versa. The above
described fusion protein may further comprise a cleavable linker or
cleavage site, which, for example, is specifically recognized and cleaved
by proteinases or chemical agents. Cleavable linker sequences include, but
are not limited to, Factor XA or enterokinase (Invitrogen, San Diego,
Additionally, said further domain may be of a predefined specificity or
function. In this context, it is understood that the protein of the
invention may be further modified by conventional methods known in the
art. This allows for the construction of fusion proteins comprising the
protein of the invention and other functional amino acid sequences, e.g.,
immunologically relevant proteins like cytokines, lymphocytes,
interferones, or protein tags (GST, GFP, h-myc peptide, FLAG, HA peptide)
which may be derived from heterologous proteins.
In yet another preferred embodiment the present invention relates to a
composition comprising at least one differentially expressed protein as
defined herein above wherein said differentially expressed protein is
biochemically, biophysically and/or recombinantly modified. Such
modifications may comprise amino acid substitutions, deletions,
insertions, additions and/or duplications wherein said modified
differentially expressed protein should still comprise at least one
antigenic fragment or epitope which is specifically recognized by an
antibody directed to, raised to and/or engineered to detect the
non-modified differentially expressed protein as defined herein above. The
non-modified amino acid sequence of a differentially expressed protein is
deducible for the person skilled in the art as described herein above,
inter alia, by employing biochemical and recombinant methods and sequence
databases. Additionally, the non-modified amino acid sequence of a
differentially expressed protein as defined herein above may be deduced
from nucleic acid sequences and/or proposed open reading frames as known
to the person skilled in the art. For example, the complete genome
sequence of M. tuberculosis H37Rv is published in Cole et al. (1998, loc.
In addition, the invention relates to a fusion protein comprising at least
two proteins as defined herein and/or (an) antigenic fragment(s) as
In a further embodiment the fusion protein of the present invention
comprises an immunostimulatory molecule.
The term "immunostimulatory molecule" denotes in accordance with the
present invention molecules or fragments thereof which, inter alia,
activate and/or stimulate the humoral and cellular response of an immune
system. They might, e.g. activate antigen-presenting cells, stimulate
natural killer cells, enhance the production of antibodies directed
against an antigen and/or a pathogen or induce the proliferation of cells
of the immune system. These molecules are known in the art and comprise,
inter alia, cytokines, lymphokines, immunoglobulins, interleukins and/or
complement factors (see, e.g. Paul, "Fundamental Immunology", Raven Press
(1989); Schaible, Adv. In Immunology 71 (1999), 261-377).
In one further preferred embodiment of the fusion protein of the present
invention said fusion protein comprises a molecule capable of optimizing
Cellular immune recognition is mediated by a special class of lymphoid
cells, T-cells. These cells do not recognize whole antigens but instead
they respond to degraded peptide fragments thereof which appear on the
surface of the target cell bound to proteins called major
histocompatibility complex (MHC) molecules (antigen processing).
Essentially all nucleated cells have MHC class I molecules, whereas MHC II
are restricted to immune cells with special presenting qualities.
Molecules which are capable of optimizing antigen processing are known in
the art and comprise, inter alia, listeriolysin, which improves MHC class
I restricted immune responses (see, e.g., Hess, PNAS 95 (1998),
The term "fusion protein" as employed hereinabove also relates to chimeric
proteins wherein said chimeric protein comprises at least one
differentially expressed protein and/or (a), preferably antigenic,
fragment(s) thereof in combination with at least one other protein,
peptide or fragment(s) thereof. Furthermore, said chimeric protein may
comprise at least two modified differentially expressed proteins as
defined herein above.
The invention also relates to a composition comprising at least one fusion
protein as defined hereinabove.
The invention further relates to a nucleic acid molecule coding for a
modified differentially expressed protein as defined herein, the antigenic
fragment as defined herein and/or a fusion protein as defined herein.
The nucleic acid molecule of the invention or employed in methods or
compositions of the invention may be DNA such as cDNA or RNA such as mRNA.
