Patent for “Method to prevent or treat M tuberculosis”

Biopharmaceuticals – Martine Marchand, GlaxoSmithKline Biologicals SA

Search Patent for “Method to prevent or treat M tuberculosis”

Abstract for “Method to prevent or treat M tuberculosis”

The present invention relates to methods for preventing the reactivation active and latent M. tuberculosis infection by administering a pharmaceutical composition that contains a nucleic acids encoding a Mtb72f or Mtb72f protein fusion protein, or an immunogenic fragment thereof. This may be combined with an adjuvant. One or more chemotherapeutic drugs can be used to treat M. tuberculosis infections. These methods can also be used to reduce the duration of a chemotherapeutic treatment for M. tuberculosis.

Background for “Method to prevent or treat M tuberculosis”

Tuberculosis, a chronic infection caused by M. tuberculosis or other Mycobacterium species, is known as “Tuberculosis.” It is a serious disease that affects both developing and developed countries. Each year, there are approximately 8 million new cases and over 3 million deaths. The disease can be mildly symptomatic for some time. However, it is most often manifested by an acute inflammation of your lungs that results in fever and a nonproductive cough. If left untreated, death is a common outcome.

Although tuberculosis is generally manageable with extended antibiotic therapy, it does not stop the spread of the disease. Although asymptomatic, infected people may become contagious for a time. It is not easy to monitor patient behavior and compliance. Patients may not finish the treatment course, which can result in ineffective treatment or drug resistance. Even after completing a course of treatment, M. tuberculosis infection can still be present.

Effective vaccination and early diagnosis are essential to stop the spread of tuberculosis. Presently, vaccines with live bacteria are the best way to instill protective immunity. Bacillus Calmette Guerin (BCG), a highly infectious strain of M. bovis, is the most commonly used mycobacterium for this purpose. BCG’s safety and efficacy are a matter of debate. Some countries, like the United States, don’t vaccinate their citizens with it.

A skin test is used to diagnose tuberculosis. This involves intradermal exposure of tuberculin PPD (protein purified derivative). Antigen-specific T cells respond to injections by causing measurable swelling at the injection site within 48-72 hours. This indicates that mycobacterial antigens have been ingested. However, this test is not sensitive or specific enough to distinguish between infected and unvaccinated individuals.

“Macrophages are not the main effectors of Mycobacterium immune response, but T cells are. It is evident that T cells play a crucial role in protecting against Mycobacterium infections. This can be seen in the high incidence of Mycobacterium infected patients with HIV. Mycobacterium-reactive CD4+ T cells have been shown to be potent producers of ?-interferon (IFN-? This, in turn, is shown to activate the anti-mycobacterial effect of macrophages in mice. Although the role of IFN? in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3 can trigger anti-mycobacterial effects in macrophages in mice. in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-? activates human macrophages to prevent M. tuberculosis infection. It is also known that IFN-1? stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Interleukin-12 (IL-12), has also been shown to be a factor in the stimulation of resistance to M. tuberculosis infections. See Chan & Kaufmann, Tuberculosis. Pathogenesis, Protection and Control (Bloom ed., 1993), Tuberculosis (2nd ed., Rom and Garay, eds.,2003), and Harrison’s Principles of Internal Medicine, Chapter150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).”

Effective treatment strategies are needed to prevent the reactivation and spread of Mycobacterium tuberculosis infection, active or latent. This invention meets these and other requirements.

“DESCRIPTION LISTED SEQUENCES.”

“SEQ ID No.1: Mtb72f, N-terminal 6 His Tag (DNA)”

“SEQ ID No.2: Mtb72f w/ N-terminal 6 His Tag (protein)”

“SEQ ID No.3: M72 (variant Mtb72f), with N-terminal 2 His insert (DNA).”

