Treatment and prevention of tuberculosis

Abstract

The invention is within the field of immunology and microbiology, more specifically the field of mycobacteriology and is related to immunotherapy and prophylaxis of tuberculosis and related diseases. The composition useful for these purposes is disclosed, including the methods of using said composition.

Claims

1. A tableted composition, to be administered orally into a host in need thereof, said composition consisting of three components: (1) at least one hydrolyzed and heat-inactivated antigen of a non-recombinant mycobacterium; (2) at least one hydrolyzed and heat-inactivated non-recombinant alloantigen derived from pooled blood of the same species as the host; and (3) a magnesium salt bound to the antigen and the alloantigen.

2. The composition of claim 1 wherein said at least one antigen of a mycobacterium is derived from non-recombinant strains of BCG, Mycobacterium (M.) tuberculosis, M. avium, M. habana, M. abscessus, M. aurum, M. bovis, M. vaccae, M. africanum, M. chelonae, M. fortuitum, M. fuerthensis, M. gastri. M. goodi, M. gordonae, M. immunogenum, M. intracellulare, M. paratuberculosis, M. lufu, M. kansasii, M. lentiflavum, M. leprae, M. w, M. mageritense, M. malmoense, M. marinum, M. massiliense, M. monacense, M. mucogenicum, M. neoaurum, M. peregrinum, M. phlei, M. porcinum, M. septicum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. tusciae, M. nonchromogenicum, M. ulcerans, M. chelonei, M. scrofulaceum, M. triviale, M. asciaticum, M. flavescens, M. genavense, or M. xenopi.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Administration of vaccines to non-infected hosts is referred to as preventive vaccinating while administration to already infected hosts is referred to as therapeutic vaccination. The term prevention includes an attempt to halt the occurrence of a disease or disorder before it happens. The term therapy includes an attempt to alleviate the disease or clinical condition of an afflicted subject.

(2) In the course of experimentations it has been discovered that when Mycobacterium tuberculosis, such as those found in the blood of one third of healthy people, are formulated into a tablet, such a preparation acquires biological activity in terms of inducing a more favorable clinical outcome when they are administered to individuals with tuberculosis or as means of prophylaxis of infection. The steps of making such a formulation are simple and straightforward. One of the steps is so-called hydrolysis, which can be easily achieved by acid treatment as the preferred mode of hydrolysis. The pH range is preferably below 6 and more preferably between 0.5 and 5.5, more preferably between 1 and 4, more preferably between 1.0 and 3.0, and even more preferably between 1.0 and 2.0. While these ranges are given as optimal ranges, they will not prevent one skilled in the art to determine additional ranges, which remain within the scope of the present invention. The proportion of hydrolyzed antigen, such as a protein, is at least 10% by weight of the non-hydrolyzed native antigen, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, and can be at least 90%. The meaning of 100% hydrolyzation is a situation when all proteins are hydrolyzed to free amino acids without any larger residue, such as a peptide, remaining after the hydrolysis reaction.

(3) The duration of hydrolysis can be anywhere between 5 minutes to several hours, more preferably they are between 10 minutes and 5 hours, even more preferably between 20 minutes and 3 hours. The duration can be a shorter period of hours, which is determined by simple experimentation under routine hydrolysis conditions well familiar to those skilled in the art. Thus, the duration of hydrolysis can be 30 minutes, one hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours, or intermediate times.

(4) The acids that are useful for hydrolysis can be, but are not limited to, inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like. Examples of organic acids include, but are not limited to, for example, acetic acid, citric acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, salicylic acid, p-toluenesulfonic acid, and the like.

(5) The source of tuberculosis antigen does not necessarily need to be from blood or blood derivatives such as plasma or serum. Sputum or other body fluids like urine, gastrointestinal secretions, liquid of tissues or tumors, synovial fluid, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, perspirations, and prostatic fluid may be used as a source of the tuberculosis antigen. These fluids can contain leukocytes, macrophages or other cells that are equally advantageous. Without limiting to fluids, solid tissues such as lung, liver, pancreas, skeletal muscle, heart, kidney, brain, bone marrow, tumor tissue, skin, myelin, collagen, feces, and other tissues may be used.

(6) Whole blood, or serum or plasma, or culture broth with mycobacterial pathogen cells present in them or recombinantly obtained mycobacterium containing antigens are treated preferably with an acid or alkali. Cell lines like HeLa, for example, can be used equally to grow mycobacterium. One skilled in the art may use other hydrolyzing agents like enzymes or detergents. Samples to be hydrolyzed are pooled or from one individual, or maintained as a separate batch.

(7) In one embodiment when the composition is still in liquid solution and in process of hydrolysis, the products of hydrolysis can be precipitated with a salt of a metal or with an alkaline metal hydroxide solution that forms a salt. This step effectively ends the hydrolysis since antigens are entrapped or embedded within the matrix of the solid matter and do not react with the hydrolytic agent. The precipitation can be run simultaneously with hydrolysis or started after the hydrolysis step, depending on the intended use of the final product. For example, an aqueous solution consisting of an antigen and alloantigen, preferably about 90% to about 20% protein, and more preferably about 80% to about 40% protein solution, is treated with an acid, until the pH is about 1 to about 6, preferably about 1 to about 4, more preferably about 1 to about 2. The solution is stirred at low speed, preferably at about 10 to about 500 rpm, more preferably about 30 to about 300, even more preferably about 60 to about 200 rpm for about 60 minutes to about 120 minutes, preferably about 45 to about 80 minutes, more preferably about 30 to about 60 minutes. A metal salt is then added to the stirring solution to create a slurry that solidifies as a solid mass. The mass is washed for about 1 minute to about 60 minutes, preferably about 5 to about 45 minutes, more preferably about 10 to about 30 minutes and then dried in an oven by heating at about 56 to about 200 C., preferably at about 70 to about 150 C., more preferably about 80 to about 140 C., more preferably about 100 to about 120 C., for about 1 hour to about 72 hours, preferably about 3 to about 48 hours, more preferably about 5 to about 12 hours. The dried solid mass is then reduced to a powder.

