WO2007091881A2

WO2007091881A2 - Methods and means for detection of mycobacterium leprae infections

Info

Publication number: WO2007091881A2
Application number: PCT/NL2006/050105
Authority: WO
Inventors: Tom Henricus Maria Ottenhof; Annemieke Geluk; Elizabeth Pereira Sampaio
Original assignee: Leiden University Medical Center
Priority date: 2005-04-29
Filing date: 2006-04-28
Publication date: 2007-08-16
Also published as: WO2007091881A3

Abstract

The current invention discloses new Mycobacterium leprae antigens to be used in methods and means for detection and diagnostics of M. leprae infections in subjects, in particular in the early stages of infection and in paucibacillary infections, which remain undetected using conventional diagnostic methods. The antigens disclosed in the invention are specific for M. leprae and the diagnostic method does not yield 'false positive' results in individuals having an immune response against other Mycobacterial species, such as M. tuberculosis, M. bovis, M. paratuberculosis, M. avium, M. smegmatis,, M. ulcerans, M. microtii, and M. marinum, or BCG vaccinated individuals.

Description

Title: Methods and means for detection of Mycobacterium leprae infections

Field of the invention The current invention pertains to the field of medicine, in particular to the fields of diagnosis and treatment of infectious diseases and mycobacterial pathogens.

Background of the invention

Although the global prevalence of leprosy has dropped dramatically coinciding with the introduction of multiple drug therapy (MDT), in many countries, only a slight reduction of incidence has been reported. According to WHO (2004), the number of leprosy patients under treatment in the world was around 460,000 at the beginning of 2004. About 515, 000 new cases were detected during 2003. Among them, 43% were multi-bacillary cases, 12% were children, and 3% were diagnosed with severe disabilities (1). One interpretation of these findings is that transmission of Mycobacterium leprae infection is not significantly affected by current leprosy control measures. In addition to delayed or missed diagnosis of infectious leprosy patients, the lack of tests to measure asymptomatic M. leprae infection in contacts prevents assessment of transmission of M. leprae. Therefore, a key priority is the development of specific and sensitive diagnostic tools that detect M. leprae infection before clinical manifestations arise and distinguish M. leprae infection from BCG vaccination or infections with other mycobacteria such as M. tuberculosis and environmental mycobacteria (2) such as M. paratuberculosis, M. avium, M. bovis, M. microti, and M. marinum. Diagnosis and classification of leprosy in the field is classically based on clinical assessment and detection of acid- fast bacilli in skin slit smears or biopsies of suspected cases. There is no specific and sensitive test available that can detect asymptomatic M. leprae infection, or predict progression of infection to clinical disease. An often used and specific test that is able to detect M. leprae infection is based on the detection of IgM antibodies against phenolic glycolipid-1 (PGL-I), an M. leprae specific cell- surface antigen. However, although this test is able to detect antibodies in most multibacillary leprosy patients, it fails to detect paucibacillary leprosy patients and their S W B i I ¹B Oe- >—w«» - - -

2 healthy household contacts, since these individuals typically develop cellular rather than humoral immunity (reviewed in 3).

Alternative tests that measure cellular rather than humoral immunity, such as skin tests, have been developed in various forms since Mitsuda (4,5). Tests that measure cellular immunity to mycobacteria historically have relied on the use of mycobacterial extracts, or purified complex mixtures of mycobacterial components. In leprosy, purified M. leprae was initially used in the lepromin skin test, followed later by the use of soluble extracts of the bacillus, designated leprosin. In tuberculosis, purified protein derivative of boiled M. tuberculosis has been used since the beginning of the last century in the classical Tuberculin Skin Test (TST). However, the diagnostic value of almost all of these tests is compromised by the presence of conserved, immunologically cross-reactive components that are shared with other mycobacteria, which results in low test specificity. For leprosy, such cross reactivity is particularly problematic in countries with high incidence rates of tuberculosis, routine BCG vaccination practice, and high levels of exposure to non-pathogenic environmental mycobacteria. Specific tests are needed which distinguish previous infection with M. tuberculosis or M. leprae from each other as well as from exposure to other mycobacteria, including BCG.

Comparative genomic analyses of the M. tuberculosis and BCG genomes have provided a blueprint for the rational design of new generation specific proteins to assess cellular immunity in blood or skin tests. Candidate M. tuberculosis proteins that were found to be lacking from the BCG genome were shown to have considerable value as potential diagnostic reagents for tuberculosis in human or cattle (20, 21). The antigens characterized in most detail are ESAT-6 (Rv3875) and CFP-10 (Rv3874). Recently several other candidate molecules were reported, which in combination with ESAT-6 and CFP-10 provided enhanced specificity and sensitivity (22-24). Scrutiny of the M. leprae genome revealed the presence of two candidate genes, ML0049 and ML0050 that encode the M. leprae homologs of ESAT-6 and CFP-10, respectively (7,8,25,26). It was reported that recombinant M. leprae ESAT-6 and CFP-10 proteins were efficiently recognized by T cells from the majority of M. leprae responsive leprosy patients (6, 7). Despite limited sequence identity with their M. tuberculosis homologues Rv3875 and Rv3874 (36% and 40%, respectively), however, significant immunologic cross reactivity (i.e. recognition by T cells from TB patients ) was detected . This clearly limits the diagnostic potential of ML0049 and ML0050 encoded proteins in leprosy endemic areas with a high prevalence of tuberculosis.

It is thus an object of the present invention to provide for M. leprae antigens that allow to distinguish between infection with M. leprae and exposure to M. tuberculosis and other mycobacteria including BCG, and that allow to immunize against infection with M. leprae.

Summary of the invention.

Detailed description of the invention

Based on the observation that antigens highly specific for M. leprae would result in a better and/or earlier detection of M. leprae infections, in particular paucibacillary infections, the inventors set out to identify candidate antigens that are unique to M. leprae and which would provide more specific targets to measure cellular immunity.

The recent publication of the genome sequences of M. leprae (9), M. tuberculosis

(10,11), M. bovis (12), and the almost completed genome sequences of several other mycobacterial species (M. avium, M. smegmatis, M. marinum, M. paratuberculosis, M. ulcerans) (28-31) provided an unprecedented opportunity to identify proteins that are unique to M. leprae. The identification of unique M. leprae proteins or peptides would help to distinguish M. leprae infection from infection with M. tuberculosis, from vaccination with BCG, or from infections or exposure any other Mycobacteria, such as environmental mycobacteria. It would provide important methods and tools for an early and highly specific diagnosis of M. leprae infection and in understanding of its transmission and for potential prophylactic and curative vaccination strategies. Based on this insight, candidate antigens were identified that are unique to M. leprae, and which provide superior targets for measuring M. leprae specific cellular immunity. Using the recently published genome sequences of M. leprae (9), M. tuberculosis and M. bovis BCG (10-12), a series of M. leprae proteins were identified that were lacking from the M. tuberculosis genome. Published comparative genomic analyses of the M. leprae and M. tuberculosis genomes had previously revealed the presence of 165 M. leprae genes with no homologue in M. tuberculosis. Based on size and the presence of promiscuous HLA-class II binding-motifs, 17 hypothetical proteins that are most unique to M. leprae and are potentially highly antigenic, shown to be expressed in vivo in infected individuals and highly antigenic in vivo, could be selected.

In a first embodiment, the current invention provides polypeptides comprising an amino acid sequence having at least 60, 70, 80, 85, 90, 95, 98 or 99 % identity with an amino acid sequence selected from SEQ ID No's 1 to 17 of M. leprae specific and/or unique polypeptides. Preferably the polypeptides are isolated polypeptides. The corresponding nucleotide sequences encoding these polypeptides are listed in SEQ ID No's 18 to 34 respectively.

In a more preferred embodiment, the invention provides M. leprae polypeptides that are encoded by SEQ ID No's 1 to 5. These encoded polypeptides are capable of eliciting a very potent IFN-γ response in peripheral blood mononuclear cells (PBMCs) from paucibacillary M. leprae infected individuals or from healthy household contacts

(HHC), i.e. individuals such as relatives or family members who are frequently and over a longer period exposed to and in close contact with leprosy patients or from multibacillary patients undergoing typical leprosy reactions (abbreviated herein as Rx).