“SEQ ID No.4: M72 (variant Mtb72f), with N-terminal 2-His (protein)

“SEQ ID No.5: Mtb72f with N-terminal His Insertion (DNA)”

“SEQ ID No.6: Mtb72f with N-terminal His Insertion (protein)”

“The present invention provides pharmaceutical compositions that contain a Mtb72f protein fusion protein or an immunogenic segment thereof from a Mycobacterium complex of tuberculosis, such as together with one or several adjuvants including AS01B or AS02A.”

The present invention was partly based on the discovery by the inventors that administration of a Mtb72f protein fusion or an immunogenic fragment thereof, together with one or two adjuvants, or a nucleic acids encoding a Mtb72f protein fusion or immunogenic fraction thereof, can prevent or treat reactivation. A preferred embodiment of the invention includes administration of a Mtb72f nucleic or fusion protein with one or more chemotherapeutic drugs that are effective against M. tuberculosis infections.

“Another aspect is that the compositions can be used in methods to prevent tuberculosis from reactivation in subjects. The method involves administering an immunologically effective amount a pharmaceutical composition consisting of a nucleic acids encoding a Mtb72f protein fusion protein or an immunogenic fraction thereof from a Mycobacterium complex tuberculosis species. In this way, Mtb72f induces an immune response against M. tuberculosisisis responsible for treatment or thereby treating or reactivation.”

“In another embodiment, the compositions can be used in methods to reduce the time course for chemotherapy against M. tuberculosis. This includes administering to a mammal infected by Mycobacterium tuberculosis one of several chemotherapeutic drugs and an immunologically efficacious amount of a pharmaceutical formulation comprising a Mtb72f protein or an immunogenic fraction thereof from a Mycobacterium complex of the tuberculosis and an adjuvant. The adju protein or said Mtb72f protein or the tuberculogenic fragment thereof, thus allowing for the treatment. The present methods can reduce the time required to treat a M. tuberculosis patient. They also increase compliance by reducing the length of the treatment.

The present invention is about compositions containing Mtb72f nucleic acid or fusion proteins, an adjuvant that can be used to treat, prevent, or delay reactivation (i.e., active) Mycobacterium infection and methods for their application. The compositions of this invention include Mtb72f fusion proteins or immunogenic fractions thereof, nucleic acids encoding Mtb72f fusion protein or immunogenic segments thereof, having components from a Mycobacterium spp. (see Harrison’s Principles of Internal Medicine Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005). The present application’s inventors discovered that compositions containing Mtb72f-fusion polypeptides or nucleic acid encoding Mtb72ffusion polypeptides (or immunogenic fragments thereof) are effective in treating, delaying, or preventing reactivation. A preferred embodiment of the invention includes administration of a Mtb72f nucleic or fusion polypeptide with one or more chemotherapy agents.

“These compositions, polypeptides and the nucleic acid that encode them, are useful in eliciting an immune reaction in mammals that is protective against the reactivation disease symptoms.”

“The Mtb72f nucleic acid and fusion polypeptides described in the present invention may further contain other components that enhance or improve their antigenicity. The addition of histidine residues at one end of the antigen may facilitate the isolation of fusion polypeptides. Additional antigens or heterologous polypeptides can be included in the compositions, polypeptides and nucleic acid of the invention. The compositions, polypeptides and nucleic acid of the invention may also contain additional copies of antigens from Mycobacterium species such as Ag85B and MTCC #2. Additional polypeptides and nucleic acid can be added to the compositions, polypeptides and nucleic acids. The compositions and fusion proteins can contain polypeptides or nucleic acid encoding polypeptides. This allows for the enhancement of expression of antigens such as NS1, an influenza virus-protein (see, e.g. WO99/40188, WO93/04175, respectively. The invention’s nucleic acid can be engineered using codon preference in a specific species, e.g. humans.

