Compounds and methods useful in tuberculosos immunotherapy and diagnostics

The invention relates generally to the detection, treatment and prevention of Mycobacterium tuberculosis infections. More particularly, the invention relates to polypeptides that are Mycobacterium tuberculosis antigen, or a portion or variant thereof, and the use of such polypeptides for diagnosis and vaccination against Mycobacterium tuberculosis infections.

Tuberculosis is a chronic infectious disease that is caused by infection with Mycobacterium tuberculosis. It is primarily a disease of developing countries, but is a growing problem in developed regions of the world, with 8 million new cases and 3 million deaths per year. Although the infection may be asymptomatic for a long time, the disease usually presents as acute pneumonia, with high fever and a non-productive cough. If left untreated, it causes serious complications and is usually fatal.

Although tuberculosis can be generally controlled with chronic antibiotic therapy, such treatment is insufficient to prevent the spread of the disease. Infected individuals may be asymptomatic but sometimes contagious. In addition, while adherence to treatment regimen is essential, patient behavior is difficult to track. Some patients do not follow a full course of treatment, which can lead to ineffectiveness and the development of drug resistance.

Inhibiting the spread of tuberculosis requires effective vaccination and thorough, early diagnosis of the disease. Currently, vaccination with live bacteria is the most effective method of inducing protective immunity. The most commonly used Mycobacterium for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a matter of controversy and some countries, such as the United States, do not vaccinate the general population. Diagnostics is usually performed using skin tests that include intradermal exposure to tuberculous PPD (purified protein fraction). The antigen-specific T-cell reaction produces a measurable reaction at the injection site 48-72 hours after injection, indicating exposure to mycobacterial antigens. Sensitivity and specificity are, however, a problem with this test, and BCG vaccinated individuals cannot be distinguished from infected individuals.

Although macrophages have been shown to act as the major effector of M. tuberculosis immunity, the main inducers of this immunity are T cells. The essential role of T cells in immunity against Mycobacterium tuberculosis is demonstrated by the frequent occurrence of Mycobacterium tuberculosis in AIDS patients due to depletion in lymphocytes. T CD4, caused by human immunodeficiency virus (HIV) infection. Mycobacterial-responsive CD4 T cells have been shown to produce significant amounts of interferon gamma (IFN-γ), which has been shown to trigger a mycobacterial killing effect in mice in macrophages. Although the role of IFN-γ in humans is less understood, studies have shown that 1,25-dihydroxy-vitamin D3, alone or in combination with IFN-γ or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Moreover, iFN-γ is known to stimulate human macrophages to produce 1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating immunity to Mycobacterium tuberculosis infection. A review of the immunology of Mycobacterium tuberculosis infection is described in Chan and Kaufmann, Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, DC, 1994.

Thus, there is a need in the art for improved vaccines and methods for the prevention, treatment and detection of tuberculosis. The invention meets these needs and provides other further advantages.

Briefly, the invention provides substances and methods for the prevention and diagnosis of tuberculosis. In one aspect, polypeptides are provided comprising an immunogenic portion of a soluble Mycobacterium tuberculosis antigen, or a variant of such antigen that differs only by conservative substitutions and / or modifications. In one embodiment of this aspect, the soluble antigen has one of the following N-terminal sequences:

(a) Asp-Pro-Val-Asp-Ala-Val-ne-Asn-Thr-Thr-Cys-Asn-Tyr-Gly-Gln-Val-Val-Ala-AlaLeu; (SEQ ID NO: 120) (b) Ala-Val-Glu-Ser-Gly-Met-Leu-Ala-Leu-Gly-Thr-Pro-Ala-Pro-Ser; (Sequence ID No.121)

186 774 (c) Ala-Ala-Met-Lys-Pro-Arg-Thr-Gly-Asp-Gly-Pro-Leu-Glu-Ala-Ala-Lys-Glu-Gly-Arg; (SEQ ID NO: 122) (d) T yr-T yr-T rp-Cys-Pro-Gly-Gln-Pro-Phe-Asp-Pro-Ala-T rp-Gly-Pro; (SEQ ID NO: 123) (e) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa-Ala-Val; (SEQ ID NO: 124) (f) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val-Pro; (SEQ ID NO: 125) (g) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Thr-Ala-Ala-Ser-Pro-Pro-Ser; (Seq. No. 126) (h) Ala-Pro-Lys-Thr-Tyr-Xaa-Glu-Glu-Leu-Lys-Gly-Thr-Asp-Thr-Gly; (SEQ ID NO: 127) (i) Asp-Pro-A] a-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala-Gln-Leu-Thr-Ser-Leu-Leu-AsnSer- Leu-Ala-Asp-Pro-Asn-Val-Ser-Phe-Ala-Asn; (SEQ ID NO: 128) (j) Xaa-Asp-Ser-Glu-Lys-Ser-Ala-Thr-Ile-Lys-Val-Thr-Asp-Ala-Ser; (SEQ ID NO: 134) (k) Ala-Gly-Asp-Thr-Xaa-Ile-Tyr-fle-Val-Gly-Asn-Leu-Thr-Ala-Asp; (Seq. No. 135) or (l) Ala-Pro-Glu-Ser-Gly-Ala-Gly-Leu-Gly-Gly-Thr-Val-Gln-Ala-Gly; (SEQ ID NO: 136) wherein Xaa can be any amino acid.

In a related aspect, polypeptides are provided comprising an immunogenic portion of a Mycobacterium tuberculosis antigen, or a variant thereof, that only differs by conservative substitutions and / or modifications, and the antigen has one of the following N-terminus sequences:

(m) Xaa-Tyr-Ile-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val-Pro-Gly-Lys-Ile-Asn-Val-HisLeu-Val; (SEQ ID NO: 137) Iub (n) Asp-Pro-Pro-Asp-Pro-His-Gln-Xaa-Asp-Met-Thr-Lys-Gly-Tyr-Tyr-Pro-Gly-GlyArg-Arg-Xaa -Phe; (SEQ ID NO: 129) wherein Xaa can be any amino acid.

In another embodiment, the antigen comprises an amino acid sequence encoded by a DNA sequence selected from the group consisting of the sequences listed as Sequence Identifiers. Nos. 1, 2, 4-10, 13-25, 52, 99 and 101, their complementary sequences and the sequences which hybridize to the sequences listed as SEQ ID NOS. Nos. 1, 2, 4-10, 13-25, 52, 99, and 101 and their complementary sequences, under moderately stringent hybridization conditions.

In a related aspect, the polypeptides include the immunogenic portion of the Mycobacterium tuberculosis antigen, or a variant of such antigen that differs only in conservative substitutions and / or modifications, and the antigen comprises an amino acid sequence encoded by a DNA sequence selected from the group consisting of the sequences listed as Sequence Identifiers. Nos .: 26-51 their complementary sequences and the sequences which hybridize to the sequences listed as Sequence Identifiers. Nos .: 26-51 and their complementary sequences, under moderately stringent hybridization conditions.

In a related aspect, also provided are DNA sequences encoding the above polypeptides, expression vectors including these DNA sequences, and host cells transformed or transfected with such expression vectors.

In another aspect, the invention provides fusion proteins comprising a first and second polypeptide of the invention, or alternatively, a polypeptide of the invention and a known Mycobacterium tuberculosis antigen.

In other aspects, the invention provides pharmaceutical compositions comprising one or more of the above polypeptides, or a DNA molecule encoding such polypeptides and a physiologically acceptable carrier. The invention also provides vaccines comprising one or more polypeptides as described above and a non-specific immune response enhancer, together with vaccines comprising one or more DNA sequences encoding such polypeptides and a non-specific immune response enhancer.

186 774

In yet another aspect, methods are provided for inducing protective immunity in a patient comprising administering to the patient an effective amount of one or more of the above polypeptides.

In further aspects of the invention, methods and diagnostic kits for detecting tuberculosis in a patient are provided. The methods include contacting skin cells of the patient with one or more of the above polypeptides and detecting an immune response on the skin of the patient. The diagnostic kits include one or more of the above polypeptides in combination with an apparatus suitable for contacting the polypeptides with the skin cells of a patient.

These and other aspects of the invention will become apparent with reference to the following detailed description and accompanying drawings. All references disclosed herein are incorporated by reference in their entirety because they are individually incorporated.

Figure 1A and B shows the stimulation of proliferation and interferon-γ production in T cells derived from a first and second Mycobacterium tuberculosis resistant donor, respectively, by the 14 kDa, 20 kDa and 26 kDa antigens described in Example 1.

Figure 2 shows the stimulation of proliferation and production of interferon-γ in T cells derived from a Mycobacterium tuberculosis resistant donor by two representative polypeptides, TbRa3 and TbRa9.