Additionally, the nucleic acid molecule of the invention may be PNA. Its
origin may be natural, synthetic or semisynthetic or it may be a
derivative, such as said peptide nucleic acid (Nielsen, Science 254
(1991), 1497-1500). Furthermore, said nucleic acid molecule may be a
recombinantly produced chimeric nucleic acid molecule comprising any of
the aforementioned nucleic acid molecules either alone or in combination.
Preferably, said nucleic acid molecule is part of a vector.
Such vectors may be, e.g., a plasmid, cosmid, virus, bacteriophage or
another vector used e.g. conventionally in genetic engineering, and may
comprise further genes such as marker genes which allow for the selection
of said vector in a suitable host cell and under suitable conditions.
Furthermore, the vectors may, in addition to the nucleic acid sequences of
the invention, comprise expression control elements, allowing proper
expression of the coding regions in suitable hosts. Such control elements
are known to the artisan and may include a promoter, translation
initiation codon, translation and insertion site for introducing an insert
into the vector. Preferably, the nucleic acid molecule of the invention is
operatively linked to said expression control sequences allowing
expression in eukaryotic or prokaryotic cells.
Control elements ensuring expression in eukaryotic and prokaryotic cells
are well known to those skilled in the art. As mentioned above, they
usually comprise regulatory sequences ensuring initiation of transcription
and optionally poly-A signals ensuring termination of transcription and
stabilization of the transcript. Additional regulatory elements may
include transcriptional as well as translational enhancers, and/or
naturally-associated or heterologous promoter regions. Possible regulatory
elements permitting expression in for example mammalian host cells
comprise the CMV-HSV thymiakine kinase promoter, SV40, RSV-promoter (Rous
sarcome virus), human elongaticn factor 1.alpha.-promoter, CMV enhancer or
SV40-enhancer. For the expression in prokaryotic cells, a multitude of
promoters including, for example, the tac-lac-promoter or the trp
promoter, has been described. Beside elements which are responsible for
the initiation of transcription such regulatory elements may also comprise
transcription termination signals, such as SV40-poly-A site or the tk-poly-A
site, downstream of the polynucleotide. In this context, suitable
expression vectors are known in the art such as Okayama-Berg cDNA
expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene),
pSPORTI (GIBCO BRL), or prokaryotic expression vectors, such as lambda
gt11. Beside the nucleic acid molecules of the present invention, the
vector may further comprise nucleic acid sequences encoding for secretion
signals. Such sequences are well known to the person skilled in the art.
Furthermore, depending on the expression system used leader sequences
capable of directing the protein/(poly)peptide to a cellular compartment
may be added to the coding sequence of the nucleic acid molecules of the
invention and are well known in the art. The leader sequence(s) is (are)
assembled in appropriate phase with translation, initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein, or a protein thereof, into the
periplasmic space or extracellular medium. Optionally, the heterologous
sequence can encode a fusion protein including an C- or N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant product.
Once the vector has been incorporated into the appropriate host, the host
is maintained under conditions suitable for high level expression of the
nucleotide sequences, and, as desired, the collection and purification of
the proteins, antigenic fragments or fusion proteins of the invention may
follow. Of course, the vector can also comprise regulatory regions from
Furthermore, said vector may also be a gene transfer or targeting vector.
Gene therapy, which is based on introducing therapeutic genes (for example
for vaccination) into cells by ex-vivo or in-vivo techniques is one of the
most important applications of gene transfer. Suitable vectors, vector
systems and methods for in-vitro or in-vivo gene therapy are described in
the literature and are known to the person skilled in the art; see, e.g.,
Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79
(1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348
(1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature
Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, Current
Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389 (1997),
239-242 and references cited therein. The nucleic acid molecules of the
invention and vectors as described herein above may be designed for direct
introduction or for introduction via liposomes, or viral vectors (e.g.
adenoviral, retroviral) into the cell. Additionally, a baculoviral system
can be used as eukaryotic expression system for the nucleic acid molecules
of the invention. In addition to recombinant production, fragments of the
protein, the fusion protein or antigenic fragments of the invention may be
produced by direct peptide synthesis using solid-phase techniques (cf
Stewart et al. (1969) Solid Phase Peptide Synthesis, WH Freeman Co, San
Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154). In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City
Calif.) in accordance with the instructions provided by the manufacturer.