“The Mtb72f protein compositions typically contain one or more adjuvants. See, U.S. Patent Publication Number. 2003/0143240; AS02A (3D phosphoryl lipid A (MPL) and QS21 in an oil-in-water emulsion); Bojang, Lancet (2001), 358:1927); ENHANZYN; 3D-MPL. A preferred embodiment of the Mtb72f-fusion polypeptide is administered in combination with one or more adjuvants from the group consisting QS21 and 3D-MPL. This liposome formulation, e.g. AS01B, MPL, and QS21 are combined with an oil-in-water emulsion (e.g. AS02A). Pichyangkul, et. al., Vaccine (2004) 22, 3831-40, further describes the adjuvants AS01B, AS02A.

“When the Mtb72f Antigen is delivered as a Nucleic Acid, it can be delivered in a viral vector (i.e. an adenovirus vector) or in a mutant Bacillus host cell (i.e. a mutant, virus-resistant Mycobacterium or Lactobacillus host cell, including Bacillus Calmette Guerin (BCG), and Lactococcus lactis).

“In one embodiment, the compositions can be used in methods of treating or preventing tuberculosis activation in a subject. The method comprises the step of administering an immunologically effective amount to a mammal infected by Mycobacterium tuberculosis. This includes an adjuvant and a Mtb72ffusion protein. This induces an immune response against M. tuberculosis. The present invention allows for the delay of M. tuberculosis reactivation (for example, by several months, years, or indefinitely).

“In one embodiment, the compositions can be used in methods of treating or preventing tuberculosis in a subject. The method comprises the step of administering an immunologically effective amount to a mammal infected by Mycobacterium tuberculosis. This includes administering an immunogenic fragment of Mtb72f protein from Mycobacterium species complex or an Mtb72f-encoding nucleic acid. In this case, Mtb72f protein stimulatesisisisisisisisis.

“In one embodiment, an individual with an active M. tuberculosis infection is given the Mtb72f nucleic acids or fusion proteins. One embodiment administers the Mtb72f nucleic acids or fusion proteins to individuals with an active or inactive M. tuberculosis disease. One embodiment administers the Mtb72f nucleic acids or fusion proteins to an individual with M. tuberculosis multi-drug resistant. One embodiment administers the Mtb72f nucleic acids or fusion proteins to an individual who has been previously immunized against Bacillus Calmette-Guerin.

“In some cases, the Mtb72f fusion protein or nucleic acid is combined with one or more chemotherapeutic drugs that are effective against M. tuberculosis. Amikacin and aminosalicylic acids, capreomycin and cycloserine are some examples of chemotherapeutic drugs. This type of chemotherapy is determined by the physician who prescribes the preferred combination of drugs. ?First-line? The first-line treatment for M. tuberculosis is isoniazid, ethambutol and streptomycin. ?Second-line? The chemotherapeutic agent used to treat M. tuberculosis infection if it has shown drug resistance to at least one or more of the?first-line’ drugs. Drugs include ofloxacin and ciprofloxacin as well as ethionamide, aminosalicylic acids, cycloserine (amikacin), kanamycin, and capreomycin.

The Mtb72f nucleic acids or fusion proteins can be administered prior to, concurrently with, and/or after the administration of one or more chemotherapeutic drugs effective against M. tuberculosis. One embodiment of the Mtb72f nucleic acids or fusion proteins is administered approximately 2 weeks after starting to administer one or more chemotherapy agents. One or more chemotherapeutic drugs are usually administered over a time period, such as for 1, 2, 3, 4 or 6 weeks, 2, 3, 4, 5, 6, or 8 months, or for 1 year.

“In some embodiments, administration of Bacillus Calmette-Guerin is increased to enhance the effects of an Mtb72f fusion protein or nucleic acid in certain cases.”