Seq. No. 1 is the TbRal DNA sequence

Seq. # 2 is the TbRal 0 DNA sequence

Seq. # 3 is the TbRal 1 DNA sequence

Seq. # 4 is the TbRal 2 DNA sequence

Seq. # 5 is the TbRal 3 DNA sequence

Seq. # 6 is the TbRal 6 DNA sequence

Seq. # 7 is the TbRal 7 DNA sequence

Seq. No. 8 is the TbRal 8 DNA sequence

Seq. # 9 is the TbRal 9 DNA sequence

Seq. No. 10 is the TbRa24 DNA sequence

Seq. No. 11 is the TbRa26 DNA sequence

Seq. No. 12 is the TbRa28 DNA sequence

Seq. ID No. 13 is the TbRa29 DNA sequence

Seq. No. 14 is the TbRa2A DNA sequence

Seq. No. 15 is the TbRa3 DNA sequence

Seq. No. 16 is the TbRa32 DNA sequence

Seq. No. 17 is the TbRa35 DNA sequence

Seq. No. 18 is the TbRa36 DNA sequence

Seq. No. 19 is the TbRa4 DNA sequence

Seq. No. 20 is the TbRa9 DNA sequence

Seq. No. 21 is the TbRaB DNA sequence

Seq. No. 22 is the TbRaC DNA sequence

Seq. No. 23 is the TbRaD DNA sequence

Seq. No. 24 is the DNA sequence of YYWCPG

Seq. No. 25 is the AAMK DNA sequence

Seq. No. 26 is the TbL-23 DNA sequence

Seq. No. 27 is the TbL-24 DNA sequence

Seq. No. 28 is the TbL-25 DNA sequence

Seq. No. 29 is the TbL-28 DNA sequence

Seq. No. 30 is the TbL-29 DNA sequence

Seq. # 31 is the DNA sequence of TbH-5

Seq. No. 32 is the DNA sequence of TbH-8

Seq. # 33 is the DNA sequence of TbH-9

Seq. No. 34 is the TbM-1 DNA sequence

Seq. No. 35 is the TbM-3 DNA sequence

Seq. No. 36 is the TbM-6 DNA sequence

186 774

Seq. ID No. 37 is the TbM-7 DNA sequence. SEQ. ID No. 38 is the TbM-9 DNA sequence. SEQ. No. 39 is the TbM-12 DNA sequence SEQ ID NO. ID No. 40 is the TbM-13 DNA sequence. SEQ. ID No. 41 is the TbM-14 DNA sequence. SEQ. ID No. 42 is the TbM-15 DNA sequence. SEQ. ID No. 43 is the DNA sequence of TbH-4 SEQ. ID No. 44 is the DNA sequence of TbH-4-FWD. Seq. ID No. 45 is the DNA sequence of TbH-12 SEQ. ID No. 46 is the DNA sequence of Tb38-1 SEQ. ID No. 47 is the DNA sequence of Tb38-4. SEQ. ID No. 48 is the TbL-17 DNA sequence. SEQ. No. 49 is the TbL-20 DNA sequence. SEQ. ID No. 50 is the TbL-21 DNA sequence. SEQ. ID No. 51 is the DNA sequence of TbH-16 SEQ. ID No. 52 is the DPEP DNA sequence. SEQ. ID No. 53 is the putative DPEP amino acid sequence. Seq. ID NO: 54 is the N-terminal protein sequence of the DPV antigen. SEQ. ID No. 55 is the N-terminal protein sequence of the AVGS antigen. Seq. ID NO: 56 is the N-terminal protein sequence of the AAMK antigen. Seq. ID No. 57 is the N-terminal protein sequence of the YYWC antigen. Seq. ID No. 58 is the N-terminal protein sequence of the DIGS antigen. SEQ. ID No. 59 is the N-terminal protein sequence of the AEES antigen. Seq. ID NO: 60 is the N-terminal protein sequence of the DPEP antigen. SEQ. ID No. 61 is the N-terminal protein sequence of the APKT antigen. Seq. ID NO: 62 is the N-terminal protein sequence of the DPAS antigen. ID No. 63 is the putative TbRal amino acid sequence. Seq. ID No. 64 is the putative amino acid sequence of TbRa1 0 Seq. ID No. 65 is the putative TbRal 1 amino acid sequence. SEQ. ID No. 66 is the putative TbRal 2 amino acid sequence. Seq. ID No. 67 is the putative TbRal 3 amino acid sequence SEQ ID. ID No. 68 is the putative TbRal 6 amino acid sequence SEQ ID. No. 69 is the putative TbRal 7 amino acid sequence SEQ ID. ID No. 70 is the putative TbRal 8 amino acid sequence SEQ ID. ID No. 71 is the putative amino acid sequence of TbRal 9 SEQ ID NO. ID No. 72 is a putative TbRa24 amino acid sequence. Seq. ID No. 73 is a putative TbRa26 amino acid sequence. Seq. No. 74 is a putative TbRa28 amino acid sequence. Seq. ID No. 75 is a putative TbRa29 amino acid sequence. Seq. ID No. 76 is a putative TbRa2A amino acid sequence. Seq. ID No. 77 is a putative TbRa3 amino acid sequence. Seq. ID No. 78 is a putative TbRa32 amino acid sequence. Seq. ID No. 79 is a putative TbRa35 amino acid sequence. Seq. No. 80 is a putative TbRa36 amino acid sequence. Seq. ID No. 81 is the putative TbRa4 amino acid sequence. Seq. ID No. 82 is a putative TbRa9 amino acid sequence. Seq. ID No. 83 is the putative TbRaB amino acid sequence. Seq. ID No. 84 is the putative TbRaC amino acid sequence. Seq. ID No. 85 is a putative TbRaD amino acid sequence. Seq. ID No. 86 is the putative amino acid sequence of YYWCPG. Seq. ID No. 87 is a putative TbAAMK amino acid sequence. Seq. ID No. 88 is a putative Tb38-1 amino acid sequence. Seq. ID No. 89 is the putative amino acid sequence of TbH-4 SEQ. No. 90 is the putative amino acid sequence of TbH-8

186 774

Seq. ID No. 91 is the putative amino acid sequence of TbH-9 SEQ ID. ID No. 92 is the putative amino acid sequence of TbH-12 SEQ. ID NO. 93 is the TbRa amino acid sequence of peptide 1 of Tb38-1. SEQ. No. 94 is the TbRa amino acid sequence of peptide 2 of Tb38-1. SEQ. No. 95 is the TbRa amino acid sequence of peptide 3 of Tb38-1 SEQ. ID No. 96 is the TbRa amino acid sequence of peptide 4 of Tb38-1. SEQ. ID No. 97 is the TbRa amino acid sequence of the Tb38-1 peptide SEQ ID. ID NO. 98 is the TbRa amino acid sequence of peptide 6 of Tb38-1. SEQ. # 99 is the DPAS DNA sequence

Seq. No. 100 is an putative DPAS amino acid sequence

Seq. # 101 is the DPV DNA sequence

Seq. ID No. 102 is the putative DPV amino acid sequence

Seq. No. 103 is the ESAT-6 DNA sequence

Seq. No. 104 is a putative ESAT-6 amino acid sequence

Seq. No. 105 is the DNA sequence of TbH-8-2

Seq. No. 106 is the DNA sequence of TbH-9FL

Seq. ID No. 107 is the putative amino acid sequence of TbH-9FL

Seq. No. 108 is the DNA sequence of TbH-9-1

Seq. No. 109 is the putative amino acid sequence of TbH-9-1

Seq. No. 110 is the DNA sequence of TbH-9-4

Seq. ID No. 111 is the putative amino acid sequence of TbH-9-4

Seq. No. 112 is the Tb38-1F2 IN DNA sequence

Seq. ID No. 113 is the Tb38-2F2 RP DNA sequence

Seq. No. 114 is a putative amino acid sequence. Tb37-FL

Seq. No. 115 is a putative Tb38-IN amino acid sequence

Seq. No. 116 is the Tb38-1F3 DNA sequence

Seq. No. 117 is the putative amino acid sequence of Tb38-1F3

Seq. No. 118 is the DNA sequence of Tb38-1F5

Seq. No. 119 is the DNA sequence of Tb38-1F6

Seq. ID No. 120 is the putative N-terminal amino acid sequence of DPV. SEQ. ID No. 121 is a putative N-terminal amino acid sequence of AVGS. Seq. ID No. 122 is a putative N-terminal amino acid sequence of AAMK

Seq. No. 123 is a putative N-terminal amino acid sequence of YYWC

Seq. ID No. 124 is a putative N-terminal amino acid sequence of DIGS. Seq. ID No. 125 is an putative N-terminal amino acid sequence of AEES. Seq. ID No. 126 is an putative N-terminal amino acid sequence of DPEP. Seq. ID No. 127 is a putative N-terminal amino acid sequence of APKT. Seq. ID No. 128 is the putative DPAS amino acid sequence. Seq. ID No. 129 is the protein sequence of the DPPD N-terminal antigen SEQ. Nos. 130-133 are the protein sequences of the four DPPD fragments from cyanogen bromide digestion

Seq. ID No. 134 is the N-terminal protein sequence of the XDS antigen. SEQ. ID No. 135 is the N-terminal protein sequence of the AGD antigen. SEQ. ID No. 136 is the N-terminal protein sequence of the APE antigen. Seq. No. 137 is the N-terminal protein sequence of an XYI antigen. As noted above, the invention relates generally to compositions and methods for the prevention, treatment, and diagnosis of tuberculosis. The compositions of the invention include polypeptides comprising at least one immunogenic portion of a Mycobacterium tuberculosis antigen or a variant of such antigen that only differs in conservative substitutions and / or modifications. Polypeptides within the scope of the invention include, but are not limited to, soluble, immunogenic M. tuberculosis antigens. “A soluble M. tuberculosis antigen is a protein derived from M. tuberculosis that is present

186 774 in a culture filtrate of M. tuberculosis. As used herein, the term "polypeptide" includes amino acid chains of any length, including full-length proteins (ie, antigens) in which amino acid residues are joined by covalent peptide bonds. Thus, a polypeptide comprising the immunogenic portion of any of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. tuberculosis may or may not be heterologous and may or may not be immunogenic.

"Immunogenic" as used herein refers to the ability to elicit an immune (eg, cellular) response in a patient, such as a human, and / or a biological sample. In particular, antigens that are immunogenic (and immunogenic portions or other variants of such antigens) are capable of promoting cell proliferation, interleukin-12 production, and / or interferon-γ production in biological samples containing one or more cells selected from the group consisting of T cells. NK, B, and macrophages, which cells are derived from an M. tuberculosis resistant individual. Polypeptides comprising at least an immunogenic portion of one or more M. tuberculosis can be used generally to detect tuberculosis or to induce protective anti-tuberculosis immunity in a patient.

The compositions and methods of the invention also include variants of the above polypeptides. "Variation" as used herein is a polypeptide that differs from the native antigen only by conservative substitutions and / or modifications such that the ability of the polypeptide to elicit an immune response is preserved. Such variants can generally be determined by modifying one of the above polypeptide sequences and testing the immunogenic properties of the modified polypeptides using, for example, representative procedures described herein.

A "conservative replacement" is one in which an amino acid is replaced with another amino acid with similar properties, such that one skilled in the art of peptide chemistry can expect the secondary structure and hydropathic nature of the polypeptide to remain substantially unchanged. In general, the following groups of amino acids are conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, cheese, thr, tyr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also be (or alternatively) modified by, for example, deletion or addition of amino acids which have little effect on the immunogenic properties, secondary structure, and hydropathic nature of the polypeptide. For example, a polypeptide may be coupled to a signal (or leader) sequence at the N-terminus of the protein that either co-translational or post-translational direct transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence to facilitate the synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance the binding of the polypeptide to a solid support. For example, a polypeptide can be fused to an Fc region of an immunoglobulin.

In a related aspect, combined polypeptides are disclosed. A "combination polypeptide" means a polypeptide comprising at least one of the above immunogenic portions and one or more additional M. tuberculosis immunogenic sequences that are linked by a peptide bond into a single amino acid chain. The sequences may be attached directly (i.e. with no spacer amino acids) or may be joined by a linker sequence (e.g. Gly-Cys-Gly), which does not significantly reduce the immunogenic properties of the constituent polypeptides.

In general, M. tuberculosis antigens, and DNA sequences encoding such antigens, can be produced using a variety of procedures. For example, soluble antigens can be isolated from M. tuberculosis culture filtrates by procedures known to those skilled in the art, including anion exchange chromatography or reverse phase chromatography. The purified antigens are then tested for their ability to elicit an appropriate immune response (e.g. cellular) by using, for example, representative methods described herein. Immunogenic antigens may then be

186 774 partially sequenced using techniques such as the traditional Edman method, see Edman and Berg, Eur. J. Biochem. 80: 116-132, 1967.

Immunogenic antigens can also be produced recombinantly, using antigen-encoding DNA sequences that have been inserted into an expression vector and expressed in a suitable host organism. DNA molecules encoding soluble antigens can be isolated by screening an appropriate M. tuberculosis expression library with antisera (e.g., rabbit) specifically raised against soluble M. tuberculosis antigens. DnA sequences encoding antigens which may or may not be soluble can be identified by screening an appropriate M. tuberculosis genomic expression library or cDNA using sera obtained from patients infected with M. tuberculosis. Such screening may generally be performed using techniques well known to those skilled in the art such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989.