Various fragments may be chemically synthesized separately and combined
using chemical methods to produce the full length molecule.
The invention in addition relates to a composition comprising at least one
nucleic acid molecule as defined herein and/or at least one nucleic acid
molecule coding for any of the differentially expressed proteins as
defined herein. Said nucleic acid molecule coding for a differentially
expressed protein, codes preferably for Rv3710, Rv1392, Rv0952, Rv2971,
Rv0068, Rv0120c, Rv2883c, Rv1463, Rv1856c, Rv2579, Rv3275c, Rv2557,
Rv3407, Rv3881c, Rv2449c, Rv0036c, Rv2005c or Rv3676.
Most preferably said nucleic acid molecule is the nucleic acid molecule as
disclosed under said Rv-number under www.sanger.ac.uk/Projects/M_tuberculosis
or bioweb.pasteur.fr/GenoList/TubercuList. However, the present invention
relates also to compositions comprising at least one Nucliec acid molecule
which hybridizes under stringent conditions to the complementary strand of
the nucleic acid molecule of any of the above cited Rv-numbers. "Stringent
conditions" are preferably conditions as described in Sambrook (Molecular
Cloning, A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
Such hybridizing sequences show preferably an identity of at least 50%,
more preferably of at least 70% and most preferably of at least 90% on the
nucleic acid level to the sequences described above. The molecules
hybridizing to the nucleic acid molecules as disclosed under the above
cited Rv-numbers or to the nucleic acid molecules of the invention thus
also comprise fragments, derivatives and allelic variants of the
above-described nucleic acid molecules which encode a differentially
expressed protein (or a fragment thereof) as described in the present
invention. In this regard, fragments are defined as parts of the nucleic
acid molecules, which are long enough in order to encode the at least one
epitope/antigenic fragment which is specifically recognized by an antibody
directed to, raised to and/or engineered to detect a differentially
expressed protein as defined herein. The term derivatives means that the
sequences of these hybridizing molecules differ from the sequences of the
above-mentioned nucleic acid molecules at one or more positions and that
they exhibit a high degree of homology to these sequences. Hereby,
homology means a sequence identity of at least 50%, in particular an
identity of at least 60%, preferably of more than 70% and still more
preferably a sequence identity of more than 90%. The deviations occurring
when comparing with the above-described nucleic acid molecules might have
been caused by deletion, substitution, insertion or recombination.
Said composition is useful, inter alia, for medical and diagnostic
purposes, in particular, for pharmaceutic and vaccination purposes.
Moreover, the invention relates to an antibody or a fragment or a
derivative thereof directed against the protein as defined herein, the
antigenic fragment of the invention, the nucleic acid molecule of the
invention or the fusion protein as defined herein. Such antibodies may
include, but are not limited to, polyclonal, monoclonal, chimeric or
single chain antibodies or fragments or derivatives of such antibodies.
The general methodology for producing antibodies is well-known and has
been described in, for example, Kohler and Milstein, Nature 256 (1975),
494 and reviewed in J. G. R. Hurrel, ed., "Monoclonal Hybridoma
Antibodies: Techniques and Applications", CRC Press Inc., Boco Raron, Fla.
(1982), as well as that taught by L. T. Mimms et al., Virology 176 (1990),
604-619. As stated above, in accordance with the present invention the
term "antibody" relates to monoclonal or polyclonal antibodies. Antibody
fragments or derivatives comprise F(ab').sub.2, Fab, Fv or scFv fragments;
see, for example, Har'ow and Lane, "Antibodies, A Laboratory Manual", CSH
Press 1988, Cold Spring Harbor, N.Y. Preferably the antibody of the
invention is a monoclonal antibody. Furthermore, in accordance with the
present invention, the derivatives can be produced by peptidomimetics.
Such production methods are well known in the art and can be applied by
the person skilled in the art without further ado.
Furthermore, the invention relates to a composition comprising at least
one antibody, a fragment or a derivative thereof as defined above. Such
antibodies, fragments or derivatives can be used for diagnostic or for
pharmaceutical purposes, i.e. for the treatment of Mycobacteria-induced
diseases or the vaccination against these pathogens.