(8) It is equally advantageous to start the precipitation and add hydrolyzant after that step. In yet another embodiment the metal salt is present prior to the hydrolysis and proteins are hydrolyzed in the presence of said metal salts, to be precipitated simultaneously and concomitantly. Representative steps for process of embedding an active ingredient such as an antigen together with alloantigen, or antigen alone without alloantigen, or alloantigen alone without the antigen, can include contacting the alloantigen with a metal salt solution to create a slurry and hydrolyzing the resulting mixture by contacting it with an acid so that this mixture is hardened and transforms into a solid phase or a solid state forma process also know as a setting of the solid matter. This solid form is then reduced to powder particles embedded within active ingredient. The sequence of these steps are easily determined before hand and can be carried out without undue experimentation as long as the final product with active ingredient is clinically effective as determined by clinical studies.

(9) The hydrolyzed mycobacterial antigens and alloantigens can be embedded in salts, including but not limited to: Lithium, Beryllium, Sodium, Silicone, Magnesium, Aluminium, Aluminum, Potassium, Calcium, Titanium, Vanadium, Chromium, Manganese, Cobalt, Nickel, Copper, Zinc, Arsenic, Zirconium, Molybdenum, Silver, Cadmium, Antimony, Barium, Osmium, Platinum, or Gold. Most suitable soluble salts include sodium chloride, potassium chloride, calcium chloride or magnesium chloride. An example of salt of a representative metal, such as for example magnesium, is Aluminium magnesium boride, Calcium magnesium acetate, Dirnagnesium phosphate, Magaldrate, Magnesium aluminide, Magnesium aspartate, Magnesium benzoate, Magnesium bromide, Magnesium carbonate, Magnesium chloride, Magnesium chloride hexahydrate, Magnesium citrate, Magnesium diboride, Magnesium diglutamate, Magnesium diuranate, Magnesium fluoride, Magnesium gluconate, Magnesium hexahydrate, Magnesium hydride, Magnesium hydroxide, Magnesium iodide, Magnesium lactate, Magnesium levulinate, Magnesium nitrate, Magnesium nitride, Magnesium orotate, Magnesium oxide, Magnesium oxychloride, Magnesium oxysulfate, Magnesium perchlorate, Magnesium peroxide, Magnesium phosphate, Magnesium pidolate, Magnesium silicide, Magnesium stearate, Magnesium sulfate, Magnesium sulfide, Magnesium sulfite, Magnesium trisilicate, Monomagnesium phosphate, Trimagnesium citrate and the like.

(10) In a further aspect of this invention two or more metals can be used simultaneously. For example, Calcium chloride and Magnesium chloride can be mixed at various acceptable ratios into which hydrolyzed antigens are embedded. The difference between present composition and prior art compositions having an antigen mainly adsorbed to the surface of an immune adjuvant such as aluminum hydroxide is that the instant antigen is securely embedded within the matrix, which allows it to keep the physical structure intact even at conditions that are commonly unfavorable, i.e., high temperature at longer periods of time.

(11) It is well known that exposure to high temperature, for example, heating at 56 C. or higher, like 80 C. for one hour or more is useful to destroy the adventitious pathogens, but not useful for making vaccines. However, surprisingly, the instant composition, when exposed to high temperatures has not lost its antigenicity and shows positive clinical benefit. Thus, instant composition can be safely used since potentially infectious virus that can be present within the composition is effectively killed by exposure to high temperatures for one hour or more. The advantage of thermal treatment, while obvious from the standpoint of elimination of infectious viruses, is not at all obvious from the standpoint of conservation of antigenicityan observation that is contrary to the consensus prevailing in the art.

(12) The instant composition is then combined with tablet excepients. An excepient is generally viewed as an inert substance which is added to an active ingredient to provide bulk, for example in tablets. These can be any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint or some other flavoring. Mixing with excipients is performed by a wet or dry granulation processes well known to formulation experts and resulting mixture is compressed into tablets. Various properties of excipients, such as moisture content, particle size and distribution, polymorphism, amorphism, crystal habit, hydration state, and lubricant and binder level of the blend that have an influence on compaction are within routine knowledge of a practitioner. Tableting speed and mechanistic aspects of tableting such as choice of punches/dies, tableting machines can be selected without undue experimentation by artisans. In addition, dosages can contain various other materials which modify the physical form, for example, coatings of sugar, shellac, or other enteric agents. The tablets can be covered with enteric coating material that is predominantly soluble in the intestinal fluid, but substantially insoluble in the gastric fluids of stomach. Preferably enteric coating material is chosen from those that are commercially available, although new materials can be selected that are with the skill of practicing artisan.

(13) The preferred dosage form is an oral solid dosage form such as a tablet or pill. Moreover, the pharmaceutical composition described herein can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, aqueous oral suspensions, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, self-emulsifying dispersions, solid solutions, liposomal dispersions, lyophilized formulations, capsules, powders, delayed release formulations, immediate release formulations, modified release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, mixed immediate release, controlled release formulations and the like.

(14) In other embodiments of the present invention, the amount of active ingredient in the tablet, i.e., antigens with alloantigens, administered to a subject via a solid dosage form, such as a tablet or pill, is the amount known in the art to achieve a therapeutically effective concentration of drug of a human or animal in need thereof. For example, the amount of one or more active ingredients may range from about 0.0001 micrograms to about 1 g. In other embodiments, the amount of active ingredients may range from about 0.001 micrograms to about 100 mg. In other embodiments, the amount of active ingredients may preferably range from about 0.01 microgram to about 10,000 microgram, from about 0.5 microgram to about 1,000 microgram or from about 1 microgram to about 500 microgram. In another embodiment, a formulation is administered in a solid dosage form at a total protein concentration of about 10 microgram to about 500 microgram. In another embodiment, the active ingredients formulation is administered in a solid dosage form at concentration of about 1-100 microgram. In other embodiment the amount of active ingredients is from about 0.00001% to about 30% based on the total weight of the composition, preferably the amount is between about 0.001% and 20%, preferably the amount is between about 0.01% and 10%, preferably the amount is between about 0.1% and 5%. The content can also vary depending on the intended use. For therapeutic use, the content can be higher by about one order of magnitude from content of the composition intended for prophylactic use. For example, if in a therapeutic composition the quantity of mycobacterial antigen is 60 microgram, then in a prophylactic version the dose can be reduced to 5 or 10 micrograms. However, this is not an absolute requirement. These and other implications related to effective dose are not intended to be limiting and are provided by a way of example only. The precise amount of active ingredients that is therapeutically effective is determined by standard clinical testing whereby the clinical effect is monitored by means well known to those skilled in the art.

