MXPA97001103A - Abundant extracelular products and methods for their production and - Google Patents

Abstract

Vaccines are presented based on combinations of extra-abundant extracellular products of pathogens and methods for their use and production. The most prevalent or largely abundant extracellular products of a target pathogen are selected regardless of their absolute molecular immunogenicity, and are used as vaccines to stimulate a protective immune response in mammalian hosts against subsequent infection by the target pathogen. The mostly abundant extracellular products can be characterized and distinguished by their respective N-terminal amino acid sequences. Since the vaccines may comprise different combinations of extracellular products, the present invention provides effective immunotherapeutic compositions of wide range. In addition to other infectious agents, the vaccines thus produced can be used to stimulate an effective immune response against intracellular pathogens, and in particular Mycobacterium tuberculosis.

Description

ABUNDANT EXTRACELÜLAR PRODUCTS AND METHODS FOR THEIR PRODUCTION AND USE Reference to a Related Request The present application is a continuation in part of the United States patent application of North America pending with serial number 156,358, filed on November 23, 1993 and incorporated herein by reference.

Reference to the Government This invention was made with the support of the Government under the Concession Number Al-31338 awarded by the Department of Health and Human Services. The Government has certain rights over this invention.

Field of the Invention The present invention relates in general to immunotherapeutic agents and vaccines against pathogenic organisms such as bacteria, protozoa, viruses and fungi. More specifically, unlike vaccines and immunotherapeutic agents of the prior art, based on pathogenic subunits or products exhibiting the greatest or most specific immunogenicity, the present invention uses the most prevalent or largely abundant determinants released by a selected pathogen such as Mycobacterium tuberculosis, to stimulate an effective immune response in mammalian hosts. Accordingly, the acquired immunity and immunotherapeutic activity produced by the present invention is directed to those antigenic markers that are most frequently displayed in infected host cells during the course of a pathogenic infection without particular consideration for the relative or absolute immunogenicity of the compound administered.

BACKGROUND OF THE INVENTION It has long been recognized that parasitic microorganisms possess the ability to infect animals by causing the same disease, and often the death of the host. Pathogenic agents have been a leading cause of death throughout history, and continue to inflict much suffering. Although the last hundred years have seen dramatic advances in the prevention and treatment of many infectious diseases, complicated host-parasite interactions still limit the universal effectiveness of therapeutic measures. The difficulties in opposing the sophisticated invasive mechanisms deployed by many pathogenic vectors are made evident by the resurgence of different diseases such as tuberculosis, as well as the appearance of numerous strains of bacteria and drug-resistant viruses. Among those agents of greatest epidemiological concern, intracellular bacteria have proved particularly intractable when faced with therapeutic or prophylactic measures. Intracellular bacteria, including the genus Mycobacterium and the genus Legionella, complete all or part of their life cycle within the 0 cells of the infected host organism, rather than extracellularly. Around the world, intracellular bacteria are responsible for millions of deaths each year and much suffering. Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of death 5 due to infectious disease around the world, with 10 million new cases and 2.9 million deaths each year. In addition, intracellular bacteria are responsible for millions of cases of leprosy, other debilitating diseases transmitted by intracellular agents include cutaneous and visceral leishmaniasis, American trypanosomiasis.

(Chagas disease), listeriosis, toxoplasmosis, histoplasmosis, trachoma, psittacosis, Q fever, and Legionellosis, including Legionnaires' disease. At this time, relatively little can be done to avoid 5 debilitating infections in susceptible individuals exposed to these organisms. Due to its inability to protect populations effectively from tuberculosis, and the inherent human morbidity and mortality caused by tuberculosis, this is one of the most important diseases that humanity faces. More specifically, human pulmonary tuberculosis caused mainly by M. tuberculosis is a leading cause of death in developing countries. Able to survive within macrophages and monocytes, M. tuberculosis can produce a chronic intracellular infection. By hiding itself within the cells primarily responsible for the detection of foreign elements and the subsequent activation of the immune system, M. tuberculosis is relatively successful in evading the normal defenses of the host organism. These same pathogenic characteristics have so far prevented the development of an effective immunotherapeutic agent or vaccine against tuberculous infections. At the same time, tubercle bacilli are relatively easy to grow and observe under laboratory conditions. Accordingly, M. tuberculosis is particularly suitable for demonstrating the principles and advantages of the present invention. Those skilled in the art will note that the following exemplary description of M. tuberculosis "" - is in no way intended to limit the scope of the present invention to the treatment of M. tuberculosis. Similarly, the teachings that are used herein are in no way limited to the treatment of tuberculous infections. On the contrary, this invention can be used to conveniently provide vaccines and safe and effective immunotherapeutic agents against the immunogenic determinants of any pathogenic agent, which express extracellular products and thereby inhibit the infectious transmission of those organisms. Currently it is believed that approximately half of the world's population is infected with M. tuberculosis, resulting in millions of cases of pulmonary tuberculosis annually. Although this disease is a particularly acute health problem in the developing countries of Latin America, Africa and Asia, it is also becoming more prevalent in the first world. In the United States of America, specific populations are at increased risk, especially urban poor, immunocompromised individuals and immigrants from areas with a high frequency of the disease. Mostly due to the AIDS epidemic, the incidence of tuberculosis in developed countries is currently increasing, often in the form of multidrug-resistant M. tuberculosis. Recently, tuberculosis resistance was reported to one or more drugs in 36 of the 50 states of the United States of America. In New York City, one third of all cases tested in 1991 was resistant to one or more major drugs. Although non-resistant tuberculosis can be cured with a long course of antibiotics, the outlook with respect to the drug-resistant strains is bleak. Patients infected with strains resistant to two or more major antibiotics have a fatality rate of about 50 percent. Accordingly, a safe and effective vaccine against such varieties of M. tuberculosis is urgently needed. Initial infections of M. tuberculosis almost always occur through the inhalation of particles dispersed in the air, since the pathogen can remain viable for weeks or months in wet or dry sputum. Although the primary site of infection is in the lungs, the organism can also cause infection of the bones, spleen, meninges and skin. Depending on the virulence of the particular strain and the resistance of the host, the The infection and the corresponding damage to the tissue may be minor or extensive. In the case of humans, the initial infection is controlled in most individuals exposed to virulent strains of the bacteria. The development of acquired immunity after the initial aggression reduces the proliferation of the bacteria, allowing by the same "" "'" to heal the lesions, and leaving the subject largely asymptomatic, but possibly contagious.When the infected individual does not control M. tuberculosis, this frequently results in extensive degradation In susceptible individuals, lesions are usually formed in the lung because the tubercle bacilli breed inside the alveolar or pulmonary macrophages.As the organisms multiply, they can spread through the system. lymphatic nodes distant from the lymph, and through the bloodstream to the apices of the lung, bone marrow, kidney and meninges that surround the brain.First, as the result of cell-mediated hypersensitivity responses, they occur characteristic gramilornatosas injuries or tubers, in proportion to the severity of the infection. They consist of epithelioid cells surrounded by monocytes, lymphocytes and fibroblasts. In most cases, a lesion or tubercle eventually becomes necrotic and undergoes caseification. Although M. tuberculosis is a significant pathogen, other species of the Mycobacterium genus also cause disease in animals, including man, and are clearly within the scope of the present invention. For example, M. bovis is closely related to M. tuberculosis, and is responsible for tuberculous infections in domestic animals such as cattle, pigs, sheep, horses, dogs and cats. In addition, M. bovis can infect humans through the intestinal tract, typically by ingesting raw milk. The localized intestinal infection eventually spreads to the respiratory tract and is soon followed by the classic symptoms of tuberculosis. Another important pathogenic vector of the genus Mycobacterium is M. leprae, which causes millions of cases of the old leprosy disease. Other species of this genus that cause disease in animals and humans include M. kansasii, M avium intracellulare, M. fortui tum, M. marinum, M. chelonei, M. africanum, M. ulcerans, M. microti and M. scrofulaceum. . Pathogenic mycobacterial species frequently exhibit a high degree of homology in their respective DNAs and corresponding protein sequences, and some species, such as M. tuberculosis and M. bovis are highly related. For obvious practical and moral reasons, initial work in humans is unfeasible to determine the effectiveness of experimental compositions, with respect to such afflictions. Accordingly, in the early development of any drug or vaccine, the standard procedure is to use appropriate animal models for reasons of safety and expense. The success of the implementation of model "" "- laboratory animals is predicated on the understanding that immunodominant epitopes are frequently active in different host species, thus, an immunogenic determinant in a species, for example, a rodent or guinea pig of Indian, will generally be immunoreactive in a different species such as in humans.Only after the appropriate animal models are sufficiently developed, clinical trials in humans will be conducted, to further demonstrate the safety and efficacy of a vaccine in man. With respect to alveolar or pulmonary infections due to M. tuberculosis, the guinea pig model closely resembles the human pathology of the disease in many aspects. Accordingly, those skilled in the art understand very well that it is appropriate to extrapolate the guinea pig model of this disease to humans and other mammals. As with humans, guinea pigs are susceptible to tuberculous infection with low doses of human pathogenic M. tuberculosis dispersed in the air. Unlike humans, where the initial infection is usually controlled, guinea pigs consistently develop disseminated disease on exposure to the pathogen dispersed in the air, facilitating subsequent analysis. In addition, both guinea pigs and humans display cutaneous hypersensitivity reactions of the delayed type, characterized by the development of a hardening or rigid area of the dense mononuclear cell at the skin test site. Finally, the characteristic tuberculous lesions of humans and guinea pigs exhibit a similar morphology, including the presence of Langhans giant cells. Since guinea pigs are more susceptible to initial infection and disease progression than humans, any protection conferred in experiments, using this animal model, provides a strong indication that the same protective immunity can be generated in humans. or other less susceptible mammals. Accordingly, for purposes of explanation only and not for purposes of limitation, the present invention will first be demonstrated in the exemplary context of the guinea pigs as the mammalian host. Those skilled in the art will appreciate that the present invention can be practiced with other mammalian hosts, including humans and domesticated animals. Any animal or human infected with a pathogenic vector and, in particular, an intracellular organism, presents a difficult challenge for the host immune system. Although many infectious agents can be effectively controlled by the humoral response and corresponding production of protective antibodies, these mechanisms are mainly effective against those pathogens located in the extracellular fluid of the body. In particular, the opsonification antibodies are fixed to foreign extracellular agents making them, by means of the same, susceptible to phagocytosis and the subsequent intracellular elimination. Still, this is not the case for other pathogens. For example, previous studies have indicated that the humoral immune response does not seem to play a significant protective role against infections by intracellular bacteria, such as M. tuberculosis. However, the present invention can generate a beneficial humoral response to the target pathogen and, as such, its effectiveness is not limited to any specific component of the invention. * - like the Ai. tuberculosis must incorporate a cell-mediated aggressive immune response component, which leads to the rapid proliferation of antigen-specific lymphocytes, which activate the committed phagocytes or eliminate them cytotoxicly. However, as will be discussed in detail below, inducing a cell-mediated immune response does not equal the induction of protective immunity. Although cell-mediated immunity may be a prerequisite for protective immunity, the production of vaccines in accordance with the teachings of the present invention requires animal-based aggression studies. This cell-mediated immune response generally involves two steps. The initial step, noting that the cell is infected, is carried out by special molecules (major histocompatibility molecules or MHC) that send pieces of the pathogen to the surface of the cell. / 'cell. These major histocompatibility molecules bind to small fragments of bacterial proteins that have been degraded inside the infected cell, and present them on the surface of the cell. Its presentation to the T cells stimulates the host's immune system to eliminate the infected host cell, or induces the host cell to eradicate any bacteria that reside inside. 5 Unlike most infectious bacteria, Mycobacterium, including M. tuberculosis, tend to proliferate in vacuoles, which are substantially sealed from the rest of the cell, by a membrane. Phagocytes naturally form these protective vacuoles, making them particularly susceptible to infection by this class of pathogens. In such vacuoles, bacteria are effectively protected from degradation, making it difficult for the immune system to present integral bacterial components on the surface of infected cells. However, the major histocompatibility molecules of the infected cell will move to the vacuole and collect any free (released) bacterial products, or move to other sites in the host cell to which extracellular extracellular bacterial products have been transported, for the normal presentation of the products on the surface of the cell. As indicated above, the presentation of foreign extracellular bacterial products will elicit the appropriate response by the host immune system. The problems that intracellular pathogens present to the immune system also constitute a special challenge for the development of a vaccine. So far, the production of an effective vaccine against infections by? FycojbacCerium and, in particular, against Af. tuberculosis has been lost to most researchers.

Currently, the only vaccine available widely against intracellular pathogens is the live attenuated BCG vaccine, an avirulent strain of Af. bovis, which is used as a prophylactic measure against tuberculosis bacilli. As late as 1988, extensive studies by the World Health Organization of India determined that the effectiveness of the best BCG vaccines was so slight as to be immeasurable. Despite this questionable efficacy, BCG vaccine has been used extensively in areas of high incidence of tuberculosis throughout the world. Complicating the matter, even individuals who have been vaccinated with BCG will often develop sensitivity to tuberculin, which negates the usefulness of the most common skin test for tuberculosis classification and control. Another serious problem involving the use of a live attenuated vaccine, such as BCG, is the possibility of initiating a life-threatening disease in immunocompromised patients. These vaccines present a particular risk for people with reduced cell-mediated immunity, due to their diminished ability to fight an induced infection that proliferates rapidly. Such individuals include those weakened by malnutrition and lower living conditions, organ transplant recipients, and people infected with HIV. In the case of the BCG vaccine, high risk individuals also include those suffering from lung disorders such as emphysema, chronic bronchitis, pneumoconiosis, silicosis or previous tuberculosis. Accordingly, the use of attenuated vaccines is limited in the same population where they have the greatest potential benefit. The use of live attenuated vaccines can also produce other undesirable side effects. Because live vaccines are reproduced in the recipient, they elicit a broad range of antibodies and a less directed cell-mediated immune response than non-infectious vaccines. Frequently this forced access tends to occlude the targeted immune response in the molecular structures most involved in cell prophylaxis. On the other hand, the use of live vaccines with an intact membrane can induce opsonification antibodies, which prepare a foreign body for effective phagocytosis. Thus, upon exposure of the host to virulent strains of the target organism, the presence of such antibodies could, in fact, increase the uptake of non-attenuated pathogens within the host cells, where they can survive and multiply. In addition, an attenuated vaccine contains thousands of different molecular species and, consequently, is more likely to contain a molecular species that is toxic or capable of provoking an adverse immune response in the patient. Other problems with live vaccines include reversion of virulence, natural spread to contacts, contaminating viruses and viral interference, and difficulty with standardization. Similarly, non-infectious vaccines, such as vaccines from dead organisms or conventional second generation subunits, directed to strongly antigenic membrane binding structures, are limited with respect to the inhibition of intracellular bacteria. Like attenuated vaccines, killed bacteria provoke an indiscriminate response that could inhibit the most effective prophylactic determinants. In addition, dead vaccines still present in large numbers of structures potentially antigenic to the immune system, thereby increasing the likelihood of toxic reactions or opsonification by the immune system. Traditional subunit vaccines that incorporate membrane fixation structures, whether synthesized or purified, can also induce a strong opsonic effect, facilitating the entry of the intracellular pathogen into the phagocytes in which they multiply. By increasing the bacterial inclusion index, killed vaccines targeting intracellular surface antigens could increase the relative virulence of the pathogenic agent. In this way, conventional attenuated or killed vaccines directed against the strongly antigenic bacterial surface components may be contraindicated in the case of intact pathogens. In order to avoid the problems associated with the use of traditional vaccines, developments have been made using extracellular proteins or their immunogenic analogues, to stimulate protective immunity against specific intracellular pathogens. For example, the inventor of U.S. Patent No. 5,108,745, issued April 28, 1992, describes vaccines and methods for producing protective immunity against Legionella pneumophila and Af. tuberculosis, as well as other intracellular pathogens. These prior art vaccines are mostly based on extracellular products originally derived from proteinaceous compounds released extracellularly by the pathogenic bacteria, within the culture of in vitro broth, and released extracellularly by the bacteria into infected host cells in vivo. As described in it, these vaccines are selectively based on the identification of extracellular products or their analogs, which stimulates a strong immune response against the target pathogen in a mammalian host. More specifically, these candidate extracellular proteins of the prior art were classified by determining their ability to elicit either a strong lymphocyte proliferative response or a delayed-type cutaneous hypersensitivity response in mammals that were immune to the mammalian pathogen. interest Although this method and the associated vaccines described avoid many of the inherent deficiencies in the use of traditional vaccines, conflicting immunoresponsive results due to cross-reactivity and host variation may complicate the selection of effective immunization agents. In this way, while immunogenicity ? ,; molecular is an indication of an effective vaccine, other factors can complicate its use by producing an effective immune response in vivo. More importantly, it was surprisingly discovered that, particularly with respect to Af. tuberculosis, Conventional methods of the prior art for identifying vaccines to induce protective immunity were difficult to manage and potentially ineffective. For example, the analysis of sodium dodecyl sulfate-polyacrylic amide gel electrophoresis of the extracellular protein of Af. tuberculosis crude, followed by conventional Western blot techniques, which aimed at identifying the most immunogenic of these extracellular components, produced inconsistent results. Repeated tests failed to identify which extracellular product would produce the strongest immunogenic response and, consistent with the thinking of "the prior art, by the same to its function as the most effective vaccine." Many of the extracellular products are well known in the art. of Af. tuberculosis, which have been identified and, in some cases, sequenced.Also, as with any foreign protein, it can be shown that these known compounds induce an immune response.However, nothing in the technique directly indicates that any of these known compounds will induce protective immunity as is traditionally identified. Accordingly, it is a principal objective of the present invention to provide vaccines or immunotherapeutic agents, and methods for their production and use by mounting an effective immune response against infectious bacterial pathogens, which do not depend on the traditional considerations of the vaccine, and selection techniques based on a highly specific operability, strongly immunogenic. It is another object of the present invention to provide vaccines or immunotherapeutic agents and methods for use, to impart acquired immunity in a mammalian host against intracellular pathogens, including Af. tuberculosis, Af. bovis, Af. kansasii, M avium intracellulare,? f. fortui tum, M. chelonei, Af. marinum, M. scrofulaceu, M. leprae, Af. africanum,? f. ulcerans and Af. microti. It is a further object of the present invention to provide vaccines and readily produced immunotherapeutic agents, exhibiting reduced toxicity relative to killed or attenuated vaccines.