The invention also relates to a composition as defined above which is a
pharmaceutical composition further comprising, optionally, a
pharmaceutically acceptable carrier.
The pharmaceutical composition may comprise the proteins of the present
invention, the fusion proteins of the present invention, antigenic
fragments of the invention and/or antibodies (or their fragments or
derivatives) of the invention, either alone or in combination. The
pharmaceutical composition of the present invention may be used for
effective therapy of infected humans and animals and/or for vaccination
The pharmaceutical composition of the present invention may further
comprise a pharmaceutically acceptable carrier, excipient and/or diluent.
Examples of suitable pharmaceutical carriers are well known in the art and
include phosphate buffered saline solutions, water, emulsions, such as
oil/water emulsions, various types of wetting agents, sterile solutions
etc. Compositions comprising such carriers can be formulated by well known
conventional methods. These pharmaceutical compositions can be
administered to the subject at a suitable dose. Administration of the
suitable compositions may be effected by different ways, e.g., by
intravenous, intraperitoneal, subcutaneous, intramuscular, topical,
intradermal, intranasal or intrabronchial administration. The dosage
regimen will be determined by the attending physician and clinical
factors. As is well known in the medical arts, dosages for any one patient
depends upon many factors, including the patient's size, body surface
area, age, the particular compound to be administered, sex, time and route
of administration, general health, and other drugs being administered
concurrently. Proteinaceous pharmaceutically active matter may be present
in amounts between 1 ng and 10 mg per dose; however, doses below or above
this exemplary range are envisioned, especially considering the
aforementioned factors. Administration of the suitable compositions may be
effected by different ways, e.g., by intravenous, intraperitoneal,
subcutaneous, intramuscular, topical or intradermal administration. If the
regimen is a continuous infusion, it should also be in the range of 1 .mu.g
to 10 mg units per kilogram of body weight per minute, respectively.
Progress can be monitored by periodic assessment. The compositions of the
invention may be administered locally or systemically. Administration will
generally be parenterally, e.g., intravenously. The compositions of the
invention may also be administered directly to the target site, e.g., by
biolistic delivery to an internal or external target site or by catheter
to a site in an artery. Preparations for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for
example, antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like. Furthermore, the pharmaceutical composition of the invention
may comprise further agents such as interleukins, interferons and/or CpG-containing
DNA stretches, depending on the intended use of the pharmaceutical
In a preferred embodiment of the present invention the pharmaceutical
composition as defined herein is a vaccine.
Vaccines may be prepared, inter alia, from one or more proteins,
derivatives of the proteins, nucleic acid molecules, fusion proteins,
antigenic fragments or antibodies, fragments of said antibodies or
derivatives of the antibodies of the invention.
For example, nucleic acid molecules of the invention may be used for gene
vaccination or as DNA vaccines. Routes for administration of gene/DNA
vaccines are well known in the art and DNA vaccination has been
successfully used to elicit alloimmune, anti-tumor and antiidiotype immune
responses (Tighe M. et al., Immunology Today 19 (1998), 89-97). Moreover,
inoculation with nucleic acid molecules/DNA has been found to be
protective in different modes of disease (Fynan, Proc. Natl. Acad. Sci.
U.S.A. 90 (1993), 11478-11482; Boyer, Nat. Med. 3 (1997), 526-532;
Webster, Vaccine 12 (1994), 1495-1498; Montgomery et al., DNA Cell Biol.
12 (1993), 777-783; Barry, Nature 311 (1995), 632-635; Xu and Liew,
Immunology 84 (1995), 173-176; Zhoug, Eur. J. Immunol. 26 (1996),
2749-2757; Luke, J. Inf. Dis. 175 (1997), 91-97; Mor, Biochem.
Pharmacology 55 (1998), 1151-1153; Donelly, Annu. Rev. Immun. 15 (1997),
617-648; MacGregor, J. Infect. Dis. 178 (1998), 92-100).
The proteins, nucleic acid molecules, fusion proteins, antigenic fragments
or antibodies, fragments or derivatives of said antibodies of the
invention used in a pharmaceutical composition as a vaccine may be
formulated e.g. as neutral or salt forms. Pharmaceutically acceptable
salts, such as acid addition salts, and others, are known in the art.