(15) In a preferred embodiment, mycobacterial antigens are present in the composition together with alloantigen(s), which will be useful for prophylactic and therapeutic applications for preventing or actively treating tuberculosis. In the manufacture of most vaccines, considerable effort is made to purify vaccine antigens. This is due to the concern that when injected, antigens other than vaccine antigens cause undesirable immune reactions that can be sometimes life-threatening. Thus, calling for alloantigens to be present in the composition together with vaccine antigens is against common wisdom prevailing in the art. The ratio between mycobacterial antigens and alloantigen(s) is determined by art known methods. Preferably, the ideal ratio is one that is found naturally, as for example in the whole blood, i.e., the ratio between a quantity of mycobacteria and other host molecules such as the amount of total proteins found in the blood. The same applies to a situation in which antigen is recombinantly expressed in propagating cells, like yeast cells or mammalian cells such as CHO cells. In situations when composition components are grown in cells of species other than human but are intended to be used in humans, the alloantigen is then termed xenoantigen, which for purposes of this invention can be called alloantigen. Notwithstanding the above reasoning about a preferred ratio, one can deliberately increase the amount of alloantigen or antigen. In any case the amount of alloantigen shall not be too low so that it could be considered as a contaminant such as that found for example in standard vaccines, such as hepatitis B vaccine made from plasma. While the amount of contaminating plasma proteins, i.e., alloantigens, in hepatitis B vaccine made from pooled plasma varies depending on a batch or manufacturer it is usually less than 1%. In the case of hepatitis A vaccine the WHO recommends the amount of contaminating albumin to be less than 50 nanograms per dose. Thus, these requirements distinguish the instant composition from classical vaccine compositions of prior art, since in the present invention the ratio of various alloantigen molecules to antigen is always higher than 1:1, preferably more 10:1, ideally more than 100:1 and always exceeds 50 nanogram/gram when albumin is used to gauge the amount of allogeneic material. As intended here more than one type alloantigen is needed. Preferably one needs as wider diversity as possible. Such compositions, especially in oral form of delivery, will be useful to a host in need thereof, such as a human host or other species in need thereof.

(16) In some embodiments, formulations provide a therapeutically effective amount of composition over an interval of about 3 hours to about 24 hours after administration, enabling, for example, once-a-day, twice-a-day (b.i.d.), or three times a day (t.i.d.) administration if desired. A simple dosing regimen of one or two tablets per day is preferred. Administration can be a single tablet administration, more preferably repeated administration for at least one week up to one month to one year, or carried out for years as deemed necessary. Treatment can be stopped after 1-3 months and repeated again after one month of break, although in some patients repeat treatment is not necessary for as long as 3 or 6 months intervals. In some instances, it is preferred to stop for one year and then repeat again. In some individuals treatment may not to be repeated at all as patients are cured and need no further dosing.

(17) In another aspect of the invention the composition is administered without an immune adjuvant. While this is the preferred mode of administration, one skilled in the art, can administer the composition with an immune adjuvant known in the art. Examples of immune adjuvants include but not limited to bacterial adjuvants; cytokine adjuvants; plant-derived adjuvants and the like.

(18) The composition of the present invention can be administered in combination with an anti-tuberculosis drug. Compositions/therapies of the instant invention may be used in combination with any other bioactive substances such as pharmaceutically effective substances, including, but not limited to, antiinflammatory drugs, analgesics, tranquillizers, antianxiety drugs, antispasmodics, antidepressants, antipsychotics, antianxiety drugs, narcotic antagonists, antiparkinsonism agents, cholinergic agonists, chemotherapeutic drugs, immunosuppressive agents, antiviral drugs, antimicrobial drugs like antibiotics, appetite suppressants, anticholinergics, antimetrics, antihistaminics, antimigraine agents, coronary, cerebal or peropheral vasodilators, hormonal agents, contraceptives, antithrombotic agents, diuretics, antihypertensive agents, cardiovascular drugs, opioids, and the like.

(19) Other features and advantages of the present invention are apparent from the additional detailed disclosure as provided below and which includes different examples. The examples provided below illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure one skilled in the art can identify and employ other components and methodology to practice the present invention, which however are within the scope of the present invention.

EXAMPLES

Example 1

(20) Upon analysis of results of phase 2 study of an oral, therapeutic hepatitis vaccine, over 94.4% of patients with chronic hepatitis C with concomitant TB and HIV had completely cleared bacilli in their sputum smears (Table 1). As a result 17 out 20 patients were discharged from the hospital within one month after treatment initiation. Statistical analysis by both parametric and non-parametric tests reveal that this phenomenon is highly significant. Once-daily vaccine tablet of the invention was administered per os to 20 patients for one month. Every patient who entered the study had enlarged liver and elevated hepatic damage markers, which at the end of study have improved in 19 out 20 (95%) patients. The reduction was highly significant, from 172.134 to 18.228.2 U/L (P=5.0 E-012) and 22.13.4 to 10.92.5 mol/L (P=5.7 E-009) for ALT and total bilirubin respectively. Enlarged liver reduced from 3.51.4 to 0.951.1 cm above normal size (P=2.9 E-009). As patients were hospitalized in a TB hospital they were treated with standard anti-TB therapy (ATT). Surprisingly, vaccine compositions of the present invention appeared to contribute to higher and faster than expected sputum conversion rate; 94.4% of smear-positive patients became negative within one month. TB-associated fever subsided within mean/median 4.1/3 days; indicators of inflammation such as elevated erythrocyte sedimentation rate and high leukocyte counts returned back to normal from 32.311.4 to 9.96.4 mm/h (P=3.7 E-008) and 14.33.9 to 4.71.4109 L (P=7.1 E-010) respectively. Average body weight gain was 7.7 kg (P=4.6 E-007) and hemoglobin levels increased from 1147.1 to 123.46.6 g/L (P=1.4 E-007). No adverse events were observed at any time.