“In some instances, priming or the first administration of Mtb72f nucleic acids or fusion polypeptides is followed by one or several?boosting? administrations of Mtb72f nucleic acids or fusion polypeptides (?prime? and boost?). method). A Mtb72f fusion nucleic acids or nucleic polypeptide can be administered first, followed by one or more subsequent Mtb72f fusion proteins or nucleic acids. One embodiment provides for a second administration of Mtb72f nucleic acids or fusion proteins. This is followed by one or several subsequent administrations with Mtb72f fuse polypeptides. One embodiment provides for a second administration of Mtb72f fusion polypeptide or nucleic acids. This is followed by one or several subsequent administrations with Mtb72f cleic acids. The first, or?priming? administration is usually the most important. Administration and the second, or?boosting? are usually given together. Administration is given approximately 2-12 weeks apart or up to 6 months apart. The next ‘booster? Administrations can be given at least six months apart or up to 1, 2, 3, 4, or 5 years apart. Conventional booster treatments (e.g., a Protein priming administration followed with a Protein Boosting Administration) are also helpful in treating or preventing M. tuberculosis activation.

“Another aspect is that the compositions can be used in methods to reduce or shorten the time course for chemotherapy against M. tuberculosis. This involves administering to a mammal infected by Mycobacterium tuberculosis one of several chemotherapeutic drugs and an immunologically efficacious amount of a pharmaceutical composition consisting of either a Mtb72f-derived fusion polypeptide, or an immunogenic fragment thereof, and an adjuvant. Most often, effective chemotherapeutic treatment against M. tuberculosis can be achieved by administering a Mtb72f nucleic acids or fusion polypeptide within six months, five months, four months, three months or less.

“The Mtb72f compositions can be administered to humans but they are also effective in other mammals, including domestic mammals (i.e. dogs, cats, rats, mice and guinea-pigs), agricultural mammals (i.e. cows, pigs (sheep, goats, goats) and horses).

“In its broadest sense, a Mtb72f protein fusion protein according the invention is a protein that contains at least one immunogenic fragment from each of the antigens Ra12?TbH9?Ra35.”

“In the nomenclature, Ra35 refers the N-terminus Mtb32A (Ra35FL) which contains at least the first 205 amino acid of Mtb32A. The nucleotide sequence and amino acid sequence are disclosed in FIG. 4 of U.S. Patent. No. 7,186,412, or the equivalent region from another Mycobacterium species. Ra35 is most commonly used to refer to the portion in SEQ ID No. 2 that corresponds to residues 535-729. It could also refer to a Ra35 variant in which the amino acids Ser and 710 in SEQID No: 2 are replaced by Ala.

“Ra12” refers to Mtb32A’s C-terminus (Ra35FL), which contains at least the last 132 of MTB32A’s amino acids. The sequence is described in SEQ ID No:4 (DNA) or SEQ ID No:66 (predicted sequence of amino acids) in U.S. Pat. No. 6,592,877 or the equivalent region from another Mycobacterium species. Ra12 is most commonly used to refer to the portion SEQ ID No. 2 disclosed in this application that corresponds to residues 8 to139.

“Mtb39” (TbH9) is a sequence that is essentially the same as what is described in SEQ ID No: 106 (cDNA full-length) and SEQID NO: 107(protein full-length) in U.S. Patent Application Ser. No. 08/658,800, Ser. No. 08/659,683, U.S. Pat. Nos. 6,290,969 and 6,338,852 respectively, as well as in the WO97/09428/WO97/09429 patents. In U.S. Pat., the sequence is also known as SEQID NO:33 (DNA), and SEQID NO:91 (amino acids). No. 5,946,926. TbH9 is most commonly used to refer to the portion SEQ ID No. 2 disclosed in this application that corresponds to residues 143-532.

“The following lists sequences of antigens that are used in compositions and fusion proteins according to the invention:

“Mtb32A” (TbRa35FL, Ra35FL), whose sequence is described in SEQ ID No:17 (cDNA), and SEQ ID No:79 (protein) respectively, are part of the U.S. Patent Application Ser. Nos. 08/523,436, 08/523,435, Ser. No. 08/658,800, Ser. No. 08/659,683, U.S. Pat. Nos. Nos.