Vaccines can be, inter alia, used for the treatment and/or the prevention
of an infection with pathogens and are administered in dosages compatible
with the method of formulation, and in such amounts that will be
pharmacologically effective for prophylactic or therapeutic treatments.
Proteins, protein fragments and/or protein derivatives used as vaccines
are well known in the art (see, e.g. Cryz, "Immunotherapy and Vaccines",
VCH Weinheim (1991); Paul (1989), loc. cit.). Furthermore, it has been
shown that even intracellular enzymes of bacterial pathogens can act as
antigenic entities which provide immunological protection (Michetti,
Gastroenterology 107 (1994), 1002; Radcliff, Infect. Immun. 65 (1997),
4668; Lowrie, Springer Semin. Immunopathol. 19 (1997), 161)
A vaccination protocol can comprise active or passive immunization,
whereby active immunization entails the administration of an antigen or
antigens (like the compositions of the present invention or proteins,
nucleic acid molecules, fusion proteins, antigenic fragments or
antibodies, fragments of said antibodies or derivatives of the antibodies
of the present invention) to the host/patient in an attempt to elicit a
protective immune response. Passive immunization entails the transfer of
preformed immunoglobulins or derivatives or fragments thereof (e.g., the
antibodies, the derivatives or fragments thereof of the present invention)
to a host/patient. Principles and practice of vaccination and vaccines are
known to the skilled artisan, see, for example, in Paul, "Fundamental
Immunology" Raven Press, New York (1989) or Morein, "Concepts in Vaccine
Development", ed: S. H. E. Kaufmann, Walter de Gruyter, Berlin, N.Y.
(1996), 243-264. Typically, vaccines are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for solution in
or suspension in liquid prior to injection also may be prepared. The
preparation may be emulsified or the protein may be encapsulated in
liposomes. The active immunogenic ingredients often are mixed with
pharmacologically acceptable excipients which are compatible with the
active ingredient. Suitable excipients include but are not limited to
water, saline, dextrose, glycerol, ethanol and the like; combinations of
these excipients in various amounts also may be used. The vaccine also may
contain small amounts of auxiliary substances such as wetting or
emulsifying reagents, pH buffering agents, and/or adjuvants which enhance
the effectiveness of the vaccine. For example, such adjuvants can include
aluminum compositions, like aluminumhydroxide, aluminumphosphate or
aluminumphospho-hydroxide (as used in "Gen H-B-Vax.RTM." or "DPT-Impfstoff
Behring"), N-acetyl-muramyl-L-th reonyl-D-isoglutamine (thr-DMP),
N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to
n-glycero-3-hydroxphaosphoryloxy)-ethylamine (CGP 19835A, also referred to
as MTP-PE), MF59 and RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM.
emulsion. Further adjuvants may comprise DNA or oligonucleotides, like,
inter alia, CpG-containing motifs (CpG-oligonucleotides; Krieg, Nature 374
(1995), 546-549; Pisetsky, An. Internal. Med. 126 (1997), 169-171).
The vaccines usually are administered by intravenous or intramuscular
injection. Additional formulations which are suitable for other modes of
administration include suppositories and, in some cases, oral
formulations. For suppositories, traditional binders and carriers may
include but are not limited to polyalkylene glycols or triglycerides. Oral
formulation include such normally employed excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate and the like. These
compositions may take the form of solutions, suspensions, tables, pills,
capsules, sustained release formulations or powders and contain about 10%
to about 95% of active ingredient, preferably about 25% to about 70%.
Vaccines are administered in a way compatible with the dosage formulation,
and in such amounts as will be prophylactically and/or therapeutically
effective. The quantity to be adminstered generally is in the range of
about 5 micrograms to about 250 micrograms of antigen per dose, and
depends upon the subject to be dosed, the capacity of the subject's immune
system to synthesize antibodies, and the degree of protection sought.