(21) TABLE-US-00001 TABLE 1 Baseline and outcome characteristics of HCV-infected patients with HIV and TB co-infections treated with TB drugs in combination with vaccine for one month. Erythrocyte No./ Months treated Smear Liver size in sedimentation Case TB drugs with ATT prior Positive cm over normal rate (ESR) No. Sex Age regimen to vaccine before after before after before after 1/602 M 33 RZSE 1 3 0 31 5 2/57 F 24 HRZSE 4 + 7 2 58 9 3/376 M 33 HRZSE 6 + 5 2 22 4 4/78 M 42 HRZSE 2 + 4 1 32 5 5/391 M 74 HRZSE 6 + 4 1 22 7 6/563 F 24 HRZSE 2 + 2 0 43 11 7/422 M 36 HRZSE 5 + + 4 4 28 32 8/502 M 34 HRZSE 3 + 5 1 32 10 9/553 M 31 HRZSE 3 + 5 2 43 18 10/579 F 34 HRZSE 3 + 2 0 40 10 11/360 M 38 HRZSE 5 2 0 45 9 12/570 M 36 HRZSE 3 + 2 0 28 7 13/605 M 42 HRZSE 1 + 4 1 28 10 14/465 F 26 HRZSE 4 + 2 1 20 4 15/519 M 33 HRZSE 3 + 4 1 18 10 16/613 M 35 HRZSE 1 + 2 0 22 5 17/654 M 38 HRZSE 1 + 3 0 34 9 18/311 M 42 HRZSE 1 + 2 1 14 5 19/558 F 41 HRZSE 2 3 0 38 11 20/641 M 26 HRZSE 3 + 5 2 48 16 20 5/15 36.1 10.6 17 1 3.5 1.4 0.95 1.1 32.3 11.4 9.9 6.4 Mean: 2.95 Fisher's exact Mean decrease 2.55 cm Mean decrease 22.4 Median: 3 2-way test P = 2.893E009 P = 3.713E008 P < 0.000001 No./ Leuko- Hb Weight change Total bilirubin ALT Case cyte 10.sup.9 L g/L kg mol/L IU/L No. before after before after before after before after before After 1/602 4.3 3.8 114 129 57 66 20 10 235 12 2/57 11.6 4.3 101 118 62 75 25 10 162 12 3/376 18.3 6 112 125 68 79 18 12 162 12 4/78 14 6.8 118 128 74 86 13 15 235 12 5/391 12.3 2.8 120 132 72 82 25 10 162 10 6/563 12 3.4 108 115 49 59 20 10 162 12 7/422 14 3.8 112 110 62 54 20 20 162 138 8/502 20 6 108 116 67 74 25 10 162 12 9/553 12 6 120 132 71 83 25 10 162 12 10/579 14.9 4 112 120 47 52 25 10 162 12 11/360 12 4 118 122 75 83 20 10 235 12 12/570 19 5 122 128 60 70 25 10 162 12 13/605 18 4 104 118 66 75 25 10 162 12 14/465 18 3 118 128 62 67 20 10 132 12 15/519 9 4 118 120 69 73 20 10 132 12 16/613 14 4 112 128 71 75 25 10 162 12 17/654 14 8 122 130 68 77 25 10 162 12 18/311 18 6 128 132 70 75 25 10 162 12 19/558 18 4 112 118 59 70 20 10 235 12 20/641 13 6 102 118 77 85 20 10 132 12 20 14.3 3.9 4.7 1.4 114 7.1 123.4 6.6 65.3 8.1 73 9.6 22.1 3.4 10.9 2.5 172.1 34 18.2 28.2 Mean decrease 9.6 10.sup.9 L Mean gain 9.3 g/L Mean gain 7.7 kg Mean decrease 11.2 mol/L Mean decrease 153.9 IU/L P = 7.162E010 P = 1.419E007 P = 4.604E007 P = 5.679E009 P = 5.027E012

(22) Following this discovery two clinical placebo-controlled studies were undertaken. These results are shown in Tables 2-5 and indicate unequivocally that claimed composition can indeed cure TB faster and more effectively regardless whether TB is drug-resistant or presents with HIV.

Example 2

(23) In the clinical trial involving 55 patients only 1 (3.7%) and 3 (10.7%) subjects in vaccine and placebo arms had first-diagnosed, drug-sensitive TB; the remaining patients had retreated TB, MDR-TB, or HIV-TB. They were divided into immunotherapy (N=27) and placebo (N=28) arms matched by age, gender, baseline body weight, and clinical manifestations. After one month, 26 out 27 patients (96.3%) became sputum smear negative in vaccine group (P<0.0000001), whereas 7 out 28 (25%) in placebo group had converted (P=0.005). The vaccine composition of the present invention contributed to the downregulation of TB-associated inflammation as shown by the normalization of high leukocyte counts, erythrocyte sedimentation rate, and faster deffervescence than in the control. Patients in both arms experienced an equally significant increase in the level of hemoglobin corresponding to 128.917.6 vs 133.114.7 g/L (P=0.03) and 112.614 vs 11711.7 g/L (P=0.03) in vaccine and placebo arms respectively. Nineteen out 28 (67.9%) placebo-receiving patients gained on average 1.07 kg (59.110 vs 60.110.4 kg; P=0.003). In contrast, every vaccine-treated patient gained mean 3.4 kg (59.78 vs 63.19 kg; P=5.7E-007). Clinical symptoms improved among all patients in the vaccine arm, while 28.6% of patients on placebo reported satisfactory results (P=0.007). No adverse or side effects attributable to the vaccine were seen at any time.