DNA sequences encoding soluble antigens can also be obtained by screening an appropriate cDNA library or genomic DNA of M. tuberculosis for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of the isolated soluble antigens. Degenerate oligonucleotide sequences for use in such screening can be designed and synthesized, followed by screening as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989 (and references cited therein). ). Polymerase chain reaction (PCR) can also be used using the above oligonucleotides in methods well known in the art to isolate a nucleic acid probe from a cDNA or genomic library. Library screening can be performed using an isolated probe.

Alternatively, genomic and cDNA libraries derived from M. tuberculosis can be screened directly using peripheral blood mononuclear cells (PBMCs) or T cell lines derived from one or more M. tuberculosis resistant individuals. In general, PBMCs and / or T cells to be used in such screening can be prepared as described below. Direct library screening can be performed by testing pools of expressed recombinant proteins for their ability to induce proliferation and / or interferon-γ production in T cells derived from a M. tuberculosis resistant individual. Alternatively, potential T cell antigens may be initially selected based on antibody reactivity as described above.

Regardless of the method of production, the antigens (and their immunogenic portions) described herein (which may or may not be soluble) have the ability to elicit an immune response. More particularly, the antigens have the ability to induce proliferation and / or cytokine production (i.e. interferon-γ and / or interleukin-12) in T, NK, B lymphocytes and / or macrophages derived from M. tuberculosis resistant individuals. The choice of the type of cell to be used in testing the immune response to an antigen should depend on the desired response. For example, interleukin-12 production is tested using preparations containing B cells and / or macrophages. A subject resistant to M. tuberculosis is one that is believed to be resistant to the development of tuberculosis due to an efficient T cell response to M. tuberculosis (ie, substantially free of disease symptoms). Such individuals can be identified by having a strong positive (ie, greater than about 10 mm in diameter) skin reaction to tuberculin proteins (PPD) and the absence of any symptoms of tuberculosis. T, NK, B lymphocytes and macrophages derived from M. tuberculosis resistant individuals can be prepared using methods known to those skilled in the art. For example, the preparation of PBMCs (peripheral blood mononuclear cells) can be used without further separation of the constituent cells. PBMCs can generally be prepared, for example, by density gradient centrifugation through Ficoll ™ (Winthrop Laboratories, NY). The T cells for use in the assays described herein can also be purified from PBMCs. Alternatively, enriched lines of T lymphocytes reactive with myco10 proteins can be used

186 774 bacteria or T cell clones reactive with individual mycobacterial proteins. Such T cell clones can be raised by, for example, culturing PBMCs from M. tuberculosis resistant individuals with mycobacterial proteins for a period of 2-4 weeks. This allows only mycobacterial protein-specific T cells to propagate, resulting in lines consisting only of such cells. Such cells can then be cloned and tested with individual proteins using methods known to those skilled in the art to determine the specificity of individual T cells in detail. and interleukin-12) made with T, NK, B lymphocytes and / or macrophages derived from an M-resistant individual. tuberculosis are considered immunogenic. Such tests can be performed using, for example, the procedures described below. Immunogenic portions of such antigens can be identified using similar assays, and may be present in the polypeptides described herein.

The ability of a polypeptide (e.g., an immunogenic antigen, or a portion or variant thereof) to induce cell proliferation is tested by contacting cells (e.g., T and / or NK lymphocytes) with the polypeptide and measuring cell proliferation. Generally, sufficient polypeptide to be assayed for is around 10⁵ the cells range from about 10 µg / ml to about 100 µg / ml and is preferably about 10 µg / ml. Typically, the polypeptide is incubated with the cells at 37 ° C for about six days. Following incubation with oolioeptγdpm, cells are examined for a proliferative response which can be tested by methods known to those skilled in the art such as exoconcing the cells to a radioactive thymidine pulse and measuring the incorporation of the tag into the cell DNA. In general, a polipeotγd that causes at least a threefold increase in proliferation above background (ie, the proliferation seen with cells cultured without ooooαptγd) is considered capable of inducing proliferation.

The ability of ooliopptγide to stimulate the production of interferon-γ and / or κterleb Uikγ-12 in cells can be tested by contacting the cells with pklippiptide and measuring poa^lomu interferon-y or iktprlpuainy-^-k you^Łcramps in the cell. Overall, the amount of polyps^yooou, which is enough to test about 10^yyOmórpk is hung from about 1 0 ng / ml to about 10 gg / ml and the fruit is about 10 gg / ml. pkliopptγdu yUomókam^l is usually performed at 37 ° C for about six days. The polioeptide may or may not be immobilized on a solid support such as beads or biodegradable microspheres such as those described in US Patent Nos. 4,897,268 and 5,075,109. Typically, incubation of the polypeptide with the cells is carried out at 37 ° C for about six days. After incubation with ooliprotide, cells are tested for interferon-γ and / or γ-terleuUikγ-12 (or one or more of its subunits), which can be tested by methods known to those skilled in the art, such as the enzyme Sγcpoγ, an immunosorbent assay (ELISA) or, in the case of a polyunit. P70 IL-12 by a bioassay such as an assay to measure T cell proliferation.⁴-10⁵ of T cells per ml) is believed to induce γ production of interferon-γ. Poiippptyide inducing the production of Jrryγnα anemia 10 pg Okduekkku P70 IL-12 and / or^at00 pg / ml add one P40 IL-12 unit to 10⁵m^γkΌ^aboutrⁿthose either B lymphocytes (or at 3x^and0^at PBMC) is considered: to stimulate wγ^rface αrαe IL-12.

Generally, α-antigens are those α antigens which promote the proliferation and / or production of cγtoUin (i.e.^andwarkkme interferonmy and / or mterleuO^liay-] 2) by Emfocyuy T, NK, B and / or tkaarofęgi ooyhodzkce from prt^rsfor the lease of 25% of the housing estate in odporkyc^about on M tuberculosis. In (among these immukogakkyfh actigomas', oolioeotides in γkrrwjkfa, the strongest therapeutic properties can be distinguished by the magnitude of the reaction in oowγszyclr testayłl and in oαγcju by the percentage of dfo individuals which are affected by the reaction.^-More specific therapies will not be^psee the proliferation and / or production of phytoUjk in vitro in posthuman tumors from more than 25% of individuals who are not immune to M. tuberculosis, thus eliminating reactions that are not specific to M. tuberculosis responsive cultures. Antigens that induce a response in a significant percentage of T, B, NK lymphocyte preparations and / or in

186 The 774 M. tuberculosis-resistant individuals (with rare reactions in cell preparations from other individuals) have the strongest therapeutic properties.

Antigens with the highest properties can also be identified based on their ability to reduce the severity of a M. tuberculosis infection in experimental animals when administered as a vaccine. Suitable vaccine formulations for use in experimental animals are described in detail below. Their effectiveness can be determined based on the ability of the antigen to cause at least about 50% reduction in bacterial counts and / or at least about 40% reduction in mortality following experimental infection. Suitable experimental animals include mice, guinea pigs, and primates.

Antigens showing the most potent diagnostic properties can generally be identified by their ability to elicit a response in a skin test performed on individuals with active tuberculosis, but not on individuals not infected with M. tuberculosis. Skin testing may generally be performed as described below, with a pH of at least 5 mm in diameter considered positive.

Immunogenic portions of the antigens described herein can be made and identified using well-known techniques such as those described by Paul, Fundamental Immunology, 2nd Ed. 3, Raven Press, 1993, pp. 243-247 and references cited therein. Such techniques include screening a portion of a native antigen polypeptide for their immunogenic properties. The representative proliferation and cytokine production assays described herein can generally be used for screening. The immunogenic part of a polypeptide is that which, in representative assays, causes an immune response (e.g., proliferation, production of interferon-y and / or interleukin-12) that is substantially similar to that induced by the full length antigen. In other words, the immunogenic portion of the antigen can elicit at least about 20%, and preferably about 100%, of the full-length antigen-induced proliferation in the cell proliferation model described herein. The immunogenic portion may also, or alternatively, promote the production of at least about 20% and preferably about 100% of the full length antigen-induced interferon-γ and / or interleukin-12 in the assay described herein.

Parts and other variants of M. tuberculosis antigens can be produced synthetically or recombinantly. Synthetic polypeptides of less than 100 amino acids, and generally less than 50 amino acids, can be made using techniques well known to those skilled in the art. For example, such polypeptides can be synthesized using commercially available solid support techniques, such as the Merrifield solid support method where amino acids are added sequentially to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Instruments for automatic polypeptide synthesis are commercially available from suppliers such as Applied Biosystems, Inc., Foster City, CA, and may be operated according to the manufacturer's instructions. Variant native antigens can generally be made using standard mutagenesis techniques, such as oligonucleotide directed site mutagenesis. Portions of the DNA sequence can also be removed using standard techniques capable of producing truncated polypeptides.

Recombinant polypeptides containing parts and / or variants of the native antigen can be readily prepared from DNA sequences encoding the polypeptide using a variety of techniques well known to those skilled in the art. For example, supernatants from appropriate host / vector systems that secrete recombinant protein into the culture medium may first be concentrated using a commercially available filter. After concentration, the concentrate can be incorporated into a suitable purification matrix such as an affinity matrix or an anion exchange resin. Finally, one or more reversed-phase HPLC steps may be used to further purify the recombinant protein.

Any of a variety of expression vectors known to those skilled in the art may be used to express the recombinant polypeptides of the invention. Expression may be achieved in any suitable host cell that has been transformed or transfected with an expression vector containing a DNA molecule encoding poly12.

186 774 recombinant peptide. Suitable host cells include prokaryotic, yeast, and higher eukaryotic cells. Preferably, the host cell used is E. coli, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring antigens, parts of natural and occurring antigens, or other variations thereof.

In general, regardless of the method of production, the polypeptides disclosed herein are produced in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure. In certain preferred embodiments, detailed below, substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.