“The following sequences contain some of the fusion proteins mentioned in the invention:

“TbH9Ra35 (Mtb59F), whose sequence is described as SEQID NO:23 (cDNA), and SEQID NO:24 (protein in U.S. Pat. No. No.

“Ra12 -TbH9 -Ra35 (Mtb72f), whose sequence is disclosed as either SEQID NO:1 (DNA) or SEQID NO:5 (protein) in this application as well as U.S. Pat. No. 6,544,522 and in WO199951748. The sequences SEQID NO: 1 and SEQUID NO:2 contain a His tag with 6 His residues.

“M72, a mutant of Mtb72f, in which the serine residue at position 710 in SEQID No. 2 has been changed from Ala to the serine residue at position 710 in SEQID No. 2. (along with 4 His residues having to be removed from the His tag at the N terminus). The sequence is disclosed in the present application as SEQID No. 3 (DNA) or SEQID No. 4 (protein). U.S. Patent discloses a variant of these sequences where the protein is tagged with 6 His residues. No. 7,186,412 as well as in WO2001098460. M72, which was replaced by Ser710 with Ala is thought to be more resistant than Mtb72f to autolysis.

“The following sequences provide additional antigens that are used in compositions and fusion proteins according to the invention:

“Mtb8.4″ (DPV), whose sequence is described as SEQID NO:101 (cDNA), and SEQID NO:102 (protein in U.S. Patent Application Ser. No. 08/658,800, Ser. No. 08/659,683, U.S.”

“U.S. Pat. Nos. Nos.

“Mtb9.8” (MSL), whose sequence is described as SEQID NO:12 (DNA), SEQID NO:109 (predicted sequence of amino acids) and SEQID NO:110 to SEQ124 (peptides), in the U.S. Patent Application Ser. No. 08/859,381, Ser. No. 08/858,998, U.S. Pat. Nos. Nos.

“Mtb9.9A, also known by MTI-A), is described as SEQID NO:3 (DNA) and as SEQID NO:4 (MTI) in U.S. Patent Application Ser. No. 08/859,381, Ser. No. 08/858,998, U.S. Pat. Nos. 6,555,653 to 6,613,881 as well as in WO199853075 or WO98053076. MTI-B, and MTI?C are two other MTI variations that exist.

“Mtb40” (HTCC #1), whose sequence is disclosed in SEQ ID No:137 (cDNA), and 138 (predicted sequence of amino acids) in U.S. Pat. Nos. Nos.

“Mtb41” (MTCC #2), whose sequence is described as SEQID NO: 140 (cDNA sequence) and SEQID NO: 142 [predicted amino acids sequence] in U.S. Pat. Nos. Nos.

“ESAT-6″ is described in U.S. Pat. as SEQID NO:103 (DNA) or SEQID NO:104 (predicted sequence of amino acids). No. 6,592,877. U.S. Pat. No. 5,955,077;”

“?-crystalline Antigen, whose sequence is described in Verbon et al. J. Bact. 174:1352-1359 (1992);”

“85 complex antigens, whose sequence is described in Content et al. Infect. & Immunol. 59:3205-3212 (1991).”

Cole et. al. also discloses each of these sequences. Nature 393:537 (1998) and can be found at, e.g., www.sanger.ac.uk and www.pasteur.fr/mycdb/.”

“The sequences described in U.S. Patent Application Ser. Nos. Nos. Nos. Nos.

“The antigens discussed herein include polymorphic variants as well as conservatively modified versions, as well inter-strain and intraspecies Mycobacterium homologs. The antigens described in this article also include subsequences and truncated sequences. Additional polypeptides and heterologous peptides may be added to fusion proteins from Mycobacterium, or from other sources. You can modify these antigens by, for instance, adding linker sequences of peptides, as explained below. These linker proteins can be placed between any one or more of the components that make up each fusion protein.