Preferably, the polypeptides according to the invention comprises at least 8 or 9 or 10 contiguous amino acids from an amino acid sequence from SEQ ID No.'s 1 to 17 (or homologues thereof), and comprise a T-cell epitope as determined by TEPITOPE 2000 analysis or by another bio informatics tool that identifies T-cell epitopes,, preferably HLA class II-restricted T cell epitopes. Polypeptides according to the invention are preferably capable of eliciting an IFN-γ response in PBMCs of a subject carrying a paucibacillary Mycobacterium leprae infection and/or a healthy subject not carrying a M. leprae infection but exposed to individuals that are carrying a M. leprae infection, abbreviated herein as HHC (Healthy Household Contacts). More preferably the polypeptides of the invention have a length in excess of 8, 9, 10, 11 or 12 amino acids such that they do not directly fit into the groove of an MHC molecule but rather must be processed before being capable of presentation in the context of an MHC molecule. These and other advantageous effects of the preferred length, 8 - 45 and even more preferably 8-35, 15-25 or 18-45 amino acids of the peptides for diagnostic- and immunization purposes can for instance be found in WO 02/070006. In the polypeptides in the invention the amino acids in excess of 8, 9, 10, 11 or 12 are not necessarily contiguous 8, 9, 10, 11 or 12 amino acids from the M. leprae antigen that comprise a T-cell epitope, the excess amino acid may be heterologous to the antigen or even to M. leprae. A polypeptide according to the invention capable of eliciting an immune response in M. leprae infected individuals, in particular recently infected individuals, paucibacillary infected individuals or HHCs, and is preferably not capable of eliciting a cellular immune response against other Mycobacteria such as but not limited to: M. tuberculosis, M. paratuberculosis, M. avium, M. bovis, M. bovis BCG, M. microtii, M. marinum or M. ulcerans. An immune response is determined by for instance IFN-γ responses, in antigen exposed PBMCs obtained from individuals carrying an infection with another Mycobacterium. In a most preferred embodiment the invention provides peptides comprising the amino acid sequences of SEQ ID No's 35-39, preferably at least 8 contiguous amino acids from SEQ ID NO's 35-39. The peptides and epitopes may also be combined in any combination of 2, 3, 4, 5, or more.

In another embodiment the invention provides a nucleic acid molecule comprising a nucleotide sequence as identified in SEQ ID No's 18 to 34, most preferably 18 to 22, or nucleotides having at least 80, 85, 90, 95, 98 or 99 % identity with these sequences. Sequences according to the invention encode polypeptides according to the invention, polypeptides having at least 60, 70, 80, 85, 90, 95, 98 or 99 % amino acid identity with any of the polypeptides according to SEQ ID No's 1 - 17, most preferably 1 to 5. Preferably the nucleic acids of the invention are isolated nucleic acids. The nucleic acid molecule according to the invention may also comprise the complementary strand which hybridizes to a nucleotide sequence according to the invention, or close homo logs thereof capable of hybridizing under stringent conditions, including oligonucleotide primers. The nucleic acid sequences may also comprise those sequences which differ due to the degeneracy of the genetic code from the previously described nucleic acid sequences encoding the polypeptides according to the invention. ^{" ■ ~ " ■" " "" "} / u u n i o o

6

Nucleic acids according to the invention may be comprised in a DNA vector, such as but not limited to a circular or linear plasmid, phage, phagemid, cosmid, transposable element, artificial chromosome or replicating sequence, virus or retrovirus. Preferably the vector is a vector capable of conferring expression of the nucleic acid according to the invention in a host cell, comprising operably linked regulatory sequences such as promoters, enhancers, terminators, initiators, IRES sequences or Kozak-sequences. Most preferably the vector is a vector capable of conferring expression in a Mycobacterium. Preferably a recombinant micro-organism according to the invention is a Mycobacterium, for instance of the species M. tuberculosis or M. bovis and most preferably M. bovis Bacillus Calmette Guerin (BCG), capable of delivering to a host the polypeptides or fragments thereof according to the invention. Recombinant BCG and methods for recombination are known in the art, for instance in W 02004094469. Such a recombinant micro-organism may be formulated as a live recombinant and/or live attenuated vaccine, as for instance in Jacobs et al. (1987, Nature, 327(6122):532-5). The vector may also be comprised in a host of bacterial origin, such as but not limited to live-attenuated and/or recombinant Shigella or Salmonella bacteria. Accordingly, the invention also provides recombinant organisms, preferably a recombinant Mycobacterium, comprising a DNA vector according to the invention capable of conferring expression of polypeptides or antigenic fragments according to the invention. Methods for the production and generation of vectors and recombinant micro-organisms such as BCG are well documented and widely known in the art and may for instance be found in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual" (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, or "Current Protocols in Molecular Biology", Ausubel F. ed., Wiley Interscience, 2004.

In another embodiment, the current invention provides a method for the identification of Mycobacterial antigens and antigenic fragments that are specific for M. leprae, the method comprising at least the steps of: (a) identification from its genome M. leprae candidate genes encoding polypeptides, the polypeptides having less than 90, 80, 70, 65, 60, 50, 40 or 30 % homology or identity with other Mycobacterial species, as determined for instance by using public database analysis such as NCBI Genbank and Blast searches; _ ^ , , . . _ _ _

7

(b) optionally identification and/or prediction of potential HLA class II T-cell epitopes comprised in the polypeptides using computer algorithms and bio informatics tools such as but not limited to Teρitope-2000, HLA_BIND, SYFPEITHI, NetMHC (references: 32 to 37) ; (c) optionally confirming expression of the candidate antigens from the candidate genes under in vivo circumstances in an infected individual; and,

(d) determining the degree of specificity of the putative antigens using a panel of subjects comprising of subjects with multibacillary leprosy, subjects with paucibacillary leprosy, leprosy patienys in reaction (Rx), healthy endemic control subjects and healthy household contact subjects that have been exposed to M. leprae infected individuals and/or leprosy patients, but who do not carry an M. leprae infection themselves or at least do not show any clinical signs of leprosy. Optionally and most preferably also subjects carrying Mycobacterial infections and diseases of other species than M. leprae and healthy BCG vaccines may be included in the panel of subjects in order to evaluate the specificity of the putative antigens for M. leprae, such as tuberculosis patients. The degree of specificity is meant to comprise the percentage of false positives, which is preferably below 50, 40, 30, 20 or 10 % as compared to (a panel of) subjects that are not infected with Mycobacteria or that are infected with species of Mycobacteria other than M. leprae. The method for identifying M. leprae antigens and antigenic fragments is highly suitable to provide suitable antigens or panels of antigens for the specific identification of new M. leprae isolates that do not crossreact with other endemic Mycobacteria for the particular isolate. Antigens and/or antigenic fragments may also be found which are unique to a specific strain or isolate and hence capable of identifying a specific M. leprae isolate, strain or serotype. The current invention provides antigenic fragments from Mycobacterium leprae genes encoded polypeptides that are not present in at least the related Mycobacterium species tuberculosis and preferably are also not present in M. bovis, M. avium, M.smegmatis, M. marinum, M. paratuberculosis and/or M. ulcerans. Antigenic fragments according to the invention are obtained from M. leprae polypeptides are expressed in vivo in an infected mammal, comprise HLA class II-restricted T-cell epitopes as determined by Tepitope 2000 or similar computer/algorithm based methods, and are capable of elicting an IFN-γ response in PBMCs from individuals carrying a M. FU 1 / IML mm # u u u i υ o

8 leprae paucibacillary infection and preferably also in PBMCs from Healthy Household Contacts of leprosy patients.

Preferably, the antigenic fragment according to the invention is or comprises at least a part of an amino acid sequence from the following list of uniquely M. leprae encoded polypeptides: ML0576 (SEQ ID No. 1), ML1989 (SEQ ID No. 2), ML1990

(SEQ ID No. 3), ML2283 (SEQ ID No. 4), ML2567 (SEQ ID No. 5), MLO 126 (SEQ

ID No. 6), ML0369 (SEQ ID No. 7), ML0573 (SEQ ID No. 8), ML0574 (SEQ ID No.

9), ML0575 (SEQ ID No. 10), ML0840 (SEQ ID No. 11), ML0927 (SEQ ID No. 12),

ML1601 (SEQ ID No. 13), ML1602 (SEQ ID No. 14), ML1603 (SEQ ID No. 15), ML1604 (SEQ ID No. 16) and ML1788 (SEQ ID No. 17).

The most preferred M. leprae antigenic fragments are those antigenic fragments capable of eliciting a strong IFN-γ response in PMBCs from a significant fraction, preferably at least 20, 30, 40 or 50%, of paucibacillary M. leprae infected individuals. Such fragments comprise at least a part of amino acid sequences from ML0576 (SEQ ID No. 1), ML1989 (SEQ ID No. 2), ML1990 (SEQ ID No. 3), ML2283 (SEQ ID No. 4) and ML2567 (SEQ ID No. 5) and in particular those in SEQ ID No's 35 - 39, or any peptides containing at least 8 contiguous amino acids selected from SEQ ID No's 35- 39.

The current invention also provides a method for diagnosing a Mycobacterium leprae infection in a mammal, comprising the step of in vivo or in vitro detection of an immune response of a subject to at least one or more M. leprae specific antigens according to the invention or an antigenic fragment according to this invention.

In a preferred embodiment the method of detection comprises the use of those antigens that are capable of eliciting a strong IFN-γ response in PMBCs from a significant fraction, preferably at least 20, 30, 40, 50 or 60 % of paucibacillary M. leprae infected individuals. A strong INF-γ response is defined herein as an induction of at least 200, 250 or 300 pg/ml, as determined in a standard assay as provided in the Examples section, or at least 4, 5, 6, 8, 10 or more times the INF-γ level detected in unstimulated control samples or cultures, using any INF-γ detection assay.