In certain particular embodiments, the invention discloses polypeptides comprising at least an immunogenic portion of a soluble M. tuberculosis antigen, including at least an immunogenic portion of a soluble M. tuberculosis antigen, having one of the following N-terminal sequences, or a variant thereof that only differs by conservative substitutions and / or modifications. :

(a) Asp-Pro-Val-Asp-Ala-Val-lle-Asn-Thr-Thr-Cys-Asn-Tyr-Gly-Gln-Val-Val-Ala-AlaLeu; (SEQ ID NO: 120) (b) Ala-Val-GlUjSerrGly-Met-LeUjAla-LeUjGly-Thr-Pro-Ala-Pro-Ssr; ((dentist

Seq. No. 121) (c) Ala-Ala-Llet-Pyr-Pro-Arg-'Γhγ-Glp-Gsy-Prly-Pro-Leu-Gla-Ala-Ala-Lyj-Glγ-GlyArg; (SEQ ID NO 122) (d) Tyγ-Tyr-Tηr-Cys-Pro-Gly-GlntPro-PPe-Asp-Pro-Ala-Trp-Giy-Pro- ((denttγfkator

Seq. No. 123) (e) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa-Ala-Val; (SEQ ID NO: 124) (f) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val-Pro; (SEQ ID NO: 125) (g) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Thr-Ala-Ala-Ser-Pro-Pro-Ser; (SEQ ID NO: 126) (h) Ala-Pro-Lys-ThrrTyyrXaa-G! U-Glu-Leu-Lys-Gly-ThrrAsp-ThrrG] y; (Identtγika-or Seq. No 127) (i) Asp-Pro-Ala-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala-Gln-Leu-Thr-Ser-Leu-Leu-AsnSer-Leu-Ala-Asp- Pro-Asn-Val-Ser-Phe-Ala-Asn; (SEQ ID NO: 128) (j) Xaa-Asp-Ser'Glu-Lys-Ser-Ala-Thr-I'le-Lγs-Val-Thr-Asp-Ala-Ser; (SEQ ID NO: 134) (k) Ala-Glγ-Asp-Thr-Xaa-Πe-Tγr-Πe-Val-Glγ-Asn-Leu-Thr-Ala-Asp; (SEQ ID NO: 5) Iub (l) Ala-Pro-Glu-Ser-Glγ-Ala-Gly-Leu-Gly-Gly-Thr-Val-Gln-Ala-Gly; (Seq. No. 136) where Xaa can be any amino acid, preferably a cysteine ​​residue. A DNA sequence encoding the antigen identified as (g) is provided as Sequence Identifier. ID No. 52 and the polypeptide encoded by SEQ ID NO: No. 52 is provided as SEQ ID NO. ID: 53. A DNA sequence encoding the antigen specified as (a) is provided as SEQ ID NO. ID No. 101, a deduced amino acid sequence thereof is provided as SEQ ID. no 102. A DNA sequence corresponding to antigen (d) is provided as Sequence Identifier. ID: 24. A DNA sequence corresponding to antigen (c) is provided as SEQ ID NO. ID: 25. The DNA sequence corresponding to the antigen (i) is provided as SEQ ID NO. No. 99, a deduced amino acid sequence thereof is provided as SEQ ID. no 100.

In a further specific embodiment, the invention discloses polypeptides comprising an immunogenic portion of a Mycobacterium tuberculosis antigen, or a variant thereof, which only differs by conservative substitutions and / or modifications, and the antigen has one of the following N-terminal sequences:

186 774 (m) Xaa-Tyr-He-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val-Pro-Gly-Lys-Ile-Asn-Val-HisLeu-Val; (SEQ ID NO: 137) or (n) Asp-Pro-Pro-Asp-Pro-His-Gln-Xaa-Asp-Met-Thr-Lys-Gly-Tyr-Tyr-Pro-Gly-GlyArg-Arg-Xaa -Phe; (SEQ ID NO: 129) wherein Xaa can be any amino acid, preferably a cysteine ​​residue.

In another specific embodiment, the invention discloses a polypeptide that includes at least an immunogenic portion of a soluble M. tuberculosis antigen (or a variant of such antigen) that includes one or more amino acid sequences encoded by (a) the sequences encoded by Sequence Identifiers. Nos. 1, 2, 4-10, 13-25, and 52; (b) their complementary sequences; or (c) DNA sequences substantially homologous to the DNA sequences of (a) or (b).

In another specific embodiment, the invention discloses a polypeptide comprising at least an immunogenic portion of a soluble M. tuberculosis antigen (or a variant of such antigen), which may or may not be soluble, which comprises one or more amino acid sequences encoded by (a) the sequences encoded by Seq Identifiers . no .: 26-52; (b) their complementary sequences; or (c) DNA sequences substantially homologous to the DNA sequences of (a) or (b).

In the specific embodiments discussed herein, the M. tuberculosis antigens include variants that are encoded by DNA sequences that are substantially homologous to one or more of the DNA sequences listed herein. "Substantial homology" as used herein refers to DNA sequences that are capable of hybridizing under moderately stringent conditions. Suitable moderately stringent conditions include a pre-rinse with 5X SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridize at 55-65 ° C in 5X SSC overnight or, in the case of interspecies homology at 45 ° C, 0.5X SSC; followed by washing twice at 65 ° C for 20 minutes in 2X, 0.5X and 0.2X SSC containing 0.5% SDS, respectively). Such hybridizing DNA sequences are within the scope of the invention, as are nucleotide sequences which, due to degeneracy, code for an immunogenic polypeptide which is encoded by the hybridizing DNA sequence.

In a related aspect, the invention provides fusion proteins comprising a first and a second polypeptide of the invention or, alternatively, a polypeptide of the invention and a known M. tuberculosis antigen, such as the 38 kDa antigen described above or ESAT-6 (SEQ ID NOS: 103 and 104). along with variants of such fusion proteins. The fusion proteins of the invention may also include a peptide linker between the first and second polypeptides.

The DNA sequence encoding the fusion protein of the invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides in an appropriate expression vector. The 3 'end of the DNA sequence encoding the first polypeptide is ligated with or without a peptide linker to the 5' end of the DNA sequence encoding the second polypeptide such that the reading frames are in the same phase, allowing the mRNA of the two DNA sequences to be translated as one fusion protein that retains the biological activity of both the first and second polypeptides.

A peptide linker sequence can be used to separate the first and second polypeptides by a distance that allows each polypeptide to fold into its secondary and tertiary structure. Such a peptide linker sequence is introduced into the fusion protein by methods known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to adopt an extended, flexible conformation; (2) failure to adopt a secondary structure that would interact with functional epitopes of the first and second polypeptides; and (3) the absence of hydrophobic or charged residues that would react with the functional epitopes of the polypeptide. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other nearly neutral amino acids such as Thr and Ala can also be used for the peptide linker sequence. Amino acid sequences which can advantageously be used as linkers are disclosed in

186 774

Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262,186; U.S. Patent Nos. 4,935,233 and 4,751,180. The linker sequence may be from 1 to 50 amino acids in length. Peptide sequences are not necessary when the first and second polypeptides have non-essential amino acid regions at the N-terminus that can be used to separate functional domains and prevent spherical interaction.

The ligated dNa sequences are operably linked to appropriate transcriptional or translational regulatory elements. Regulatory elements responsible for expression of DNA are located exclusively 5 'from the DNA sequence encoding the first polypeptide. Likewise, stop codons required to complete translation and transcription termination signals are present only at the 3 'end of the DNA sequence encoding the second polypeptide.

In another aspect, the invention provides methods for using one or more of the above polypeptides or fusion proteins (or DNA molecules encoding such polypeptides) to induce protective immunity in a patient against tuberculosis. As used herein, "patient" refers to any warm-blooded animal, preferably a human. The patient may be afflicted with the disease or may be free from detectable disease and / or infection. In other words, protective immunity can be induced to prevent or treat tuberculosis.

In this aspect, the polypeptide, fusion protein or DNA molecule is generally present in a pharmaceutical composition and / or a vaccine. The pharmaceutical compositions may include one or more polypeptides, each of which may contain one or more of the above sequences (or variations thereof), and a physiologically acceptable carrier. Vaccines may include one or more of the above polypeptides and a non-specific enhancer of the immune response, such as an adjuvant or liposome (into which the polypeptide is incorporated). Such pharmaceutical compositions and vaccines may also contain other M. tuberculosis antigens, incorporated into the combination polypeptides or present in separate polypeptides.

Alternatively, the vaccine may contain DNA encoding one or more polypeptides as described above, such that the polypeptides are generated in situ. In such vaccines, the DNA can be in any of the delivery systems known to those of skill in the art, including nucleic acid expression systems, viral and bacterial expression systems. Suitable nucleic acid expression systems include DNA sequences necessary for expression in the patient (such as appropriate promoter and termination signals). Bacterial delivery systems involve the administration of bacteria (such as Bacillus Calmette-Guerin) that express on their surface an immunogenic portion of a polypeptide. In a most preferred embodiment, the DNA can be introduced using viral expression systems (e.g. Vaccinia virus or other pox virus, retrovirus or adenovirus), which may involve the use of non-pathogenic (defective) replication competent viruses. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can also be "naked" as described in, for example, Ulmer et al., Science 259: 1745-1749, 1993 and Cohen, Science 259: 1691-1692, 1993. Uptake of naked DNA can be enhanced by coating the DNA of biodegradable beads, which are efficiently transported inside the cells.

In a related aspect, a DNA vaccine as described above may be administered simultaneously with or sequentially with a polypeptide of the invention or a known M. tuberculosis antigen, such as the 38 kDa antigen described above. For example, administration of DNA encoding a polypeptide of the invention, naked or in a delivery system as described above, may precede administration of the antigen to enhance the protective immunity effect of the vaccine.

The route and frequency of administration, as well as the doses, will vary from subject to subject and may be similar to those currently used in immunization with BCG. Generally, the pharmaceutical compositions and vaccines can be administered by injection (e.g., intradermally, intramuscularly, intravenously or subcutaneously), intranasally (e.g., by aspiration), or orally. Over a period of 1-36 weeks, 1 to 3 doses may be administered. Preferably 3 doses are administered,

186 774 at intervals of 3-4 months after which periodic booster doses may be given. A different protocol may be used for specific patients. A suitable dose is an amount of polypeptide or DNA which, when administered as described above, is capable of eliciting an immune response in an unimmunized patient sufficient to protect the patient from infection with M. tuberculosis for at least 1-2 years. Generally, the amount of polypeptide present in a dose (or generated in situ by the DNA in a dose) is from about 1 µg to about 100 mg per kg of patient, usually from about 10 µg to about 1 mg, and preferably from about 100 µg to about 1 µg. pg. Suitable dose levels may vary depending on the size of the patient, but will typically range from about 0.1 ml to about 5 ml.

While suitable carriers known to practitioners of the field can be used in the pharmaceutical compositions of the present invention, the nature of the carrier depends on the mode of administration. For parenteral administration, such as subcutaneous administration, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above carriers or a solid carrier such as manrntol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate can be used. Biodegradable microspheres (e.g., dolylactogalactide) can also be used as a carrier for the pharmaceutical composition of the invention. Suitable biodegradable microspheres are disclosed in, for example, US Patent Nos. 4,897,268 and 5,075,109.

Any of a number of αvuants may be used in the vaccines of the invention to enhance the non-specific immune response. Most aOuvants contain substances designed to prevent rapid catabolism of the antigen, such as aluminum hydroxide, and non-specific immune enhancers such as lipiOA, Bordetella pertussis, or Mycobacterium tuberculosis. Suitable aljuvants are commercially available, for example, incomplete Freund's formula and complete Freund's formula (Difco Laboratories) and Merck Adjuvant 65 (Merck & Co. Inc., Rahway, NJ). Other suitable αuvants include alum, biodegradable microspheres, monophosphoryl lipid A, and Quil A.