(24) TABLE-US-00002 TABLE 2 Baseline and outcome characteristics of TB patients receiving vaccine in combination with ATT for one month TB form Months treated Hemoglobin ERS (* = HIV + with ATT prior Therapy g/L (mm/h) No. Sex Age status) to vaccine regimen before After before after 1 M 37 Retreated 3 Individual 140 155 16 30 2 M 33 Retreated* 1 Individual 138 136 15 11 3 M 33 Retreated 4 Individual 150 151 6 4 4 M 24 MDR-TB 12 Individual 110 122 15 9 5 M 35 MDR-TB* 2 Individual 149 150 7 8 6 M 47 Retreated 4 Individual 121 97 5 13 7 M 26 MDR-TB 9 Individual 146 142 17 5 8 M 59 Retreated 2 Individual 131 137 43 17 9 M 35 Retreated 1 Individual 152 150 3 4 10 M 45 Retreated 1 Individual 119 115 31 28 11 F 28 Retreated* 8 Standard 124 122 20 8 12 M 32 Retreated* 7 Individual 140 138 21 11 13 F 42 Retreated* 6 Individual 113 128 10 8 14 M 56 1stDx 4 Standard 120 128 21 9 15 M 40 Retreated 3 Standard 124 126 28 4 16 M 33 Retreated 3 Standard 150 142 6 8 17 M 34 Retreated 2 Standard 145 143 6 7 18 M 55 Retreated 2 Standard 142 145 48 11 19 M 45 Retreated 4 Individual 124 143 18 4 20 M 41 Retreated 11 Individual 136 138 23 9 21 M 54 Retreated 3 Standard 117 125 21 11 22 M 32 1stDx* 2 Standard 114 122 42 18 23 M 36 MDR-TB 4 Individual 142 148 12 8 24 M 23 1stDx* 3 Standard 145 148 24 8 25 F 24 1stDx* 4 Standard 101 118 58 9 26 M 42 Retreated* 1 Standard 104 118 28 10 27 M 37 Retreated* 1 Standard 84 108 47 11 3/24 Mean 38.1 10 Mean 4 3 128.9 17.6 133.1 14.7 21.9 14.8 10.5 6.4 Median 36 Median 3 P = 0.03 P = 0.0003 Leukocyte Smear Axillary temperature Body weight count 10.sup.9 L positive ( C.) (kg) No. before after before after before after before after 1 8.7 7.2 + 38 36.8 58 60 2 6.7 4.1 + + 38.3 36.8 60 61 3 5.2 4.8 + 37.8 36.8 65 70 4 11 8 + 38 36.8 55 57 5 9 8 + 37.6 36.8 60 61 6 11.3 3.7 + 37.8 36.8 55 57 7 6.1 6.4 + 38 36.8 42 45 8 8.1 7.8 + 37.3 36.8 57 59 9 12.4 10.8 + 37.4 36.8 63 65 10 7.9 11.3 + 37.9 36.8 50 51 11 7.1 5.1 + 36.8 36.8 53 58 12 14 6 + 36.8 36.8 63 68 13 6.9 6.1 + 38 36.8 50 55 14 5.9 5.3 + 38.3 36.8 48 50 15 12.2 7.4 + 37.1 36.8 65 66 16 5.2 5.6 + 37.1 36.8 65 66 17 16.7 6.3 + 37.8 36.8 58 60 18 11.5 7 + 37.4 36.8 67 69 19 9 5.9 + 36.8 36.8 71 79 20 7.7 5 + 37.8 36.8 73 78 21 12.8 8.1 + 36.8 36.8 50 55 22 6.3 7.1 + 37.4 36.8 59 63 23 7.8 7.2 + 38 36.8 67 71 24 8.6 4 + 37.3 36.8 69 73 25 11.6 4.3 + 38 36.8 62 75 26 18 4 + 38 36.8 66 75 27 11.6 9.2 + 38 37.2 56 58 9.6 3.4 6.5 2 0/27 26/1 4/23 27/0 59.7 8 63.1 8.6 37.6 0.48 36.8 0.08 P = 0.0002 P < 0.0001 P = 2.6E009 P = 5.7E007