SUMMARY OF THE INVENTION The present invention accomplishes the above-mentioned and other objectives by providing compounds for use as vaccines and / or immunotherapeutic agents, and methods for their production, for generating protective or therapeutic immune responses in mammalian hosts against infection by pathogens. In a broad aspect, the invention provides the elements for inducing a protective or therapeutic immune response against infectious vectors, producing extracellular compounds. Although the compounds of the present invention are particularly effective against pathogenic bacteria, they can also be used to generate a protective or therapeutic immune response against any pathogen by producing mostly extracellular, abundant products. For the purposes of the present invention, the term "mostly abundant" should be understood as a relative term that identifies those extracellular products released in the largest amount by the pathogen of interest. For example, with respect to Af. Tuberculosis growing under different culture conditions at an optical density of about 0.5, one skilled in the art should expect to obtain a largely abundant cell product in the order of 10 μg / L. Thus, out of the exemplary total of 4 mg / L, the total yield of extracellular product for M. tuberculosis that grows under normal conditions or heat shock, approximately fifteen to twenty (alone or in combination) of the hundred more or less, of the extracellular products will constitute approximately ninety percent of the total amount. These are the mostly abundant extracellular products contemplated within the scope of the present invention, and are readily identifiable as the broad bands appearing on sodium dodecyl sulfate gels-polyacrylic amide gel electrophoresis. In addition, the extracellular products of interest can also be characterized and differentiated by amino acid sequencing. The remaining extracellular products are smaller. Those skilled in the art will also note that the relative quantitative abundance of specific higher extracellular products may vary, depending on culture conditions. However, in most cases, the identification of an extra-cellular product that is mostly abundant will not change. Accordingly, the present invention can be used to protect a mammalian host against infection by viral, bacterial, fungal or protozoan pathogens. It should be noted that in some cases, such as in viral infections, the infected host cell can generate extracellular products that are mostly abundant. Although they are active against all microorganisms that release largely abundant extracellular products, the vaccines and methods of the present invention are particularly effective for the generation of protective immunity against intracellular pathogens, including : Different species and serogroups of the genus Mycobacterium. The vaccines of the present invention are also effective as immunotherapeutic agents for the treatment of existing disease conditions. Surprisingly, this inventor has found that Immunization with the more or more abundant products released extracellularly by bacterial pathogens or their immunogenic analogues can elicit an effective immune response regardless of the absolute immunogenicity of the compound administered. Due to its release from the body and, consequently, to their availability for host molecules involved in the processing and presentation of the antigen, and due to their naturally high concentration in the tissue during infection, the extra-abundant extracellular products of a pathogenic agent are processed and presented at host immune system more frequently than other bacterial components. In the case of intracellular pathogens, the mostly abundant extracellular products are the principal immunogenic determinants presented on the surface of the infected host cells and, therefore, exhibit a greater presence in the surrounding environment. Accordingly, the acquired immunity against the largely abundant extracellular products of a pathogenic organism allows the host defense system to rapidly detect pathogens sequestered inside host cells and effectively inhibit them. More particularly, it seems that the main or mostly abundant products released by the pathogenic bacteria are processed by phagocytes and other mechanisms the host immune system at a higher rate than the pathogenic components less prevalent or fixed to the membrane, regardless of their activity or immunogenic specificity respective. This disparity in the immunoprocess is particularly significant when the pathogenic agent is a sequestered intracellular bacterium of normal immune activity. By virtue of their profuse and continuous presentation to the host immune system, the most prevalent bacterial extracellular products or their immunogenic analogs elicit a vigorous immune response regardless of their individual molecular immunogenic characteristics.

The mostly abundant extracellular products are the main constituents of proteins and other molecular entities that are released by an objective pathogen within the surrounding environment. Current research indicates that in some cases a single, mostly abundant, extracellular product can comprise up to 40 percent by weight of the products released by a microorganism. More frequently, extracellular, mostly abundant, individual products account for between about 0.5 percent to about 25 percent of the total products released by the infectious pathogen. On the other hand, it can be found that the five or six major mostly extracellular products comprise between 60 percent and 70 percent of the total mass released by a microorganism. Of course, those skilled in the art will note that the relative levels of extracellular products can fluctuate over time, as can the absolute or relative amount of products released. For example, pH, oxidants, osmolality, heat and other stress conditions in the organism, stage or life cycle, the state of reproduction and the composition of the surrounding environment, may alter the composition and quantity of products released. In addition, the absolute and relative levels of extracellular products can differ greatly from species to species, and even between / * * strains within a species. In the case of extracellular products of intracellular pathogens, they seem to expand the population of specifically immune lymphocytes, capable of detecting and exerting an antimicrobial effect against macrophages containing live bacteria. Furthermore, by virtue of their repeated display on the surface of infected cells, extracellular, mostly abundant or major products function as effective antigenic markers. In accordance, according to the teachings of The present invention, the vaccination and induction of protective immunity directed at the largely abundant extracellular products of a pathogenic bacterium or its immunogenically equivalent determinants, incites the host immune system to mount a rapid immune response and efficient with a strong cell-mediated component when subsequently infected with the target pathogen. In direct contrast to the immunization activities of the prior art, which had focused mainly on vaccine production and stimulation In the case of immune responses based on the highly specific molecular antigenicity of individual classified pathogenic components, the present invention conveniently exploits the relative abundance of bacterial extracellular products or their immunogenic analogues (rather than their specificities).

Immunogenic) to establish or induce protective immunity with compounds that may in fact exhibit less immunogenic specificity than less prevalent extracellular products. For the purposes of this description, an immunogenic analogue is a molecule or compound sufficiently analogous to at least one extracellular, largely abundant product, expressed by the target pathogen, or any fraction thereof, to have the ability to stimulate a protective immune response in a mammalian host, on subsequent infection by the target pathogen. In short, the vaccines of the present invention will be identified or produced by selecting the product or products largely abundant released extracellularly by a specific pathogen (or molecular analogues capable of stimulating a substantially equivalent immune response), and isolating them in a relatively pure form . Then, the desired prophylactic immune response can be produced to the target pathogen, by formulating one or more immunoreactive isolated products, using techniques well known in the art, and immunizing a mammalian host prior to infection by the target pathogen. It is anticipated that the present invention will consist of at least one, two, or possibly even many well-defined immunogenic determinants. As a result, the present invention produces standardized, consistent vaccines that can be developed, tested and administered relatively easily and quickly. In addition, the use of a few well-defined molecules that correspond to the largely abundant secretory or extracellular products, greatly reduces the risk of adverse side effects associated with conventional vaccines, and eliminates the possible occlusion of effective immunogenic markers. Similarly, because the present invention is not an attenuated or killed vaccine, the risk of infection during production, purification or administration is effectively eliminated. As such, the vaccines of the present invention can be administered safely to immunocompromised individuals, including patients with asymptomatic tuberculosis, and those infected with HIV. On the other hand, since the humoral immune response is directed exclusively to products released by the target pathogen, there is little likelihood of generating a harmful opsonic immune component. Accordingly, the present invention allows the stimulated humoral response to aid in the elimination of the target pathogen from the susceptible areas of the antibody. Another beneficial aspect of the present invention is the ease with which the vaccines are subsequently harvested or produced and purified. For example, predominantly abundant extracellular products can be obtained from cultures of the target pathogen, including Af. tuberculosis or the Af. Bovis, with little effort. As the desired compounds are released into the medium during growth, they can be easily separated from the intrabacterial components and bound to the membrane of the target pathogen, using conventional techniques. More preferably, the desired immunoreactive constituents of the vaccines of the present invention can be produced and purified from genetically engineered organisms, JLJ within which the genes that express the specific extracellular products of Af have been cloned. tuberculosis, Af. Bovis, the Af. leprae or any other pathogen of interest. As is known in the art, such designed organisms can be modified to produce higher levels of the selected extracellular products or modified immunogenic analogues. Alternatively, immunoprotective products, portions thereof or analogs thereof can be chemically synthesized using techniques well known in the art. Whatever the If the source of production is used, the immunogenic components of the predominant or largely abundant extracellular products can be separated and subsequently formulated into vaccines that can be sent using common biochemical procedures such as fractionation, chromatography or other purification methodology and conventional formulation techniques. For example, in an exemplary embodiment of the present invention, the target pathogen is Af. tuberculosis and the largely abundant products released extracellularly by Af. tuberculosis within the broth culture are separated from other bacterial components, and are used to produce an immune response in mammalian hosts. The individual proteins or protein groups are then used in animal-based aggression experiments to identify those that induce protective immunity, making them suitable for use as vaccines, in accordance with the teachings of the present invention. More specifically, after the growth and harvest of the bacteria, by virtue of their physical abundance, the main ethalcellular products are separated from the intrabacterial and other components, through centrifugation and filtration. If desired, the resulting crude filtrate is then subjected to fractionation, using precipitation of ammonium sulfate with subsequent dialysis, to give a mixture of extracellular products, commonly called EP. The extracellular products solubilized in the dialyzed fractions are then purified to substantial homogeneity, using suitable chromatographic techniques, as is known in the art, and as more fully described below.

These exemplary procedures result in the production of fourteen individual, larger proteinaceous extracellular products of Af. tuberculosis that have molecular weights that vary from 110 kilo Daltons (KD) to 12 kilo Daltons. After purification, each individually abundant extracellular product exhibits a band corresponding to its respective molecular weight, when subjected to polyacrylic amide gel electrophoresis, - < • allowing through the same thing that products are identified '10 individual or groups of products corresponding to the most abundant extracellular products, and which are prepared for use as vaccines, in accordance with the teachings of the present invention. The mostly abundant purified extracellular products are also can characterize and distinguish by determining all or parts of their respective amino acid sequences, using techniques common in the art. Sequencing may also provide information regarding possible structural relationships between extracellular products plentiful. Subsequently, immunization and stimulation of acquired immunity in a mammalian host system can be carried out through the teachings of the present invention, using a series of subcutaneous injections or intradermal of these purified extracellular products, "• -" * over a period of time. For example, an injection with a purified mostly abundant bacterial extracellular product or products may be used, in incomplete Freund's adjuvant, followed by a second injection in the same adjuvant, approximately three weeks later, to produce a protective response on a subsequent aggression with the virulent pathogen. Other exemplary immunization protocols within the scope and teachings of the present invention may include a series of three or four injections of purified extracellular product or products or their analogues, in Syntex Adjuvant Formulation (SAF) for a period of time. Although a series of injections can generally be proved more effective, the single administration of a most abundantly selected extracellular product, or its subunits or the like, can impart the desired immune response, and is also contemplated as being within the scope of the present invention. Such exemplary protocols can be demonstrated using laboratory models accepted in the art, such as guinea pigs. For example, as will be discussed in detail, immunization of many guinea pigs with a combination of five mostly abundant extracellular products (purified from Af. Tuberculosis as described above) was performed with a series of three immunization injections of the bacterial products, in Syntex Adjuvant Formulation, with the corresponding false immunization of control animals. The exemplary doses of each protein ranged from 100 μg to 2 μg. After the last vaccination, all animals were exposed to an infectious and potentially lethal dose of Af. tuberculosis dispersed by aerosol, and were monitored for an extended period of time. The control animals showed a significant loss in weight compared to the animals immunized with the combination of the mostly abundant extracellular products of the Af. tuberculosis On the other hand, half of the control animals died during the observation period, while none of the immunized animals succumbed to tuberculosis. The autopsies conducted after this experiment revealed that the non-immunized control animals had significantly more colony forming units (CFU) and corresponding damage in their lungs and spleens than the protected animals. Ten and seven additional combinations of mostly abundant purified extracellular products, provided immunoprophylaxis when tested, demonstrating by the same the scope of the present invention and the wide range of vaccines that can be formulated in accordance with the teachings thereof. However, it should be emphasized that the present invention is not restricted to combinations of secretory or extracellular products. For example, many alternative experimental protocols demonstrate the ability of a single abundant extracellular product to induce mammalian protective immunity, in accordance with the teachings of the present invention. In each experiment guinea pigs were immunized with a single, mostly abundant, extracellular product, purified from extracellular Af products. tuberculosis, using the chromatography protocols detailed herein. In one example these animals were vaccinated in multiple experiments with an adjuvant composition containing a purified abundant secretory product, having a molecular weight corresponding to 30 kilo Daltons. In another example of the present invention, different guinea pigs were vaccinated with an adjuvant composition containing a plentiful extracellular product isolated from Af. tuberculosis, having a molecular weight corresponding to 71 kilo Daltons. After their respective immunizations, both groups of animals and their appropriate controls were exposed to lethal doses of Af. tuberculosis dispersed by aerosol, to determine the effectiveness of the vaccine. More particularly, in one experiment six guinea pigs were immunized with 100 μg of 30 kilo Dalton protein in Syntex Adjuvant Formulation, three times over a period of six weeks. "Control animals" were simultaneously "vaccinated" with corresponding amounts of a crude extracellular protein (EP) or pH buffer preparation. Three weeks after the final vaccination, these animals were attacked with a lethal dose dispersed by aerosol of Af. tuberculosis and were monitored over a period of 14 weeks. Guinea pigs immunized with 30 kilo Daltons and those immunized with crude extracellular preparation had survival rates of 67 percent and 50 percent, ~ or respectively (illustrating the unexpectedly superior performance of the extracellular product mostly abundant against extracellular proteins), while the falsely immunized animals had a survival rate of only 17 percent. After the completion of the experiments were sacrificed and the animals examined to look for viable tuberculosis bacilli. As expected, the non-immunized animal showed markedly higher concentrations of Af. tuberculosis in the lungs and spleen. Similar experiments were carried out in those animals vaccinated with 71 kilo Dalton protein. In one experiment six guinea pigs were vaccinated with a Syntex Adjuvant Formulation composition containing 100 μg of purified 71 kilo Dalton protein, twice, over a period of three weeks. They were immunized in a Similar to other animals with a crude preparation of '"' - unpurified extracellular proteins or extracellular products, to be used as a positive control, and with a pH regulator to be used as a negative control, after exposure to lethal doses of bacilli. Tuberculosis dispersed by aerosol, the guinea pig weight was monitored for a period of 6 months Once again animals immunized with the purified form of abundant extracellular product developed protective immunity with respect to virulent tuberculosis. At the end of that period, the animals immunized with the pH regulator showed a significant loss in weight when compared with the immunized animals, while the positive controls and the animals immunized with 71 kilo Daltons had survival rates of 63%. percent and 50 percent, respectively, the non-immunized animals died before the end of the observation period. It is important to note that the formulation of the vaccine is not critical to the present invention, and it can be optimized to facilitate its administration. The solutions of the purified immunogenic determinants, derived from the largely abundant pathogenic extracellular products, can be administered alone or in combination designed in any way to generate a protective immune response. The purified protein solutions can be sent alone, * "" "- or formulated with an adjuvant before being administered The specific exemplary adjuvants that are used in the present invention to increase the activity of the selected immunogenic determinants are the Adjuvant Formulation Syntex, Adjuvants containing Monophosphoryl Lipid A, Incomplete Freund's Adjuvant and Freund's Complete Adjuvant Containing Dead Bacteria Additional adjuvants that may be useful in the present invention are water-in-oil emulsions, mineral salts (e.g. ), u * and nucleic acids, block polymer surfactants, and microbial cell walls (peptide glycolipids). While not limiting the scope of the invention, it is believed that adjuvants can increase immune responses due to slow release of injection site antigens 5 Other objects will be obvious to those skilled in the art. The features, features, and advantages of the present invention will be discussed, by a consideration of the following detailed description of the preferred exemplary embodiments thereof, taken in conjunction with the figures which will first be briefly described.

Brief Description of the Drawings Figure 1 is a representation of 4 gels stained with coomassie blue, labeled from IA to ID, 5 illustrating the purification of extracellular products - * "" - mostly abundant specimens of Af. tuberculosis as identified by sodium dodecyl sulfate-polyacrylic amide gel electrophoresis (SDS-PAGE). Figure 2 is a tabular representation identifying the five N-terminal amino acids of twelve exemplary abundant extracellular products of Af. tuberculosis, and the apparent molecular weight for fourteen such products. Figure 3 is a tabular representation of the extended N-terminal amino acid sequence of three exemplary abundant secretory products of the? F. tuberculosis that did not distinguish the five N-terminal amino acids shown in Figure 2. Figure 4 is a graphical comparison of the survival rate of guinea pigs immunized with the secretory product of 30 kilograms Dalton mostly abundant exemplary, purified, the? f. tuberculosis against positive controls immunized with a crude extracellular protein preparation of the prior art, and unimmunized negative controls, after exposure to a lethal dose dispersed by? f aerosol. tuberculosis Figure 5 is a graphical comparison of the average guinea pig body weight of the animals immunized with 71 kilo Dalton extracellular product mostly abundant, purified, against positive controls * - immunized with a preparation of extracellular proteins from the Af. crude tuberculosis of the prior art, and unimmunized negative controls, after exposure to a lethal dose dispersed by aerosol of the Af. tuberculosis. Figure 6 is a graphical comparison of the survival rate of the guinea pigs immunized in Figure 5 with extracellular product of 71 kilograms Dalton mostly abundant exemplary, purified from Af. j * tuberculosis, against positive controls immunized with a preparation of extracellular proteins of Af. crude tuberculosis of the prior art, and unimmunized negative controls, after exposure to a lethal dose dispersed by aerosol of the Af. tuberculosis Figure 7 is a graphic comparison of the average guinea pig body weight of the animals immunized with 71 kilo Dalton extracellular product, mostly abundant, purified, and unimmunized negative controls, after exposure to a dose lethal dispersed by spray from the Af. tuberculosis in a second separate experiment. Figures 8A and 8B are graphical comparisons of lymphocyte proliferative responses to the 71 kilo Dalton extracellular product mostly abundant exemplary, purified, in human subjects PPD + (indicative of "" - infection with M. tuberculosis) and PPD-. Figure 8A is a graph of the values measured at 2 days after the incubation of lymphocytes with this antigen, while the Figure 8B is a graph of the measured values at 4 days after incubation. Figure 9 is a graphical comparison of the average guinea pig body weight of animals immunized with the vaccine, comprising a combination of extracellular products produced in accordance with the teachings of the present invention and non-immunized controls, after exposure to a vaccine. lethal dose dispersed by aerosol of Af. tuberculosis Figure 10 is a graphical comparison of average guinea pig body weight of animals immunized with three different doses of a vaccine, comprising a combination of extracellular products produced in accordance with the teachings of the present invention and non-immunized controls, after exposure to a lethal dose dispersed by aerosol of Af. tuberculosis Figure 11 is a graphical comparison of the average guinea pig body weight of animals immunized with vaccines, which comprises six different combinations of extracellular products produced in accordance with the teachings of the present invention and non-immunized controls, after exposure to a lethal dose dispersed by aerosol of the? f. tuberculosis Detailed Description The present invention is directed to compounds and methods for their production and use against pathogenic organisms, such as vaccines and immunotherapeutic agents. More specifically, the present invention is directed to the production and use of mostly abundant extracellular products, released by pathogenic organisms or their immunogenic analogues, such as vaccines or immunotherapeutic agents, and to methods associated to generate protective immunity in mammalian hosts against infection. These compounds will be referred to as vaccines throughout this application for simplicity purposes. In the exemplary embodiments, which illustrate the teachings of the present invention, the largely abundant extracellular products of the? F were distinguished and subsequently purified. tuberculosis Guinea pigs were immunized with purified forms of these mostly prevalent extracellular products, without determination of the specific molecular immunogenicity of the individual product. In addition, exemplary immunizations were carried out using the purified extracellular products alone or in combination, and with different doses and routes of administration. Those skilled in the art will recognize that the above strategy can be used with any pathogenic organism or bacterium to practice the method of the present invention and, accordingly, the present invention is not specifically limited to vaccines and methods directed against the? . tuberculosis In these exemplary embodiments, the largely abundant extracellular products of the? F were separated and purified. tuberculosis using column chromatography. The t . >; Determination of relative abundance and purification of extracellular products was performed using polyacrylic amide gel electrophoresis. After purification of the vaccine components, guinea pigs were vaccinated with the mostly abundant extracellular products alone or in combination, and subsequently attacked with the? F. tuberculosis As will be discussed in detail, in addition to the development of the expected responses that could be measured, to these extracellular products after immunization, the vaccines of the present invention unexpectedly conferred an effective immunity in these laboratory animals against subsequent lethal doses of ?F. tuberculosis dispersed by aerosol. Although these exemplary embodiments used purified forms of the extracellular products, those skilled in the art will appreciate that the present invention can be readily practiced using immunogenic analogues that are produced through recombinant means or other forms of chemical synthesis, using techniques well known in the art. The technique. In addition, the selected immunogenic analogues, homologues or segments of the extra-abundant products may be used in place of naturally occurring products within the scope and teaching of the present invention.