In another aspect, the invention provides methods of using one or more of the polypeptides described above for the diagnosis of tuberculosis using skin testing. As used herein, a "skin test" is a test performed directly on a patient that measures a delayed-type hypersensitivity (DTH) reaction (such as swelling, redness, or dermatitis) after injection of one or more of the above-described polypeptides. Such injection may be performed using any device known to contact the polypeptides or polydeptides with the skin cells of the patient, such as, for example, a tuberculin syringe or a 1 ml syringe. Preferably, the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.

The DTH response is a cellular immune response which is stronger in patients exposed to the test antigen (i.e., the immunogenic part of the polydndeptide used, or a variant thereof). The reaction can be measured optically using a ruler. Generally, a reaction greater than about 0.5 cm in diameter, preferably greater than about 1 cm in diameter, is a positive response, indicative of tuberculosis infection, which may or may not be manifested by an active disease.

The polypeptides of the invention are preferably formulated for use in a skin test as pharmaceutical compositions comprising the polypeptide and a physiologically acceptable carrier as described above. Such compositions typically contain one or more of the above dolidedtyts in an amount ranging from about 1 µg to about 100 µg, preferably from about 10 µg to about 50 µg in a volume of 0.1 ml. Preferably, the carrier used in such pharmaceutical compositions is saline with a suitable preservative such as phenol and / or Tween 80 ™.

In a preferred embodiment, the polydytes used in the skin test are of sufficient size to remain at the injection site for the duration of the reaction period. In general, a solidide of at least 9 amino acids in length is sufficient. PćlidnptyO is also included

186 774 is preferably degraded by macrophages within hours of injection to allow presentation to T lymphocytes. Such polypeptides may contain repeats of one or more of the above sequences and / or immunogenic or non-immunogenic sequences.

The following examples are provided for illustrative purposes and not for limitation.

Example 1

Purification and characterization of polypeptides from M. tuberculosis culture filtrate

This example illustrates the preparation of soluble M. tuberculosis polypeptides from the culture filtrate. In the example below, all percentages are by weight by volume, unless otherwise stated.

M. tuberculosis (H37Ra, ATCC No. 25177 or H37Rv, ATCC No. 25618) was grown in sterile GAS media at 37 ° C for 14 days. Then, the media was filtered under vacuum (separating most of the cells) through a 0.45P filter into a sterile 2.5L bottle. The medium was then filtered through a 0.2P filter into a 4L sterile bottle and 0.04% NaN3 was added. The bottles were placed in a 4 ° C cooler.

The culture filtrate was concentrated by placing it in a 12 liter sterilized container and loading it into a 400 ml amicon stirred cell which was rinsed with ethanol, equipped with a 10,000 kDa MWCO membrane. The pressure was maintained at 60 psi with nitrogen. The procedure reduced the volume of the 12 L to approximately 50 ml.

The culture filtrate was dialyzed against 0.1% ammonium bicarbonate using an 8000 kDa cellulose ester membrane with two changes of ammonium bicarbonate solution. Protein concentration was determined using a commercially available BCA assay (Pierce, Rockford, IL).

The dialyzed culture filtrate was lyophilized and the polypeptides suspended in distilled water. The polypeptides were dialyzed against 0.01 mM 1,3 bis [tris (hydroxymethyl) methylamino] propane, pH 7.5 (Bis-Tris propane buffer), initial anion exchange chromatography conditions. Fractionation was performed using profusion gel chromatography on a POROS 146 II Q / M 4.6 mm X 100 mm anion exchange column (Perseptive BioSystems, Framingham, MA) equilibrated with 0.01 mM Bis-Tris propane buffer pH 7.5. Polypeptides were eluted with a linear 0-0.5 M NaCl gradient in the above buffer. The eluent was monitored at a wavelength of 220 nm.

The pools of polypeptides eluted from the anion exchange column were dialyzed against distilled water and lyophilized. The resulting material was dissolved in 0.1% trifluoroacetic acid (TFA) pH 1.9 in water, then the polypeptides were purified on a Delta-Pack C18 column (Waters, Milford, MA), pore size 300 L, particle size 5 μ (3, 9 X 150 mm). Polypeptides were eluted from the column with a linear gradient of 0-60% dilution buffer (0.1% TFA in acetonitrile). The flow rate was 0.75 ml / min and the HPLC eluent was monitored at 214 nm. Fractions containing eluted polypeptides were collected to maximize the purity of individual samples. About 200 purified polypeptides were obtained.

Purified polypeptides were screened for the ability to induce proliferation of T cells in the PBMC preparations. PBMCs from donors known to be positive in the skin PPD test and whose T cells have been shown to proliferate in response to PPD and MTB soluble crude proteins were cultured in RPMI 1640 containing medium supplemented with 10% pooled human serum and 50 μg / ml gentamicin. Purified polypeptides were added in duplicate at a concentration of 0.5 to 10 µg / ml. After six days of culture in 96-well round-bottom plates in a volume of 200 µl, 50 µl of medium was withdrawn from each well for determination of IFN-γ levels as described below. Plates were then pulsed with 1 µCi / well of irritated thymidine for a further 18 hours, harvested and tritium uptake determined using a scintillation counter. Fractions giving rise to both replicate proliferation greater than the threefold proliferation observed on cells grown in medium alone were considered positive.

IFN-γ was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with mouse monoclonal antibodies directed against human IFN-γ (PharMingen, San Diego, GA) in PBS for 4 hours at room temperature. Next,

186 774 wells were blocked with PBS containing 5% (w / v) skim milk powder for one hour at room temperature. The plates were then washed six times in PBS / 0.2% Tween-20 and samples diluted 1: 2 with culture medium were incubated in the ELISA plates overnight at room temperature. The plates were washed again and polyclonal rabbit serum against human IFN-γ, diluted 1: 3000 in PBS / 10% goat serum, was added. The plates were then incubated for two hours at room temperature, washed and anti-rabbit IgG-conjugated horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) at a dilution of 1: 2000 in PBS / 5% non-fat dry milk was added. After a further two hours of incubation at room temperature, the plates were washed and TMB substrate was added. The reaction was stopped after about 20 minutes with 1 N sulfuric acid. Optical density was determined at 450 nm using 570 nm as the reference wavelength. Fractions giving in both replicates an OD twice the mean OD of cells grown in medium alone plus three standard deviations were considered positive.

For sequencing, the polypeptides were separately dried on Biobrene ™ treated glass fiber filters (Perkin-Elmer / Applied Biosystems Division, Foster City, CA). The polypeptide filters were inserted into the Perkin-Elmer / Applied Biosystems Division Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminus using the Edman reaction. The amino acid sequence for each polypeptide was determined by comparing the retention time of the amino acid PTH derivative to the corresponding standard of the PTH derivative.

Using the procedure described above, antigens with the following N-terminal sequences were isolated:

(a) Asp-Pro-V al-Asp-Ala-V al-Ile-Asn-Thr-Thr-Cys-Asn-T yr-Gly-Gln-V al-V al-Ala-AlaLeu; (SEQ ID NO: 54) (b) Ala-Val-Glu-Ser-Gly-Met-Leu-Ala-Leu-Gly-Thr-Pro-Ala-Pro-Ser; (SEQ ID NO: 55) (c) Ala-Ala-Met-Lys-Pro-Arg-Thr-Gly-Asp-Gly-Pro-Leu-Glu-Ala-Ala-Lys-Glu-GlyArg; (SEQ ID NO: 56) (d) Tyr-Tyr-Trp-Cys-Pro-Gly-Gln-Pro-Phe-Asp-Pro-Ala-Trp-Gly-Pro; (SEQ ID NO: 57) (e) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa-Ala-Val; (Seq.

No. 58) (f) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val-Pro; (SEQ ID NO: 59) (g) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Thr-Ala-Ala-Ser-Pro-Pro-Ser; (SEQ ID NO: 60) (h) Ala-Pro-Lys-Thr-Tyr-Xaa-Glu-Glu-Leu-Lys-Gly-Thr-Asp-Thr-Gly; (SEQ ID NO: 61) wherein Xaa can be any amino acid.

Using a small diameter HPLC purification step, an additional antigen was isolated in addition to the procedures described above. Specifically, the 20 µl fraction containing the antigen mixture from the above-described chromatographic purification step was purified on an Aquapore C18 column (Perkin-Elmer / Applied Biosystems Division, Foster City, CA) 7 µ pore size 1 mm X 100 mm column in Perkin HPLC -Elmer / Applied Biosystems Division Model 172. Fractions were eluted from the column with a linear gradient of 1% / min acetonitrile (containing 0.05% TFA) in water (0.05% TFA) at a flow of 80 µl / min. The eluent was monitored at a wavelength of 250 nm. The initial fraction was split into 4 major peaks plus other small components and the resulting polypeptide had a molecular weight of 12.054 kDa (as determined by mass spectrometry) and the following N-terminal sequence:

(i) Asp-Pro-Ala-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala-Gln-Leu-Thr-Ser-Leu-Leu-AsnSer-Leu-Ala-Asp-Pro-Asn -Val-Ser-Phe-Ala-Asn; (Seq ID No.62)

This polypeptide induced proliferation and production of IFN-γ by PBMC under the assay conditions described above.

186 774

Additional soluble antigens were isolated from the M. tuberculosis filtrate as follows. The M. tuberculosis culture filtrate was prepared as described above. After dialysis against Bis-Tris propane buffer, pH 5.5, fractionation was performed using anion exchange chromatography on a POROS QE 4.6 mm X 100 mm column (Perseptive BioSystems) equilibrated in Bis-Tris propane buffer pH 5.5. The polypeptides were eluted with a linear gradient of 0-1.5 M NaCl in the above buffer at a flow rate of 10 ml / min. The eluent leaving the column was monitored at a wavelength of 214 nm.

Fractions eluted from the ion exchange column were pooled and subjected to reverse phase chromatography using a Poros R2 4.6 x 100 mm column (Perseptive BioSystems). The polypeptides were eluted from the column using a linear gradient of 0-100% acetonitrile (0.1% TFA at 5 ml / min. The eluent was monitored at 214 nm.

Fractions containing eluted polypeptides were lyophilized and resuspended in 80 μΐ aqueous 0.1% TFA and subjected to further reverse phase chromatography on a Vydac C4 4.6 X 150 mm column (Western Analytical, Temecula, CA) with a linear gradient of 0-100% acetonitrile (0.1% TFA) at a flow of 2 ml / min. The eluent was monitored at 214 nm.