(25) TABLE-US-00003 TABLE 3 Baseline and outcome characteristics of TB patients receiving placebo in combination with ATT for one month Tb form Months treated Hemoglobin ESR (* = HIV + with ATT prior Therapy (g/L) (mm/h) No. Sex Age status) to placebo regimen before After before after 1 M 29 Retreated* 8 Individual 102 104 23 20 2 M 35 Retreated* 6 Individual 126 130 13 11 3 M 44 Retreated 2 Standard 108 112 14 10 4 M 35 Retreated 2 Individual 118 120 18 15 5 M 28 MDR-TB 4 Individual 120 132 32 11 3 M 24 Retreated 1 Individual 115 117 22 18 7 M 22 Retreated 1 Standard 121 122 28 24 8 M 44 MDR-TB 3 Individual 128 130 16 9 9 F 78 1.sup.st Dx 3 Standard 116 124 14 9 10 M 28 MDR-TB 3 Individual 128 122 16 11 11 F 41 1.sup.st Dx 3 Standard 113 122 21 10 12 M 41 1.sup.st Dx 3 Standard 120 132 14 8 13 M 47 Retreated 4 Standard 126 132 24 10 14 M 44 Retreated* 4 Standard 118 128 18 9 15 M 33 Retreated* 3 Individual 100 103 22 16 16 F 21 MDR-TB 4 Individual 102 100 14 11 17 M 24 MDR-TB 10 Individual 110 112 15 10 18 M 30 1.sup.st Dx* 1 Standard 112 110 26 22 19 M 33 Retreated* 2 Individual 116 108 15 12 20 M 31 1.sup.st Dx* 3 Standard 122 120 17 15 21 F 50 Retreated 4 Individual 130 118 10 18 22 F 42 Retreated 1 Individual 83 90 38 35 23 M 51 Retreated 12 Individual 65 96 47 21 24 M 52 Retreated 1 Individual 110 112 26 20 25 M 53 Retreated 1 Individual 119 120 35 28 26 M 45 Retreated 1 Individual 117 115 18 9 27 M 36 Retreated 1 Individual 108 110 15 11 28 M 31 Retreated 4 Individual 100 135 21 27 5/23 Mean 38.3 12 Mean 3.4 2.7 112.6 14 117 11.7 21.1 8.6 15.4 7 Median 35.5 Median 3 P = 0.03 P = 7.9E005 Leukocyte Smear Axillary temperature Body weight count 10.sup.9 L positive ( C.) (kg) No. before after before after before after before after 1 17 17 + + 38 37.8 60 60 2 11 8 + 38.3 36.8 68 70 3 9 8 + + 37.8 37.4 61 62 4 12 11 + + 38 37.5 69 70 5 12 9 + 37.6 36.8 68 72 3 8 9 + + 37.8 37.6 70 71 7 14 11 + + 38 37.4 67 67 8 9 8 + + 37.3 36.8 72 73 9 8 8 + 37.4 36.8 52 54 10 13 9 + + 37.9 36.8 60 61 11 12 8 + 36.8 36.8 47 50 12 9 5 + 36.8 36.8 78 81 13 11 6 + 38 36.8 72 71 14 13 8 + 38.3 36.8 61 63 15 11 10 + + 37.1 37.7 57 57 16 9 8 + + 37.1 37.2 47 48 17 11 9 + + 37.8 37.5 55 55 18 7 9 + + 37.4 38 63 63 19 11 10 + + 36.8 37.4 60 55 20 8 8 + + 37.8 37.4 65 65 21 7.8 7.5 + + 37.3 37.5 55 56 22 7.7 8 + + 37.5 36.8 35 36 23 9.4 9.2 + + 38 36.8 58 60 24 5.5 6 + + 38 37.5 55 55 25 11.2 12.3 + + 37.3 37.1 40 41 26 11.5 9 + + 37.5 37 55 55 27 16.4 12 + + 38 37.3 49 50 28 5.1 7.8 + + 37.5 37.1 55 57 10.3 2.9 9 2.3 0/28 7/21 3/25 11/17 59.1 9.9 60.1 10.4 37.6 0.4 37.2 0.4 P = 0.002 P = 0.01 P = 0.0005 P = 0.003

Example 3

(26) In the third study conducted in 34 adult, there were 18 first-diagnosed (52.9%), 6 relapsed TB (17.6%), and 10 MDR-TB (29.4%) cases. The immunotherapy (N=24; Table 4) and placebo (N=10; Table 5) arms received once-daily tablet of vaccine or placebo in addition to conventional anti-TB therapy (ATT) administered under directly observed therapy (DOT). The enlarged liver, total bilirubin, erythrocyte sedimentation rate, lymphocyte and leukocyte counts improved significantly in vaccine recipients (P equal to 0.002; 0.03; 8.3E-007; 2.8E-005; and 0.002 respectively) but remained statistically unchanged in the placebo group (0.68; 0.96; 0.61; 0.91; and 0.43). The changes in hemoglobin and ALT levels in both treatment arms were not significant. The body weight increased in all vaccine-treated patients by an average 3.51.8 kg (P=2.3E-009), while 6 out 10 patients on placebo gained mean 0.90.9 kg (P=0.01). Mycobacterial clearance in sputum smears was observed in 78.3% and 0% of patients on vaccine and placebo (P=0.009). The conversion rate in vaccine-receiving subjects with MDR-TB (87.5%) seemed to be higher than in first diagnosed TB (61.5%) but the difference was not significant (P=0.62). Scoring of sputum bacillary load (range 0-3) at baseline and post-treatment revealed highly significant decrease in vaccine group (from mean/median 2.2/3 to 0.3/0; P=6E-010) but not in placebo (1.9/1.5 vs. 1.8/1; P=0.34). No adverse effects or TB reactivation were seen at any time during follow-up. Thus, the vaccine of the present invention is safe as an immune adjunct to chemotherapeutic management of TB and has a potential to shorten the duration of treatment.