Y . An additional understanding of the - V present invention for those skilled in the art by the following non-limiting examples, which illustrate exemplary protocols for the identification, isolation, production and use of mostly abundant extracellular products (alone and in combination) as vaccines. Example 1 Isolation and Production of Extracellular Proteins (EP) in Raw from Mycobacterium tuberculosis The Erdman strain of the? F was obtained. tuberculosis (ATCC 35801) of the American Tissue Culture Collection (Rockville, Md.). The lyophilized bacteria were reconstituted in Middlebrook 7H9 culture medium (Laboratories Difco, Detroit, Mich.) And remained on Middlebrook agar 7H11. 7H11 agar was prepared using Bacto Middlebrook 7H10 agar (Difco), OADC Enrichment Medium (Difco), 0.1% casein enzymatic hydrosylate (Sigma), and glycerol, as previously described by Cohn (Cohn, ML Am. Rev. Respir Dis. 98: 295-296 ) incorporated herein by reference. After sterilization by autoclaving, the agar was dosed in bacteriological Petri dishes (100 by 15 millimeters) and allowed to cool. Then the? F was put on plates. tuberculosis using sterile techniques and grew at 37 ° C in 5 percent C02-95 percent air, 100 percent humidity. After culturing in 7H11 for 7 days, the colonies were scraped from the plates, suspended in broth from 7H9 to 108 colony forming units / milliliter and aliquoted into 1.8-milliliter Nunc cryotubes (Roskilde, Denmark). Each liter of the broth was prepared by re-hydrating 4.7 grams of Bacto Middlebrook 7H9 powder with 998 milliliters of distilled water, and 2 milliliters of glycerol (Sigma Chemical Co., St. Louis, Mo.), before adjusting the mixture. at a pH value of 6.75 and autoclaving the broth for 15 minutes at 121 ° C. Then they were slowly frozen and stored at -70 ° C these cells were aliquoted. Cells stored under these conditions remained viable indefinitely and were used as needed. Crude extracellular protein (EP) preparations were obtained from? F cultures. tuberculosis that grew in the Middlebrook 7H9 broth done as stated < "" - "- above After reconstitution, aliquots of 150 milliliters of the broth were autoclaved for 15 minutes at 121 ° C and dosed into 225-centimeter-square vented tissue culture flasks. Tuberculosis cells stored at -70 ° C, as described in the previous paragraph, and used to inoculate 7H11 agar plates.After culturing for 7 days, the colonies were scraped from the plates, suspended in few milliliters of 7H9 broth, and were sonicated in a water bath to form a single cell suspension Af. tuberculosis cells were suspended in the sterile aliquots of 150 milliliters at an initial optical density of 0.05, as determined by a spectrometer Perkin-Elmer Junior model 35 (Norwalk, Conn) Cells were incubated at 37 ° C in 5 percent C02-95 percent air, for 3 weeks, until the suspension showed a high density Optical density from 0.4 to 0.5 These crops were used as supply bottles for subsequent crops also in 7H9 broth. The supply bottles were sonified in a water bath, to form a single cell suspension. The Ai cells were then diluted. tuberculosis in 7H9 broth at an initial optical density of 0.05, and incubated at 37 ° C in 5 percent C02-95 percent air for 2 1/2 to 3 weeks, until the suspension showed an optical density of 0.4 to 0.5. The supernatant of the r1"- culture was then decanted and filter sterilized sequentially through 0.8 μm and 0.2 μm low-binding-to-protein filters.

(Gelman Sciences Inc., Ann Arbor, Mich.). The filtrate was then concentrated approximately 35 times in a Filtron Minisette with an Omega membrane having a cut of 10 kilo Daltons and stored at 4 ° C. Analysis of crude extracellular protein preparation by sodium dodecyl sulfate-polyacrylic amide gel electrophoresis (SDS-PAGE) revealed a multi-band protein composition. HE / *. 0 prepared a mixture of crude extracellular protein (EP) by obtaining a cut of ammonium sulfate at 40-95 percent of the culture filtrate.

Example 2 Purification of Major Abundant Extracellular Products of Afycobacterium tuberculosis Ammonium sulfate (grade I, Sigma) was added to the sterile culture filtrate of Example 1, in concentrations ranging from 10 percent to 95 percent at 0 ° C, and stirred gently to fractionate the proteins. The suspension was then transferred to plastic bottles and centrifuged in a rotating pan rotor at 3,000 revolutions per minute in a Sorvall Centrifuge to give the pellet to the resulting precipitate. The supernatant fluid was decanted and, depending on the product of interest, the supernatant fluid or pill was subjected to further purification. When the product of interest was contained in the supernatant fluid, a second cut of ammonium sulfate 5 was executed by increasing the salt concentration on that of the first cut. After a period of gentle agitation, the solution was centrifuged as described e, to precipitate the desired product, and the second supernatant was subjected to further purification. After centrifugation, the precipitated proteins were re-solubilized in the appropriate cold pH buffer and extensively dialyzed in a Spectrapor dialysis membrane (Spectrum Medical Industries, Los Angeles, California) with a molecular weight cutoff of 6,000. to 8,000 to remove the salt. The concentration of extracellular protein was determined by a bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, Illinois) and the components of the fraction were determined using sodium dodecyl sulfate-amide gel electrophoresis polyacrylic. The fractions were then applied to chromatography columns for further purification. Using the general scheme described e, fourteen extracellular products of crude extracellular protein filtrate obtained by the process were purified. detailed in Example 1. The exact ammonium sulfate precipitation procedure and the chromatography protocol for each isolated extracellular product are detailed below.

A. Extracellular Product of 110 kilograms Daltons 1. A precipitate of 50-100 percent ammonium sulfate was obtained, as described e. 2. The resolubilized precipitate was dialyzed and applied to a DEAE-Sepharose ion exchange column. : -f CL-6B or QAE-Sepharose, in column pH regulator, consisting of 10 percent sorbitol, potassium phosphate lOmM, a pH of 7, 2-mercaptoethanol 5mM, and 0.2 mM EDTA, and was levigated with a gradient of sodium chloride. Fractions containing 110 kilo Dalton protein were leviginated to approximately 550 mM salt and were collected. 3. The collected fractions were applied to a size fractionation column of Sepharose S200 in PBS buffer (phosphate regulated saline). The protein was levigated as a 110 kilo Dalton protein homogeneous.

B. Extracellular Product of 80 kilograms Daltons 1. Discarded the ammonium sulfate cut at 0-25 percent (1 hour at 0 ° C) and the sulfate cut was retained Ammonium 25-60 percent (overnight at 0 ° C), as - "*** - described e 2. A CL-6B column of DEAE (Pharmacia) was loaded with 25mM Tris, with a pH of 8.7, containing NaCl IM and balanced with 25 mM Tris, with a pH of 8.7, 10 mM NaCl, and dialyzed the protein sample against 25 mM Tris, with a pH of 8.7, NaCl lOmM and was applied to the column. The column was washed overnight with the same regulator. A first salt gradient of NaCl lOmM at 200 mM in Tris mM, with a pH of 8.7, was run through the column to levigate other proteins. f - -j ^. A second salt gradient (200 to 300 mM NaCl) was run through the column, and the 80 kilo Dalton protein at approximately 275 mM NaCl was levitated. 3. An HP column of Q-Sepharose was loaded with 25mM Tris, with a pH of 8.7, NaCl IM and was rebalanced to Tris 25mM, with a pH of 8.7, NaCl lOmM. The protein sample was dialyzed against 25 mM Tris, with a pH of 8.7, lOmM NaCl and applied to the column. The column was washed in the same regulator and then levigated with 200-300 mM NaCl in Tris mM, with a pH of 8.7. The fractions containing the protein were collected of 80 kilograms Daltons and dialyzed against 25 mM Tris, with a pH of 8.7, lOmM NaCl and then concentrated in a Speed-Vac concentrator at 1-2 milliliters. The protein sample was applied to a Superdex 75 column, and levigated with 25 mM Tris, with a pH of 8.7, 150 mM NaCl. The protein was levig 80 kilo Daltons as a homogeneous protein.

C. Extracellular product of 71 kilograms Daltons 1. A precipitate of ammonium sulfate was obtained at 40-95 percent, as described above, with the exception that the 71 kilo Dalton product was cultivated in 7H9 broth at a pH of 7.4 and in 0% C02, and heat was applied at 42 ° C for 3 hours once a week. The precipitate was dialyzed against the Initial pH Regulator (20 M Hepes, 2 mM MgAc, 25 mM KC1, 10 mM (NH4) 2 SO4, 0.8 mM DL-Dithiothreitol, with a pH of 7.0). 2. The resolubilized precipitate was applied to a column of balanced Agarose ATP with Initial Regulator. The effluent was collected and reapplied to the Agarose ATP column. The 71 kilo Dalton protein was fixed to the column. 3. Subsequently the column of Agarose was washed ATP, first with Initial Regulator, then with KC1 1 M, then with Initial Regulator. 4. The homogeneous 71 kilo Dalton protein of the column was lightened with 10 mM ATP and dialyzed against phosphate buffer.

D. Extracellular Product of 58 kilo Daltons 1. A precipitate of ammonium sulfate was obtained at 25-50 percent, as described above. 2. The resolubilized precipitate was dialysed and applied to a DEAE-Sepharose CL-6B or QAE-Sepharose column and levigated with NaCl. The collected fractions containing the 58 kilo Dalton protein were levigated at approximately 400 mM NaCl. 3. The collected fractions were applied to a size fractionation column of Sepharose CL-6B. The protein was levigated at approximately 670-700,000 Daltones. 4. The levigated protein was applied to a thiopropyl-sepharose column. The protein of 58 kilograms homogeneous Daltones -CA was levigated to approximately 2-mercaptoethanol 250-350 mM. The levigated protein was monitored using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis, and exhibited the only band shown in Figure IA, column 2.

E. Extracellular product of 45 kilograms Daltones 1. a. A cut of 0-25 percent ammonium sulfate was discarded (1 hour at 0 ° C). b. The cut of ammonium sulfate was retained -60 percent (overnight at 0 ° C). 20 2. a. A column of DEAE CL-6B was loaded (Pharmacia) with 2.5 mM Tris, with a pH of 8.7, containing 1 M NaCl and balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialyzed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column. The column was then washed overnight with the same regulator. c. The column was levigated with a salt gradient (10 mM to 200 mM) in 25 mM Tris buffer, with a pH of 8.7. The 45 kilo Dalton protein was levigated at approximately 40 mM NaCl. 3. a. An HP column of Q-Sepharose (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and resumed with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing using the same regulator. c. The column was levigated with 10-150 mM NaCl in 25 mM Tris, with a pH of 8.7. 4. a. The fractions containing the 45 kilo Dalton product were collected, grouped and dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 before concentration to 1 milliliter in a Speed Vac concentrator. b. The concentrate was applied to a column Superdex 75 balanced with 25 mM Tris, 150 mM NaCl, with a pH of 8.7. The product was levighed as a homogeneous protein. The levigated protein was monitored using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis, and resulted in the only band shown in Figure IB, column 2.

F. Extracellular Product of 32 kilo Daltons (A) 1. a. A cut of ammonium sulfate 5 at 0-25 percent (1 hour at 0 ° C) was discarded. b. The ammonium sulfate cut was retained at 25-60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B, (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing NaCl * * • * - ^ J 1 M and then balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialyzed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and was applied to the column with subsequent washing overnight with the same regulator. c. The column was levigated with a salt gradient (10 mM to 200 mM) in 25 mM Tris buffer, with a pH of 8.7. The 32 kilo Dalton protein was levigated to approximately 70 mM NaCl. 20 3. a. Fractions containing the product of 32 kilo Daltons were collected, pooled and dialyzed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 before concentrating the protein sample at 1 milliliter in a Speed Vac Concentrator. 25 b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with this regulator. The 32 kilo Dalton product was levigó as a homogeneous protein. 4. a. An HP column of Q-Sepharose (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and resumed with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing in the same buffer. c. The column was levigated with a gradient of 100-300 mM NaCl. Labeled with 32A, the homogeneous protein is levigated at approximately 120 mM NaCl, and is shown as a single band in Figure IB, column 4. G. Extracellular Product of 32 kilo Daltons (B) 1. a. A cut of 0-25 percent ammonium sulfate was discarded (1 hour at 0 ° C). b. The cut of ammonium sulfate was retained -60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B was loaded (Pharmacia) with 25 mM Tris, with a pH of 8.7, containing NaCl 1 M and then balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the "" - column with subsequent washing overnight with the same regulator. c. A preliminary salt gradient of 10 mM NaCl at 200 mM in 25 mM Tris was run, with a pH of 8.7, levigating many proteins. After the column balance, a second salt gradient was run (200 to 300 mM NaCl). The 32 kilo Dalton protein was levigated at approximately 225 mM NaCl. 3. a. An HP column of Q-Sepharose (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing NaCl 1 M and was rebalanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing in the same buffer. c. The column was levigated with a gradient of 200-300 mM NaCl in the same regulator. 4. a. Fractions containing the product of 32 kilo Daltons were collected, pooled and dialyzed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 before concentrating the protein sample at 1 milliliter in a Speed Vac Concentrator. b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with the same regulator. The 32-kilo Dalton product, labeled 32B to distinguish it from the 32-kilogram protein Daltones separated using protocol H, was levigó as a homogeneous protein and is shown as a single band in Figure IB, column 3. 5 H. Extracellular Product of 30 kilograms Daltones 1. a. A cut of 0-25 percent ammonium sulfate was discarded (1 hour at 0 ° C). b. The cut of ammonium sulfate was retained at *. 25-60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and then balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. 15 b. The protein sample was dialysed against mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing overnight with the same regulator. c. The column was levigated with a salt gradient (10 M to 200 M) in 25 mM Tris buffer, with a pH of 8. 7. The 30-kilo Dalton protein was levigated at approximately 140 mM NaCl. 3. a. The fractions containing the product of 30 kilo Daltons were collected, grouped and dialyzed against 25 mM Tris, 10 M NaCl, with a pH of 8.7, before concentrating the protein sample at 1 milliliter in a Speed Vac Concentrator. b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with this regulator. The 30 kilo Dalton product was levigated as a homogeneous protein and is shown as a single band in Figure IB, column 5.

I. Extracellular product of 24 kilograms Daltones 1. a. A cut of 0-25 percent ammonium sulfate was discarded (1 hour at 0 ° C). b. The ammonium sulfate cut was retained at 25-60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and then balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing overnight with the same regulator. c. A preliminary salt gradient of 10 mM NaCl at 200 mM in 25 mM Tris was run, with a pH of 8.7, levigating many proteins. After the column balance, a second salt gradient (NaCl 200 to 300 mM) of balance was run. The 24 kilo Daltons were levigated to approximately 250 mM NaCl. 3. a. An HP column of Q-Sepharose (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and resumed with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against mM Tris, 10 mM NaCl, with a pH of 8.7 and was applied to the column with subsequent washing in the same regulator. c. The column was lightened with a gradient of 200-300 mM NaCl in the same regulator. 4. a. The fractions containing the product of 24 kilo Daltons were collected, grouped and dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7, before concentrating the protein sample at 1 milliliter in a Speed Vac Concentrator b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with the same regulator. The 24 kilo Dalton product was levigó as a homogeneous protein and is shown as a single band in Figure IB, column 7.

J. 23.5 kilo Extracellular Product Daltones 1. a. A cut of 0-25 percent ammonium sulfate was discarded (1 hour at 0 ° C). - 'b. The cut of ammonium sulfate was retained -60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and then rocked with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column before subsequent washing overnight with the ^ _ same regulator. c. The column was levigated with a salt gradient (10 mM to 200 mM) in 25 mM Tris buffer, with a pH of 8.7. The 23.5 kilo Dalton protein was levigated at approximately 80 mM NaCl. 15 3. a. An HP column of Q-Sepharose was loaded (Pharmacia) with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and was rebalanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialyzed against 25 M Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing in the same regulator. c. The column was levigated with 100-300 mM NaCl in 25 mM Tris, with a pH of 8.7. 4. a. Fractions containing the 23.5 kilo Dalton product were collected, pooled and "" - "dialyzed against 25 mM Tris., 10 mM NaCl, with a pH of 8.7, before concentrating the protein sample at 1 milliliter in a Speed Vac Concentrator. b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with the same regulator. The product of 23.5 kilo Daltons was levigó like homogenous protein. The levigated protein was monitored using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis, and resulted in the only band shown in Figure IB, column 6.K. 23 kilo Dalton Extracellular Product 1. a. Ammonium sulfate cuts of 0-25 percent (1 hour at 0 ° C) and 25-60 percent (overnight at 0 ° C) were discarded. b. A cut of 60-95 percent ammonium sulfate was retained. 2. a. A column of DEAE CL-6B 20 (Pharmacia) was loaded with 50 mM Bis-Tris, with a pH of 7.0, containing 1 M NaCl and balanced with 50 mM Bis-Tris, 100 M NaCl, with a pH of 7.0. b. The protein sample was dialyzed against 50 M Bis-Tris buffer, with a pH of 7.0, 100 mM NaCl, and was applied to the column before washing the column during the night with the same regulator. c. The column was levigated with a linear gradient of 100 to 300 mM NaCl, in 50 mM Bis-Tris, with a pH of 7.0. 5 d. The fractions containing the 23 kilo Dalton protein were harvested, which was levigated at approximately 100-150 mM NaCl. 3. a. The protein fractions were dialyzed against 25 mM Tris, with a pH of 8.7, 10 mM NaCl, and they were concentrated to 1-2 milliliters in a Savant Speed Vac Concentrator. b. The concentrate was applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 M NaCl, with a pH of 8.7. The product is levigated as a homogeneous protein, 5 as shown in Figure IB, column 8.

L. 16 kilo Dalton Extracellular Product 1. a. A cut of 0-25 percent ammonium sulfate was discarded (1 hour at 0 ° C). 0 b. The cut of ammonium sulfate was retained -60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B (Pharmacia) was charged with 2.5 mM Tris, with a pH of 8.7, containing 1 M NaCl and then balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialyzed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column with subsequent washing overnight in the same regulator. 5 c. The column was levigated with a salt gradient (10 mM to 200 mM) in 25 mM Tris buffer, with a pH of 8.7. The 16 kilo Dalton protein was levigated at approximately 50 mM NaCl. 3. a. Fractions containing * the 16 kilo Dalton product, were pooled and dialyzed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7, before concentrating the protein sample at 1 milliliter in a Speed-Vac Concentrator b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with the same regulator. The 16 kilo Dalton product was levigó as a homogeneous protein. The levigated protein was monitored using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis and resulted in the only band shown in Figure IB, column 9.