The bioactive fractions were separated into one major peak and several minor components. Western blot analysis on the PVDF membrane showed three major bands with molecular weights of 14 kDa, 20 kDa and 2.6 kDa. The polypeptides had the following N-terminal sequences:

(j) Xar-Asp-SertGlu-^Nys-Ser-Ala-Thr-Iln-Lys-Val-Thr-Asp-Ala-Snr; (SEQ ID NO: 134) (k) Ala-Gly-Asp-Thr-Xaa-Ile-Tq-Ile-Val-Gly-Asa-Lnu-Thr-Ala-Asp; (SEQ ID NO: 135) or (l) Ala-Pro-Glu-Snr-Gly-Ala-Gly-Lnu-Gly-Gla-Thr-Val-Gln-Ala-Gla: (SEQ ID No 136)

Using the assays described above, these polypeptides have been shown to induce proliferation and IFN-γ production by PBMC preparations. Figures 1A and B show the results of such tests performed with the PBMC preparations from the first and second donors, respectively.

iSekrncencjaz DNA encodes the genes marked with nako (a), er), with^e) ^about and g) played by genomic history / W. tubeacutneis scything cdegenerated olinonpkleprye ^, zn0.oaven) no Ooó ^ ui²P, corresponding to the sequences of bagpipes N and making the preterial codons Μ. tuberculoti3. Prkasz.u0iwenc oji ożcaiN soa ^ cW ojipowiodejnrkj źa) allowed to identify ϋοπ in ąSazmnej as IstkaPnnkatos Sej

Cw. ge o 01. Poliptyd Ι ^ οΟο) /.!^. ' pnwz Ideoiyfikato) nakw, ot łon iSsCkkand j ^ o kdenOyfikWor S ekw. no. Praerodkiwani e π / Οοη! / ^ are the orycik wondy corresponding to the antigen kj) iro ^ wWó In ziNentyfϊnowrρ century clone ^ νεΏρίί onko ^^ pi ^^ i ^ i ^ tj ^ r S eCw. LV 5W.

Pol iOkpWΌ coded ρ ^ ζ Idrntnfikator Snkw. No. 52 pcka / ano lkantyfika1or nrkw.

ot 5Ó. We used the srndy to answer the question (d) let the aident ^ y0nwwr Hen e) ekwaaaji ookawżąj u ako Iąon) pfiOaior ScŃ. nm ge, the name of the word `` natyoen '', it was expressed with the use of the eonda of the voice of the `` natyoen '' Sek, which made it possible to de-doicify the notion as Idkd00okatoc ąckw. nm) ϊ.

Driving so0wencjn ^ mmokev; tejoi ^^ 'co^abouthave been equated with c known amino acid sequences in the eme bank using the DNA STna ^^. Search data ορ ^ ϊι ^^ Ια

17W000 i wost Cembmk)) ą0aD NAnych SWPrS, 0Ik wanc from ο.ΐο.ηηο'ίοο ^ ϊ barrel twkrtj a 77). 'Nothing confirmed iotc ^ -to ^ j ^ ιπιοΚ'.π of the sequence omino0wasownr0 dic aotygeaów (w) - (hji (1).

The amino acid sequence of antigen (i) appeared to be homologous to that of M. leprae. The sequence CO / nom ^^^ ej at the synonymy with the term of DNW and ^ tc ^ taking the sequences {^ c ^^ n ^^ to ^^ e from GENBANK. ^ Rwecc ^ ta / (^^ ι o) ethosovon) dD Nom mesueing the question ^ r ^ t ^ ki M. tubeuoulosib opusAnniCnj in arsąktadzkc C iu: cokcko in this junctionb ^ kiwokią bP. tubdrρulosis of full length CtnsntlżnSaton Seq. ot 99).

Emission sequence of antiquat O Syphnmological with the known protein M. tuberculod1sle ^ aosjocooii} kr with skOeqaoi DNA. On the basis of the better knowledge that the protein has been shown to have T-cell stimulating activity beforehand, it has been shown to stimulate T lymphocytes. The amino acid sequence of antigen (k) was related to that of M. leprae.

In the proliferation and IFN-γ assays described above, using the three PPD-positive donors, the results shown in Table 1 were obtained:

Table 1

Results of PBMC and IFN-γ proliferation tests

<td>Sequence</td><td>Proliferation</td><td>IFN-γ</td>

<td>(and)</td><td> +</td><td> -</td>

<td>(c)</td><td> +++</td><td> +++</td>

<td>(d)</td><td> ++</td><td> ++</td>

<td>(g)</td><td> +++</td><td> +++</td>

<td>(h)</td><td> +++</td><td>+ -H-</td>

In Table 1, responses giving an induction index (SI) between 2 and 4 (compared to cells grown with medium alone) were rated +, a SI of 4-8 or 2-4 at a concentration of 1 pg or less was rated ++, and SI greater than 8 was rated +++. Sequence (i) was found to show a high SI (+++) against the first donor and a low SI (++ and +) against the two other donors in the proliferation and IFN-γ assays. These results indicate that these antigens are capable of inducing proliferation and / or producing IFN-γ.

Example 2

The use of patient sera for the isolation of M. tuberculosis antigens

This example shows the isolation of antigens from a M. tuberculosis lysate by screening with sera from individuals infected with M. tuberculosis.

Dried M. tuberculosis H37Ra (Difco Laboratories) was added to a 2% NP40 solution and alternately homogenized and sonicated three times. The resulting suspension was centrifuged at 13,000 rpm in the tubes and the supernatant was passed through a 0.2 µ syringe filter. The filtrate was bound to Macro Prep DEAE beads (BioRad, Hercules, CA). The beads were washed extensively with 20 mM Tris pH 7.5 and bound proteins were eluted with 1 M NaCl. The eluate was dialyzed overnight against 10 mM Tris, pH 7.5. The dialyzed solution was treated with DNase and RNase at a concentration of 0.05 mg / ml for 30 minutes at room temperature, followed by aD-mannosidase, 0.5 U / mg at pH 4.5 for 3-4 hours at room temperature. After adjusting to pH 7.5, the material was fractionated by FPLC on a Bio Scale-Q-20 column (BioRad). Fractions were pooled into 9 pools, concentrated in Centriprep 10 (Amicon, Beverley, MA), and then screened by Western blot for serological activity using serum pools from M. tuberculosis infected patients who were not immunoreactive with the other antigens of the invention.

The most reactive fraction was subjected to SDS-PAGE and transferred to a PVDF membrane. A band of approximately 85 kDa was excised, giving the sequence:

(m) Xaa-Tyr-Ile-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val-Pro-Gly-Lys-Ile-Asn-Val-HisLeu-Val; (SEQ ID NO: 137) wherein Xaa is any amino acid.

Comparison of this sequence with a gene bank as described above showed no significant homology with known sequences.

Example 3

Generation of DNA sequences encoding M. tuberculosis antigens

This example shows the production of DNA sequences encoding M. tuberculosis antigens by screening a M. tuberculosis expression library using sera obtained from patients infected with M. tuberculosis, or antisera raised against soluble M. tuberculosis antigens.

A. Production of soluble M. tuberculosis antigens using rabbit antisera

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Genomic DNA was isolated from M. tuberculosis strain H37Ra. DNA was randomly cut and used to construct an expression library using the Lambda ZAP expression system (Stratagene, La Jolla, CA). Rabbit antisera were induced against the secretion proteins of M. tuberculosis strains H37Ra, H37Rv and Erdman by immunizing rabbits with concentrated M. tuberculosis culture supernatants. Specifically, a rabbit was immunized subcutaneously with 200 µg of the protein antigen in a ml total volume containing 10 µg of muramyl dipeptide (Calbiochem, La Jolla, CA) and 1 ml of Freund's incomplete adjuvant. Four weeks later, the rabbit was boosted subcutaneously with 100 µg of the antigen in Freund's incomplete adjuvant. Finally, the rabbit was immunized intravenously four weeks later with 50 pg of the protein antigen. The immune sera were used to screen the expression library as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. Plaques of bacteriophages expressing immunoreactive antigens were cleaned. The phagemid was saved from the plaques and the nucleotide sequence of the M. tuberculosis clones was deduced.

32 clones were purified. Among them, 25 were sequences not previously identified in human M. tuberculosis. Recombinant antigens were expressed and the purified antigens were used for immunoassay described in Example 1. proteins were induced with IPTG and purified by gel elution as described in Seiky et al., J. Exp. Med. 181: 1527-1537, 1995. Representative sequences of DNA molecules identified by this screening are shown as SEQ ID NOS. no. 1-25. The corresponding predicted amino acid sequences are shown as SEQ ID NOS. no. 63-87.

When these sequences were compared with known sequences in a gene bank using the databases described above, it was found that the clones hereinafter referred to as TbRA2A, TbRAl 6, TbRA18 and TbRA29 (SEQ ID Nos. 76, 68, 70 and 75) show some homology to the sequences described above. previously in M. leprae but not in M. tuberculosis. TbRA11, TbRA26, TbRA28 and TbDPEP (SEQ ID NO: 65, 73, 74, 53) have been previously identified in M. tuberculosis. No significant homology was found with TbRA1, TbRA3, TbRA4, TbRA9, TbRA10, TbRA13, TbRA17, TbRA19, TbRA29, TbRA32, TbRA36 and overlapping clones TbRA35 TbRA12 (SEQ ID NO: 63, 77, 81, 82, 64, 67, 71) , 75, 78, 80, 79, 66). The TbRA24 clone overlapped the TbRA29 clone.

Results of PBMC and IFN-γ proliferation assays performed with representative recombinant antigens and with T lymphocyte preparations from several different M-resistant patients. tuberculosis is presented in Table 2 and 3, respectively.

186 774

Results of PBMC proliferation in response to soluble antigens

<td rowspan="13">Patient</td><td></td><td> 1</td><td>vol</td><td>IN c</td><td> 1</td><td>G.</td><td>vol</td><td>G.</td><td>G.</td><td>* -&gt; G.</td><td>G.</td><td>G.</td><td>G.</td><td></td><td>G.</td><td>S.</td><td>+ - » G.</td><td>G.</td><td></td>

<td>ΓΊ</td><td> +1</td><td> 1</td><td>c</td><td>-H</td><td> +1</td><td>Ł</td><td>G.</td><td>in G.</td><td>G.</td><td>G.</td><td>and</td><td>G.</td><td>G.</td><td>* -&gt; G.</td><td>-H</td><td> +</td><td>-H</td><td> 1</td>

<td></td><td> +</td><td> 1</td><td>g</td><td>-H</td><td> + +</td><td> +</td><td>G.</td><td>* -&gt; G</td><td>G.</td><td>ABOUT</td><td>and</td><td>G.</td><td>G.</td><td>ABOUT</td><td>G.</td><td>ABOUT</td><td>-H</td><td> 1</td>

<td>about</td><td> 1</td><td></td><td>in G.</td><td> + +</td><td> +</td><td> •</td><td>G.</td><td>G.</td><td>4— » G.</td><td>G.</td><td>vol</td><td>- * -&gt; G.</td><td>G.</td><td>G.</td><td> •</td><td> 1</td><td> +</td><td> 1</td>

<td>about</td><td> 1</td><td>-H</td><td>G.</td><td> 1</td><td> + +</td><td>-H</td><td>G.</td><td>ABOUT</td><td>G.</td><td>G.</td><td>and</td><td>G.</td><td>G.</td><td>G.</td><td> 1</td><td> 1</td><td>-H</td><td> 1</td>