“Definitions”

“Tuberculosis Reactivation” is the term used to describe this condition. The later appearance of disease symptoms in an individual who tests positive for tuberculin but has not yet shown any symptoms. Individual is suffering from M. tuberculosis and may have had active symptoms in the past that were sufficiently treated to make tuberculosis inactive or latent. However, there are ways to prevent or treat tuberculosis activation in cases where active symptoms have been identified.

“?Primary tuberculosis? Refers to clinical illness (manifestation disease symptoms) immediately following M. tuberculosis infection. Harrison’s Principles of Internal Medicine Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).”

“?Secondary tuberculosis? or?postprimary tuberculosis? Refers to the activation of an inactive, dormant or latent M. tuberculosis disease. See Harrison’s Principles of Internal Medicine, supra.

“An active infection of M. tuberculosis” Refers to M. tuberculosis infections with symptoms.

“An inactive, dormant, or latent M. tuberculosis infection.” Refers to M. tuberculosis infections that do not manifest symptoms.

“A ?drug resistant? M. tuberculosis refers to an infection that is resistant or not able to be killed by one or more of the so-called “front-line” drugs. M. tuberculosis infection can be treated with chemotherapeutic drugs (e.g., isoniazid and rifampin), which are effective in killing the infecting strain.

“A ?multi-drug resistant? M. tuberculosis refers to an M. tuberculosis infected with a strain that is resistant to at least two of the?front-line’ drugs. M. tuberculosis infection can be treated with chemotherapeutic drugs.

“A?chemotherapeutic drug that is effective in the treatment of a M. tuberculosis-related infection.” The term refers to all pharmacological agents that are known to be effective in treating M. tuberculosis. Amikacin is one example of pharmacological agents that can be used to treat M. tuberculosis. ?First-line? The first-line treatment for M. tuberculosis is isoniazid, ethambutol and streptomycin. ?Second-line? The chemotherapeutic agent used to treat M. tuberculosis infection. It has been shown to be resistant to at least one of the?first-line’ drugs. Drugs include ofloxacin and ciprofloxacin. These pharmacological agents can be found in Chapter 48 of Goodman & Gilman’s The Pharmaceutical Basis of Therapeutics, Hardman & Limbird, 2001.

“?FL? “?FL” refers to full length, which is a polypeptide with the same length as the wild type polypeptide.

“?His tag? A string of His residues is inserted at the N terminus. It can be 6 residues or more. These are often not homologous to the native sequence, but they are incorporated because they facilitate the isolation of immobilised metal affinity chromatography (IMAC) resins by improving protein binding. The presence or absence of a his tag is generally not significant in terms of triggering an immune response against the antigenic proteins to be elicited. It is best to reduce the His tag’s length to four or less residues in the event of an adverse immune reaction.

“The term “immunogenic fragment of it?” A polypeptide that contains an epitope that is recognized and processed by cytotoxic or helper T lymphocytes as well as B cells. An immunogenic fragment of Mtb72f is typically a polypeptide that contains 500 or more amino acid eg 600, eg 700, or more amino acid. The invention can also include multiple fragments, eg overlapping fragments that together cover all or substantially all of (eg 500 amino acids or more eg 600 amino acids or 700 amino acids) of a Mtb72F sequence protein.

“The term Mycobacterium species in the tuberculosis compound? This includes species which are traditionally thought to cause the disease tuberculosis. It also includes Mycobacterium environmental or opportunistic strains that can cause it. 953-966 (16th ed., Braunwald, et al., eds., 2005).”

An adjuvant is a component in a vaccine, or therapeutic composition, that increases the immune response to an antigen. (See, e.g. Edelman, AIDS Res. Hum Retroviruses 8 : 1409-1411 (1992). Adjuvants induce immune responses of the Th1 and Th2 types. Th1-type cytokines, e.g. IFN-? and IL-2, tend to favor inducing cell-mediated immune responses to administered antigens, while Th2 type cytokines, e.g. IL-4 and IL-5, Il-6 and IL-10, tend to favor the inducing of cell-mediated immune responses to an administered antigen. Favor the induction and maintenance of humoral immune responses. “WO 94/00153 and W 95/17209 describe adjuvants that can preferentially stimulate a Th-1-cell-mediated immune response.