M. Extracellular Product of 14 kilo Daltons 1. a. A cut of ammonium sulfate 25 was discarded at 0-25 percent (1 hour at 0 ° C). b. The ammonium sulfate cut was retained at 25-60 percent (overnight at 0 ° C). 2. a. A column of DEAE CL-6B (Pharmacia) was loaded with 25 mM Tris, with a pH of 8.7, containing 1 M NaCl and then balanced with 25 mM Tris, 10 mM NaCl, with a pH of 8.7. b. The protein sample was dialysed against 25 mM Tris, 10 mM NaCl, with a pH of 8.7 and applied to the column before subsequent washing overnight in the same regulator. c. The column was levigated with a salt gradient (10 mM to 200 mM) in 25 mM Tris buffer, with a pH of 8.7. The 14 kilo Dalton protein was levigated at approximately 60 mM NaCl. 3. a. An HP column of Q-Sepharose was loaded with Tris 25 mM, with a pH of 8.7, containing 1 M NaCl and was rebalanced with 25 mM NaCl, with a pH of 8.7. b. The protein sample was dialyzed against 25 mM Tris, 10 M NaCl, with a pH of 8.7 and applied to the column with subsequent washing in the same buffer. c. The column was levigated with 10-150 mM NaCl in 25 mM Tris, with a pH of 8.7. d. Steps 3a to 3c were repeated. 4. a. The fractions containing the product of 14 kilo Daltons were collected, grouped and dialyzed - "against Tris 25 mM, 10 mM NaCl, with a pH of 8.7, before concentrating the protein sample at 1 milliliter in a Speed-Vac Concentrator. b. The concentrate was then applied to a Superdex 75 column equilibrated with 25 mM Tris, 150 mM NaCl, with a pH of 8.7 and levigated with this regulator. The product of 14 kilo Daltones was levigó as a homogeneous protein. The levigated protein was monitored using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis, and resulted in the only band shown in Figure IC, column 2.

N. 12 kilo Dalton Extracellular Product 1. A precipitate of 0-10 percent ammonium sulfate was obtained (overnight at 4 ° C). 2. The resolubilized precipitate was applied to a fractionation column of size S200 Sephacryl, levigating the protein as a 12 kilo Dalton molecule. 3. The protein fractions were applied to an ion exchange column of DEAE-Sepharose CL-6B or QAE-Sepharose, and was levigated with a NaCl gradient as described above. Fractions containing two homogeneous proteins having molecular weights of approximately 12 kilo Daltons were levigated at approximately 300-350 mM NaCl, and harvested. The proteins were labeled as 12A and 12B, and purified as a doublet shown in Figure ID, column 2. As illustrated in the profile of sodium dodecyl sulfate-polyacrylic amide gel electrophoresis of Figure 1, the main or mostly abundant extracellular proteins of the? f were purified. tuberculosis for homogeneity, through the use of the protocols detailed in the above Examples 2A-2N. More particularly, Figure 1 illustrates four 12.5 percent exemplary acrylic amide gels, developed using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis and labeled IA, IB, IC, and ID. The standard in row 1 of gels 1A-1C has proteins with molecular weights of 66, 45, 36, 29, 24, 20, and 14 kilo Daltons. In gel ID the standard in row 1 contains proteins with molecular weights of 68, 45, 31, 29, 20, and 14 kilo Daltons. The rows containing the respective purified extracellular products show essentially one band in the reported molecular weight of the individual protein. It should be noted that in the ID gel the 12 kilo Dalton protein runs as a visible doublet in row 2. The sequence analysis shows that the 12 kilo Dalton lower band (or 12B KD) is equivalent to the 12 kilo Dalton band superior (or 12A KD), except that it lacks the first 3 amino acids with terminal N. In Figure 2 provides an additional analysis - '* of these extracellular products mostly abundant individual copies. More particularly, Figure 2 is a tabular compilation of the N-terminal sequence data obtained from these purified extracellular products, showing that most of the isolated products are certainly indistinct. The proteins 32A, 32B and 30 have the same 5 amino acids with N-terminus, therefore, more sequencing was necessary to characterize them and differentiate them completely. Figure 3 shows the extended N-terminal amino acid sequences for these three purified secretory products. The different amino acids at positions 16, 31 and 36 show that these isolated proteins are distinct from one another despite their similar molecular weight. In addition to proteins 30, 32A and 32B, N-terminal amino acid sequences extended from other, largely abundant, extracellular products were determined to provide primary structural data and to discover possible protein relationships. Sequencing was performed on the purified extracellular products, according to Example 2, using techniques well known in the art. Subsequently, the variant lengths of the N-terminal amino acid sequence, determined for each individual extracellular product, identified by the apparent molecular weight of the intact protein, and represented using standard one-letter abbreviations for the naturally occurring amino acids are shown. When adhering to the established rules of notation, the sequences with terminal N are written from left to right, in the direction of the amino terminus to the carboxy term. Those positions where the identity of the determined amino acid is less than certain, are underlined. Where the amino acid in a particular position is unknown or ambiguous, the position in the sequence is represented by a hyphen. Finally, where two amino acids are separated by a diagonal, the correct constituent has not been explicitly identified and either can occupy the position in that sequence.

PROTEIN AMINO ACID SEQUENCE WITH TERMINAL N 5 10 15 20 25 30 35 40 12 KD FDTRL MRLED EMKEG RYEVR AELPG VDPDK DVDIM VRDGQ 45 LTIKA ERT 10 15 20 25 30 14 KD ADPRL QFTAT TLSGA PFDGA S / NLQGK PAVLW 5 10 15 20 25 30 35 16 KD AYPIT GKLGS ELTMT DTVGQ WLGW KVSDL F / YKSTA 40 45 VIPGY TV-EQ Ql 10 15 20 23 KD AETYL PDLDW DYGAL EPHIS GQ 10 23.5 KD APKTY -EELK GTD 10 15 20 25 30 35 40 24 KD APYEN LMVPS PSMGR DIPVA FLAGG PHAVY LLDAF NAGPD 45 50 55 60 VSNWV TAGNA MMTLA -KGIC / S 10 15 20 25 30 35 40 30 KD FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG NNSPA VYLLD 10 15 20 25 30 35 40 32A KD FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG ANSP-LYLLD 10 15 20 32B KD FSRPG LPVEY LQVPS A-MGR DI 5 10 15 20 25 30 45 KD DPEPA PPVPD DAASP PDDAA APPAP ADPP- 10 15 20 58 KD TEKTP DDVFK LAKDE KVLYL 71 KD ARAVG I 80 KD TDRVS VGN 10 15 20 110 KD NSKSV NSFGA HDTLK V-ERK RQ These sequence data, combined with the physical properties investigated using sodium dodecyl sulfate-polyacrylic amide gel electrophoresis, allow these mostly abundant extracellular products representative of the present invention to be characterized and distinguished. The analysis described indicates that these proteins constitute the majority of the extracellular products of the Ai. tuberculosis, with the products of 71 KD, 30 KD, 32A KD, 23 KD and 16 KD comprising approximately 60 percent by weight of the total available cell product. It is also estimated that the 30 kilo Dalton protein can constitute up to 25 percent by weight of the total products released by the Af. tuberculosis In this way, extracellular products mostly abundant individual copies of the Ai. Tuberculosis, useful in the practice of the present invention can fluctuate anywhere from about 0.5 percent to about 25 percent of the total weight of the extracellular products. As previously described, after the "" or inability of the traditional Western blot analysis to consistently identify the most immunogenically specific extracellular products, the present inventor decided to analyze the immunogenicity of the largely abundant extracellular products, based on their abundance and consequent 5 ease of identification and isolation Surprisingly, these extracellular, largely abundant products were found to unexpectedly induce effective immune responses, leading this inventor to conclude that these can function as vaccines This surprising discovery led to the development of the non-limiting functional theory of This invention, discussed above In order to demonstrate the efficacy of the present invention, further experiments were conducted, using individual mostly abundant extracellular products, and combinations thereof in different exemplary doses, for induce protective immunity, in laboratory models accepted in the art. More specifically, extra purified, mostly pure, extracellular products were used to induce protective immunity in guinea pigs which were then attacked with Af. tuberculosis On the demonstration that these proteins were capable of inducing protective immunity, combinations of five mostly abundant purified extracellular products were tested in a similar manner, using different routes of administration. In particular, the abundant 30 kilo Dalton extracellular product was used to induce protective immunity in the accepted animal model, as was the purified form of the 71 kilo Dalton extracellular product. As with the exemplary mostly abundant individual extracellular products, the combination vaccines of five mostly abundant extracellular products also confer protection against aggression with lethal doses of Af. tuberculosis The results of different studies of these exemplary vaccines of the present invention are presented below. In all experiments involving immunogenic aggressions or aerosol with? F. tuberculosis, Hartley guinea pigs of the specific pathogen-free male Hartley strain (Charles River Breeding Laboratories, North Wilmington, Massachusetts) were used. Two or three animals were housed in a stainless steel cage and were allowed free access to standard feed for standard guinea pigs and water. After arriving at the animal facility, the guinea pigs were observed for at least one week before starting each experiment, to make sure they were healthy. Initial experiments were conducted using extracellular, mostly abnt individual products, which were believed to comprise between 3 percent and 25 percent of the total extracellular proteins normally present. These experiments showed that the extra-abnt extracellular products produce an effective immune response. More particularly, it was shown that the 30 kilo Dalton and 71 kilo Dalton extracellular products are individually capable of generating a cell-mediated immune response, which protected the guinea pigs from exposure to lethal doses of the? F. tuberculosis as follows.

EXAMPLE 3 Skin Test of 30 KD Protein Purified for Cell-mediated Immunity of Guinea pigs Immunized with 30 kd To illustrate that a measurable immune response can be induced by purified forms of "~, - abundant extracellular products , a cutaneous hypersensitivity test was carried out. Guinea pigs were immunized with the secretory product of 30 kilograms Dali de Ai. most abundant tuberculosis, purified according to Example 2 and believed to comprise approximately 25 percent of the total extracellular product of Ai. tuberculosis In three independent experiments, guinea pigs were immunized three times with three weeks of separation, with 100 μg of 30 kilogram Daltones protein substantially purified in Syntex Adjuvant Formulation.

Control animals were also injected with pH regulator in Syntex Adjuvant Formulation. Three weeks after the last immunization the guinea pigs were attacked with the 30 kilogram protein Daltones in a cutaneous hypersensitivity assay. The guinea pigs were shaved in the back and injected intradermally with 0.1, 1 and 10 μg of 30 kilo Dalton protein, with erythema (skin irritation) resulting and hardening was measured after 24 hours, as is shown in the following Table A. Data were reported in terms of average measurement values for the group ± standard error (SE), as determined using traditional methods. ND indicates that this particular aspect of the invention was not made.

Table A Erythema (mm) at 30 KD (Average + SE) State of the Guinea Pig n 0.1 μg 1.0 μg 10.0 μg Exp. 1 Immunized 6 1.2 ± 0.5 3.9 ± 0.8 6.9 ± 1.0 Controls 5 ND ND 3.0 ± 0.9 Exp. 2 Immunized 6 0.5 ± 0.5 5.4 ± 0.7 8.1 ± 0.6 Controls 3 O ± O 2.5 ± 0 1.7 ± 0.8 Exp. 3 Immunized 6 ND 1.7 ± 1.1 6.2 ± 0.3 Controls 3 ND ND 2.0 ± 0.0 Hardening at 30 KD (Average + SE) State of the Guinea Pig n 0.1 μg 1.0 μg 10.0 μg Exp. 1 Immunized 6 O ± O 3.3 ± 0.3 5.6 ± 0.9 Controls 5 ND ND 1.6 ± 1.0 Ex. Immunized 6 O ± O 3.8 ± 0.7 4.9 ± 1.2 Controls 3 O ± O 0.8 ± 0.8 1.7 ± 0.8 Exp. Immunized, 6 ND ll ± ll 4.7 ± 0.4 Controls 3 ND O ± OO ± O As shown in Table A, the guinea pigs immunized with the 30 kilogram secretory product Exemplary Daltones exhibited a strong immune response mediated by cells, as evidenced by marked erythema and hardening. In contrast, the control animals exhibited a minimal response. To confirm the immunoreactivity of the 30 kilo Dalton secretory product, and to show its applicability to infectious tuberculosis, guinea pigs not immunized with? F were infected. tuberculosis and were attacked with this protein as follows.

Example 4 30 KD Purified Protein Test for Immune Responses Mediated by Cells of Guinea Pigs Infected With Af. Tuberculosis To obtain bacteria to be used in experiments that require the infection of guinea pigs, the? f was first cultured. tuberculosis on 7H11 agar and once passed through a lung of a guinea pig to make sure it was virulent. For this purpose, guinea pigs were attacked by aerosol with a suspension of 10 milliliters of bacteria in 7H9 broth containing approximately 5 x 104 bacteria / milliliter. After the guinea pigs became ill, the animals were sacrificed and the lungs removed, containing prominent lesions by Af. tuberculosis Each lung was seeded and cultured on 7H11 agar for 7 days to 10 days. Bacteria were scraped from the plates, diluted in 7H9 broth containing 10 percent glycerol, sonified in a water bath to obtain a single cell suspension, and frozen slowly at -70 ° C, at a concentration of approximately 2. x 107 viable bacteria / milliliter. The viability of the frozen cells was measured by thawing the bacterial suspension and culturing serial dilutions of the suspension on 7H11 agar. Just before an assault, a bottle of bacterial cells was thawed, and diluted to the desired concentration in 7H9 broth. The guinea pigs were exposed to aerosols of the Af. viable tuberculosis in a specially designed lucite spray chamber. The aerosol chamber measured 35.56 by 33.02 by 60.96 centimeters and contained two 15.24 cm diameter inlets on opposite sides, to introduce or remove the guinea pigs. The aerosol inlet was located in the center of the ceiling of the chamber. One pump (Gast Mfg. Co., Benton Harbor, Michigan) released air at 2,1093 kilograms / square centimeter to a venturi nebulizer unit (Mes Inc., Burbank, California), and an aerosol was generated from a suspension of 10 milliliters. of bacilli. At one end of the chamber was a 0.2 μm breathing circuit filter unit (Pall Biomedical Inc., Fajardo, Puerto Rico), to balance the pressure inside and outside the array. Due to safety considerations, aerosol aggressions were conducted inside the chamber placed completely inside a laminar flow hood. The animals were exposed to pathogenic spray for 30 minutes, during which time the suspension of bacilli in the nebulizer was completely expelled. Each aerosol was generated from the suspension of 10 milliliters containing approximately 5.0 x 104 bacterial particles per milliliter. Previous studies have shown that guinea pig exposure to this concentration of bacteria consistently produces infections in unprotected animals. After the aerosol infection, the guinea pigs were housed in stainless steel cages contained within a laminar flow biohazard safety housing (Airo Clean Engineering Inc., Edgemont, Pennsylvania) and were observed for signs of disease. Free access to the animals was allowed to standard feed for guinea pigs and water throughout the experiment. In this experiment, the infected guinea pigs were sacrificed and the proliferation of spleen lymphocytes was measured in response to different concentrations of the 30 kilo Dalton protein. More specifically, spleen lymphocytes were obtained and purified as described by Brieman and Horowitz (J. Exp. Med. 164: 799-811) which is incorporated herein by reference. The lymphocytes were adjusted to a final concentration of 107 / milliliter in RPMI 1640 (GIBCO Laboratories, Grand Island, New York) containing penicillin (100 Units / milliliter), streptomycin (100 μg / milliliter), and fetal calf serum at 10 ° C. percent (GIBCO), and incubated with different concentrations of purified 30 kilogram secretory product Daltones, in a total volume of 100 μl in micro-test wells (round bottom tissue culture plate with 5 96 wells; Falcon Labware, Oxnard, California), for 2 days at 37 ° C in 5 percent C02-95 percent air and 100 percent humidity. Uninfected animals were used as negative controls. At the end of the incubation period, 0.25 μCi of [3 H] thymidine (New England Nuclear, *? Or Boston, Massachusetts) to each well, and the cells were further incubated for 2 hours at 37 ° C in 5 percent C02- 95 percent air at 100 percent moisture. An automated multi-sample cell harvester was used (Skatron Inc., Sterling, Virginia) to wash each well, and passed the effluent through a filter mat (Skatron). Sections of the filter mat were placed representing separate micro-test wells in scintillation flasks, and 2 milliliters of Ecoscint H liquid scintillation cocktail (National Diagnostics, Manville, New Jersey). The emission of beta particles was measured in a beta scintillation counter (Beckman Instruments Inc., Fullerton, California). Trials were performed on tissue samples from infected and uninfected guinea pigs against 1 and 10 μg / milliliter of 30-kilogram secretory protein Daltones "- ** - isolated, after which the samples were monitored for their ability to incorporate [* H] imidine The results of these tests were tabulated and presented in Table B Subsequently, data such as a stimulation index were reported which, for purposes of this description, is defined as: average incorporation of [l] thymidine of lymphocytes incubated with antigen / average incorporation of [*?] thymidine from lymphocytes incubated without antigen.

Table B Stimulation rates at 30 KD (Average + SE) Guinea pig status n 1.0 μg / ml 10.0 μcr / ml Infected 6 2.2 ± 0.2 9.7 ± 4.6 Controls 6 1.5 ± 0.3 2. O ± O.8 As shown in Table B, the cells of the infected animals exhibited a strong response to the 30 kilogram protein Daltones exemplary, as manifested by the proliferation of lymphocytes of the spleen dependent on the dose, in response to exposure to this secretory product mostly abundant. Conversely, uninfected control animals showed little lymphocyte proliferation. Accordingly, the 30-kilo Dalton secretory product clearly induces a cell-mediated immune response in mammals infected with Ai. tuberculosis To illustrate the protective aspects of the vaccines of the present invention, guinea pigs were immunized with purified 30 kilo Dalton protein, and exposed to the? F. tuberculosis, as follows.

Example 5 Aggression of 30 kilo Dalton Guinea Pigs Immunized with Af. Tuberose Dyspersed by Aerosol As was done previously, the animals were immunized at least three times at three week intervals with 100 μg of the 30 kilogram secretory protein Daltones in adjuvant formulation of Syntex. Guinea pig guinea pigs were immunized with 120 μg of crude extracellular protein or were immunized in fake with pH adjuster in the same adjuvant. Three weeks after the last immunization, the animals were attacked with M. tuberculosis dispersed by aerosol as described in Example 4. The survival ranges were verified for the three groups of animals and presented graphically in Figure 4. An absolute mortality was determined 14 weeks after the assault, which is presented in Table C below.