<td>o. o</td><td>-H</td><td> + +</td><td>+ - » G.</td><td>-H</td><td> 1</td><td>-H</td><td>ABOUT</td><td>G.</td><td>G.</td><td>G.</td><td>Ł</td><td>* -&gt; G.</td><td>G.</td><td>G.</td><td> 1</td><td> 1</td><td>ί</td><td> 1</td>

<td>r-</td><td>-H</td><td> 1</td><td>4— » G.</td><td>G.</td><td>G.</td><td> +</td><td>G.</td><td>+ - » G.</td><td>G.</td><td>4— » G.</td><td>G.</td><td>G.</td><td>s</td><td>G.</td><td>g</td><td>G.</td><td>* -&gt; G.</td><td> 1</td>

<td></td><td> 1</td><td> 1</td><td> + +</td><td> +</td><td> +</td><td> + +</td><td> +</td><td> 1</td><td> 1</td><td> 1</td><td>and</td><td> »</td><td></td><td> 1</td><td> •</td><td></td><td></td><td> 1</td>

<td></td><td> 1</td><td>-H</td><td> + +</td><td>-H</td><td> + +</td><td>-H</td><td> 1</td><td> 1</td><td> 1</td><td>f</td><td>vol</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td>Ł</td><td> 1</td><td> 1</td>

<td></td><td>and</td><td> 1</td><td>4— » G.</td><td> +1</td><td> ++</td><td> +</td><td>G.</td><td>G.</td><td>G.</td><td>G.</td><td>and</td><td>G.</td><td>G.</td><td>G.</td><td> 1</td><td> 1</td><td>and</td><td> 1</td>

<td></td><td>-H</td><td> ++</td><td>G.</td><td>-H</td><td> +</td><td> +</td><td>G.</td><td>G.</td><td>G.</td><td>ABOUT</td><td> + +</td><td></td><td>G.</td><td>G.</td><td>-H</td><td> •</td><td>vol</td><td>vol</td>

<td></td><td> 1</td><td>-H</td><td> 1</td><td></td><td>-H</td><td></td><td>G.</td><td>G.</td><td> +</td><td>•in G.</td><td>G.</td><td>s</td><td>G.</td><td>G.</td><td> 1</td><td> 1</td><td> +</td><td> 1</td>

<td> -</td><td> 1</td><td></td><td></td><td> «</td><td>-H</td><td> 1</td><td>G.</td><td>G.</td><td> 1</td><td>G.</td><td> + +</td><td>G.</td><td>G.</td><td>G.</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>Antigen</td><td></td><td>TbRal</td><td>f2</td><td>TbRa9</td><td>TbRalO</td><td>TbRal 1</td><td>TbRal2</td><td>TbRaló</td><td>s</td><td>TbRa26</td><td>TbRa29</td><td>TbRa35</td><td>TbRaB</td><td>TbRaC</td><td>TbRaD</td><td>AAMK</td><td>YY</td><td>DPEP</td><td>control</td>

nt = not tested

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Results of PBMC's production of interferon-γ in response to soluble antigens

<td rowspan="13">Patient</td><td></td><td>Ł</td><td> 1</td><td> 2</td><td>vol</td><td>4— » G.</td><td>Ł</td><td> 2</td><td>· * -&gt; G.</td><td>4— » G.</td><td> 2</td><td> 2</td><td>· * - » G.</td><td> 2</td><td> 2</td><td>* - » G.</td><td> 2</td><td> 2</td><td> 1</td>

<td>CN</td><td>-H</td><td> 1</td><td> 2</td><td>-H</td><td>-H</td><td> 1</td><td>G.</td><td> 2</td><td> 2</td><td>· * - » G.</td><td>and</td><td>· * - » G.</td><td>· * - » G.</td><td>G.</td><td>-H</td><td> +</td><td>-H</td><td>and</td>

<td></td><td> +</td><td></td><td>c</td><td>-H</td><td>ί</td><td> +</td><td>· * - » ABOUT</td><td>· «-» ABOUT</td><td> 2</td><td> 2</td><td>and</td><td> 2</td><td>s</td><td> 2</td><td> 2</td><td> 2</td><td>-H</td><td> 1</td>

<td> 2</td><td> 1</td><td> 1</td><td> 2</td><td> +</td><td>ί</td><td> 1</td><td> 2</td><td> 2</td><td> 2</td><td> 2</td><td>and</td><td> 2</td><td>· * - » G.</td><td>in G.</td><td> 1</td><td>AND</td><td> +</td><td> 1</td>

<td>about</td><td> 1</td><td>-H</td><td>-M c</td><td></td><td>ί</td><td>-H</td><td> 2</td><td> 2</td><td> 2</td><td>· * - » G.</td><td>ί</td><td> 2</td><td> 2</td><td> 2</td><td> 1</td><td> 1</td><td>-H</td><td> 1</td>

<td> 00</td><td>-H</td><td>vol</td><td> 2</td><td>-H</td><td> 1</td><td>-H</td><td> 2</td><td> 2</td><td> 2</td><td>in G.</td><td>vol</td><td>4— » G.</td><td> 2</td><td> ·*-» 0</td><td> 1</td><td> 1</td><td>-H- +</td><td> 1</td>

<td></td><td></td><td> 1</td><td>c</td><td> 2</td><td> 2</td><td> +</td><td> 2</td><td> 2</td><td>· * - » G.</td><td> 2</td><td> 2</td><td> 2</td><td> 2</td><td> 2</td><td> 2</td><td>· * -&gt; G.</td><td>in α</td><td> &gt;</td>

<td></td><td>vol</td><td> 1</td><td> •</td><td> +</td><td> +</td><td>and</td><td> +</td><td> 1</td><td> +</td><td> 1</td><td>and</td><td> +</td><td> +</td><td> +</td><td> 1</td><td> -</td><td> 1</td><td> 1</td>

<td></td><td> +</td><td>-H</td><td>vol</td><td>-H</td><td>and</td><td> +1</td><td> +</td><td> +</td><td> +</td><td> +</td><td>ΐ</td><td>vol</td><td> +</td><td> +</td><td> 1</td><td> 1</td><td> +</td><td></td>

<td></td><td>and</td><td>AND</td><td> 2</td><td>-H</td><td>and</td><td> +</td><td>with</td><td>-ł- » with</td><td>with</td><td>with</td><td>and</td><td>Nt</td><td>with</td><td></td><td></td><td> 1</td><td>ΐ</td><td> (</td>

<td>cn</td><td></td><td>and</td><td>G.</td><td>-H</td><td> +</td><td> +</td><td> 2</td><td> 2</td><td> 2</td><td> 2</td><td>and</td><td> 2</td><td> 2</td><td> 2</td><td>-H</td><td>AND</td><td> +</td><td></td>

<td>CN</td><td>and</td><td>-H</td><td> +</td><td> +</td><td>-H</td><td> 1</td><td> 2</td><td>about</td><td>and</td><td>4— » G.</td><td> 2</td><td> 2</td><td> 2</td><td> 2</td><td></td><td>Ł</td><td> +</td><td> 1</td>

<td> -</td><td> +</td><td> 1</td><td>vol</td><td> +</td><td></td><td> 1</td><td>s</td><td> 2</td><td>and</td><td> 2</td><td>and</td><td> 2</td><td> 2</td><td>G.</td><td> 1</td><td> 1</td><td> +</td><td> 1</td>

<td>Antigen</td><td></td><td>TbRal</td><td>at 4 JO H.</td><td>about £ H.</td><td>TbRalO</td><td>TbRal 1</td><td>CN s</td><td>TbRal 6 1 1</td><td>TbRa24</td><td>(= 4 λ H.</td><td>TbRa29</td><td>IT) cont | 2</td><td>TbRaB</td><td>TbRaC</td><td></td><td>AAMK</td><td>YY</td><td>DPEP</td><td>control</td>

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In Tables 2 and 3, responses that showed a stimulation index (SI) between 1.2 and 2 (compared to cells grown in medium alone) were scored as ±, SI 2-4 scored +, SI 4-8 or 2-4 at a concentration of 1 pg or less was scored ++ and SI greater than 8 was scored + + +. In addition, the effect of concentration on proliferation and intnrfnron-γ production is shown in the accompanying Figure for two of the above antigens. For proliferation and interferon-γ production, TbRa3 was scored ++ and TbRa9 was +.

These results show that soluble antigens are able to induce proliferation and / or production of IFN-γ droeo T cells derived from M. tuberculosis resistant individuals.

B. Apply euśowlc ραο ^ ι ^^ 'to wee ^ tyi ^ and ^^ icji DNAs encoding M. tuberculosis genes

The gecomal DNA library and an additional H37Rv library were screened using pooled serum from patients with active tuberculosis. To generate the H37Rv library, genomic DNA of M. tuberculosis strain H37Rv was isolated, partially digested with Sau3A, and used to construct an expression library using the Lambda ZAP expression system (Stratagene, La Jolla, CA). Three different pools of sera, each containing sera obtained from about three individuals with active pulmonary or epithelial tuberculosis, were used for expression droning. Pools were labeled TbL, TbM, and TbH according to reactivity with H37Ra lysate (ie, TbL = weak reactivity, TbM = intermediate reactivity and TbH = high reactivity) in ELISA and immunoblot format. A fourth pool of sera oO seven patients with active pulmonary tuberculosis was also used. All sera were devoid of increased reactivity with the recombinant 38 kDaM phosphate binding protein. tuberculosis.

All pools were preadsorbed onto E. coli lysate, and used to screen H37Ra and H37Rv expression libraries, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. Plaque cleaned bacteriophages expressing immunoreactive antigens. Plaque phagemid was rescued and the sequences of the sacchleotide of M. tuberculosis clones were determined.

32 clones were purified. Among them, 31 were identified as yet unknown in human M. tuberculosis. Representative sequences of DNA molecules are given in SEQ ID NO. Nos. 26-51 and 105. Among them, TbH-8 and TbH-8-2 (SEQ ID NO: 105) are discontinuous sequences of the same clone, and TbH-4 (SNKW ID NO: 43) and TbH-4-FWD (SEQ ID NO: 44) are discontinuous sequences of the same clone. The amino acid sequences for the antigens identified by Oalej as Tb38-1, TbH-4, TbH-8, TbH-9, and TbH-12 are shown as the Snkw Identifier. no. 88-92. Comparison of these sequences with the evaluating sequences in the databank using the mn databases described above showed significant homology with TbH-4, TbH-8, TbH-9 and TbM-3, although weak TbH-9 homologins were found. TbH-12 was found to be homologous to the 34 kDa antigenic protein previously identified in M. tuberculosis (# S28515). Tb38-1 was found to be 34 base pairs above the reading frame for the ESAT-6 antigen previously described in M. bovis (No. U34848) and M. tuberculosis (Sorenstn et al., Infect. Immun. 63: 17101717 (1995).