“?Nucleic acid? “Nucleic acid” refers to single- and double-stranded deoxyribonucleotides, ribonucleotides, and polymers thereof. This term includes nucleic acids that contain known nucleotide analogues or modified backbone linkages. These are synthetic, naturally occurring and non-naturally occurring and have similar binding properties to the reference nucleic Acids. They are also metabolized in a similar way to the reference nucleotides. These analogs include, but are not limited to, phosphorothioates and phosphoramidates as well as methyl phosphonates and chiral-methylphosphonates.

“Unless otherwise stated, a specific nucleic acid sequence implicitly includes conservatively modified variants (e.g. degenerate codon substitutes) and complementary sequences as well as the sequence specifically indicated. Degenerate codon substitutes can be made by substituting the third position of one or several selected codons with mixed-base or deoxyinosine (Batzer and colleagues, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91?98 (1994). Nucleic acid can be used interchangeably with gene, DNA, mRNA and polynucleotide.

“Polypeptide” is a term that refers to a protein. ?peptide? ?peptide? and?protein? are interchangeable herein. These terms are interchangeable to mean a polymer of amino acids residues. These terms refer to amino acids polymers in which one of the amino acid residues is an artificial chemical mimetic to a naturally occurring amino Acid.

“Amino acid” is a generic term that refers to amino acids. “Amino acid” can be used to refer to both naturally occurring and synthetic amino acid, as well as amino-amino mimetics and amino acid analogs that function in a similar way to naturally occurring amino acids. The genetic code encodes naturally occurring amino acids, along with those that have been modified in some way, such as hydroxyproline, O-phosphoserine, and?-carboxyglutamate. Amino acids analogs are compounds with the same basic chemical structure and function as naturally occurring amino acids. This means that they have a carbon bound to a hydrogen, carboxyl or amino group and an R group (e.g. homoserine. norleucine. methionine.methyl.sulfonium. These analogs may have modified R (e.g. norleucine), or modified peptide-backbones but maintain the same basic chemical structure of an naturally occurring amino acid. Amino acids mimetics are chemical compounds with a structure different from that of an amino acid but that function in a similar way to naturally occurring amino acids.

“Amino acids can be referred to by their three-letter symbols, or the one-letter symbols suggested by the IUPAC/IUB Biochemical Nomenclature Commission. The commonly accepted single-letter codes for nucleotides may also be used to refer to them.

“Conservatively modified versions? This applies to both nucleic acid and amino acid sequences. Conservatively modified variants are those nucleic acids that encode identical or almost identical amino acid sequences. It also applies to nucleic acids that do not encode an amino sequence. These nucleic oils can be used to create essentially identical sequences. Due to the degeneracy in the genetic code, many functionally identical nucleic acid sequences encode any one protein. The amino acid alanine is encoded by the codons GCC, GCC and GCG, for example. The codons that specify alanine can be changed to any of the other codons without affecting the encoded polypeptide at any position. These nucleic acids variations are called?silent variants? These are one type of conservatively modified variants. Each sequence of nucleic acids that encodes a polypeptide describes all possible silent variations. Any one with skill will be able to recognize that every codon in a nucleic acids (except AUG which is ordinarily only codon of methionine and TGG which is ordinarily only codon of tryptophan), can be modified to produce a functionally identical molecule. Each silent variant of a nucleic acids that encodes a polypeptide can be implicitly included in the described sequence.

“Conservatively modified variants are those that alter, add or delete one amino acid in a sequence of amino acids. where an alteration results in the substitution or addition of an amino acid by a chemically identical amino acid. The art is well-versed in the use of conservative substitution tables that provide functionally identical amino acids. These conservatively modified variants can be used in conjunction with and without exclusion of polymorphic variants, interspecies homologs, or alleles according to the invention.