Table C Condition Guinea pigs Survivors / Percent of Indians Assaulted Survival Immunized 30 KD 4/6 67% Immunized EP 3/6 50% Immunized in False 1/6 17% As shown in Figure 4, guinea pigs Indians immunized three times with 30 kilo protein Exemplary Daltones, they were protected against death. Approximately 67 percent of guinea pigs immunized with the 30-kilogram Dalton protein survived, while only 17 percent of the guinea pigs immunized to control false survived. Weight retention of immunized animals was also verified (data not shown) and further illustrates the prophylactic ability of vaccines incorporating largely abundant extracellular products produced by pathogenic bacteria as taught in the present invention. While it seems that the immunized animals maintained their weight, the high death rate of the animals immunized in false makes graphic comparison between the immunized animals and the control animals impossible. After the conclusion of the weight verification study, the surviving animals were sacrificed and the spleen and the right lung of each animal were tested to look for? F. viable tuberculosis. The animals were immersed in a 2 percent ampile solution (National Laboratories, Montavale, New Jersey), and the lungs and spleens were aseptically removed. The number of macroscopic primary surface lesions in the lungs was enumerated by optical inspection. The colony formation units (CFU) of Ai were determined. tuberculosis in the right lung and in the spleen by homogenizing each organ in 10 milliliters of 7H9 with a mortar and pistachio and 90 mesh Norton Alundum (Fisher), serially diluting the tissue homolog in 7H9, and cultivating the solutions in duplicate plates of 7H11 agar by using 0.1 milliliter / drop drops. All plates were stored in modular incubator chambers and incubated for 12 to 14 days at 37 ° C in 5 percent C02, 95 percent air and 100 percent humidity. The assays were conducted using this protocol and Table D below shows the results of the counts in terms of mean colony formation units (CFU) ± standard error (SE).

Table D Mean CFU + SE Condition Guinea pig n Right lung Spleen Immunized 30 KD 4 3 .4 ± 1. 7 x l7 7. 7 ± 3. 9 x l06 - Immunized in false 1 1.8 x 108 8.5 x 107 Difference of Reg 0.73 1.04 As shown in Table D, immunization with the secretory protein of 30 kilograms Daltones limited the growth of? F. tuberculosis in the lung and spleen. Although only data from the only animal immunized in false survivor were available for comparison purposes, the four animals immunized with 30 kilo Daltons ,. survivors had a 0.7 lower registry of units of ^ ú colony formation in their lungs and 1 smaller record of colony formation units in their spleens than animals immunized in false survivors. Based on previous demonstrations of a high correlation between counts of colony formation units and mortality, the animal The survivor probably had fewer units of colony formation in the lungs and in the spleen than the animals that died before an analysis of colony formation units could be made. Again this reduction of colony formation units in the lungs and spleens of the immunized animals conclusively demonstrates the scope and operability of the present invention. The immunoprotective potential of another extracellular product, mainly abundant from M. tuberculosis, the extracellular product of 71 kilo Daltons, was tested. its isolated form to demonstrate its immunoprotective capacity. - ** > EXAMPLE 6 Skin Test of Protein 71 KD Purified from Guinea Pigs Immunized with a Crude Preparation of EP 5 To demonstrate the potential of the 71 kilo Dalton protein to elicit an effective immune response in animals, this extracellular product was used mostly abundant for skin tests in guinea pigs immunized with a crude preparation of proteins -e.0 extracellular (EP) of Ai. tuberculosis in a cutaneous hypersensitivity trial. As discussed above, the crude extracellular proteins will impart acquired immunity against infection by Ai. tuberculosis but to a lesser degree than the vaccines in this invention. Guinea pigs were immunized twice with a "three week" interval, with 120 μg of a crude extracellular protein preparation prepared as detailed in Example 1. The vaccine was prepared in a Incomplete Freunds adjuvant with fake immunized animals that received a pH regulator instead of the extracellular proteins. Three weeks after the last vaccination, the guinea pigs of each group were shaved on their back and they were given a skin test with an injection intradermal of 0.1, 1.0 and 10 μg of protein of 71 kilograms Daltones. 10 μg of pH buffer was used as a control and all injections were made using a total volume of 0.1 milliliters. After 24 hours the erythema and hardening diameters were measured with the results as shown in Table E below. Data are reported in terms of mean measurement values for the group ± standard error (SE) as determined using traditional methods.

Table E - O Erythema (mm) to 71 KD (Medium + SE) Guinea Pig Condition n Q-i μg í.o μg 10-0 q Immunized 4 6.5 ± 0.7 11.9 ± 1.4 18.9 ± 2.2 Controls 3 2.5 ± 1.4 5.0 ± 2.9 11.8 ± 2.1 Hardening (mm) to 71 KD (Medium + SE) Condition Guinea pig n o- μg i-Q μq 10.0 μq Immunized 5.5 ± 1.1 6.8 ± 1.1 11.6 ± 0.8 Controls 3 0.7 ± 0.7 3.7 ± 0.9 7.8 ± 1.0 The responses of the immunized animals were almost twice the response of the guinea pigs attacked with the pH regulator alone and were comparable to those attacked with extracellular proteins. identical to those used to immunize animals (data not shown).

"" ". To additionally confirm that the largely abundant extracellular product of 71 kilo Daltons produces immediate immune responses, the guinea pigs immunized with crude extracellular proteins were sacrificed and the proliferation of the splenic lymphocyte was measured in response to various concentrations of the 71 kilo Dalton protein. Following the protocol of Example 4, the lymphocytes were incubated with and without protein and then assayed for their ability to incorporate [3 H] thymidine., 7 The data are reported in terms of indices of stimulation calculated as in Example 4. In Table F below, the results of this aggression of 71 kilo Daltons are shown.

Table F Stimulation rates for 71 KD (Medium + SE) Condition Guinea Pig n 0.1 μg / ml 1.0 μg / ml 10.0 μg / ml Immunized 4 1.5 ± 0.1 2.3 ± 0.5 8.1 ± 2.2 0 Controls 2 1.7 ± 0.6 1.6 ± 0.4 2.5 ± 0.6 Stimulation indexes for EP (Medium + SE) Condition Guinea Pig n 0.1 μg / ml 1.0 μg / ml 10.0 μg / ml Immunized 4 1.5 ± 0.1 2.2 ± 0.3 5.3 ± 1.4 Controls 2 1.4 ± 0.2 1.5 ± 0.2 1.2 ± 0.1 '"*'" - As shown in Table F, the stimulation indices for the lymphocyte proliferation assay were comparable to the results obtained in the cutaneous hypersensitivity test. Both the samples tested of 71 5 kilo Daltons in gross and the extracellular proteins showed responses between two and three times greater than those obtained with the controls indicating that the extra abundant extracellular product of 71 kilo Daltons is able to provoke a immune response mediated by cells "r in animals immunized with extracts of Ai. tuberculosis However, it should again be emphasized that the mostly abundant or major extracellular product is free from the problems associated with the prior art or raw compositions and adapts more rapidly to the Synthetic and commercial production making the vaccines of the present invention superior to the prior art. More particularly, the crude preparation can not be easily fabricated on a large scale by modern biomolecular techniques. Any commercial production of These crude unrefined preparations containing all the extracellular products would include the cultivation of large quantities of the target pathogen or a closely related species and harvest the resulting supernatant fluid. Such production methodology is highly susceptible to contamination by the pathogenic agent, toxic by-products or other parasitic agents. In addition, the large amount of immunogenic determinants in such a preparation is much more likely to cause a toxic immune reaction in a susceptible segment of the immunized population. 5 The use of these raw unrefined preparations also nullifies the use of the most popular skin tests currently used for the classification and control of tuberculosis. In direct contrast, vaccines of the present? < The invention can be mass produced with relative safety using high-throughput transformed hosts. Similarly, the vaccines of the present invention can be produced in identical batches, easy to standardize as opposed to the more widely variable production of the raw extracellular products. On the other hand, since the number of immunogenic determinants presented to the host immune system is relatively small, toxic reactions and the possibility of invalidating common classification tests are greatly reduced. EXAMPLE 7 Protein Skin Test 71 KD Purified from 71 KD Immunized Guinea Pigs The following demonstrates that the largely abundant extracellular product of 71 kilo Daltons generates a cell-mediated immune response in animals immunized with crude extracellular proteins. , it was shown that the purified form of this largely abundant product was z-capable of inducing an immune response mediated by cells 5 in animals immunized with 71 kilo Daltons. The guinea pigs were vaccinated twice with 100 μg of purified 71 kilo Dalton protein in Syntex adjuvant formulation at a three week interval. The control animals were immunized falsely with l < and pH regulator in adjuvant formulation of Syntex with this same schedule. Three weeks after the last immunization, both groups of animals were attacked intradermally with 1 and 10 μg of isolated 71 kilo Dalton protein. After 24 hours the erythema and the hardening were measured results with the results shown in Table G below.

Table G Erythema (mm) at 71 KD (Medium + SE) Condition of the Guinea Pig n 0.1 μg 1.0 μ 10.0 μq Immunized 3 O ± O 6.5 ± 1.5 15.0 ± 1.5 Controls 3 O ± O 2.7 ± 1.3 6.7 + 1.3 Hardening (mm) to 71 KD (Medium + SE) Condition Guinea pig n 0.1 μg 1.0 μg 10.0 μq Immunized 3 O ± O 3.0 ± 1.0 9.3 ± 0.3 Controls 3 O ± OO ± O 1.3 ± 1.3 The extent of hardening and erythema was much greater in the immunized animals than in the non-immunized control animals, demonstrating that a strong cell-mediated immune response to the 71 kilo Dalton protein by the vaccination protocol of the present invention. To further confirm the ability of this abundant extracellular product to induce an effective immune response by itself in accordance with the teachings of the present invention, lymphocyte proliferation assays were performed. The immunized animals were sacrificed as in Table G and spleen lymphocyte proliferation assays were run using the protocol set forth in Example 4. The tissue samples of the guinea pigs immunized with 71 kilo Daltons and those of the guinea pigs were assaulted. Indies of control with 0.1, 1 and 10 μg / milliliter of protein of 71 kilograms Daltones and their capacity to incorporate [3H] thymidine was verified. Stimulation rates were calculated as described above. In Table H below, the X results of these tests are shown.

Table H Stimulation indexes for 71 KD (Medium + SE) Condition Guinea Pig n 0.1 μg / ml 1.0 μg / ml 10.0 μg / ml Immunized 3 4.0 ± 1.3 5.6 ± 2.5 12.2 ± 5.1 Controls 3 1.3 ± 0.3 1.3 ± 0.3 3.2 ± 1.5 As with the cutaneous hypersensitivity test **, animals immunized with 71 kilo Daltons showed a much larger response of 71 kilo Daltons purified that the controls immunized in false. Although a foreign protein was expected, such results clearly show that a largely abundant extracellular product has the ability to induce a cell-mediated immune response. After establishing that a largely abundant extracellular protein will induce an effective cell-mediated immune response, further experiments were conducted to confirm that any such response is cross-reactive against tuberculosis bacilli as follows.

Example 8 Protein Aggression 71 KD Purified from Guinea Pigs 5 from Indians Infected with Ai. Tuberculosis Guinea pigs not immunized with M. tuberculosis dispersed by aerosol were infected as reported in Example 4. A purified protein derivative (PPD-CT68; Connaught Laboratories, Ltd.) was used as the positive control to ensure that the animals infected will demonstrate a cell-mediated immune response indicative of? f. tuberculosis Widely used in the Mantoux test for exposure to tuberculosis, PPD is generally prepared by fractionation of ammonium sulfate and comprises a mixture of small proteins having an average molecular weight of approximately 10 kilo Daltons. The immune responses to PPD are substantially analogous to those elicited by the fractions of crude extracellular proteins isolated in Example 1. Three weeks after infection, guinea pigs were challenged intradermally with 0.1, 1 and 10 μg of the extracellular protein of 71 kilo Dalton mostly abundant purified. The uninfected animals used as controls with the isolated protein were similarly assaulted. After 24 hours, the extent of erythema and hardening was measured with the results reported in Table I below.

Table I Erythema (mm) at 71 KD (Medium + SE) Condition Guinea pig n 0.1 μg í.o μq iQ.o μg Immunized 9.5 ± 1.7 13.4 ± 1.3 19.7 ± 1.3 Controls 2.3 ± 2.3 3.5 ± 2.2 7.8 + 1.9 Hardening (mm) at 71 KD (Medium + SE) Condition Cone- • -. or jillo de Indias n 0.1 μg 1.0 μg 10.0 μg Immunized 7 5.3 ± 1.8 8.7 ± 1.6 13.4 ± 1.1 Controls 6 O ± O 0.8 ± 0.8 0 + 0 As shown in Table I, they are present strong immune responses in the infected animals assaulted with the exemplary purified abundant extracellular protein of the present invention. These answers are in the order of three to four times larger for the - Erythema and more than 10 times larger for hardening than those of the uninfected animals, confirming that the 71 kilo Dalton extracellular protein prominently induces a strong immune response mediated by cells in animals infected with Af. Tuberculosis To corroborate these results additionally, The infected animals and the uninfected animals were sacrificed and subjected to a lymphocyte proliferation assay in accordance with the protocol of Example 4. Tissue samples from both groups of guinea pigs were tested against 0.1, 1 and 10. μg / milliliter of protein of 71 kilograms Dalton isolated and PPD. The samples were then checked for their ability to incorporate [3 H] thymidine as described above with the results of this test presented in Table J below. Table J Stimulation rates for 71 KD (Medium + SE) Condition Guinea pig n 0.1 μg / ml 1.0 μg / ml 10.0 μg / ml Immunized 3 2.4 ± 0.5 6.2 ± 1.8 29.1 ± 16.2 Controls 3 1.1 ± 0.1 2.6 ± 0.8 18.2 ± 6.1 Stimulation indexes for PPD (Medium + SE) Condition Guinea Pig n 0.1 μg / ml 1.0 μg / ml 10.0 μg / ml Immunized 3 4..0 ± 0.1 4.0 ± 1.5 11.4 ± 3.4 Controls 3 0.9 ± 0.2 0.9 ± 0.03 3.2 ± 1.5 As with the results of the skin sensitivity test, Table J shows that the stimulation rates were much higher for the infected tissue than for the uninfected samples. More specifically, the mean peak stimulation index of infected animals was 2 times higher than that of the 71 kilogram Dalton protein and 3 times higher than the PPD than it was for the uninfected controls, confirming that the strong response cell-mediated immune is induced in animals infected with M. tuberculosis by the exemplary copious extracellular protein vaccines of the present invention. Following this demonstration of cross-reactivity between the mostly abundant 71 kilogram purified Daltones protein and the? F. tuberculosis, further experiments were performed to demonstrate that an effective immune response could be stimulated by these exemplary purified samples of the largely abundant extracellular products as described by the present invention. Example 9 Aggression of Guinea Pigs Immunized with 71 KD Con? F. TB Dispersed by Aerosol To demonstrate the immunoprotective capacity of the mostly abundant or major extracellular protein vaccines, guinea pigs were immunized twice, with an interval of three weeks, with 100 μg of the 71 kilogram Dalton protein, mostly abundant exemplary, purified according to Example 2. All the animals were immunized using the adjuvant of the adjuvant formulation of Syntex Three weeks after the last immunization, the guinea pigs immunized with the 71 kilogram Dalton protein were subjected to a skin test with 10 μg of the material to assess whether a cell-mediated immune response had developed or not. The control animals and guinea pigs immunized with 71 kilo Daltons with Af were then infected. tuberculosis dispersed by aerosol as detailed in Example 4. After infection, the animals were checked and weighed for six months. The graph of Figure 5 contrasts the weight loss experienced by the falsely immunized group with the relatively normal weight gain shown by the animals immunized with 71 kilo Daltons and the crude extracellular proteins. The data are the average weights ± standard error for each group. The mortality curves for the same animals are shown in the graph of Figure 6. In Table K below, the absolute mortality averages are reported for the study. Table K Condition of Survivors / Percentage Guinea Pigs Assaulted Survival Immunized 71 KD 3/6 50% Immunized EP 5/8 62.5% Immunized in False 0/6 0% Both the weight loss curves and the mortality averages clearly show that the mostly abundant extracellular ~~ proteins of the present invention confer a prophylactic immune response. This is emphasized by the fact that 100 percent of the non-immunized animals died before the end of the verification period. Example 10 Aggression of Guinea Pigs Immunized with 71 KD With Af. TB Dispersed by Aerosol A similar experiment was conducted to verify the results of the previous Example and showed that the administration of an exemplary principal extracellular protein can confer a protective immune response in animals. In this experiment, guinea pigs were again immunized Indies three times, with an interval of 3 weeks, with 100 μg of the extracellular protein of 71 kilograms Daltones in adjuvant formulation of Syntex. They were immunized in false guinea pigs Indies of control with pH regulator in adjuvant formulation of Syntex. Three weeks after the last immunization, the animals were assaulted with Af. tuberculosis dispersed by aerosol and weighed weekly for 13 weeks. Mean weights ± standard error for each group of 6 guinea pigs were calculated and plotted in Figure 7. This curve shows that animals falsely immunized lost a considerable amount of weight during the verification period while immunized animals They maintained a clearly consistent body weight. Since the loss of body mass or "wasting" is one of the classic side effects of tuberculosis, these results indicate that the growth and proliferation of the tuberculosis bacillus was inhibited in animals immunized by means of the exemplary vaccine of the present invention. Protective immunity having been developed in guinea pigs through vaccination with an abundant extracellular product in an isolated form, experiments were carried out to demonstrate the interspecies immunoreactivity of the vaccines of the present invention and to further confirm the validity and applicability of the guinea pig model.

EXAMPLE 11 Human Immunity-mediated PPD Positive PPD Test With Purified 71 KD Protein To assess the cell-mediated component of a human immune response to the largely abundant protein of 71 kilo Daltons, the proliferation of peripheral blood lymphocytes was studied from PPD-positive and PPD-negative individuals to the protein in the standard lymphocyte proliferation assay as reported in Example 4 above. A positive PPD response, or tuberculin response, is well known - "". in the technique as being indicative of a prior exposure to Ai. tuberculosis The proliferative response and the corresponding incorporation of [3 H] thymidine were measured at two and four days. The data for these studies are shown in Figures 8A and 8B. Figure 8A shows the response at different levels of 71 kilo Daltons after two days, while Figure 8B shows the same responses at four days. ^ As illustrated in Figures 8A and 8B, the index of ± 0 mean peak stimulation of the positive PPD individuals was twice as high for the 71 kilo Dalton protein and three higher folds for the PPD than that of the negative PPD individuals. Among the positive PPD individuals, there was an interlinear correlation between the peak stimulation indices for the 71 kilo Dalton protein and for the PPD, demonstrating that a strong cell-mediated response is stimulated by the most prominent or largely abundant extracellular products of M. tuberculosis in humans previously exposed to M. tuberculosis. These dates correspond to the profile of reactivity observed in guinea pigs and confirms the applicability of the guinea pig model to other mammals subjected to infection. In this way, as well as with the exemplary protein of 30 kilo Dalton discussed above, the development of a strong immune response to the 71 kilo Dalton extracellular product mostly abundant demonstrates the broad scope of the present invention as evidenced by the fact that the 71 kilo Dalton product is also effective in stimulating cell-mediated immunity in humans. Again, it should be emphasized that the present invention is not limited to the extracellular products of Ai. tuberculosis or to the use of the protein of 71 kilograms Daltones. Rather, the teachings of the present invention are applicable to any extracellular product that is largely abundant as demonstrated in the examples. Additional studies were carried out to be able to ascertain whether the combinations of the extra-abundant extracellular products of? F. tuberculosis would also provide protective immunity. In general, these studies used guinea pigs that had been immunized three times, either intradermally or subcutaneously with different doses of vaccines comprising combinations of purified extracellular proteins of M. tuberculosis in adjuvant formulation of Syntex, with intervals of 3. or 4 weeks. The first protein combination used by the immunization process, labeled Combination I, consisted of proteins of 71 kilograms Dalton, 32 kilo kilograms Dalton, 30 kilo Daltons, 23 kilo Daltons, and 16 kilo Daltons purified of f "conformity with the protocols described in Example 2. It is believed that this combination comprises up to 60 percent of the total extracellular protein normally present in culture supernatants of tuberculosis. selected for use in Combination I, are identified with an asterisk in Figure 2. The vaccine was administered to the Combination I containing 100 μg, 20 μg, or 2 μg of each protein intradermally with the adjuvant of the Syntex adjuvant formulation. It was also administered -x Subcutaneously the vaccine of Combination I that contained μg of each protein in similar experiments. False guinea pigs were immunized falsely with equivalent volumes of Syntex adjuvant formulation and pH regulator at the same time while positive controls were immunized using 120 μg of the crude extracellular protein preparation of Example 1 in Syntex adjuvant formulation. All injection volumes were standardized using pH regulator.