The detection method may alternatively comprise measuring other types of immune responses than the preferred IFN-γ production or release upon contacting PBMCs with one or more of the antigenic fragments or polypeptides according to the PCT/ML ^'/UUd ' u u u i u D

9 invention. Such responses comprise but are not limited to IFN-α β, TNF-α, TGF-β, IL- 2, IL-4, IL4-52, IL-6, IL-IO, IL-12 (p70), IL-15, GM-CSF, MIP-Ib. Up to 17 of these cytokines may be simultaneously, for instance by a Luminex machine or analogous commercially available means and techniques (38, 39). In another embodiment, the method of detection comprises the use of M. leprae specific antigens ML0576 (SEQ ID No. 1), ML1989 (SEQ ID No. 2), ML1990 (SEQ ID No. 3), ML2283 (SEQ ID No. 4) and ML2567 (SEQ ID No. 5), that are capable of inducing an INF-γ response of more than 200 pg/ml in the stimulation assay as determined in the assay in the Examples section. The method of detection may preferably comprise the use of more than one, preferably at least 2, 3, 4, 5, 10, 17 or more antigenic fragments or polypeptides according to the invention, derived of one or more of the sequences having at least 80% identity to SEQ ID No's 1 to 17, preferably SEQ ID No's 1 to 5. The combined use of several antigens will increase both the sensitivity and the specificity of the method of detection of M. leprae infections according to the invention. In particular one or more of the peptides sequences of SEQ ID No's 35 - 39 and peptides comprising at least 8 contiguous amino acids from these sequences are highly preferred for the methods of the invention.

The method of detection according to the invention is may be used on samples of a bodily fluid and is preferably used on whole blood samples, blood fractions, blood containing compositions or isolated peripheral blood monocytes (PBMCs). Isolated PBMCs may upon isolation be frozen and/or stored before use, cultured or propagated in vitro, or sorted using cell sorting methods and means known in the art, prior to performing the method of detection of a cellular and specific immune response against M. leprae according to the invention.

Alternatively method for diagnosing may comprise in vivo detection of cellular immunity to M. leprae, in which one or more of the M. leprae specific antigens of the invention are e.g. used in skin tests as have been described by Mitsuda (4,5), or variants of the classical lepromin-, leprosin- or tuberculin skin tests that have been modified to include one or more of the M. leprae specific antigens of the invention, and preferably excluding antigens that crossreact with non-M leprae mycobacteria. In such skin tests the M. leprae specific antigens of the invention are applied under the top layer of skin and after several days (usually 2 days) the site of injection is inspected for the rUVNL ZOOB / U U 0 1 05

10 occurrence of a firm red bump at the injection site. In the methods the antigens may be applied by injection under the top layer of skin using a syringe and needle, or the may be applied using multiple tines wherein a small "button" that has several short needles (tines) coated with antigen is pressed into the skin (usually on the inner side of the forearm), forcing the antigen-coated tines into skin. The inventionthus also relates to the use of the M. leprae specific antigens of the invention for the manufacture of a preparation for diagnosis of M. leprae, preferably for use in a skin test as described above.

The current invention also provides a kit of parts, the kit comprising at least one or more polypeptides according to the invention, and optionally means for the detection of an immune response in blood cells. Alternatively the kit may comprise compenents for in vivo skin tests, such as a container for the antigens, needles, syringes and/or multiple tine "buttons". The kit will allow rapid detection of a M. leprae infection in a subject, in particular detection of paucibacillary infections in a subject. Such a kit may also comprise a carrier with one or more antigenic fragments according to the invention, such as a (micro)well plate or a protein (micro)array.

In yet another embodiment, the current invention provides a medicament for the prevention or the treatment of Mycobacterium leprae infections. The antigenic fragments according to the invention that are present in the polypeptides according to the invention may be used for the manufacture of a medicament. Such a medicament according to the invention may comprise an immunogenic composition to be administered to subjects at risk of acquiring a Mycobacterium leprae infection or already infected with Mycobacterium leprae, in particular a paucibacillary M. leprae infection and healthy household contacts. The immunogenic composition may comprise at least one and preferably more; i.e. 2, 3, 4, 5, 6, 10, 17 or more polypeptides or antigenic fragments according to the invention and optionally one or more pharmaceutical excipients such as carriers, fillers, solutes, stabilizers and adjuvants. Excipients for pharmaceutical preparations, pharmaceutical delivery systems and methods of administration are easily and appropriately selected by the skilled artisan using textbooks such as in Remington; The Science and Practice of Pharmacy, 21^st

Edition 2005, University of Sciences in Philadelphia. Adjuvants may similarly be TO 1 / IML £UUO / U U U I U O

11 selected and may for instance be found in Current Protocols in Immunology, Wiley Interscience, 2004. Preferred adjuvants for use in the pharmaceutical compositions, medicaments or vaccines according to the current invention are preferably selected from the following list of adjuvants: cationic (antimicrobial) peptides and Toll-like receptor ligands, such as but not limited to: poly(I:C), CpG motifs, LPS, lipid A, lipopeptide Pam3Cys and bacterial flagellins or parts thereof, including their derivatives having chemical modifications. Other preferred adjuvants for use in the method and in compositions according to the invention are: mixtures with BCG or parts thereof, immunoglobulin complexes with the said latency antigens or parts thereof, IC31 (from www.intercell.com; in WO03047602), QS21/MPL (US2003095974), DDA/MPL (WO2005004911), DA/TDB (WO2005004911; Holten-Andersen et al, 2004 Infect Immun. 2004 Mar;72(3): 1608-17.) and soluble LAG3 (CD223) ( from www.Immunotep.com; US2002192195).

The method and the composition for immunization according to the current invention may further comprise the use and/or addition of a CD40 binding molecule in order to activate dendritic cells and enhance a CTL response, thereby enhancing the therapeutic effects of the methods and compositions of the invention. The use of CD40 binding molecules is described in WO 99/61065, incorporated herein by reference. The CD40 binding molecule is preferably an antibody or fragment thereof or a CD40 Ligand or a variant thereof, and may be added separately or may be comprised within a composition according to the current invention. The method and the composition for immunization according to the current invention may further comprise the use and/or addition an agonistic anti-4-lBB antibody or a fragment thereof, or another molecule capable of interacting with the 4- IBB receptor. The use of 4- IBB receptor agonistic antibodies and molecules is described in WO 03/084999, incorporated herein by reference. 4- IBB agonistic antibodies may be used with or without the addition of CD40 binding molecules, in order to enhance CTL immunity through triggering / stimulating the 4-1BB and/or CD40 receptors. The 4-1BB binding molecule or antibody may be added separately or may be comprised within a composition according to the current invention.

The polypeptides may be isolated from their natural source, i.e. M. leprae, or from recombinant organisms, such as recombinant bacteria or mycobacteria. The _ . , . _„ _ _ -

12 polypeptides may also be chemically synthesized using standard techniques. Polypeptides according to the invention having a length between 18 and 45 amino acids have been observed to provide superior immunogenic properties as is described in WO 02/070006. Polypeptides may advantageously be chemically synthesized and may optionally be (partially) overlapping and/or may also be ligated to other molecules, peptides or proteins. Polypeptides may also be rased to form synthetic proteins, as in PCT/NL03/00929 and in Welters et al.,Vaccine. 2004 Dec 2;23(3):305-ll. It may also be advantageous to add to the amino- or carboxy-terminus of the polypeptide chemical moieties or additional (modified or D-) amino acids in order to increase the stability and/or decrease the biodegradability of the polypeptide. To improve the immunogenicity / immuno-stimulating moieties may be attached, e.g. lipidation. To enhance the solubility of the peptide, addition of charged or polar amino acids may be used, in order to enhance solubility and increase stability in vivo.

The current invention also comprises a method of treatment, the method comprising the step of administering to a subject suffering from or at risk of a M. leprae infection the medicament or immunogenic composition / vaccine according to the invention.

Definitions:

"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence ro i / iML ium / u u u i u b

13

Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN₅ and FASTA (Altschul, S. F. et al., J. MoI. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wisconsin. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and iso leucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, FUI /ML ZUUb^' / U U U l U D

14 tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having acidic side chains is aspartic acid and glutamic acid and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; GIn to asn; GIu to asp; GIy to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

Antigen herein is a property of a molecule, or fragment thereof, that is capable of inducing an immune response in a mammal. The term includes immunogens and regions responsible for antigenicity or antigenic determinants or epitopes. An antigen is a chemical or biochemical structure, determinant, antigen or portion thereof that is capable of inducing the formation of a cellular (T-cell) or humoral (antibody) immune response.

An immune response in vivo or in vitro may be determined and/or monitored by one of the methods provided in this specification, but also by many other methods that are known and obvious to the skilled person and which may for instance be found in Current Protocols in Immunology, Wiley Interscience 2004. A cellular immune response can be determined by induction of the release of a relevant cytokine such as IFN-γ or IL-10 from (or the induction of proliferation in) lymphocytes withdrawn from a mammal, currently or previously infected with (virulent) mycobacteria or immunized with, but not limited to, polypeptide(s). For monitoring cell proliferation the cells may be pulsed with radioactive labeled thymidine or counted in a (flow)cytometer or under a microscope. Induction of cytokines can be monitored by various immuno-chemical methods such as, but not limited to ELISA or Elispot assays. An in vitro cellular response may also be determined by the use of T cell lines derived from a healthy rv i / iNt. mm i u u u I 0 5

15 subject or a Mycobacterium-infected mammal where the T cell lines have been driven with either live and/or killed, attenuated or recombinant Mycobacteria, latent Mycobacteria or selected antigens derived or obtained thereof.