(27) TABLE-US-00004 TABLE 4 Baseline and outcome characteristics of TB patients receiving vaccine in combination with TB drugs for 30 days Months Liver size Erythrocyte No./ on ATT Smear in cm sedimentation Case prior to TB drugs status over normal rate (mm/h) No. Sex Age vaccine Dx regimen before after before after before after 1/80 M 43 0 1.sup.stDx HRZE 3 1 2 0 50 30 2/146 M 39 0 1.sup.stDx HRZE 3 1 0 1 17 16 3/184 M 44 1 1.sup.stDx HRZES 1 0 1 0 21 15 4/34 M 60 3 1.sup.stDx HRZES 3 0 1 0 27 20 5/186 F 27 1 1.sup.stDx HRZES 3 2 1 0 58 40 6/165 M 26 3 1.sup.stDx HRZES 3 1 3 1 15 6 7/179 M 57 1 1.sup.stDx HRZES 2 0 0 0 32 9 8/225 M 31 0 1.sup.stDx HRZES 1 0 0 0 36 26 9/115 M 39 0 1.sup.stDx HRZES 2 1 0 0 50 30 10/805 M 31 3 1.sup.stDx HRZES 3 0 0 0 37 2 11/106 M 59 0 1.sup.stDx HRZES 1 0 1 1 25 24 12/65 M 26 0 1.sup.stDx HRZES 3 0 3 1 19 9 13/12 M 51 1 1.sup.stDx HRZES 3 0 1 2 15 6 14/192 F 46 1 RTB HRZES 3 0 0 0 17 3 15/94 F 54 0 RTB RZEO 3 0 3 1 16 8 16/177 F 35 1 RTB HRZEA 3 0 0 0 32 15 17/5 M 49 1 MDR HRZEO 3 0 1 1 55 40 18/755 M 29 9 MDR HEAOC 3 1 5 3 25 14 19/866 F 53 2 MDR ZEPasAO 0 0 0 0 19 17 20/139 M 29 0 MDR ZEPasAO 2 0 1 0 10 8 21/807 M 29 4 MDR ZEPasAO 2 0 3 0 25 17 22/171 M 49 1 MDR ZEPasO 1 0 2 0 24 17 23/156 M 52 0 MDR ZPasAO 1 0 3 1 24 18 24/469 M 22 1 MDR CsCiGF 1 0 0 0 23 18 5/19 Mean Mean 1st 1/232.2/3 18/60.3/0 1.3 0.5 28 17 40.8 12 1.6 2.5 Dx = 13 1.4/1 0.8/0 13.4 10.4 Median = 41 Median 1 RTB = 3 P = P = P = MDR = 8 0.000027 0.0018 8.3E007 Leukocyte Weight Total No./ Lymphocytes count Hb change bilirubin Case (%) (10.sup.9/L) (g/L) (kg) (mol/L) ALT No. before after before after before after before After before after before after 1/80 18 23 11.7 9.2 129 122 64 65 8.8 8.8 0.4 0.1 2/146 28 25 4.4 5.4 142 139 65 66 9.9 8.8 0.2 0.6 3/184 19 29 6.1 5.6 127 116 74 78 8.8 9.9 0.6 0.5 4/34 29 31 6.1 6 144 122 71 74 8.8 8.8 0.7 0.1 5/186 16 22 11 8 101 110 43 49 8.8 8.8 0.7 0.7 6/165 30 37 11.6 6 158 140 66 69 8.8 12.1 0.9 1.7 7/179 21 34 5.3 5.1 109 123 60 63 12.1 9.9 0.1 0.5 8/225 21 29 7.5 6.6 134 136 62 68 8.8 9.9 0.3 0.4 9/115 14 17 11.7 9.6 154 144 63 67 8.8 8.8 0.2 0.2 10/805 33 46 9.7 4 145 151 64 67 9.9 8.8 0.4 0.4 11/106 27 29 6.6 5.2 138 136 61 66 18.1 12.1 0.3 0.5 12/65 24 21 5.7 6.0 156 153 70 73 8.8 8.8 1.3 0.7 13/12 16 29 14.2 6.9 142 150 81 81 12.1 8.8 0.6 0.9 14/192 17 21 8.3 7.7 103 110 52 58 8.8 8.8 0.3 0.1 15/94 24 34 6.1 5.1 143 128 58 59 8.8 8.8 1.2 0.6 16/177 18 37 6.7 5.5 142 136 62 67 8.8 8.8 0.2 0.2 17/5 18 37 20.4 7.5 138 100 73 75 12.1 9.9 0.8 0.8 18/755 11 26 7.9 7.9 129 138 59 62 8.8 8.8 0.5 0.9 19/866 35 31 4.4 5.8 139 167 55 59 14.3 10.4 0.3 0.4 20/139 33 35 10 6.2 147 148 65 69 8.8 8.8 0.6 0.6 21/807 26 37 10.1 6.8 135 133 57 60 9.9 8.8 1.3 0.6 22/171 12 26 5.3 4.9 159 137 72 74 14.2 9.9 1 0.6 23/156 24 26 5 4.2 124 128 68 73 13.5 8.8 1.4 0.7 24/469 25 27 19.6 7.2 121 131 66 73 8.8 8.8 0.1 0.2 22.5 29.5 9 6.4 135.8 133.3 63.8 67.3 10.4 9.4 0.6 0.54 6.8 6.7 4.3 1.4 15.8 15.5 7.9 7.3 2.5 0.99 0.4 0.35 P = P = 0.0024 P = 0.39 P = P = 0.03 P = 0.47 2.8E005 2.3E009 TB drugs used in this arm are abbreviated as follows: Isoniazid (H), Rifampicin (R), Pyrazinamide (Z), Ethambutol (E), Streptomycin (S), Ofloxacin (O), Amikacin (A), Capreomycin (C), Para-aminosalicylic acid (Pas), Cs (Cycloserine), Cilastatin (Ci), Gatifloxacin (G), Metronidazole (F), Prothionamide (Pt)