Example 12 Response of the Guinea pigs of Combination I to Aggression with the Combination Vaccine I A cutaneous hypersensitivity test was performed to determine whether the animals had developed a immune response that could be measured after vaccination with the mixture of Combination I of the major extracellular products. The guinea pigs were shaved on the back and injected intradermally with 1.0 μg and 10.0 μg of the same combination of the five purified extracellular proteins. 10.0 μg of pH buffer was used as a control and all injections were made using a total volume of 0.1 milliliter. At 24 hours after the injection, the erythema and hardening diameters were measured at the skin test sites. The results of the measurements are presented in Table L below. The data is reported again in terms of the mean measurement values for the group ± standard error (SE) as determined using traditional methods. ND indicates that this particular aspect of the experiment was not done.

Table L Erythema (mm) (Medium + SE) Condition Guinea pig n P PDD 1.0 μg 10.0 μg Immunized 6 0 11.4 ± 4.6 17.4 ± 2.6 Controls 6 0 ND 6.0 ± 0.5 Hardening (mm) (Medium + SE) Condition Guinea pig n PD 1.0 μg 10.0 μg Immunized 6 0 7.3 + 0.8 11.6 ± 1.2 Controls 6 0 ND 4.2 ± 0.3 The data clearly demonstrate that a strong cell-mediated immune response was generated to the extracellular proteins of Combination I by the vaccinated animals. The immunized guinea pigs showed measures of erythema and hardening almost three times higher than the control animals. t * 5 'Example 13 Immunoprotective Analysis of the Vaccine of Combination I Against Af. TB Dispersed by Aerosol Three weeks after the last immunization, guinea pigs used for the test of previous hypersensitivity with Af. tuberculosis of the Erdman strain dispersed by aerosol, and weighed weekly for 10 weeks. This aerosol aggression was performed using the protocol of Example 4. They were assaulted in a manner - Simultaneously six animals immunized with 100 μg of the main extracellular products of Combination I, together with groups with the same number of positive and negative controls, with? F. Tuberculosis dispersed by aerosol, positive controls were immunized three times with 120 μg of extracellular proteins in adjuvant formulation of Syntex.

An autopsy was performed on guinea pigs who died before the end of the observation period and were examined for evidence of serious tuberculosis injuries. These lesions were found in all the animals that died during the study. The differences between the immunized and control animals in the average weight profiles after the aerosol aggression were analyzed by repeated measures variation analysis (ANOVA). The differences between the guinea pigs immunized and control in terms of survival after the aggression were analyzed by the exact Fisher's test of two extremities. The data are the average weights ± standard error (SE) for each group of six guinea pigs. Figure 9 shows the results of the weekly weight determinations after the aggression. In Compared with guinea pigs immunized with the combination of extracellular products, the animals immunized in fake lost 15.9 percent of their weight ___ total body The weights of the positive controls were similar to those of the animals immunized with the combination of five purified extracellular proteins. Body weights were normalized immediately before the assault. The difference between the animals immunized with Combination I and the controls immunized in false was significantly higher with p < .0000001 by repeated ANOVA measures.

The mortality was determined ten and a half weeks after the attack. The three animals falsely immunized died at an interval of three days between each one between ten and ten and a half weeks after the aggression. Table 5 below gives the results of the mortality of the experiment.

Table M Condition of Survivors / Percentage i * Guinea pigs Survived Survival Immunized with Comb. 6/6 100% Immunized with EP 5/6 83% Immunized in False 3/6 50% After the conclusion of the verification study of weight, the surviving animals were sacrificed by means of hypercarbia and the right lung and the spleen of each animal are tested for searching. viable tuberculosis using the protocol of Example 5. Table N below shows the results of the counts, including the 3 animals that died the last week of the experiment, in terms of training units of 'colony (CFU) means ± standard error (SE). Table N CFU means + SE Condition Guinea pig Right Lung Sick Immunized in False 8.9 + 5.4 xlO '1.3 ± 0.7 xlO7 Immunized 6 3.4 ± 1.7 xlO60 i1.8o ±, n0. c6 Immunized with EP 6 1.7 ± 0.7 xlO7 5.0 ± 2.8 x 106 The difference in the record between the concentration of bacilli in the lungs of animals immunized with the combination of purified proteins and that of animals immunized in false was 1.4 while the difference in the bacilli in the spleen was 0.9. Making a parallel of this, in the autopsy inspection, the immunized animals had a markedly reduced involvement of the lung with tuberculosis compared to the controls falsely immunized. The positive control animals immunized with the crude extracellular preparation (EP) of the Example 1 showed a record of 0.7 more bacilli in the lung and a record of .5 more bacilli in the spleen than animals immunized with the mixture of Combination I of purified extracellular proteins.

Example 14 Vaccine Immunoprotection Analysis of Combination I at Low Doses by Intradermal and Subcutaneous Transfer While Example 13 confirmed that Combination I proteins demonstrated immunoprotection in animals immunized 3 times intradermally with 100 μg of each protein ( 30 + 32A + 16 + 23 + 71), with an interval of 4 weeks, an alternative study was conducted to demonstrate the immunoprotective capacity of lower doses of the Combination I proteins, specifically 20 μg or 2 μg of each protein. As in Example 13, guinea pigs (6 animals per group) were immunized with Combination I proteins (30 + 32A + 16 + 23 + 71) intradermally in an adjuvant formulation of Syntex with a range of 3 weeks. The animals were already receiving 20 μg r-. of each protein per immunization or 2 μg of each protein per i. immunization. They were immunized in false control animals using the previous protocol. Three weeks later, the animals were assaulted with? F. tuberculosis dispersed by aerosol and weights were measured weekly for 9 weeks. All the immunized animals survived until the end of the experiment while a falsely immunized animal died before the completion of the experiment. As illustrated by the following results, doses 5 times and even 50 times lower than those of Example 13, protected the immunized animals from the? F. tuberculosis dispersed by aerosol and that the intradermal and subcutaneous transfer was effective. Compared with guinea pigs immunized with 20 μg of each protein of Combination I, the animals immunized in fake lost 12 percent of their weight total body during the 9 weeks of the experiment (the weights were normalized until just before the aggression). Compared with guinea pigs immunized with 2 μg of each Combination I protein, the animals immunized falsely lost 11% of their normalized total body weight. In this way, guinea pigs immunized intradermally with low doses of Combination I proteins were protected against weight loss after aerosolization with M. tuberculosis. Similarly, guinea pigs immunized intradermally with low doses of Combination I proteins were also protected against splenomegaly associated with the spread of Af. tuberculosis in the spleen. As shown in Table 0, while animals immunized with 20 μg or with 2 μg of each protein of combination I have spleens weighing an average of 4.6 ± 1.2 grams and 4.0 ± 0.8 grams (Mean ± SE), respectively , animals falsely immunized had spleens weighing an average of 9.6 ± 1.8 grams (Table 1), or more than twice that amount.

Table O Condition of Spleen Connections (g) from India n Medium + SE Immunized in False 5 9.6 ± 1.8 Immunized (20 μg) 6 4.6 ± 1.2 Immunized (2 μg) 6 4.0 ± 0.8 Guinea pigs immunized intradermally with low doses of the proteins of the Combination I also had fewer colony forming units of? F. tuberculosis in his spleens. As shown in Table P, when compared to animals falsely immunized, guinea pigs immunized with 20 μg or with 2 μg of each protein of Combination I, had an average of 0.6 and 0.4 less than units of colony formation, respectively, in their spleens.

Table P Condition ConeCFU in spleen Difference of Indian jelly n Medium + SE Immunized record in False 5 3.1 ± 2.3 x 106 Immunized (20μg) 6 8.1 ± 2.4 x 105 -0.6 Immunized (2μg) 6 1.2 ± 0.6 x 106 -0.4 Moreover , guinea pigs immunized subcutaneously with the Combination I proteins were also protected against weight loss, splenomegaly, and growth of M. tuberculosis in the spleen.

In the same experiment described in Example 14, guinea pigs were also immunized four times subcutaneously, rather than intradermally with 20 μg of the Combination I proteins, with a range of 3 weeks. These animals were protected from aggression "'" almost as much as animals immunized intradermally with 20 μg of the Combination I proteins.

Example 15 Response of Guinea Pigs Immunized with Combination I and Combination II to Aggression with Combination I and Combination II Additional studies were conducted to confirm whether other extracellular combinations of mostly abundant Ai. tuberculosis would also provide protective immunity. One study used guinea pigs that were immunized with a vaccine that comprised two combinations - Combination I (71, 32A, 30, 23 and 16) and Combination II (32A, 30, 24, 23 and 16). It is believed that Combination II comprises up to 62 percent of the total extracellular protein normally present in Ai supernatants. tuberculosis Animals were immunized (6 per group) four times with 100 μg of each Combination I or II protein in Syntex adjuvant formulation, with an interval of 3 weeks. The negative control animals were falsely immunized with equivalent volumes of Syntex adjuvant formulation and pH regulator at the same time. As in Example 12, the animals were tested for delay type hypersensitivity to determine whether the animals developed an immune response that could be measured after vaccination. The animals immunized with Combination II had an erythema of 16.8 ± 1.3 millimeters (Mean ± SE) and a hardening of 12.8 ± 1.2 millimeters in response to the skin test with Combination II while the animals immunized falsely had an erythema of only 1.3 ± 0.8 millimeters and a hardening of 0.3 ± 3 millimeters in response to Combination II. In this way, animals immunized with Combination II had more than 12 times more erythema and more than 40 times more hardening than controls. By way of comparison, animals immunized with Combination I had an erythema of 21.3 ± 2.0 millimeters and a hardening of 2.6 ± 0.7 millimeters in response to Combination I. In this way, animals immunized with Combination I had more than 3 times more erythema and more than 6 times more hardening than controls. The difference of the controls for the proteins of the Combination II was even greater than that for the proteins of the Combination I. In the same experiment, the animals immunized with lower doses of the proteins of the Combination II (20 μg of each protein against 100 μg) also develop a strong cutaneous hypersensitivity to Combination II. These had an erythema of 21.0 ± 2.0 millimeters and a hardening of 15.3 ± 0.9 millimeters in response to Combination II, while the animals immunized in false had an erythema of only 1.3 ± 0.8 millimeters and a hardening of 0.3 ± 0.3 millimeters, as It was noticed earlier. In this way, the animals immunized with a lower dose of the Combination II proteins had more than 16 times more erythema and more than 50 times more hardening than the controls, a difference that was even greater than that of the animals immunized with the highest dose of Combination II proteins.

Example 16 Immunoprotective Analysis of the Vaccine of Combination I and II Against Af. TB Dispersed by Aerosol Three weeks after the last immunization, the guinea pigs that were used for the previous hypersensitivity test with Ai were attacked. aerosol-dispersed tuberculosis, strain Erdman as in Example 13 and weighed weekly for 7 weeks. As in Example 13, 6 animals were used in each group. During the first 7 weeks after the assault, animals falsely immunized lost an average of 19.5 grams. In contrast, animals immunized with Combination II (100 μg of each protein) gained 52.4 grams and animals immunized with Combination II with smaller doses, (20 μg of each protein), gained an average of 67.2 grams. By way of contrast, the animals immunized with Combination I gained 68 grams. Thus, compared to guinea pigs immunized with Combination II (100 μg), animals falsely immunized lost 11% of their total body weight. Compared with guinea pigs immunized with Combination II at a smaller dose (20 μg), animals falsely immunized lost 14% of their total body weight. Compared with the animals immunized with Combination I, animals immunized falsely also lost 14% of their total body weight.

Example 17 Response of Immunized Guinea Pigs with Combinations III to XII to Aggression with the Same Vaccine or Its Components Additional experiments were performed to demonstrate the effectiveness of different combinations of mostly abundant extracellular products of Af. tuberculosis In these studies, Hartley-type guinea pigs were immunized in an intradermal manner with vaccines comprising combinations of 2 or more mostly abundant extracellular products purified as in Example 2. The purified extracellular products are identified using their apparent molecular weight as determined by the sodium dodecyl sulfate-polyacrylic amide gel electrophoresis. The guinea pigs were immunized with the following combinations of mostly abundant extracellular products.

Combination Constituents of Protein III 30 + 32A + 32B + 16 + 23 IV 30 + 32A V 30 + 32B VI 30 + 16 VII 30 + 23 VIII 30 + 71 IX 30 + 23.5 X 30 + 12 XI 30 + 24 XII 30 + 58 Each combined vaccine included 100 μg of each protein listed. The combined vaccines were volumetrically adjusted and injected intradermally into the adjuvant of the Syntex adjuvant formulation. As was done previously, the guinea pigs were immunized four times, with an interval of three weeks. A cutaneous hypersensitivity assay was performed to determine if the animals had developed an immune response that could be measured after vaccination with Combinations III through XII. Groups of 6 guinea pigs were shaved on the back and injected intradermally with the same combination of purified extracellular products to which they were immunized. For this aggression, 10 μg of each of the proteins in the combination was injected. All injections were carried out using a total volume of 0.1 milliliter. The fake immunized controls, which had been immunized only with the adjuvant formulation of Syntex, were also subjected to the skin test with Combinations III to XII, again using 10 μg of each protein in the respective combination. The diameters of erythema and hardening were measured at the skin test sites, 24 hours after the injection as described in Example 3. In Table Q below, the results of these measurements are presented. Data are reported again in terms of mean measurement values for the group ± standard error (SE) as determined using traditional methods. Table Q Diameter of the Skin Reaction (mm) Combination Combination Test Skin Erythema Hardening III I I I 12. 2 ± 2. 0 6 8 ± 0. 8 IV IV 9. 9 ± 0. 5 6. 3 ± 0. 2 V V 13.0 ± 1.1 8.1 ± 0.7 VI VI 19.2 ± 1.2 12.4 ± 0.5 VII VII 14.3 ± 1.0 8.7 ± 0.4 VIII VIII 18.9 ± 1.1 12.6 ± 0.8 IX IX 17.0 ± 0.9 12.1 ± 0.9 X X 19.3 ± 1.4 13.6 ± 1.2 XI XI 18.3 ± 1.2 12.4 ± 0.8 XII XII 17.7 ± 0.9 14.0 ± 1.2 E Enn ffaallssoo 4.8 ± 0.9 2.0 ± 0.0 False IV 4.3 ± 1.1 2.0 ± 0.0 False V 5.0 ± 0.5 2.0 ± 0.0 Fake VI 4.5 ± 0.3 2.0 ± 0.0 False VII 4.5 ± 0.3 2.0 ± 0.0 E Enn ffaallssoo 3.3 ± 0.3 2.3 ± 0.3 False IX 3.7 ± 0.3 2.0 ± 0.0 False X 3.7 ± 0.4 2.0 ± 0.0 False XI 3.7 ± 0.2 2.0 ± 0.0 False XII 3.8 ± 0.2 2.0 ± 0.0 The results clearly demonstrate that a strong cell-mediated immune response was generated to each of the combinations of the purified extracellular proteins.

The immunized guinea pigs showed erythema at least twice and usually three times or more that of the controls for all combinations. In addition, the immunized guinea pigs showed hardening of at least 3 times those of the controls for all combinations.

Example 18 Immunoprotective Analysis of Combinations III-XII Contra Af. TB Dispersed by Aerosol To demonstrate the prophylactic efficacy of these exemplary combinations of purified extracellular products, guinea pigs immunized with Combinations III to XII were attacked with Af. tuberculosis three weeks after the last immunization using the protocol of Example 4. Consistent with the above results, all guinea pigs immunized with Combinations III to XII were protected against death after aggression. At 4 weeks after the attack, 2 or 6 animals immunized in false (33 percent) died, compared to 0 animals in the groups immunized with Combinations IV-XII and 1 or 6 animals (17 percent) in the group immunized with Combination III. At 10 weeks after the assault, 50 percent of the animals falsely immunized had died compared to 0 deaths in the animals of the groups immunized with Combinations IX and XII (0 percent), 1 in 6 deaths ( 17 percent) in the animals of the groups immunized with Combinations III, IV, V, VI, X and XI, 1 of 5 deaths (20 percent) in animals immunized with Combination VIII, and 2 of 6 deaths ( 33 percent) in animals immunized with Combination VII. The guinea pigs that died before the end of the observation period were examined and examined for evidence of injuries of serious tuberculosis. Injuries were found in all the animals that died during this study. After the conclusion of the mortality study, the surviving animals were sacrificed by , < - hypercarbia and the spleens of each animal were tested to search? f. viable tuberculosis using the protocol of Example 5. The results are presented in Table R below in terms of colony formation units (CFU) means together with the decrease of registration of animals immunized in false. An asterisk next to the CFU values indicates that the spleen counts were zero in one animal in each group. For calculation purposes, zero counts were treated as 103 units of colony formation per spleen or 3 records.

Table R Group Vacuna CFU in Spleen Decrease Reg (Average Record) of False III 5.99 .5 IV 5.41 1.1 V 6.27 .3 25 VI < 5.80 * > .7 VII < 5.61 * > .9 VIII 6.47 .1 IX < 5.85 * > .7 X < 5.74 * > .8 XI 5.93 .6 XII 6.03 .5 False 6.53 Animals immunized with Combinations III, IV, VI, VII, IX, X, XI and XII had at least one record 0.5 less colony formation units of? F. tuberculosis in its spleens on the average of the controls immunized in false. In particular, combinations IV and VII proved to be especially effective, reducing the average number of colony formation units by approximately a factor of ten. Animals immunized with Combinations V and VIII had 0.3 and 0.1 fewer colony forming units (CFU), respectively, in their spleens on average, than controls falsely immunized. This dramatic reduction in colony forming units in accordance with the teachings of the present invention once again illustrates the immunoprotective operability of the present invention.