The terms vaccine or immunogenic composition is used herein to describe a composition useful for stimulating a specific immune response in a mammal, optionally comprising adjuvants and other active components to enhance or to direct a particular type of immune response, preferably a CTL or Th or Ab response.

The term subject as used herein refers to living multi-cellular vertebrate organisms, a category that includes both non-human and preferably human mammals. The term subject includes both human, veterinary or laboratory subjects. Laboratory and livestock animals such as mice, rabbits, cows, pigs, dogs, cats, goats, sheep, horses and camels are included.

A nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

A vector as used herein refers to a nucleic acid molecule as introduced into a host cell thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may for instance be a plasmid, phagemid, phage, cosmid, virus, retrovirus, episome or transposable element. A vector may also include one or more selectable (antibiotic resistance) or visual (e.g. GFP, immuno-tag) marker genes and other genetic elements known in the art.

Proteins, peptides and polypeptides are linear polymeric chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of rwi / IVL IUW^Q / U U U 1 U t)

16 one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term amino terminus (N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal end of the peptide, or to the alpha amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. The term carboxy terminus (C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminal end of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide.

A synthetic polypeptide refers to a polypeptide formed, in vitro, by joining amino acids in a particular order, using the tools of organic chemistry to form the peptide bonds. Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminus and increasing in the direction toward the carboxy terminus of the peptide.

Figure legends

Figure IA: Ex vivo IFN-γ production against five unique M. leprae antigens by PBMC of (o) healthy controls (Controls; n = 30), (•) MB patients (n = 19), (T) PB patients and MB patients with leprosy reactions (n = 28), ( A) healthy household contacts and endemic controls (n = 35) and ( ) tuberculosis patients without anti-PGL-I Ab or M. leprae reactive T cells (n = 11), and (^■) tuberculosis patients that show significant T cell responses to M. leprae extracts and/ or are seropositive for anti-PGL-1 Ab (n = 5). Median values are indicated by horizontal lines. P values are calculated by the non- parametric Mann Whitney U test.

Figure IB: Ex vivo IFN-γ production against two M. leprae antigens with homologues in M. tuberculosis. See legend Figure IA.

Figure 2: Added value of the use of in vitro T cell responses to selected M. leprae antigens ML0567, ML1989, ML1990, ML2283 and ML2567. T cell responses were considered positive if IFN-γ production in response to one of these antigens was > 200 pg/ ml. Significant differences are indicated.

Figure 3: M. leprae specific candidate antigens.

Figure 4: See legend Figure IA, Ex vivo IFN-γ production against twelve unique M leprae antigens by PBMCs from the 5 groups of individuals as in Figure IA.

Figure 5: Interferon gamma responses in Brazilian Leprosy patients (PB/Rx) using ML1989, ML2283 and ML2567 peptides separately and combined.

Figure 6: As figure 5, responses in Dutch Healthy Controls.

Figure 7: CD4+ and CD8+ T cells are activated by M.leprae peptides, demonstrated for the ML1990 peptide.

Examples

Materials and methods

Purification and reverse transcription of M. leprae RNA

Two M. leprae isolates from geographically distinct regions of the world were used for this study. M. leprae Thai-53 was originally isolated from a skin lesion of an untreated MB (multibacillary) leprosy patient in Thailand in 1982. The other M. leprae isolate (NHDP98) was obtained from a lesion of an untreated, Mexican-born, MB leprosy patient in 1990. M. leprae was purified from hind foot pads of BALBc athymic nude mice (Hsd:Athymic Nude, Harlan, Indianapolis, IN) at the Laboratory Research Branch of the National Hansen's Disease Programs, Baton Rouge, LA as described previously (14). A punch biopsy from a skin lesion of a MB leprosy patient from the United States, previously obtained for diagnostic testing and stored frozen at -70 ⁰C in OCT cryoprotective medium (Tissue Tek, Columbus, OH), was thawed at room temperature and removed from the cryopreservative medium. The tissue was washed x 3 in 1 ml of sterile phosphate-buffered saline (PBS; Sigma, St. Louis, MO, USA) and minced finely. M. leprae RNA was extracted from purified bacteria or from minced fp\/l / INH. £ϋUU ' u υ u I U u 18 biopsy tissue using a previously described, single-tube homogenization/RNA extraction protocol (15). cDNA was made from 10 μl of M. leprae RNA using random hexamer primers (15), the Advantage cDNA Polymerase Mix and Advantage RT-for-PCR Kit in a final volume of 50 μl according to manufacturer's recommendations (BD Biosciences, Clontech, Palo Alto, CA). Controls for DNA contamination consisted of total M. leprae RNA incubated with the reverse transcription reagents excluding reverse transcriptase. Specificity controls consisted of cDNA made from BALB/c mouse spleen total RNA (BD, Biosciences, Clontech) and human PBMC-derived RNA.

PCi? amplification and DNA sequencing

DNA was purified from 2 x 1010 mouse foot pad-derived M. leprae Thai-53 (15). DNA concentration was obtained by spectrophotometric means and total DNA was stored at -80°C. PCR primers and amplification protocols were designed for 19 M leprae genes by acquiring gene sequences from the M. leprae genome database (http://www.Sanger.ac.uk/ Projects/M_ leprae) and using OmigaTM 2.0 Primer Design software (Oxford Molecular Ltd., www.gcg.com). PCR assays were initially characterized using 1 ng M. leprae Thai-53 DNA and mouse and human cDNA as specificity controls. PCR fragments were separated by gel electrophoresis (15). The PCR products were purified using QIAquick PCR purification kit spin columns (QIAGEN, Carlsbad, CA) and the DNA sequence of each PCR fragment was obtained using automated DNA sequencing (GeneLab, SVM, LSU, Baton Rouge, LA). Initially, cDNA from M. leprae isolates was analyzed for the presence of a 507 bp fragment of the M. leprae gap transcript using 30 cycles of PCR. cDNA templates from all M. leprae preparations were adjusted to yield equivalent amounts of cDNA for subsequent PCR analysis based on these amplification results. mRNA expression analysis was then performed for all genes using cDNA from each M. leprae preparation and 40 cycles of each PCR assay.

Production and testing of M. leprae-specific recombinant antigens Nineteen M. leprae candidate genes (see Results section for selection) were amplified by PCR from genomic DNA of M. leprae (generously provided by the late Dr. M. J. Colston) and cloned using the Gateway technology platform (Invitrogen, Carlsbad, CA) with pDEST17 expression vector containing an N-terminal histidine tag (Invitrogen). Sequencing was performed on selected clones to confirm identity of all cloned DNA fragments. Recombinant proteins were overexpressed in E. coli BL21(DE3) and purified as described to remove possibly present endotoxin (16). Each purified M. leprae protein was analyzed by 12% SDS-PAGE followed by Coomassie Brilliant Blue staining and Western-blotting with an anti-His antibody (Invitrogen) to confirm size and purity. Endotoxin contents were below 50 IU/ mg recombinant protein as tested using a Limulus Amebocyte Lysate (LAL) assay (Cambrex, East Rutherford, NJ). AU proteins were tested to exclude antigen non-specific T cell stimulation using PBMC based IFN-γ release assays of different healthy, M. leprae-unexposed and BCG- negative donors. Possible cellular toxicity of each recombinant protein batch was evaluated in the same assays as described (16).

Study subjects

Brazilian leprosy patients were recruited from the Leprosy Out-Patient Unit, Leprosy Laboratory (Oswaldo Cruz Institute, Rio de Janeiro). Among these were 15 paucibacillary (PB) leprosy patients, 19 multibacillary (MB) leprosy patients, 13 patients with leprosy reactions (Rx), and 21 healthy household contacts of MB leprosy patients (HHC). Leprosy patients were diagnosed and classified based on clinical, bacteriological, and sometimes histopathological findings. AU MB patients were skin slit smear-positive whereas all PB patients were skin slit smear negative. The 43 endemic healthy controls were blood donor volunteers attending the Blood Bank of the Santa Casa da Misericordia, Rio de Janeiro, and consisted of 30 M. leprae T cell nonresponders and 13 M. leprae T cell responders, as determined by their T cell response (IFN-γ) to whole M. leprae bacterial extracts (see below). AU controls were examined for clinical signs of leprosy and the presence of a BCG vaccination scar. An additional 16 Brazilian tuberculosis patients were recruited from the Ambulatory Service, District Hospital Raphael de Paula e Souza, Rio de Janeiro. AU patients tested in this study were HIV-I seronegative. Written informed consent was obtained from all individuals before participating in the study. Ethical approval of the study protocol was obtained through the Institutional Review Boards. Details of all studied groups are summarized in Table 1. TABLE 1. Brazilian study subjects

ML-

MB PB Rx HHC ML+ controls contrals TB

Individuals per group 19 15 13 21 13 30 16

Clinical leprosy diagnosis + + + - - - -

T cell response to M leprae^ 0% 100% 92% 62% 100% 0% 31%

Detected withanti-PGL-1 IgM 100% 13% 69% 14% 0% 0% 19%

Detected using M leprae Ag² 0% 93% 85% 62% 100% 7% 50%

¹ T cell responses to M leprae extracts were considered positive if IFN-g production levels in response to one or more antigens were > 200 pg/ml ^τ T cell responses to 8 individual M leprae Ag (ML0576, ML0S40, ML0927, MLl 601, ML2283, ML 2567, ML1989 and ML1990) were analyzed Individuals were considered positive if they recognized one or more Ag (> 200 pg/ml)