(28) TABLE-US-00005 TABLE 5 Baseline and outcome characteristics of TB patients receiving placebo in combination with TB drugs for 30 days Months Liver size Erythrocyte No./ on ATT TB Smear in cm sedimentation Lymphocytes Case prior to drugs status over normal rate (mm/h) (%) No. Sex Age placebo Dx regimen before after before after before After Before after 1/191 M 22 1 1.sup.st Dx HRZSE 1 1 1 0 3 6 22 36 2/215 F 35 0 1.sup.st Dx HRZSE 3 3 1 0 22 41 8 10 3/234 M 40 0 1.sup.st Dx HRZSE 1 1 0 1 35 32 12 11 4/239 F 30 0 1.sup.st Dx HRZSE 1 1 0 1 8 10 21 16 5/227 M 20 0 1.sup.st Dx HRZSE 3 3 0 0 50 35 27 26 6/149 M 53 2 RTB HRZSE 2 1 2 1 23 27 19 23 7/242 M 42 0 RTB HRZSE 3 3 1 1 52 49 19 17 8/236 M 48 0 RTB HRZSE 3 3 0 0 47 38 40 37 9/189 M 22 1 MDR HRZSE 1 1 1 1 31 20 17 13 10/136 M 36 1 MDR ZAPas PtO 1 1 3 3 33 30 27 25 Mean Mean 1.9 1.8 0.9 0.8 30.4 28.8 21.2 21.4 34.8 3.58 0.5 0.7 0.31 0.29 5.30 4.27 8.9 9.7 Median 35.5 Median 0 P = 0.34 P = 0.68 P = 0.61 P = 0.91 Leukocyte Weight Total No./ count Hb change bilirubin ALT Case (10.sup.9/L) (g/L) (kg) (mol/L) (mM/h/ml) No. before after Before after before after before after before After 1/191 6.1 8.7 155 150 74 75 12.1 9.9 0.3 0.3 2/215 8 14.1 75 80 45 45 8.8 8.8 0.4 0.6 3/234 11.8 11.1 158 150 62 62 9.9 9.9 0.4 0.6 4/239 7.2 7.4 126 120 69 69 8.8 14 0.2 0.6 5/227 9.2 10.9 116 110 64 66 8.8 8.8 0.3 0.2 6/149 5.5 4.6 86 89 84 86 12.1 8.8 0.3 0.1 7/242 10 9.8 113 115 60 61 8.8 8.8 0.1 0.4 8/236 11 10.3 139 129 60 61 8.8 17.2 0.1 0.4 9/189 8.8 7.7 136 146 70 72 8.8 8.8 0.4 0.6 10/136 10.1 9.1 107 131 69 69 21 12.1 0.7 1.7 8.77 9.37 121.1 122 65.7 66.6 10.79 10.71 0.32 0.55 0.65 0.80 8.64 7.69 3.25 3.39 1.21 0.90 0.05 0.14 P = 0.43 P = 0.79 P = 0.01 P = 0.96 P = 0.055 TB drugs used in this arm are abbreviated as follows: Isoniazid (H), Rifampicin (R), Pyrazinamide (Z), Ethambutol (E), Streptomycin (S), Amikacin (A), Para-aminosalicylic acid (Pas), Prothionamide (Pt), Ofloxacin (O)

(29) The findings in Tables 4 and 5 support previous clinical trials of vaccine demonstrating favorable outcome in TB patients (Tables 1-3). These studies report a range of beneficial effects including better quality of life, body weight gain, reversal of ATT-associated hepatotoxicity, reduced inflammation, faster deffervescence, and higher clearance rate of M. tuberculosis in sputum smears. Furthermore, the adjunct immunotherapy resulted in much shorter duration of treatment than among those who received standard ATT. The normalization of inflammatory indices is considered to have favorable effect on the course of the TB disease. Contrary to the outcome in placebo group the markers of inflammation such as elevated leukocyte counts and prolonged erythrocyte sedimentation rate have been significantly reduced in vaccine recipients. A favorable change in the blood picture is supported by the increase in total lymphocyte percentage among vaccine recipients, but not in the control group. The restoration of suppressed lymphocyte counts and decrease in leukocyte counts is associated with positive treatment outcome. Thus, changes in relative and absolute lymphocyte and leukocyte numbers hold promise as surrogate markers of treatment response.

(30) The hepatotoxicity induced by anti-TB drugs has serious adverse consequences to treated patients and imposes limitations on treatment options. Addition of instant composition appeared to reduce baseline bilirubin and ALT levels, as well as the abnormal liver size when compared to placebo regimen. For this reason the use of vaccine in combination with ATT is advisable to prevent or reverse iatrogenic liver damage.

(31) The immunotherapy has shown clear benefit in reversing body weight loss. The average gain in vaccine and placebo groups was 3.5 kg and 0.9 kg which is almost identical to the results of placebo controlled trial involving a comparable group of 55 TB patients, i.e., 3.4 kg (59.78 vs 63.19 kg; P=5.7E-007) and 1.07 kg (59.110 vs 60.110.4 kg; P=0.003) respectively. In contrast, the mean weight gain observed in the earlier conducted, open-label trial involving 20 patients with HIV-TB was 7.7 kg (P=4.6E-007). This almost two-fold discrepancy is perhaps due to the fact that all these patients had HIV infectiona condition that is associated with worsened wasting.

(32) Conversion of sputum smear from positive to negative is a main indicator of the efficacy of anti-TB intervention. The vaccine accelerates and significantly enhances bacillary clearance as compared to control group on AU. The difference in outcome between placebo and vaccine recipients was also significant. In the prior placebo-controlled study the conversion in placebo arm was seen in 25% of patients while in this trial 0% converted.

(33) The instant vaccine is shown to be safe and capable of reversing ATT-associated hepatotoxicity. In addition, vaccine seems to reduce the inflammation as evidenced by several hematological and biochemical markers. Weight gain and sputum smear conversion rate have been significantly enhanced as compared to conventional ATT. As evidenced by obtained improvements the combination of vaccine and ATT can shorten the duration of treatment.

Example 4

(34) Prophylactic use. Pooled blood from latent or active M. tuberculosis carrying donors are hydrolyzed in their entirety, heat-inactivated, embedded into metal salt matrix and made into solid form tablets, which is then fed to Swiss mice at a dose equivalent to dose proven effective in therapeutic use. After one month the Swiss mice are challenged with wild-type H37Rv mycobacterium strain by aerosol inhalation. The analysis reveals that 12 out of 13 of vaccinated mice failed to develop the TB infection by not showing any detectable bacilli. In contrast, all unvaccinated mice in control group had shown acute TB infection. The difference in outcome between control and vaccinated groups is highly significant as tested by Fisher's 22 exact test. Thus, vaccination of mice with instant vaccine elicits the protective immune response resulting in prevention of TB infection. The same results are obtained when recombinant yeast expressing mycobacteria antigens and alloantigens is fed to mice instead of mycobacterium from natural source.

(35) All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

(36) As will be apparent to those skilled in the art to which the invention pertains, the present invention may be embodied in forms other than those specifically disclosed above, without departing from the spirit or essential characteristics of the invention. The particular embodiments of the invention described above, are, therefore to be considered as illustrative and not restrictive. The scope of the present invention is as set forth in the appended claims rather than being limited to the examples contained in the foregoing description.