Example 19 Response of the Immunized Guinea Pigs with 3 Different Dose of the Combination XIII to an Aggression with the Combination XIII Guinea pigs were immunized to further define the operability and scope of the present invention as well as to demonstrate the effectiveness of the additional combinations of purified extracellular products, as was done previously using alternative vaccination doses. Specifically, 50 μg, 100 μg and 200 μg of an alternative combination of 3 mostly abundant extracellular products identified as Combination XIII and comprising proteins of 30 kilo Daltons, 32 (A) kilo Daltons and 16 kilo Daltons. As in the previous examples, groups of animals were immunized four times intradermally, with an interval of 3 weeks with the alternative doses of Combination XIII in the adjuvant formulation of Syntex. A cutaneous hypersensitivity assay was performed to determine if the animals had developed an immune response that could be measured after vaccination. The animals were shaved on the back and injected intradermally with Combination XIII containing 10.0 μg of each of the purified extracellular products. All injections were made using a total volume of 0.1 milliliter. The fake immunized controls were also skin tested with the same dose of Combination XIII. Erythema and hardening diameters were measured at the skin test sites 24 hours after the injection. The results are presented in Table S below in terms of mean measurement values for the group ± standard error (SE) as determined using traditional methods.

Table S Skin Reaction Diameter (mm) Compression Dose Vaccine Erythema Hardening Vaccine (μq) XI I I 50 17. 8 ± 1. 3 13.2 ± 1.0 XI I I 100 11. 2 ± 0. 9 7.3 ± 0.4 XI II 200 10. 0 ± 0. 7 7.0 + 0.4 False 0 5.7 ± 0.5 0.2 ± 0.2 Once again, these results clearly demonstrate that a strong immune response mediated by cells was generated to Combination XIII in animals immunized with each of the three doses of Combination XIII. The immunized animals exhibited erythema approximately two or three times than the controls. Even more impressive, the immunized animals exhibited a hardening of at least 35 times that of the control animals that exhibited minimal response in all cases.

Example 20 Immunoprotective Analysis of Combination XIII in Three Different Dose Contra? F. Aerosol Dispersed Tuberculosis To further demonstrate aspects of the protective immunity of the vaccines of the present invention at different doses, immunized guinea pigs (6 per group) used for the above cutaneous hypersensitivity assay were assaulted with? f. tuberculosis dispersed by spray three weeks after the last immunization. The aerosol aggression was performed using the protocol detailed in Example 4. A control group of 12 falsely immunized animals was simultaneously attacked. Figure 10 shows graphically the results of the weekly weight determinations after the aggression and shows in a distinctive way that guinea pigs immunized with each of the three doses of Combination XIII were protected against weight loss. . Animals immunized with the highest doses of Combination XIII (100 to 200 μg) actually showed a net gain in weight and animals immunized with the lowest doses (50 μg) showed a relative weight loss. In contrast, animals falsely immunized lost approximately 22 percent of their total body weight in the weeks immediately after the attack and averaged a loss of 182 grams over the 10-week observation period. Table U below illustrates the weight change percentage for the immunized and control animals as determined by taking the average weight at the end of the aggression, subtract the average weight at the beginning of the aggression and divide the result by the average weight at the beginning of the aggression. Similarly, percent protection was determined by subtracting the average weight loss percent of controls from the percent gain or mean weight loss of immunized animals.

Table U Immunogen Dose% Change Weight% Weight Loss Protection Combination XIII 50 -4% 18% Combination XIII 100 + 7% 29% Combination XIII 200 + 5% 27% Fake At -22% false Table U shows that animals immunized falsely lost a considerable amount of weight (18 percent - 29 percent) during the verification period compared to immunized animals. Figure 10 provides a more graphical illustration of net weight loss for each group of animals immunized against animals immunized in counterfeit at weekly intervals during the ten week verification period. Since loss of body mass or "wasting" is one of the classic side effects of tuberculosis, these results indicate that the growth and proliferation of the tuberculosis bacillus in the immunized animals was inhibited by the three different doses of the vaccine of exemplary combination of the present invention.

Example 21 Immunoprotective Analysis of Combinations XIV-XVIII against Aggression with Combinations XIV-XVIII To further demonstrate the scope of the present invention and the wide range of effective vaccines that can be formulated in accordance with the teachings thereof, they were tested five additional combination vaccines, Combinations XIV to XVIII, in guinea pigs. Identified by the apparent molecular weight of purified extracellular products determined by the use of sodium dodecyl sulfate-polyacrylic amide gel electrophoresis, the composition of each of the combination vaccines is given below.

Combination Constituents of Protein XIV 30, 32A, 16, 32B, 24, 23, 45 XV 30, 32A, 16, 32B, 24, 23, 45, 23.5, 12 XVI 30, 32A, 16, 32B, 24, 23 XVII 30, 32A, 16, 32B, 24, 71 XVIII 30, 32A, 32B I 30, 32A, 16, 23, 71 In addition to the new combination vaccines and appropriate controls, Combination I was also used in this series of experiments Guinea pigs were immunized intradermally with 50 μg of each of the proteins of the XIV or XV combination and with 100 μg of each of the proteins of Combinations I, XVI, XVII, and XVIII, all in adjuvant of the formulation of Syntex adjuvant. The animals were immunized a total of four times, with an interval between each injection of three weeks. A cutaneous hypersensitivity assay was performed to determine whether the animals had developed an immune response that could be measured after vaccination using the protocol discussed above. The guinea pigs were shaved on the back and injected intradermally with the same combination of purified extracellular proteins to which they were immunized. The appropriate combination vaccine containing 10 μg was injected for each aggression. All injections were made using a total volume of 0.1 milliliter. The immunized controls were also subjected to a skin test with the same dose of each combination. The diameters of erythema and hardening were measured at the skin test sites at 24 hours after the injection as described in Example 3. In Table V below, the results of these measurements, reported in of mean measurement values for the group ± standard error (SE) as determined using traditional methods.

Table V Diameter of the Skin Reaction (mm) Combination Combination Test Skin Erythema Hardening XIV XIV 13.3 ± 0.7 9.1 ± 0.4 XV XV 10.4 ± 0.4 6.5 ± 0.4 XVI XVI 8.0 ± 1.8 5.1 ± 1.0 XVII XVII 9.4 ± 0.9 6.1 ± 1.1 XVIII XVIII 13.6 ± 1.2 8.7 ± 0.7 I I 10.0 ± 0.3 6.7 ± 0.2 False XIV 5.5 ± 1.6 0.4 ± 0.2 False XV 6.1 ± 0.5 0.4 ± 0.2 False XVI 4.6 ± 1.4 0.4 + 0.2 False XVII 5.7 ± 1.2 0.2 ± 0.2 False XVIII 2.1 ± 1.1 O ± O False I 6.0 ± 1.2 0.6 ± 0.2 These results clearly demonstrate that a strong cell-mediated immune response was generated to Combinations XIV to XVIII, and, as before, to Combination I. The immunized animals exhibited erythema approximately twice as much as the controls. Even more surprising, the f "> .- immunized animals exhibited a hardening at least 10 times higher than the fake immunized controls that exhibited minimal response in all cases.

Example 22 15 Immunoprotective Analysis of Combinations XIV-XVIII and the Combination I Against Af. TB Dispersed by Aerosol To confirm the immunoreactivity of the combination vaccines of Example 21 and to demonstrate their applicability for infectious tuberculosis, they were assaulted the immunized guinea pigs used for the previous hypersensitivity test, with? F. tuberculosis dispersed by aerosol three weeks after the last immunization and verification using the protocol of Example 4. A control group of .12 immunized animals was similarly changed. in false, the same as those used in Example 20. In Figure 11 the results of this aggression are plotted and are shown in Table W below. The weight change percent was determined by taking the average weight at the end of the aggression, subtracting the average weight at the beginning of the aggression and dividing the result between the average weight at the beginning of the aggression. Similarly, the percent protection was determined by subtracting the average percent of the weight loss of the controls from the average percent of gain or weight loss of the immunized animals.

Table W Immunogen% Change Weight% Protection of Weight Loss Combination XIV 3% 25% Combination XV - 4% 18% Combination XVI - 15% 7% Combination XVII - 11% 11% Combination XVIII - 12% 10% Combination I - 11% 11 % Fake -22Í As shown in Table W, the guinea pigs immunized with each of the combination vaccines were protected against weight loss. Animals falsely immunized lost approximately 22 percent of their total combined body weight. In contrast, the prophylactic effect of the combination vaccines actually resulted in weight gain for one of the test groups and a reduced amount of weight loss in the others.

Specifically, animals immunized with Combination XIV showed a 3% weight gain while animals immunized with the other combinations lost only 4% to 5% of their total combined weight. These results are shown graphically in Figure 11 which marks the weekly weight determinations in terms of net weight gain or loss for each group of animals after the spray dispersed aggression.

This statistically significant difference between the net weight loss for the immunized animals and the fake immunized controls shown in the Figure 11, provides additional evidence for the immunoprophylactic response generated by the combination vaccines of the present invention.

EXAMPLE 23 Cell Mediated Immunity in Guinea Pigs Immunized with Three Different Adjuvants In order to further demonstrate the broad applicability and versatility of the vaccine formulations of the present invention, immunogenic studies were carried out using different adjuvants, namely three different immunogens, 30-kilo Dalton proteins, Combination I (30, 32A, 16, 23, 71) and Combination XIII (30, 32A, 16) were each formulated using three different adjuvants, 5 Adjuvant Formulation of Syntex I (SAF) , Adjuvant Freunds Incomplete (IFA) and Lipid A Monophosphoryl containing adjuvant (MPL) It is generally known that these adjuvants increase the immune response of an organism when , _, administer with an immunogen. Guinea pigs were immunized intradermally with 100 μg of each protein containing Combinations I and XIII and about 100 μg of purified 30 kilo Dalton protein in each of the adjuvant formulations. The guinea pigs were immunized with each formulation a total of three times with injections at intervals of three weeks. After the immunization, a cutaneous hypersensitivity test was conducted to determine if the guinea pigs had developed an immune response that could be measure. The guinea pigs are shaved on the back and injected intradermally with the same immunogen to which they had been immunized. For the aggression, 10 μg of each protein was injected in Combinations I and XIII or 10 μg of protein of 30 kilograms Daltones purified in a total volume of 100 μl. The guinea pigs immunized in fake, vaccinated with one of these three adjuvants, were skin tested with each of the immunogenic formulations containing the same adjuvant. Erythema and hardening diameters were measured at the skin test sites 24 hours after the assault as described in Example 3. The results of these measurements are presented in Table X below. As discussed above, the data are reported in terms of mean measurement values for the group ± standard error as determined using accepted statistical techniques.

Table X Adjuvant vaccine Reac. Skin Test Dia. Reac. Skin (mm) Erythema Hardening 30 SAF 30 10.7 ± 1.6 5.8 ± 1.5 30 IFA 30 8.8 ± 0.7 4.6 ± 0.7 30 MPL 30 10.2 ± 1.7 5.3 ± 1.5 XIII SAF XIII 7.3 ± 0.5 4.1 ± 0.5 XIII IFA XI II 6.8 ± 0.9 3.5 ± 0.5 XIII MPL XIII 6.3 ± 0.4 3.4 ± 0.3 I SAF I 6.9 ± 0.6 4.0 ± 0.3 I IFA I 6.8 ± 0.2 3.6 ± 0.3 I MPL I 7.4 ± 0.4 3.9 ± 0.5 Fake SAF 30 0.7 ± 0.7 1.0 + 0 False IFA 30 O ± O 0 + 0 False MPL 30 O ± O 0 + 0 False SAF XIII 1.0 ± 1.0 1.0 ± 0 False IFA XIII O ± O 0.3 ± 0.3 False MPL XIII O ± O O ± O False SAF I 4.7 ± 0.3 1.0 ± 0 Ifi False IFA I 2.0 ± 1.0 0.7 + 0.3 False MPL I 1.0 ± 1.0 0.7 ± 0.3 As shown in the data presented in Table X, combination vaccines and extracellular products Purified of the present invention provide a strong immunogenic cell-mediated response when formulated with different adjuvants. On the other hand, each of the three adjuvants gave approximately the same immunogenic response for each respective immunogen. In Overall, the immunized guinea pigs showed erythema diameters of approximately seven to ten times those of the guinea pigs immunized falsely while hardening were approximately four to six times greater than those measured in the animals of control. The ability of the present invention to elicit a strong immunological response in combination with different "" "adjuvants facilitates the optimization of the vaccine. That is, the adjuvants used can be selected to produce effective vaccine formulations in accordance with the teachings herein, based largely on the consideration of secondary criteria such as stability, lack of side effects, cost and ease of storage. These and other criteria, not directly related to the stimulation of an immune response, are particularly important when developing formulations of vaccines for extended use under relatively primitive conditions.

Example 24 Immunoprotective Analysis of Combinations XIX-XXVIII 15 against Aggression with Combinations XIX-XXVIII The broad scope of the present invention was further demonstrated by the generation of an immune response using ten additional combination vaccines, Combinations XIX to XXVIII. In addition to the new combination vaccines and appropriate controls, Combinations IV and XIII were also used as positive controls to elicit an immune response in guinea pigs. Identified by the apparent molecular weight of the purified extracellular products determined using sodium dodecyl sulfate-25 polyacrylic amide gel electrophoresis, the composition of each of the combination vaccines is given below.

Combination C Co-constituents of Protein XIX 30, 32A, 23 XX 30, 32A, 23.5 XXI 30, 32A, 24 XXII 30, 32A, 71 XXIII 30, 32A, 16, 23 XXIV 30, 32A, 16, 23.5"1-J XXV 30, 32A, 16, 24 XXVI 30, 32A, 16, 71 XXVII 30, 32A, 16, 32B XXVIII 30, 32A, 16, 45 IV 30, 32A 15 XIII 30, 32A, 16 The guinea pigs were immunized a total of four times, with each injection with an interval of three weeks. Each combination vaccine used to immunize the 20 animals consisted of 100 μg of each protein in adjuvant of the Syntex adjuvant formulation to provide a total volume of 0.1 milliliter. Using the protocol discussed in Example 3, a cutaneous hypersensitivity study was performed To determine if the animals had developed an immune response that could be measured after vaccination with the selected combination vaccine. The guinea pigs were shaved on the back and injected intradermally with the same combination of purified extracellular proteins with those that were immunized. The protein combinations used to attack the animals consisted of 10 μg of each protein. A skin test was also performed on the controls immunized in fake with the same dose of each combination. As in Example 3, the diameters of erythema and hardening were measured at the skin test sites at 24 hours after injection. Table Y below shows the results of these measures, reported in terms of average measurement values for the group of animals ± standard error.