³ T MB multibacillary, PB paucibacillary, Rx leprosy patients with reaction, HHC healthy household control, ML+ controls healthy endemic controls showing significant T cell responses to M leprae extracts, ML- controls showing no T cell responses to M leprae extracts, TB tuberculosis patients

Cell culture and stimulation

Venous blood was obtained from study participants in heparinized tubes and PBMC isolated by Ficoll density centrifugation. PBMC (2 x 106 cells/ ml) were plated in duplicate or triplicate cultures in 96-well flat bottom plates (Costar Corporation, Cambridge, Mass.) in 200 μl/well of Adoptive Immunotherapy medium (AIM-V; Gibco) supplemented with 100 U/ ml penicillin, 100 μg/ ml streptomycin and 2 mM L- glutamine (Invitrogen). Recombinant protein or irradiated armadillo-derived M. leprae sonicate (Colorado State University, Fort Collins) were added at final concentrations of 10 μg/ ml. After 5 days of culture at 37°C at 5% CO2, 70% relative humidity, 75 μl supernatants were removed from each well, pooled and frozen in aliquots at —20 ⁰C. IFN-γ levels were determined by ELISA (R&D Systems Inc. Minneapolis, MN). The cut-off value to define positive responses was set beforehand at 200 pg/ ml. The level of sensitivity was 20 pg/ml. Values for unstimulated cell cultures (usually < 50 pg/ ml) were subtracted from the values found for stimulated cultures in all analyses. As a positive control phytoheamagglutinin (1%) or PPD (Tuberculin, RT49, Statens Serum Institute, Copenhagen, Denmark) of M. tuberculosis (5 μg/ ml) were used. Due to the sometimes- limited numbers of PBMC that could be isolated, not all 19 M leprae antigens could be tested for all individuals.

Ann PGL-I IgM antibodies

For detection of IgM Ab directed against phenolic glycolipid I (PGL-I) of M. leprae by ELISA as described (17). Briefly, blood was allowed to clot for 30 min. and serum was harvested by centrifugation. Serum samples were kept frozen at -20oC until use. The antigen used in ELISA was NT-P-BSA and cut-off values for positive responses were set beforehand at OD450 of 0.200 (17).

Statistical analysis

Differences in IFN-γ levels between groups of patients, controls and antigens were analyzed with the SPSS software package using the Mann-Whitney U test. P- values were corrected for multiple comparisons. The statistical significance level used was p<0.05.

Example 1, Selection of candidate proteins unique to M. leprae.

The M. leprae genome contains 165 candidate genes with no orthologue in M. tuberculosis (9; http://genolist.pasteur.fr/ Leproma/). While 29 of these genes have attributable functions, the remaining 136 were previously described as belonging to "functional classification VI" (i.e. with unknown function) and contained no similarity to known genes at the time of selection (9).

Short hypothetical proteins encoding fewer than 70 amino acids were excluded from further analysis. The remaining 126 hypothetical proteins were analyzed by the TEPITOPE-2000 program (http://www.vaccinome.com/), which uses virtual matrices of peptide-binding pocket profiles to predict promiscuous T cell epitopes that bind multiple HLA-DR alleles (13). Twenty four hypothetical proteins that contained multiple T cell epitopes predicted to bind > 75% of the tested HLA-DR alleles were scanned for sequence homology with M. tuberculosis using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Hypothetical proteins showing an overall homology of > 30% were excluded, resulting in ten candidate proteins that were unique to M. leprae. In addition, seven hypothetical group VI proteins were added that were located in a single putative operon with one of the above selected proteins. This strategy yielded 17 candidate M. leprae antigens. In addition, two candidate M. leprae antigens, ML0007 and ML2649, for which homologs were found in several other mycobacteria (Figure 3), were included as specificity controls to examine the level of cross reactivity in tuberculosis patients.

During the course of this work, information from other mycobacterial genome projects became available, notably the completed sequence of M. bovis (12), and the I V^ I I I Y U. --w» - - — — • w -

22 almost completed or draft genome sequences of M. avium, M. smegmatis, M. marinum, M. paratuberculosis and M. ulcerans. In figure 3, we have reanalyzed the above selected hypothetical M. leprae unique proteins against all currently available genome sequences. The results indicate that ML0369, ML0840, ML0925 and ML1601, which do not have orthologues in M. tuberculosis, do have orthologues in M. avium paratuberculosis (Figure 3). It remains unknown, however, whether these hypothetical genes are expressed in M. avium paratuberculosis (whereas our results indicate they are in M. leprae, see below). Following these analyses, ML0573, ML0574, ML0575, ML0576, ML1602, ML1603, ML1604, ML1788, ML1989, ML1990, ML2283 and ML2567 were found to remain unique to M. leprae (Figure 3).

Example 2, Expression of M. leprae-spedfic candidate genes in infected murine and human tissue.

Since the above selected 19 candidate genes were of unknown function, we first assessed their expression in M. leprae bacilli isolated from nude mice. M. leprae RNA was extracted from the hind footpad tissue of nude mice six months post infection with M. leprae. Specific mRNA for all genes could be detected by RT-PCR analysis in M. leprae from this source (Figure 3), indicating that these genes were indeed expressed by M. leprae. To analyze whether these genes are also expressed during M. leprae infection in humans, their transcriptional activity was determined in M. leprae obtained from a skin lesion of a MB leprosy patient. Since limited amounts of M. leprae mRNA derived from human tissues were available, only a selected number of genes could be tested (Figure 3). All nineteen genes tested were expressed at the mRNA level in human leprosy tissue.

Example 3, Grouping of individuals according to T cell response to whole M. leprae extracts

PBMC of all individuals included in the study (n =127) were tested first for IFN-γ responses to crude M. leprae extracts (Table 1). Thirty of the 43 healthy control individuals did not show any detectable in vitro T cell response to M. leprae. Since they also lacked detectable anti-PGL-I IgM levels (see above), they most likely had not been M. leprae infected and were therefore regarded as negative endemic controls in further data analysis (Controls; Table 1 and Fig 1). The other 13 healthy endemic controls showed strong responses to M. leprae and therefore were classified as potential M. leprae exposed controls. These 13 individuals were combined with the 21 healthy household contacts of leprosy patients (HHC), of whom the majority (n=13; 62%) were M. leprae responders, and together were designated as (H)HC group (Figure 1 and Table 1).

All leprosy patients were tested similarly for responses to M. leprae extracts. Both PB leprosy patients and patients with a history of leprosy reactions (Rx) showed significant IFN-γ production in response to M. leprae extract. Since these two groups did not differ significantly in their IFN-γ response to M. leprae, they were combined (PB/Rx) for further data analysis. None of the 19 MB leprosy patients showed a significant IFN-γ response to M. leprae extract as expected.

Of note, five of the 16 analysed tuberculosis patients responded significantly to M. leprae extracts whereas the remaining 11 showed no significant response (Table 1). These five M. leprae responsive tuberculosis patients are indicated separately in Figure IA.

Example 4, T cell recognition of M. leprae antigens in Brazilian leprosy patients and controls

T cell recognition of all 19 M. leprae candidate antigens was evaluated by measuring IFN-γ production by PBMC in response to each individual recombinant protein (data shown for seven proteins in Fig. IA and B). The results show that most MB patients did not respond to any of the 19 tested antigens (median < 30 pg/ ml), as expected. Similar results were found for the negative control group (median < 25 pg/ ml in response to any of the antigens with the exception of ML1602, data not shown). In contrast, PBMC of PB/Rx patients were highly responsive to several of the M. leprae hypothetical antigens examined (Fig.l and Table 1). When individually analyzed, four types of responses could be distinguished. First, against five of the M. leprae proteins (ML0576, ML1989, ML1990, ML2283 and ML2567, Fig. IA), IFN-γ responses were observed in PB/Rx leprosy patients that were significantly higher than those seen in negative controls (p < 0.001) or in MB patients (p <0.05). Median values differed between PB/Rx and tuberculosis patients for all five proteins (250 vs. < 30 pg/ ml), but differences reached significance only for ML1989, ML2283 and ML2567, probably as a result of the relatively low numbers tested in the current screening study (Fig. 1). Eleven of the 16 tuberculosis patients (62%) did not show a clear response to the M. leprae antigens tested, which is in agreement with their lack of response to M. leprae extracts. In contrast, the five tuberculosis patients that responded to the M. leprae proteins also responded to M. leprae extracts, whereas three of them had antibodies to the M. leprae specific PGL-I (Table 1 and Fig. 1), suggesting previous exposure to M. leprae. Importantly, all five proteins unique to M. leprae were recognized by the (H)HC group. Two of these, MLl 989 and ML 1990, induced higher responses among the latter group compared to PB/Rx leprosy patients, but these differences did not reach statistical significance. Secondly, three antigens, ML1602, ML1604 (Figure 4) and the control antigen

ML2649 (shown as a representative example in Figure IB) which has a homologue in M. tuberculosis (RvO264), induced responses that were not specifically associated with M. /eprøe-infection: ML 1602 and ML 1604 were recognized well by the majority of the negative control group that did not respond to M. leprae extracts, whereas ML2649 induced responses in 63% of the tuberculosis patients. These results identify ML2649, and likely also its M. tuberculosis orthologue (RvO264) as a new T cell stimulatory antigen in tuberculosis. Thirdly, intermediate responses (median 100 - 150 pg/ ml) were found in PB patients to ML0575 and MLl 603 (figure 4). Finally, IFN-γ responses to the six remaining proteins (M. tuberculosis homologue ML0007 (shown as a representative example in Figure IB), ML0126, ML0369, ML0573, ML0574 and ML1788 (data not shown) overall appeared to be insignificant in all groups, with median IFN-γ responses < 100 pg/ ml for ML0573 and ML0574 and < 50 pg/ ml for all other antigens tested. None of these antigens displayed any inhibitory activity in control cellular stimulation assays (see Materials and Methods).