“The following eight groups contain amino acids that can be used as conservative substitutes for each other:

“1) Alanine, Glycine (G);

“2) Aspartic acid, Glutamic acid (D),”

“3) Asparagine (N), Glutamine (Q);”

“4) Arginine (R), Lysine (K);”

“5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);”

“6. Phenylalanine, Tyrosine, Tyrosine (Y), Tryptophane (W);

“7) Serine, Threonine (T), and

“8) Cysteine (C), Methionine (M)”

“(see, e.g., Creighton, Proteins (1984)).”

“Heterologous” is a term that refers to a combination of subsequences. When referring to portions of a nucleic acids, it means that they contain two or more subsequences which are not in the same relationship to one another in nature. The nucleic acids are often recombinantly made, meaning that they contain two or more sequences of unrelated genes, which are arranged to create a new functional nucleic Acid. A heterologous protein, on the other hand, indicates that the protein contains two or more sequences that are not in the same relationship in nature (e.g. a fusion protein).

“?Fusion polypeptide? or ?fusion protein? A protein that contains at least two Mycobacterium species sp. polypeptides that are covalently linked either directly or through an amino acid linker. Although the polypeptides that make up the fusion protein are usually linked C?terminus-N-terminus (although they can also be connected N-terminus-N-terminus or C?terminus-C-terminus), they can also be linked N-terminus-N-terminus or N?terminus-C-terminus. You can have any number of polypeptides in the fusion protein. This term can also be used to refer to conservatively modified versions, polymorphic variations, alleles and mutants as well as subsequences and interspecies homologs for the antigens in the fusion protein. Cole et. al., Nature 393 537 (1998) describes the Mycobacterium tuberculosis antigens. The complete sequence of Mycobacterium tuberculosis can also be found at www.sanger.ac.uk and at www.pasteur.fr/mycdb/(MycDB). You can identify antigens from other Mycobacterium species which correspond to M. tuberculosis by using sequence comparison algorithms (described herein) or other methods that are well-known to those skilled in the art, such as hybridization assays or antibody binding assays.

“Exemplary Mtb72f Fusion Proteins of Use in the Present Invention Are:

“Proteins consisting of residues 8-729 in the sequence of SEQ ID Number: 2;”

“Proteins consisting of or consisting in the sequence of SEQ ID Number 2 (Mtb72F), optionally without the His Tag forming residues 2-7 or with a different His tag;”

“Fusion proteins consisting of the sequence of SEQID No: 2 optionally with or without the His tag forming residues 2-4 of said sequence, or with a different His tag (e.g. A protein containing residues 8-729 from the sequence of SEQID No: 2 together with one or several M. tuberculosis antibodies, such as one or more of these proteins or an immunogenic fragment thereof;

“Proteins consisting of residues 4-725 in the sequence of SEQ ID Number 4 (Mtb72F);

“Proteins consisting of or consisting in the sequence of SEQID No: 4 (Mtb72F), optionally without the His tags forming residues 2-3 of said sequence, or with a different His tag;”

“and”

“Fusion proteins consisting of the sequence of SEQID No: 4 without or with a different His tag (e.g. A protein containing residues 4-725 from the sequence of SEQID No: 4, together with one or several M. tuberculosis antibodies, such as one or more of these proteins or an immunogenic fragment thereof;

“Exemplary immunogenic pieces of a Mtb72f Fusion Proteins that are used in the present invention include:

“Proteins consisting or consisting in the sequence TbH9?Ra35 (Mtb59F); TbH9; Ra35; Ra12; and

“Fusion proteins consisting of said sequences and one or more M. tuberculosis Antigens, such as one or more of these proteins or an immunogenic fragment from any one of them.”

“Further exemplary immunogenic pieces of a Mtb72f Fusion Proteins of Use in the Present Invention include:

Summary for “Method to prevent or treat M tuberculosis”