Precise amounts of active ingredient required to be administered also may
depend upon the judgment of the practitioner and may be unique to each
subject. The vaccine may be given in a single or multiple dose schedule. A
multiple dose is one in which a primary course of vaccination may be with
one to ten separate doses, followed by other doses given at subsequent
time intervals required to maintain and/or to reinforce the immune
response, for example, at one to four months for a second dose, and if
required by the individual, a subsequent dose(s) after several months. The
dosage regimen also will be determined, at least in part, by the need of
the individual, and be dependent upon the practitioner's judgment. It is
contemplated that the vaccine containing the immunogenic compounds of the
invention may be administered in conjunction with other immunoregulatory
agents, for example, with immunoglobulins, with cytokines or with
molecules which optimize antigen processing, like listeriolysin.
In a preferred embodiment, the composition of the present invention is a
diagnostic composition further comprising, optionally, suitable means for
For diagnosis and quantification of pathogens like Mycobacteria,
pathogenic fragments, their derivatives, their (poly)peptides (proteins),
their polynucleotides, etc. in clinical and/or scientific specimens, a
variety of immunological methods, as well as molecular biological methods,
like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immuno
Assays (DEIA; Mantero et al., Clinical Chemistry 37 (1991), 422-429) have
been developed and are well known in the art. In this context, it should
be noted that the nucleic acid molecules of the invention may also
comprise PNAs, modified DNA analogs containing amide backbone linkages.
Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization. The
proteins of the invention may be, inter alia, useful for the detection of
anti-pathogenic (like, e.g., anti-bacterial or anti-viral) antibodies in
biological test samples of infected individuals. It is also contemplated
that antibodies and compositions comprising such antibodies of the
invention may be useful in discriminating acute from non-acute infections.
The diagnostic composition optionally comprises suitable means for
detection. The proteins, antigenic fragments, fusion proteins and
antibodies or fragments or derivatives thereof described above are, for
example, suitable for use in immunoassays in which they can be utilized in
liquid phase or bound to a solid phase carrier. Solid phase carriers are
known to those in the art and may comprise polystyrene beads, latex beads,
magnetic beads, colloid metal particles, glass and/or silicon chips and
surfaces, nitrocellulose strips, membranes, sheets, animal red blood
cells, or red blood cell ghosts, duracytes and the walls of wells of a
reaction tray, plastic tubes or other test tubes. Suitable methods of
immobilizing nucleic acids, (poly)peptides, proteins, antibodies,
microorganisms etc. on solid phases include but are not limited to ionic,
hydrophobic, covalent interactions and the like. Examples of immunoassays
which can utilize said proteins, antigenic fragments, fusion proteins,
antibodies or fragments or derivatives of said antibodies of the invention
are competitive and non-competitive immunoassays in either a direct or
indirect format. Commonly used detection assays can comprise radioisotopic
or non-radioisotopic methods. Examples of such immunoassays are the
radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western
blot assay. Furthermore, these detection methods comprise, inter alia,
IRMA (Immune Radioimmunometric Assay), EIA (Enzym Immuno Assay), ELISA
(Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno. Assay), and CLIA (Chemioluminescent
Immune Assay). Other detection methods that are used in the art are those
that do not utilize tracer molecules. One prototype of these methods is
the agglutination assay, based on the property of a given molecule to
bridge at least two particles.
The proteins, antigenic fragments, antibodies, nucleic acid molecules
and/or fusion proteins of the invention can be bound to many different
carriers. Examples of well-known carriers include glass, polystyrene,
polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,
nylon, amyloses, natural and modified celluloses, polyacrylamides,
agaroses, and magnetite. The nature of the carrier can be either soluble
or insoluble for the purposes of the invention.
Appropriate labels and methods for labeling are known to those of ordinary
skill in the art. Examples of the types of labels which can be used in the
present invention include inter alia, fluorochromes (like fluorescein,
rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase,
.beta.-galactosidase, alkaline phosphatase), radioactive isotopes (like
.sup.32P or .sup.125I), biotin, digoxygenin, colloidal metals, chemi- or
bioluminescent compounds (like dioxetanes, luminol or acridiniums).