Example 5, T cell responses to M. leprae antigens in relation to anti-PGL-I IgM titers

All 43 endemic control individuals were seronegative for M. leprae-specific anti- PGL-I IgM. Anti-PGL-I IgM antibodies were present in all 19 MB patients, in 2 out of the 15 PB patients, in 9 out of the 13 Rx patients, in 3 out of the 21 household contacts and in 3 out of the 16 Brazilian tuberculosis patients (Table 1; OD450 values > 0.200, range: 0.210 - 3.922).

An issue of considerable significance is whether the five most promising M. leprae proteins studied above (Fig. IA) could provide added diagnostic value for

25 detecting M. leprae infection, compared to already available assays that measure humoral immunity, such as anti-PGL-I IgM titers. T cell reactivity to these five M. leprae proteins was therefore analyzed versus humoral immune responses against M. leprae as assessed by IgM Ab levels to M. /eprae-specific PGL-I (Fig. 2). In the (H)HC group 31 out of 34 individuals did not have detectable levels of anti-PGL-I IgM. Interestingly, 22 (71%) of these exposed individuals responded to at least one of the five proteins unique to M. leprae (Fig. 2). In the PB/Rx group 17 of the 28 patients did not have detectable levels of anti-PGL-I IgM Ab. Sixteen of these 17 individuals (94%) recognized 1 to 5 of these antigens. Only two individuals in the negative control group responded to M. leprae antigens and in both cases only one antigen was recognized.

The results from our antigen discovery approach show that several of the M. leprae proteins thus selected are recognized efficiently by T cells from M. leprae responsive donors, but not M. leprae unresponsive individuals, as evaluated by IFN-γ production by PBMC. Paucibacillary and reactional leprosy patients and healthy household contacts of leprosy patients produced significant levels of IFN-γ in response to the five unique M. leprae antigens encoded by ML0576, ML1989, ML1990, ML2283 and ML2567. These proteins were not recognized by negative endemic controls, MB patients, M. leprae unresponsive tuberculosis patients from the same endemic area or by healthy, M. /eprαe-unexposed and BCG-negative donors. Only tuberculosis patients that likely had been exposed to M. leprae as evidenced by the presence of T cell responses to M. leprae and/or IgM antibodies to M. leprae PGL-I, responded well in T cell assays to these M. leprae specific proteins. These responses thus are likely due to latent co-infection with M. leprae.

An important observation was that 71% (22 out of 31) of the (H)HC group that lacked detectable levels of anti-PGL-I IgM responded to at least one of the five proteins. Only 9% of these likely M. leprae exposed individuals would have been detected by the anti-PGL-I IgM test. This increase in the number of detectable M. /eprøe-exposed individuals shows that newly identified M. leprae antigens have added diagnostic value in identifying M. leprae infection, next to existing humoral assays. In contrast to the above five antigens, the proteins encoded by MLl 602, MLl 604 and the control ML2649 (homologue to M. tuberculosis RvO264) were not specifically recognized by M. leprae-expossd individuals: MLl 602 and MLl 604 gene products were often recognized by M. leprae unresponsive controls, perhaps due to crossreaction with unknown antigens (figure 4), whereas that of ML2649 induced responses in 63% of the tuberculosis patients. These results identify ML2649, likely together with its M. tuberculosis orthologue RvO264, as a new T cell stimulatory antigen in tuberculosis.

Despite the presence of multiple HLA class II binding motifs, six other candidate M. leprae proteins (encoded by ML0126, ML0369, ML0573, ML0574, ML1788 and the M. tuberculosis homologue ML0007) induced no significant IFN-γ responses.

These results underscore the specificity of recognition of the above-identified five proteins.

In summary, the 12 M. leprae unique genes and 5 M. leprae genes with less than 30% identity with M. tuberculosis, have no known functions and thus encode hypothetical proteins. Their expression was examined and confirmed at the transcriptome level. The results show that mRNA derived from these hypothetical M. leprae-specific genes is indeed transcribed in M. leprae, both in the mouse footpad as well as in multibacillary leprosy patients' lesions. These results show that the selected M. leprae genes are expressed and that they could encode functional proteins with a potential for use in M. leprae specific tests.

The M. leprae genes selected for immunologic evaluation were also selected on the presence of amino acid motifs predicting high affinity binding to multiple HLA class II molecules, thus increasing the likelihood that single antigens are recognized by T cells from multiple donors. Moreover, additional comparative genome analyses were performed using mycobacterial genome sequence data that had become available after the initial selection of the above mentioned proteins.

The current invention provides more specific tests to detect M. leprae infections. It is anticipated that such tests will contribute to the understanding of M. leprae transmission in affected populations, as well as to help diagnosing disease at early onset and increase the specificity of the test for M. leprae and distinguish from other Mycobacterial infections or earlier contacts, including BCG vaccination. Furthermore, longitudinal studies in healthy M. leprae infected contacts will help to develop biomarker profiles that discriminate between resistance versus susceptibility to developing clinical disease, based on specific T cell response profiles against the M. leprae antigens. Since the M. leprae antigens described here were recognized well by several leprosy patients with reactions, it will be of interest to monitor T cell responses to M. leprae antigens over time and determine their temporal relationship with the development of typical leprosy reactions in order to evaluate their prognostic value in predicting leprosy reactions. Finally, the invention provides valuable tools for preparing pharmaceutical and immunological compositions which will help to prevent and treat M. leprae infections.

Example 6

As the use of whole proteins incorporates the risk of detecting unspecific, cross- reactive T cell responses (despite the lack of substantial overall sequence homology with other proteins), the M. leprae genes encoding for the proteins that were highly immunogenic in M.leprae infected individuals only ML0576, ML1989, ML1990, ML2283 and ML2567 were analysed in more detail. The analysis provided peptides derived from M. leprae unique Ags, which have the advantage over whole proteins that they offer the possibility to identify epitopes that do not induce T cell cross-reactivity. Definition of optimal peptide epitopes and the use of (mixtures of) relevant M. leprae peptides, will improve the specificity of the tests for early diagnosis of leprosy and detection of M. leprae infection.

Overlapping peptides (n = 50) covering the 5 most promising proteins (ML0576, ML1989, ML1990, ML2283 and ML 2567) were synthesized (table 1). The individual peptides assayed are shown in table 2 below.

Table 1

Designation size peptides (pools)

ML0576 8 kDa n = 7 (1)

ML1989 13 kDa n = l l (2)

ML 1990 9 kDa n = 7 (1)

ML2283 12 kDa n = 10 (1)

ML2567 17 kDa n = 15 £21

Total n = 50 7

Table 2

Peptides ML0576 n=7 MASGVNILQDVFAVIVEVLL VFAVIVEVLLLSVGTGNART LSVGTGNARTAEDWPGLAVL AEDWPGLAVLSAGLVAVLTL SAGLVAVLTLLIHEQQEDWV LIHEQQEDWVCQPVTRLCWG • * I « It—

28

CQPVTRLCWGRSTVDR Peptides ML1989 n=ll

VARFGDCLFGDRLSGSMAIW DRLSGSMAIWRAIAPDVYLH RAIAPDVYLHRTWAIRDCI RTVVAIRDCILILFRYAGHQ LILFRYAGHQNEAAIRAADG NEAAIRAADGNLLLQQMVRL NLLLQQMVRLALVWSAKTTS ALVWSAKTTSNSPSDDLLHS NSPSDDLLHSNPQPEIYSTK NPQPEIYSTKSTNYVNLSTI STNYVNLSTIGIKLKI

Peptides ML1990 n=7

LFSVMLFILCIREIYRNPTA IREIYRNPTAITVSMTVIAV ITVSMTVIAVGFLAQAAAVS GFLAQAAAVSIATNWATGR IATNWATGRHNTINFNVDT HNTINFNVDTVGFVNAWSLL VGFVNAWSLLFYPPQGWESP Peptides ML 2283 n=10

VNEPHHAILTDLLNTFRFSE DLLNTFRFSEVLQHIDEYQL VLQHIDEYQLRQSIAGQKVT RQSIAGQKVTPLARELYRKI PLARELYRKIDCYSEEDLVE DCYSEEDLVEIAAKSLKATI IAAKSLRATIALHSAVGQPQ ALHSAVGQPQTSSGIIDITI TSSGIIDITIPRGLWTPQIK PRGLWTPQiKiPiR