SonOy is derived from Tb38-1 and TbH-9 isolated from the H37Ra library, used to identify clones in the H37Rv library. Tb38-1 hybridized with Tb38-1F2, Tb38-1F3, Tb38-1F5 and Tb38-1F6 (SEQ ID NO: 112, 113, 116, 118 and 119). (Snkw ID Nos. 112 and 113 are discontinuous sequences of the Tb38-1F2 clone). Two reading frames were deduced from clone Tb38-1F2; one corresponds to Tb37FL (Seq. No. 114), Orange, a partial sequence, may be a Tb38-1 homologue and named as Tb38-IN (SEQ ID NO: 115). The deduced amino acid sequence of Tb38-1F3 is shown in IOectyfikatoroe Snkw. No. 117. The TbH-9 probe identified three clones in the H37Rv library; TbH-9-FL (SEQ ID NO: 106), which may be hćmologenm TbH-9 (R37Ra), TbH-9-1 (SEQ ID NO: 108) and TbH-9-4 (SEQ ID NO: 110), all of which are closely related to TbH-9. The deduced amino acid sequences for Ola of these three clones are shown in Identifier Snkw. no.107, 109 and 111.

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The results of T-cell assays performed on Tb38-1, ESAT-6 and other representative recombinant antigens are shown in Tables 4A, B and 5 below, respectively:

Table 4A

Results of PBMC proliferation in response to representative antigens

<td>Antigen</td><td colspan="11">Giver</td>

<td></td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td>

<td>Tb38 1</td><td> +++</td><td> +</td><td> -</td><td> -</td><td> -</td><td> ++</td><td> -</td><td> +</td><td> -</td><td> ++</td><td> +++</td>

<td>ESAT-6</td><td> +++</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td><td> +</td><td> +</td><td> ++</td><td> +++</td>

<td>TbH-9</td><td> ++</td><td> ++</td><td> -</td><td> ++</td><td> ±</td><td> ±</td><td> +</td><td> ++</td><td> ++</td><td> ++</td><td> ++</td>

Table 4B

Results of the production of IFN-γ by PBMC in response to representative antigens

<td>Antigen</td><td colspan="11">Giver</td>

<td></td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td>

<td>Tb38.1</td><td> +++</td><td> +</td><td> -</td><td> +</td><td> +</td><td> +++</td><td> -</td><td> ++</td><td> -</td><td> +++</td><td> +++</td>

<td>ESAT-6</td><td> +++</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> +</td><td>+ -H-</td><td> +++</td>

<td>TbH-9</td><td> ++</td><td> ++</td><td> -</td><td>+ -H-</td><td> ±</td><td> ±</td><td> +++</td><td> +++</td><td> ++</td><td> +++</td><td> ++</td>

Table 5

Summary of T cell responses to representative antigens

<td>Antigen</td><td colspan="3">Proliferation</td><td colspan="3">Interferon-γ</td><td>sum</td>

<td></td><td>patient 4</td><td>patient 5</td><td>patient 6</td><td>Patient 4</td><td>patient 5</td><td>patient 6</td><td></td>

<td>TbH9</td><td> ++</td><td> ++</td><td> +—+</td><td> +++</td><td> ++</td><td> ++</td><td> 13</td>

<td>TbM7</td><td> -</td><td> +</td><td> -</td><td>-H-</td><td> +</td><td> -</td><td> 4</td>

<td>TbH5</td><td> -</td><td> +</td><td> +</td><td> ++</td><td> ++</td><td> ++</td><td> 8</td>

<td>TbL23</td><td> -</td><td> +</td><td> ±</td><td> ++</td><td> ++</td><td> +</td><td> 7,5</td>

<td>TbH4</td><td> -</td><td> ++</td><td> ±</td><td> ++</td><td> ++</td><td> ±</td><td> 7</td>

<td>control</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 0</td>

These results indicate that both the M. tuberculosis antigens of the invention and ESAT-6 are able to induce proliferation and / or interferon-γ production by T cells derived from M. tuberculosis resistant individuals. To the best of the inventors' knowledge, ESAT-6 has not been shown to stimulate the human immune response so far.

A set of six overlapping peptides covering the amino acid sequence of the Tb38-1 antigen was constructed using the method described in Example 4. The sequences of these peptides, hereinafter referred to as pepl-6, are shown in SEQ ID, respectively. No. 9398. The results of the T cell assays using these peptides are shown in Tables 6 and 7. These results support the presence and help localize T-cell epitopes within Tb38-1 capable of inducing proliferation and interferon-γ production by T cells derived from M. tuberculosis resistant individuals.

186 774 c3 &lt;υ χ

C0

Η

<td rowspan="13">1 Patient</td><td>ΓΊ</td><td> +</td><td> +</td><td>-Η</td><td> +</td><td> +</td><td> +</td><td></td>

<td>CN</td><td> 1</td><td> •</td><td> -</td><td> 1</td><td>AND</td><td></td><td></td>

<td></td><td>AND</td><td> 1</td><td> 1</td><td> 1</td><td> «</td><td> 1</td><td> 1</td>

<td>Ο</td><td>-Η</td><td>-Η</td><td>vol</td><td>-Η</td><td> 1</td><td> +</td><td>vol</td>

<td>ο</td><td> 1</td><td>-Η</td><td>-Η</td><td>-Η</td><td>-Η</td><td>-Η</td><td> 1</td>

<td> 00</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> &gt;</td><td> 1</td>

<td>ο</td><td> -</td><td> 1</td><td> 1</td><td> +</td><td> +</td><td>-Η</td><td> 1</td>

<td></td><td></td><td> 1</td><td> 1</td><td> -</td><td></td><td> •</td><td></td>

<td>1 / Ί</td><td> +1</td><td>-Η</td><td>vol</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>Tf</td><td></td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>ΓΊ</td><td> 1</td><td> 1</td><td> •</td><td> -</td><td> 1</td><td></td><td> 1</td>

<td>ΓΝ</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td>-Η</td><td>ί</td><td> 1</td>

<td> -</td><td> 1</td><td>-Η</td><td> 1</td><td>-Η-</td><td>-Η-</td><td> •</td><td> 1</td>

<td>Peptide</td><td></td><td>&lt;υ CL</td><td>ΓΜ CX ϋ CŁ</td><td>ΓΊ Cu (ϋ Λ</td><td>&lt;Χ</td><td>νΊ Cu ϋ α</td><td>Cu &lt;υ &lt;Χ</td><td>control</td>

186 774

Γ03 &lt;υ

X&gt;

Λ

Η &gt;&gt;

% &amp;

D§ 'Ξ £

<td rowspan="13">Patient</td><td>Γ * Ί</td><td> +</td><td> +</td><td>-Η</td><td> +</td><td> +</td><td> +</td><td> 1</td>

<td>CS</td><td> 1</td><td> 1</td><td> 1</td><td> •</td><td> 1</td><td> 1</td><td> 1</td>

<td></td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>ο</td><td> +</td><td>• Ή</td><td> »</td><td> +</td><td> 1</td><td> +</td><td> 1</td>

<td>σ&gt;</td><td> 1</td><td>-Η</td><td> +</td><td>-Η</td><td>Ή</td><td>-Η</td><td> 1</td>

<td> 00</td><td> 1</td><td>vol</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>Γ-</td><td> 1</td><td> 1</td><td> 1</td><td> +</td><td> +</td><td> +</td><td> 1</td>

<td></td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>• τι</td><td> +(</td><td>-Η</td><td></td><td> 1</td><td> 1</td><td>and</td><td> 1</td>

<td>τΤ</td><td> 1</td><td> 1</td><td> 1</td><td></td><td> 1</td><td> 1</td><td> •</td>

<td>m</td><td> •</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td>

<td>CS</td><td> 1</td><td> 1</td><td>AND</td><td> »</td><td>-Η</td><td>-Η-</td><td>vol</td>

<td> -</td><td> +</td><td></td><td> 1</td><td>φ</td><td>Φ</td><td> +</td><td> 1</td>

<td>Peptide</td><td></td><td>&lt;υ α</td><td>CS (X &lt;υ α-</td><td>ΠΊ CU 8,</td><td>pep4</td><td>ΙΤ) α. (AT ο &lt;</td><td>&amp; Cm</td><td>control</td>

186 774

Example 4

Purification and characterization of the polypeptide from the purified protein derivative of tuberculin

M. tuberculosis polypeptide was isolated from the purified tuberculin protein derivative (PPD) as follows.

PPD was prepared as described in Seibert et al., Tuberculin purified protein derivative. Preparation and analyzes of large quantity for standard. The American Review for Tuberculosis 44: 9-25, 1941, with minor modifications.

The M. tuberculosis strain Rv was grown for 6 weeks on synthetic medium in roller bottles at 37 ° C. The bottles containing the bacteria were heated to 100 ° C in steam for 3 hours. The cultures were sterile filtered using a 0.22 µ filter and the liquid phase was concentrated 20 fold using a 3 kDa cut-off membrane. Proteins were precipitated once with 50% ammonium sulfate solution and eight times with 25% ammonium sulfate solution. The resulting proteins (PPD) were fractionated by reverse phase chromatography (RP-HPLC) using a C18 column (7.8 x 300 mm; Waters, Milford, Ma) in a Biocad HPLC system (Perseptive BioSystems, Framingham, MA). Fractions were eluted from the column with a linear gradient of 0-100% buffer (0.1% TFA in acetonitrile). The flow was 10 ml / minute and the eluent was monitored at 214 nm and 280 nm.

Six fractions were collected, dried, suspended in PBS, and tested individually in M. tuberculosis infected guinea pigs for delayed-type hypersensitivity (DTH) reactions. One of the fractions was found to induce a strong DTH reaction, and it was further fractionated by RP-HPLC on a small diameter Vydac C18 column (Cat # 218TP5115) in an HPLC model 172 Perkin-Elmer / Applied Biosystems Division, Foster City, CA. Fractions were eluted with a linear gradient of 5-100% buffer (0.05% TFA in acetonitrile) at a flow of 80 µ / min. The eluent was monitored at 215 nm. Eight fractions were collected and tested individually for the induction of DTH in M. tuberculosis infected guinea pigs. One of the fractions was found to induce a strong DTH with a pH of about 16 mm. The other fractions did not induce any detectable DTH. The positive fraction was subjected to SDS-PAGE and found to contain a single protein band with a molecular weight of approximately 12 kDa.

This polypeptide, hereinafter referred to as DPPD, was sequenced from the amino terminus using the Perkin-Elmer / Applied Biosystems Division Procise 492 protein sequencer as described above, and found the N-terminus sequence shown in SEQ ID NO. No. 129. Comparison of this sequence with known sequences in a databank as described above showed no significant homology. Four DPPD fragments from the digestion of cyanogen bromide were isolated and found to have the sequences shown in SEQ ID NOS. no. 130-133.

The ability of the DPPD antigen to stimulate human PBMC to proliferate and produce IFN-γ was tested as described in Example 1. As shown in Table 8, DPPD was found to promote proliferation and induce the production of significant amounts of IFN-γ; more potent than a commercial PPD formulation.