A variety of techniques are available for labeling biomolecules, are well
known to the person skilled in the art and are considered to be within the
scope of the present invention and comprise, inter alia, covalent coupling
of enzymes or biotinyl groups, iodinations, phosphorylations,
biotinylations, random priming, nick-translations, tailing (using terminal
transferases). Such techniques are, e.g., described in Tijssen, "Practice
and theory of enzyme immuno assays", Burden, RH and von Knippenburg (Eds),
Volume 15 (1985), "Basic methods in molecular biology"; Davis L G, Dibmer
M D; Battey Elsevier (1990), Mayer et al., (Eds) "Immunochemical methods
in cell and molecular biology" Academic Press, London (1987), or in the
series "Methods in Enzymology", Academic Press, Inc.
Detection methods comprise, but are not limited to, autoradiography,
fluorescence microscopy, direct and indirect enzymatic reactions, etc.
Said diagnostic composition may be used for methods for detecting a
pathogenic organism in a biological and/or medical sample and/or for
detecting expression of a protein or a nucleic acid molecule of the
invention by detecting the presence of mRNA coding for a protein of the
invention which comprises, for example, obtaining mRNA from pathogen
preparations (like bacterial or viral preparations) and contacting the
mRNA so obtained with a probe/primer comprising a nucleic acid molecule
capable of specifically hybridizing with a nucleic acid molecule of the
invention under suitable conditions and detecting the presence of mRNA
hybridized to the probe/primer. Further diagnostic methods leading to the
detection of nucleic acid molecules in a sample comprise, e.g., polymerase
chain reaction (PCR), ligase chain reaction (LCR), Southern blotting in
combination with nucleic acid hybridization, comparative genome
hybridization (CGH) or representative difference analysis (RDA). These
methods for assaying for the presence of nucleic acid molecules are known
in the art and can be carried out without any undue experimentation.
The invention relates further to a method for the production of a vaccine
against a virulent strain of the genus Mycobacterium comprising the steps
of (a) recombinant expression of a differentially expressed protein as
defined above, an antigenic fragment as defined above or the fusion
protein of the invention, and (b) combining said recombinantly expressed
differentially expressed protein, antigenic fragment or fusion protein
with a pharmaceutically acceptable carrier.
Furthermore, the invention relates to a method for the production of a
vaccine against a virulent strain of the genus Mycobacterium by combining
a vector comprising a nucleic acid molecule encoding a differentially
expressed protein, an antigenic fragment or the fusion protein of the
invention with a biologically acceptable carrier, wherein said nucleic
acid molecule in said vector is placed under the control of an expression
Moreover, the invention relates to the use of a nucleic acid molecule
encoding a differentially expressed protein, an antigenic fragment as
defined above or the fusion protein of the invention for the methods as
The invention further relates to the use of at least one of the proteins,
an antigenic fragment, a nucleic acid molecule, a fusion protein or the
antibody or fragments or derivatives thereof as defined herein for the
preparation of a composition for the treatment of a Mycobacteria-induced
The invention further relates to the use of at least one of the proteins,
an antigenic fragment, a nucleic acid molecule, a fusion protein or the
antibody or fragments or derivatives thereof as defined herein for the
preparation of a vaccine for vaccination against a Mycobacteria-induced
In a preferred embodiment of the use of the present invention said
Mycobacteria induced disease is selected from the group consisting of
tuberculosis, leprosy, tropical skin ulcer, ulceration, abscess, pulmonary
disease, granulomatous (skin) disease, opportunistic infections with non-tuberculous
mycobacteria as well as from diseases elicited by atypical mycobacteria
such as M. avium including pulmonary disease, lymphadenitis, cutaneous and
disseminated diseases, e.g. in immunocompromised patients. The use is not
restricted to Mycobacteria-induced diseases in humans but comprises also
the use of the present invention in animal diseases, like bovine
Claim 1 of 16 Claims
1. An isolated or purified nucleic acid
molecule coding for a protein selected from the group consisting of
oxidoreductase (Rv0068) (SEQ ID NO: 1) from M. tuberculosis, hypothetical
protein (Rv3407) (SEQ ID NO: 2) from M. tuberculosis, and a fusion protein
comprising said Rv0068 or said Rv3407 protein or a combination of Rv0068
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