Peptides ML2567 n=15

VRPGEFPDKEMAAQIGEPGV MAAQIGEPGVTRKYPGCTSQ TRKYPGCTSQRNRKHMQVRP RNRKHMQVRPGKPINNTNTT GKPINNTNTTPAATLVASVG PAATLVASVGVSVTPASPPR VSVTPASPPRKNHAVSSDFK KNHAVSSDFKTRSTNTENRV TRSTNTENRVIPLARRTVKI IPLARRTVKILRPLPSNITE LRPLPSNITEGTNASKSPQP GTNASKSPQPAPTLTSADIA APTLTSADIAARLASLRQPD ARLASLRQPDGSARHANHPF GSARHANHPFDQPSSRMCG

Each peptide is analyzed separately for its ability to induce IFN-γ secretion by PBMC derived from Brazilian leprosy patients, Dutch TB patients, Dutch BCG- vaccinated individuals and Dutch PPD-negative individuals. Peptides that uniquely activate T cells from leprosy patients but not from all other groups will be selected as they may be used for diagnosis of M. leprae infection in healthy household contacts (HHC) from endemic countries. We have analyzed the peptides in Brazilian leprosy patients (4MultiBaα, 6PauciBac. and 4 Rx) and in 11 Dutch healthy individuals. Based on these results, 5 promising peptides were selected that activate T cells of Brazilian leprosy patients but not those of Dutch controls:

STNYVNLSTIGIKLKI . ML1989 (SEQ ID No . 35) VGFVNAWSLLFYPPQGWESP ML1990 (SEQ ID No. 36) PLARELYRKIDCYSEEDLVE ML2283 (SEQ ID No. 37) DCYSEEDLVEIAAKSLRATI ML2283 (SEQ ID No. 38) KNHAVSSDFKTRSTNTENRV ML2567 (SEQ ID No. 39)

The use of single peptides induces a decrease in T cell response compared to the use of recombinant proteins, as not all infected individuals respond to the same peptides because of HLA-polymorphism. Thus a combination of these selected, immunogenic peptides was tested (Figures 5 and 6) and compared to the responses directed against single peptides. The combined peptides induced a more sensitive diagnostic tool in a leprosy endemic setting as all tested individuals, and none of the controls, reacted to a mixture of these peptides. Figures 5 and 6 illustrate this for the peptides STNYVNLSTIGIKLKI/ ML1989, PLARELYRKIDCYSEEDLVE/ ML2283 and KNHAVSSDFKTRSTNTENRV/ ML2567 separately and in combination (last lane: mix) . Simultaneously, proliferation of CD4⁺ and CD8⁺ T cells, induced by these peptides in leprosy patients, was measured by FACs analysis of CFSE labeled PBMC. The data obtained show peptide-induced activation of both CD4⁺ and CD8⁺ T cells, which is illustrated in Figure 7 for the VGFVNAWSLLFYPPQGWESP/ML1990 - peptide.

References

1. WHO Fact Sheet N⁰IOl, Weekly Epidemiological Report, 2002, 77(1):1.

2. Technical Bulletin. The ILA Technical Forum - Implications for leprosy control programmes. 2002, 16.

30

3. Oskam, L., E. Slim, and S. Bϋhrer-Sekula. 2003. Serology: recent developments, strength, limitations and prospects: a state of the art overview. Lepr. Rev. 74:196.

4. Breπnan, P. 2000. Skin test development in leprosy: progress with first- generation skin test antigens, and an approach to the second generation. Review

Lepr Rev. 71: Suppl:S50-54.

5. Mitsuda, K. 1919. On the value of a skin reaction to a suspension of leprosy nodules. Jap. J. Der. Urol. 19:697.

6. Dockrell, H.M., S. Brahmbhatt, B. D. Robertson, S. Britton, U. Fruth, N. Gebre, M. Hunegnaw, R. Hussain, R. Manandhar, R., L. Murillo, M. C. V. Pessolani,

P. Roche, J. L. Salgado, E. P. Sampaio, F. Shahid, J. E. R. Thole, and D. B. Young. 2000. A postgenomic approach to identification of Mycobacterium /eprøe-specific peptides as T-CeIl Reagents. Infect Immun. 68:5846.

7. Geluk, A., K. E. van Meijgaarden, K. L. M. C. Franken, B. Wieles, B., S. M. Arend, W. R. Faber, B. Naafs, and T. H. M. Ottenhoff. 2004. Immunological cross reactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis. Scand. J. Immunol. 59:66.

8. Geluk, A., K. E. van Meijgaarden, K. L. M. C. Franken, Y. W. Subronto, B. Wieles, S. M. Arend, E. P. Sampaio, T. de Boer, W. R. Faber, B. Naafs, and T. H. M. Ottenhoff. 2002. Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T cell cross reactivity with Mycobacterium tuberculosis. Infect. Immun. 70: 2544.

9. Cole, S. T., K. Eiglmeier, J. Parkhill, K. D. James, N. R. Thomson, P. R. Wheeler, N. Honore, T. Gamier, C. Churcher, D. Harris, K. Mungall, D. Basham, D. Brown, T. chillingworth, R. Connor, R. M. Davies, K. Devlin, S.

Duthoy, T. Feltwell, A. Fraser, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, C. Lacroix, J. Maclean, S. Moule, L. Murphy, K. Oliver, M. A. Quail, M. -A. Rajandream, K. M. Rutherford, S. Rutter, K. Seeger, S. Simon, M. Sirnrnonds, J. Skelton, R. Squares, S. Squares, K. Stevens, K. Taylor, S. Whitehead, J. R. Woodward, and B. G. Barrell. 2001. Massive gene decay in the leprosy bacillus.

Nature 409:1007.

10. Cole, S.T., R. Brosch, J. Parkhill, T. Gamier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, F. Tekaia, K. Badcock, D. Basham, rui/ϊML mm / u u u i u o

31

D. Brown, T. Chillingworth, R. Connor, R., Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, B. G. Barrell. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537. 11. Fleischmann, R.D., D. Alland, J. A. Eisen, L. Carpenter, O. White, J. Peterson,

R. DeBoy, R. Dodson, M. Gwinn, D. Haft, E. Hickey, J. F. Kolonay, W. C. Nelson, L. A. Umayam, M. Ermolaeva, S. L. Salzberg, A. Delcher, T. Utterback, J. Weidman, H. Khouri, J. Gill, A. Mikula, W. R. Bishai Jacobs, Jr., J. C. Venter, and C. M. Fraser. 2002. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J. Bacteriol.

184:5479.

12. Gamier, T., K. Eiglmeier, J. C. Camus, N. Medina, H. Mansoor, M. Pryor, S. Duthoy, S. Grondin, C. Lacroix, C. Monsempe, S. Simon, B. Harris, R. Atkin, J. Doggett, R. Mayes, L. Keating, P. R. Wheeler, J. Parkhill, B. G. Barrell, S. T. Cole, S. V. Gordon, and R. G. Hewinson. 2003. The complete genome sequence of Mycobacterium bovis. Proc. Natl. Acad. ScL USA. 100:7877.

13. Sturniolo, T., E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler, F. Gallazzi, M. P. Protti, F. Sinigaglia, and J. Hammer. 1999. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17:555.

14. Truman, R.W., and J. L. Krahenbuhl. 2001. Viable M. leprae as a research agent. Int. J. Leprosy and Other Mycobact. Dis. 69:1.

15. Williams, D.L., S. Oby-Robinson, T. L. Pittman, and D.M. Scollard. 2003. Purification of Mycobacterium leprae RNA for gene expression analysis directly from biopsy specimens of leprosy patients. BioTechniques 35:534.

16. Franken, K.L.M.C., H. S. Hiemstra, K. E. van Meijgaarden, Y. W. Subronto, J. den Hartigh, T. H. M. Ottenhoff, and J. Drijfhout. 2000. Purification of His- tagged proteins by immobilized chelate affinity chromatography (IMAC): the benefits from the use of organic solvent. Protein expression and Purification. 18:95.

17. Buhrer-Sekula, S., E. N. Sarno, L. Oskam, S. Koop, I. Wichers, J. A. Nery, L. M. Vieira, H. J. de Matos, W. R. Faber, and P. R. Klatser. 2000. Use of ML dipstick as a tool to classify leprosy patients. Int. J. Lepr. 68:456. K %j a / a «■_ ••»*»»«^> - - - - • -^■ —

32

18. Aagaard, C, I. Brock, A. Olsen, T. H. M. Ottenhoff, K. Weldingh, and P. Andersen. 2004. Mapping immune reactivity towards Rv2653 and Rv2654: two novel low-molecular-mass Ag found specifically in the Mycobacterium tuberculosis complex. J. Inf. Dis. 189: 812. 19. Wucherpfennig, K. W., and J. L. Strominger. 1995. Molecular mimicry in T cell- mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80:695. 20. Andersen P., M. E. Munk, J. M. Pollock, and T. M. Doherty. 2000. Specific immune-based diagnosis of tuberculosis. Lancet 356:1099. 21. Arend S.M., P. Andersen, K. E. van Meijgaarden, R. L. Skjot, Y. W. Subronto,

J. T. van Dissel, and T. H. M. Ottenhoff. 2000. Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10. J Infect Dis. 181:1850.