Tab! The and Diameter of the. Skin Reaction (mm) Combination Combination Vaccine Test Skin Erythema Hardening XIX XIX 8.5 ± 0.6 3.9 ± 0.3 XX XX 8.2 ± 0.3 3.7 ± 0.3 XXI XXI 11.1 ± 1.1 4.5 ± 0.4 XXII XXII 9.4 ± 0.8 4.3 ± 0.4 XXIII XXIII 8.3 ± 1.1 3.0 ± 0.3 XXIV XXIV 8.5 + 0.9 3.4 ± 0.5 XXV XXV 7.9 ± 0.5 3.2 ± 0.4 XXVI XXVI 8.9 ± 0.7 3.3 ± 0.5 XXVII XXVII 7.2 ± 1.0 2.8 ± 0.5 XXVIII XXVIII 8.5 ± 0.5 2.8 ± 0.3 IV IV 9.0 ± 0.9 4.1 ± 0.3 XIII XIII 9.4 ± 0.9 4.3 ± 0.3 False XIX 4.0 ± 2.6 1.0 ± 0 False XX 1.3 ± 1.3 1.0 ± 0 False XXI 3.5 ± 1.0 1.3 ± 1.3 False XXII 1.3 ± 1.3 1.0 ± 1.0 False XXIII O ± O 1.0 ± 0 False XXIV O ± O 1.0 ± 0 False XXV O ± O 1.0 ± 0 False XXVI 2.3 ± 2.3 2.0 ± 0.0 False XXVII O ± O 1.0 ± 0 False XXVIII 2.0 ± 1.2 1.0 ± 0 False IV 2.8 ± 1.6 1.0 ± 0 False XIII 1.5 ± 1.5 1.0 + o The results presented in Table Y show explicitly that a strong immune response mediated by cells was generated to Combinations XIX to XXVIII when they were attacked with the same immunogens. As above, a strong cell-mediated immune response was also elicited by Combinations IV and XIII. The erythema exhibited by the immunized guinea pigs was at least doubled, and generally proved to be more than four times greater than, the reaction provoked in the control animals immunized in false. Similarly, the hardening exhibited by the immunized animals was at least doubled, and generally three to four times greater than that of the non-immunized controls. The substantially stronger immune responses generated among the animals immunized according to the teachings of the present invention, illustrate once more the immunoprotective operability of the combination vaccines of the present invention. Those skilled in the art will also appreciate the additional benefits of the vaccines and methods of the present invention. For example, because individual compounds or selected combinations of highly purified molecular species are used for subject vaccines in place of bacteria or compounds thereof, it is considerably less likely that the vaccines of the present invention will elicit a toxic response when compared with the killed or attenuated bacteria vaccines of the prior art. Furthermore, the molecular vaccines of the present invention do not threaten the life of immunocompromised individuals. In fact, the compositions of the present invention can be used therapeutically to stimulate a direct immune response to a pathogen in an infected individual. The selective use of the largely abundant extracellular products or their immunogenic analogues also prevents the development of a humoral opsonization response which may increase the pathogenesis of the intracellular bacteria. Because the protective immunity generated by this invention is directed against unfixed proteins, any opsonic response will simply result in phagocytosis and destruction of the extracellular product mostly abundant rather than the expeditious inclusion of the parasitic bacterium. On the other hand, the selective use of purified extracellular products reduces the potential to generate a response which makes the use of widely used control and classification techniques based on the host recognition of immunogenic agents impossible. Unlike prior art vaccines, classification tests could still be performed using an immunoreactive molecule which is expressed by the pathogen but is not included in the vaccines manufactured in accordance with the present invention. The use of such an immunogenic determinant would only elicit a response in those individuals who have been exposed to the target pathogen by allowing appropriate measures to be taken. Another advantage of the present invention is that extracellular products purified in large quantities are readily obtained and rapidly isolated using techniques well known in the art as opposed to the components of attenuated and bacterial bacteria of prior art vaccines. Since the immunoreactive products of the present invention are naturally released extracellularly within the surrounding medium for most of the organisms of interest, the removal of intracellular contaminants and cell debris is simplified. In addition, since more prominent or largely abundant extracellular products or immunogenic analogs thereof are used to stimulate the desired immune response, expression levels and product culture concentrations that can be harvested are generally raised in most protein systems. production. Accordingly, whatever form of production is used, large-scale isolation of the desired products is easily achieved by routine biochemical procedures such as chromatography or ultrafiltration. These inherent attributes and molecular characteristics of the immunogenic determinants used in the present invention greatly facilitate the production of a high quality, consistent, standardized composition for large scale use. Alternatively, the use of purified molecular compounds based on the immunogenic properties of the most prominent or largely abundant extracellular products of target pathogens also makes the large-scale synthetic generation of the immunoactive vaccine components of the present invention relatively simple . For example, the extracellular products of interest or their immunogenic analogues can be cloned into a non-pathogenic host bacterium using recombinant DNA technology and safely harvested. Cloning techniques well known in the art can be used to isolate and express the DNA corresponding to the extracellular products of interest, their homologues or any segments thereof in high expression vectors selected for insertion into the host bacterium such as Escherichia coli. . Exemplary techniques can be found in II R. Anon, Synthetic Vaccines 31-77 (1987), Tam et al., Incorporation of T and B Epi topes of the Circumsporozoi te Protein in a Chemically Defined Synthetic Vaccine Against Malaria, 171 J. Exp Med 299-306 (1990), and Stover et al., Protective Immuni ty and Elici ted by Recombinant Bacille Calmette -Guerin (BCG) Expressing Outer Surface Protein a (OspA) Lipoprotein: A Candidate Lyme Disease Vaccine, 178 J. Exp. Med. 197-209 (1993). Similarly, extracellular proteins, their analogs, homologs or large-scale immunoreactive protein subunits can be chemically synthesized in a relatively pure form using common laboratory techniques and sequence technology. This manner of production is particularly attractive for the construction of peptide subunits or lower molecular weight analogs corresponding to antigenic determinants of the extracellular products. Exemplary techniques for the production of smaller protein subunits are well known and can be found in II R. Anon, Synthetic Vaccines 15-30 (1987), and in A. Streit ieser, Jr., Introduction to Organic Chemistry 935-55 (3rd ed., 1985). Alternative techniques can be found in Gross et al., "Nonenzimatic Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic Ribonuclease," 237 The Journal of Biological Chemistry No. 6 (1962), Mahoney, "High-Yield Cleavage of Tryptophanyl peptide Bonds. by o-Iodosobenzoic Acid, "18 Biochemistry No. 17 (1979), and Shoolnik et al.," Gonococcal Pili, "159 Journal of Experimentl Medicine (1984). Other immunogenic techniques such as anti-idiotype or direct molecular evolution can also be employed using peptides, nucleotides or other molecules such as mimetics, to generate effective, immunoreactive compounds capable of producing the desired prophylactic response. The prior art techniques for the use of naked DNA as a vaccine in Robinson, Protection Against a Lethal Influenza Virus Challenge by Im unization wi th a Hemagglutinin-Express in Plasmid DNa, 11 Vaccine 9 (1993), and in Ulmer et al., Heterologous Protection Against Influenza by Injection or DNA Encoding to Viral protein, 259 Science (1993). Alternatively, techniques for the fusion of a highly immunogenic protein limb have been described in Tao et al. Idiotype / Granuloci t -Macrophage Colony-Sti ulating Factor fusion Protein as a Vaccine for B-Ceo Lymphoma, 362 Nature (1993) , and for T-cell epitope mapping in Good and collaborators Human T-Cell Recognition of the Circumsporozoi Protein of Plasmodium falciparum: Immunodominant T-Cell Domains Map to the Polymorphic Regions of the Molecule, 85 Proc. Nati Acad. Sci. USA (1988), and Gao et al., Identification and Characterization of T Helper Epipes in the Nucleoprotein of Influenza a Virus, 143 The Journal of Immunology No. 9 (1989). Since many genera of bacteria exhibit homology, the foregoing examples are provided for purposes of illustration and are not intended to limit the scope and content of the present invention or to restrict the invention to the Mycobacterium genus or to particular species or serogroups thereof or to vaccines. against tuberculosis only. It should also be re-emphasized that the prevalence of interspecies homology in the DNA and corresponding proteins of microorganisms allows the vaccines of the present invention to induce cross-reactive immunity. Because the immunodominant epitopes of the largely abundant extracellular products can provide cross-protection immunity against aggression with other serogroups and species of the selected genera, those skilled in the art will appreciate that vaccines directed against a species can be developed using products extracellular or immunogenic analogues of other species. For example, the? F. bovis is between 90 percent and 100 percent homologous with the? F. tuberculosis and has a very high cross-reaction in terms of causing an immune response. Accordingly, vaccines based on abundant extracellular products of Af. Bovis or other Mycobacterium can offer several degrees of protection against Ai infection. tuberculosis and vice versa. Therefore, it is considered as being within the scope of the present invention to provide an immunoprophylactic response against different bacterial species of the same genera using a high homologous immunogenic determinant of a largely abundant extracellular product. It should also be emphasized that the selected immunogenic determinant can be used to practice the present invention in several different ways to produce an effective immune response. Thus, the presentation of one or more immunogenic determinants of the extra abundant extracellular products selected to the host immune system is not critical and can be altered to facilitate production or administration. For example, vaccines of the present invention can be formulated using complete extracellular products or any immunostimulatory fraction thereof including peptides, protein subunits, immunogenic analogues and homologs as noted above. The smaller protein subunits of the largely abundant extracellular products and the molecular analogues thereof are within the scope of the present invention as long as these ,, - provoke an effective immunoprophylaxis. On the other hand, the XO recombinant protein products such as fusion proteins or extracellular products modified by known molecular recombinant techniques are entirely compatible with the teachings of the present invention. In addition, they are also within the scope of the invention analogues immunogenically generated from the selected immunoactive determinants such as anti-idiotype antibodies, or peptides and derived nucleotides using direct evolution. Similarly, the formulation and presentation of The immunogenic agent to the host immune system is not limited to solutions of proteins or their analogs in adjuvants. For example, the immunogenic determinant derived from the appropriate extracellular proteins can be expressed on different species of bacteria, phages, mycoplasmas or viruses that are not pathogenic and modify them using recombinant technology. In such cases the whole living organism can be formulated and used to stimulate the desired response. Conversely, large-scale vaccination programs in hostile environments may require very stable formulations without involving adjuvants or additives. In addition, the formulation of vaccines can be directed to facilitate the stability or immunoreactivity of the active component when subjected to difficult conditions such as lyophilization or oral administration or encapsulation. In accordance, the x? present invention encompasses many different formulations of the immunogenic determinants comprising the subject vaccines depending on the intended use of the product. Those skilled in the art will appreciate that vaccine doses will be determined for each pathogen and host using routine experimentation. So far, it is believed that the lowest practical doses will be in the order of 0.1 μg up to doses of 2.0 μg, 20.0 μg, 100 μg and up to 1 milligram may be optimal for the appropriate system. The appropriate dose can be administered using any technique and conventional immunization sequence known in the art. Those skilled in the art will further appreciate that embodiments of the present invention can be made in other specific forms without departing from the spirit or central attributes thereof. Because the above description 5 of the present invention describes only exemplary embodiments thereof, it should be understood that other variations are contemplated as being within the scope of the present invention. Accordingly, the present invention is not limited to the particular embodiments that have been described in detail therein. Rather, reference should be made to the appended claims as indicative of the scope and content of the present invention.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: The Regents of the University of California (ii) TITLE OF THE INVENTION: Abundant Extracellular Products and Methods for Their Production and Use (iii) NUMBER OF SEQUENCES: 15 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Kurt A. MacLean (B) STREET: 2029 Century Park East, Suite 3800 (C) CITY: Los Angeles (D) STATE: California (E) COUNTRY: United States of America * (F) ) POSTAL CODE: 90067 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: 5 1/2 (B) disk COMPUTER: IMB compatible with PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Reeléase # 1.0, Version # 1.25 (vi) CURRENT REQUEST DATA: 5 (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: 424 (viii) ATTORNEY / AGENT INFORMATION: (A) ) NAME: MacLean, Kurt A X.XJ (B) REGISTRATION NUMBER: 31,118 (C) REFERENCE NUMBER / LAWYER: 104-223-1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 310-788-5000 (B) TELEFAX: 310-277-1297 15 (2) INFORMATION PER SEQ. ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear 20 (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Asn Ser Lys Val Ser 1 5 25 (2) INFORMATION PER SEQ. ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: Thr Asp Arg Val Ser 1 5 (2) INFORMATION PER SEQ. ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3: Ala Arg Ala Val Gly 1 5 (2) INFORMATION PER SEQ. ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (v) TYPE OF FRAGMENT: with terminal N (vi) ORIGINAL SOURCE: (A) ORGANISM: Mycobacterium tuberculosis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Thr Glu Lys Thr Pro 1 5 (2) INFORMATION PER SEQ. ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 Asp Pro Glu Pro Wing 1 5 (2) INFORMATION PER SEQ. ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 Phe Ser Arg Pro Gly 1 5 (2) INFORMATION PER SEQ. ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Phe Ser Arg Pro Gly 1 5 (2) INFORMATION PER SEQ. ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Phe Ser Arg Pro Gly 1 5 5 (2) INFORMATION PER SEQ. ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear r- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Ala Pro Lys Glu Asn 1 5 (2) INFORMATION PER SEQ. ID NO: 10 (i) SEQUENCE CHARACTERISTICS: 15 (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 Ala Pro Lys Thr Tyr 20 1 5 (2) INFORMATION PER SEQ. ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid 25 (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 Wing Glu Thr Tyr Leu 1 5 (2) INFORMATION PER SEQ. ID NO: 12: (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 Wing Tyr Pro lie Thr -p- 1 5 (2) INFORMATION PER SEQ. ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid 5 (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 Ala Asp Pro Arg Leu 1 5 ( 2) INFORMATION PER SEQ. ID NO: 14: 0 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 5 Phe Asp Thr Arg Leu 1 5 (2) INFORMATION PER SEQ. ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser 5 10 15 Pro 1 Ser Met Gly Arg Asp lie Lys Val Gln Phe Gln Ser Gly Gly Asn 20 25 30 Asn Ser Pro Ala Val Tyr Leu Leu Asp 35 40

Claims (47)

1. A vaccination agent to be used to promote a protective immune response, in a mammalian host, against a pathogen of the Mycobacterium genus, said vaccination agent comprising a 30 kDa protein of M. tuberculosis, or a protein sufficiently analogous to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

2. The vaccination agent of claim 1, characterized in that said pathogen is selected from the group consisting of? F. tuberculosis, M. bovis,? f. marinum,? f. kansasii, Af avium intracellulare,? f. fortuitum,? f. chelonei,? f. scrofulaceum and? f. leprae

3. The vaccination agent of claim 1, characterized in that the 30 kDa protein of? F. purified tuberculosis has a N-terminal amino acid sequence of 5 10 15 20 25 30 35 40 FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG NNSPA VYLLD written from left to right in the direction of the amino terminus to the carboxy terminus.

4. The vaccination agent of claim 1, characterized in that it also comprises the 110 kDa protein of Af. purified tuberculosis, having an N-terminal amino acid sequence of 5 10 15 NSKSV NSFGA HDTLK written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently similar protein to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

5. The vaccination agent of claim 1, characterized in that it also comprises the 80 kDa protein of Ai. purified tuberculosis, having a N-terminal amino acid sequence of 5 TDRVS VGN written from left to right in the direction of the amino terminus to the carboxy terminus, or a protein sufficiently similar to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

6. The vaccination agent of claim 1, characterized in that it also comprises the 71 kDa protein of Af. purified tuberculosis, having an N-terminal amino acid sequence of 5 ARAVG I written from left to right in the direction of the amino terminus to the carboxy terminus, or a protein sufficiently similar to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

7. The vaccination agent of claim 1, characterized in that it also comprises the 58 kDa protein of Af. purified tuberculosis, having an N-terminal amino acid sequence of 5 10 15 20 TEKTP DDVFK LAKDE KVLYL written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

8. The vaccination agent of claim 1, characterized in that it also comprises the 45 kDa protein of? F. purified tuberculosis, having a N-terminal amino acid sequence of 5 10 15 20 25 DPEPA PPVPD DAASP PDDAA APPAP written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently similar protein to have the ability to stimulate a response protective immune system in the mammalian host, against subsequent infection by said pathogen.

9. The vaccination agent of claim 1, characterized in that it also comprises the 32A kDa protein of Ai. purified tuberculosis, having an amino acid sequence with terminal N of 5 10 15 20 25 30 FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently similar protein to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

10. The vaccination agent of claim 1, characterized in that it also comprises the 32B kDa protein of Af. purified tuberculosis, having a N-terminal amino acid sequence of 5 10 15 FSRPG LPVEY LQVPS written from left to right in the direction of the amino terminus to the carboxy terminus, or a protein sufficiently similar to have the ability to stimulate a protective immune response in the mammalian host, against infection "" ** "'subsequent to said pathogen

11. The vaccination agent of claim 1, characterized in that it also comprises the purified 24 kDa protein of purified tuberculosis, having an amino acid sequence with terminal N of 5 10 15 20 25 30 35 APYEN LMVPS PSMGR DIPVA FLAGG PHAVY LLDAF written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in the mammalian host , against subsequent infection by said pathogen

12. The vaccination agent of claim 1, character bristled because it also comprises the 23.5 kDa protein of? f. purified tuberculosis, having an N-terminal amino acid sequence of 5 10 APKTY -EELK GTD written from left to right in the direction of the amino terminus to the carboxy terminus, or a protein sufficiently similar to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

13. The vaccination agent of claim 1, characterized in that it also comprises the 23 kDa protein of Af. purified tuberculosis, having an amino acid sequence with terminal N of 5 10 15 20 AETYL PDLDW DYGAL EPHIS GQ written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently similar protein to have the ability to stimulate an immune response protective in the mammalian host, against subsequent infection by said pathogen.

14. The vaccination agent of claim 1, characterized in that it also comprises the 16 kDa protein of Af. purified tuberculosis, having an amino acid sequence with terminal N of 5 10 15 20 25 AYPIT GKLGS ELTMT DTVGQ WLGW written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficiently similar protein to have the ability to stimulate a response protective immune system in the mammalian host, against subsequent infection by said pathogen.

15. The vaccination agent of claim 1, characterized in that it also comprises the 14 kDa protein of? F. purified tuberculosis, having an N-terminal amino acid sequence of 5 10 15 20 ADPRL QFTAT TLSGA PFDGA written from left to right in the direction of the amino terminus to the carboxy terminus, or a protein sufficiently similar to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

16. The vaccination agent of claim 1, characterized in that it also comprises the 12 kDa protein of Ai. purified tuberculosis, having an amino acid sequence with terminal N of 5 10 15 20 25 30 35 40 FDTRL MRLED EMKEG RYEVR AELPG VDPDK DVDIM VRDGQ 45 LTIKA ERT written from left to right in the direction of the amino terminus to the carboxy terminus, or a sufficient protein analogous to have the ability to stimulate a protective immune response in the mammalian host, against subsequent infection by said pathogen.

17. The vaccination agent of claim 1, characterized in that it also comprises the 32A kDa protein of Af. purified tuberculosis, and the 16 kDa protein of? f. purified tuberculosis.

18. The vaccination agent of claim 1, characterized in that it also comprises the 32A kDa protein of? F. purified tuberculosis, and the 23.5 kDa protein of Ai. purified tuberculosis.

19. The vaccination agent of claim 1, characterized in that it also comprises the 23.5 kDa protein of Af. purified tuberculosis, and the 16 kDa protein. purified tuberculosis.

20. The vaccination agent of claim 1, characterized in that it also comprises the 32A kDa protein of Af. purified tuberculosis, the 23.5 kDa protein of Ai. purified tuberculosis, and the 16 kDa protein of Ai. purified tuberculosis.

21. The vaccination agent according to any of claims 1-16, characterized in that the protein sufficiently analogous to have the ability to stimulate a protective immune response, is selected from the group of analogues consisting of protein fragments, peptides, homologs , fusion proteins and glycosylates.

22. The vaccination agent of any of claims 1-21, characterized in that the protein is presented to the immune system of a mammalian host by inoculating said host with a nucleic acid molecule encoding said protein.

23. The vaccination agent of any of claims 1-21, characterized in that the protein is presented to the immune system of a mammalian host, in a delivery system selected from the group consisting of modified avirulent bacteria and viruses expressing said protein.

24. The vaccination agent of any of claims 1-21, characterized in that it also comprises an adjuvant.

25. A method for producing a vaccine, for use in a mammalian host, against a pathogen of the Mycobacterium genus, said method comprising the steps of producing a 30 kDa protein of? F. tuberculosis or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

26. The method, according to claim 25, characterized in that said pathogen is selected from the group consisting of? F. tuberculosis,? f. bovis,? f. marinum, M. kansasii, M avium intracellulare, Af. fortui tum, M. chelonei,? f. scrofulaceum and? f. leprae

27. The method, according to claim 25, characterized in that the 30 kDa protein of? F. purified tuberculosis has a N-terminal amino acid sequence of 5 10 15 20 25 30 35 40 FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG NNSPA VYLLD written from left to right in the direction of the amino terminus to the carboxy terminus.

28. The method, in accordance with the claim 25, characterized in that it also comprises the step of producing a 110 kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

29. The method according to claim 25, characterized in that it also comprises the step of producing an 80 kDa protein of Af. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

30. The method according to claim 25, characterized in that it also comprises the step of producing a 71 kDa protein of? F. tuberculosis, or a protein sufficiently analogous to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen, and to substantially purify said protein.

31. The method, according to claim 25, characterized in that it also comprises the step of producing a 58 kDa protein of? F. tuberculosis, or a protein sufficiently analogous to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen, and to substantially purify said protein.

32. The method according to claim 25, characterized in that it also comprises the step of producing a 45 kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

33. The method, in accordance with the claim 25, characterized in that it also comprises the step of producing a 32A kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

34. The method according to claim 25, characterized in that it also comprises the step of producing a 32B kDa protein of Ai. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

35. The method according to claim 25, characterized in that it also comprises the step of producing a 24 kDa protein of? F. tuberculosis, or a protein sufficiently analogous to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen, and to substantially purify said protein.

36. The method, according to claim 25, characterized in that it also comprises the step of producing a 23.5 kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

37. The method according to claim 25, characterized in that it also comprises the step of producing a 23 kDa protein of? F. tuberculosis, or a protein sufficiently analogous to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen, and to substantially purify said protein.

38. The method according to claim 25, characterized in that it also comprises the step of producing a 16 kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

39. The method according to claim 25, characterized in that it also comprises the step of producing a 14 kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

40. The method according to claim 25, characterized in that it also comprises the step of producing a 12 kDa protein of? F. tuberculosis, or a sufficiently analogous protein to have the ability to stimulate a protective immune response in a mammalian host, against subsequent infection by said pathogen; and substantially purifying said protein.

41. The method according to any of claims 25-40, characterized in that said protein sufficiently similar to have the ability to stimulate a protective immune response, is selected from the group of analogues consisting of protein fragments, peptides, homologs, fusion proteins and glycosylates.

42. The method, according to any of claims 25-40, characterized in that said protein is produced synthetically.

43. The method, according to any of claims 25-40, characterized in that said protein is produced using recombinant techniques.

44. A vaccine produced according to the method of any of claims 25 to 43.

45. A method for immunizing a mammalian host against a pathogen of the Mycobacterium genus, comprising administering to said mammalian host a protective amount of an agent. of vaccination, according to any of claims 1-24.

46. The method, in accordance with the claim 45, characterized in that the vaccination agent is a nucleic acid molecule encoding a vaccination agent, according to any of claims 1-24.

47. The method, according to claims 45 or 46, characterized in that the mammalian host is a human host.