22. Agger, E. M., I. Brock, L. M. Okkels, S. M. Arend, C. S. Aagaard, K. N. Weldingh, and P. Andersen. 2003. Human T-cell responses to the RDl -encoded protein TB27.4 (Rv3878) from Mycobacterium tuberculosis. Immunology 110:507.

23. Liu, X. Q., D. Dosanjh, H. Varia, K. Ewer, P. Cockle, G. Pasvol, and A. Lalvani. 2004. Evaluation of T-cell responses to novel RDl- and RD2-encoded Mycobacterium tuberculosis gene products for specific detection of human tuberculosis infection. Infect Immun. 72:2574.

24. Brock, L, K. Weldingh, E. M. Leyten, S. M. Arend, P. Ravn, and P. Andersen. 2004. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection. J. CHn. Microbiol. 42:2379. 25. Spencer, J. S., M. A. M. Marques, M. C. B. S. Lima, A. P. Junqueira-Kipnis, B.

C. Gregory, R. W. Truman, and P. J. Brennan. 2002. Antigenic specificity of the Mycobacterium leprae homologue of ESAT-6. Infect. Immun. 70:1010.

26. Spencer, J. S., H. J. Kim, A. M. Marques, M. C. B. S. Lima, V. D. Vissa, M. L. Gennaro, S-N. Cho, S. T. Cole, and P. J. Brennan. 2004. Comparative analysis of B- and T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture filtrate protein 10. Infect. Immun. 72:3161.

27. Booth, R. J., D. L. Williams, K. D. Moudgil, L. C. Noonan, P. M. Grandison, J. J. McKee, R. L. Prestidge, and J. D. Watson JD. 1993. Homologs of « V l / Di U fc.ω uu • w w w i \j \j

33

Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria. Infect. Immun. 61:1509.

28. (http://www.sanger.ac.uk/Projects/.

29. http://www.tigr.org/tdb/mdb/mdbinprogress.html. 30. http://www.ncbi.nlm.nih.gov/genomes/.

31. http://genopole.pasteur.fr/Mulc/BuruList.html.

32. Parker, K. C, M. A. Bednarek, and J. E. Coligan. 1994. Scheme for ranking potential HLA- A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152:163. HLA BIND (http://bimas.cit.nih.gov/mo lbio/hla bind/)

33. Rammensee, Friede, Stevanovic, MHC ligands and peptide motifs: 1st listing, Immunogenetics 41, 178-228, 1995; SYFPEITHI (http://www.syfpeithi.de/) and Rammensee, Bachmann, Stevanovic:MHC ligands and peptide motifs. Landes Bioscience 1997 (International distributor - except North America: Springer Verlag GmbH & Co. KG, Tiergartenstr. 17, D-69121 Heidelberg.

34. Buus S, Lauemoller SL₅ Worning P, Kesmir C, Frimurer T, Corbet S, Fomsgaard A, Hilden J₅HoIm A, Brunak S. Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee' artificial neural network approach, in Tissue Antigens., 62:378-84, 2003; NetMHC (http://www.cbs.dtu.dk/services/NetMHC/)

35. Nielsen M₅ Lundegaard C₅ Worning P, Lauemoller SL₅ Lamberth K₅ Buus S₅ Brunak S, Lund 0., Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein ScL₅ 12:1007-17, 2003.

36. Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach, Nielsen M, Lundegaard C, Worning P₅ Hvid CS₅ Lamberth

K, Buus S₅ Brunak S₅ Lund 0.₅ Bioinformatics, 20(9):1388-97, 2004.

37. Sturniolo, T. et al.₅ Nature Biotechnology 17, 555-562, 1999, Generation of tissue-specific and promiscuous HLA ligand databases using DNA chips and virtual HLA class II matrices; TEPITOPE (http ://www. vaccinome.com/pages/597444/) .

38. Oliver, K.G., J.R. Kettman, and RJ. Fulton, Multiplexed analysis of human cytokines by use of the FlowMetrix system. Clin Chem, 1998. 44(9): p. 2057- 60. 39. de Jager, W., et al., Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin Diagn Lab Immunol, 2003. 10(1): p. 133-9.

Claims

I W I / I M !_ £.UUU ' w w \J I KJ yJ35 Claims

1. A polypeptide having a length of 8 - 35 amino acids comprising an amino acid sequence stretch of at least 8 contiguous amino acids having at least 80 % identity with a sequence selected from SEQ ID No's 1 to 5.

2. The polypeptide according to claim 1 wherein the length of the peptide is between 15 and 25 amino acids.

3. The polypeptide according to claims 1 or 2, wherein the at least 8 contiguous amino acids comprise a T-cell epitope.

4. The polypeptide according to any of claims 1 to 3, wherein the polypeptide is capable of eliciting an IFN-γ response in blood cells of a subject carrying a paucibacillary Mycobacterium leprae infection.

5. A polypeptide according to any of the preceding claims comprising at least 8 contiguous amino acids from the sequences in SEQ ID No's 35 - 39.

6. A nucleic acid molecule comprising a nucleotide sequence selected from:

(a) a nucleotide sequence encoding a polypeptide as defined in any one of claims 1 to 5;

(b) a nucleotide sequence that has at least 80 % nucleotide identity with any of SEQ ID No's 18 to 22; (c) a nucleotide sequence the complementary strand of which hybridizes to a nucleotide sequence of (a) or (b); and,

(d) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of (a-c) due to the degeneracy of the genetic code.

7. A nucleic acid vector comprising the nucleic acid sequence according to claim 6.

8. A recombinant organism, preferably a recombinant Mycobacterium, comprising the DNA vector according to claim 7.

9. An antigenic fragment from Mycobacterium leprae that: i) is not present in related Mycobacterium species tuberculosis, paratuberculosis and avium; ii) is expressed in vivo in an infected mammal; iii) comprises T cell epitopes; and iv) is capable of elicting an INF-γ response in individuals carrying a M. leprae infection; or, v) comprises an amino acid sequence stretch of at least 8 contiguous amino acids having at least 80% identity with an amino acid sequence selected from the group of uniquely M. leprae encoded polypeptides: ML0576 (SEQ ID No. 1), ML1989 (SEQ ID No. 2), MLl 990 (SEQ ID No. 3), ML2283 (SEQ ID No. 4), ML2567 (SEQ ID No. 5).

10. A method for diagnosing a Mycobacterium leprae infection in a mammal, comprising the step of in vivo or in vitro detection of an immune response of a subject to at least one or more M. leprae antigens, selected from the group of M. leprae specific antigens ML0576 (SEQ ID No. 1), ML1989 (SEQ ID No. 2), ML1990 (SEQ ID No. 3), ML2283 (SEQ ID No. 4), ML2567 (SEQ ID No. 5).

11. The method according to claim 10 wherein the in vitro detection takes place on a sample of peripheral blood or on isolated peripheral blood monocytes (PBMCs).

12. The method according to any of claims 10 or 11 wherein the detection comprises measuring an T helper response comprising production or release of a compound selected from the group IFN-γ, IFN-α, IFN-β, TNF-cc, TGF-β, IL-2, IL-4, IL-6, IL-10, IL-12 (p70), IL-15, GM-CSF, MIP-Ib, upon contacting PBMCs with one or more of the antigens or fragments thereof.

13 The method according to claim 10 wherein the in vivo detection is a skin test comprising application of the antigen under the top skin.

14. The method according to any of claims 10 to 13 wherein the immune response is detected using an antigenic fragment as defined in claim 8 or a polypeptide according to any of claims 1 to 5. f*O | / I^L ZUUO / U U U

37

15. The use of an antigenic fragment as defined in claim 9 or a polypeptide as defined in any of claims 1 to 5 for the manufacture of a medicament.

16. The use according to claim 15 wherein the medicament is for the prevention, treatment or diagnosis of M. leprae infections.

17. An immunogenic composition, comprising one or more polypeptides as defined in any one of claims 1 to 5 or an antigenic fragments as defined in claim 8, optionally comprising at least one pharmaceutically acceptable excipient or adjuvant.

18. The immunogenic composition according to claim 17, wherein the adjuvant is selected from the group of adjuvants consisting of poly(I:C), LPS, lipid A, CD40 binding CD40 ligand or antibody, agonistic anti-4-lBB antibody, CpG motifs, lipopeptide Pam3Cys, IC31, QS21/MPL, DDA/MPL, DA/TDB, soluble LAG3 and bacterial flagellins.

19. Kit of parts comprising at least one or more polypeptides as defined in any one of claims 1 to 4, and optionally means for the detection of an immune response in blood cells.

20. A method for the identification of Mycobacterial antigens that are specific for M. leprae, comprising at least the steps of:

(a) identification from its genome M. leprae candidate genes encoding peptides, the peptides having less than 30 % homology with other Mycobacterial species;

(b) identification of potential HLA class I and/or class II T-cell epitopes using computer algorithms;

(c) confirming expression of the candidate antigens in vivo;

(d) determining the specificity of the putative antigen using a panel of subjects consisting of subjects carrying multibacillary M. leprae infections, subjects carrying a paucibacillary infection, healthy endemic control subjects and healthy household control subjects that have been exposed to M. leprae infected individuals but do not show any clinical signs of leprosy themselves and optionally subjects carrying Mycobacterial infections of other species than M. leprae.