KR101680052B1 - Phamaceutical composition for prevention or treatment of anthrax - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating anthrax, and a method of preventing or treating anthrax.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF ANTHRAX [0002]

The present invention relates to a pharmaceutical composition for preventing or treating anthrax, and a method of preventing or treating anthrax.

Anthrax is a common infectious disease caused by gram-positive bacillus, Bacillus anthracis, which infects mainly livestock but also affects humans. Anthrax has been developed as a biological weapon for a long time. Recently, it has been deployed in actual operations, and the anthrax vaccine has been inoculated to US troops overseas in the Gulf War of 1990 and in 1998. In 2001, The use of biochemical terrorism has focused on more effective measures against the disease. It is known that anthrax caused by anthrax infection is highly fatal.

The pathogenicity of such anthrax is known to be determined by its ability to produce toxins and the formation of the cavernosum. Proteins that affect the toxin production ability of double anthrax are protective antigen (PA), edema factor (EF). Lethal factor (LF) is known to be under study. (PA) and edema (EF) combine to form edema toxin (EdTx, edema toxin), while protective antigen (PA) and lethal factor (LF) (LeTx, ethal toxin). Here, PA binds to the receptor protein (ATR) on the surface of the infected cell line such as macrophages and is then cleaved into PA20 and PA63 by a proteolytic enzyme (furin family protease). PA63 is heptamerized and binds to LF or EF, (Toxin delivery channel). These complexes enter the cell via the endocytosis mechanism, and when the pH of the endosomal is lowered, the structural change of the PA63 heptamer occurs and pores are formed in the endosome membrane. As a result, LF or EF is released into the cytoplasm and becomes toxic.

Currently, the US approved human anthrax vaccine AVA requires six consecutive immunizations during the first 18 months and one additional immunization every year to maintain immunity. It is also known that there are problems such as local side effects accompanied by pain or edema due to unidentified cellular components and defective effects against some pathogenic anthrax. In addition, the protective antigen (rPA) vaccine under clinical trials is effective against neutralization of toxin secreted by anthrax spores after being infected with anthrax spores because it induces toxin neutralizing antibodies. However, in the early stages of anthrax spores infection, The development of vaccine antigens that can block germination is still in short supply. Korean Patent Registration No. 0974485 discloses a therapeutic agent for anthrax, composition and its related method, etc. However, development of a vaccine antigen capable of effectively blocking spore germination at the initial stage of anthrax spore infection is still required .

The present invention relates to a pharmaceutical composition comprising a novel vaccine candidate substance which can be inhibited or inhibited the germination of anthrax spores at an early stage of anthrax infection, which is found by analyzing an immunogen of anthrax using an immunoproteomical approach. And to provide a method for preventing or treating anthrax.

However, the problems to be solved by the present invention are not limited to the problems described above, and other problems not described can be clearly understood by those skilled in the art from the following description.

A first aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of anthrax, comprising a protein encoded by a nucleotide sequence comprising the sequence of SEQ ID No. 1 or SEQ ID No. 2, and a pharmaceutically acceptable carrier.

(A) an anthrax alkylhydroperoxide reductase subunit C protein or an anthrax maroon CoA-acyl transfer protein acyltransferase protein; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating anthrax is provided.

A third aspect of the invention provides a method of preventing or treating anthrax, comprising administering the pharmaceutical composition of either the first or second aspect of the invention to an animal other than a human.

Anthrax is caused by the spore-forming bacteria anthracnose (Bacillus anthracis) and has also been used as a biochemical weapon. Recent vaccines against anthrax are also effective, but they require continuous immunity for long periods of time and often cause side effects. Therefore, the development of improved vaccines against anthracnose with high mortality rates is a top priority. In order to identify novel vaccine candidate substances, the present inventors have attempted an immunoproteomics approach. Using the serum of guinea pigs or human patients recovered from anthrax, 34 immunogenic proteins against malignant anthrax H9401 were identified. To evaluate candidate vaccine candidates, six of these were expressed as recombinant proteins and tested in vivo. Two types of proteins, rGBAA_0345 (alkylhydroperoxide reductase subunit C) and rGBAA_3990 (maronyl CoA-acyl transfer protein acyl transferase), provided partial protection against anthrax in a malignant-spore assay. In addition, a combined vaccine of rGBAA_0345 and rPA (protective antigen) showed an improved effect on anthrax mortality. Finally, the inventors have demonstrated that GBAA_0345 is ubiquitous in anthrax spores and bacilli. Experimental results show that GBAA_0345 is likely to be a component of a multivalent anthrax vaccine and may improve the efficacy of the rPA vaccine. This is the first attempt to study the proteome of malignant anthrax vegetative cells using sera from anthrax patients.

FIGS. 1A to 1C are images showing the result of performing 2-dimensional gel electrophoresis (2DE) using anthrax nutritional cell protein according to an embodiment of the present invention.
FIGS. 2A and 2B are graphs showing mass spectrometry results of anthrax vaccine candidates selected according to an embodiment of the present invention. FIG.
3 is an image showing Western blot analysis results using anthrax extract and guinea pig serum according to one embodiment of the present invention.
FIG. 4 is an image showing the result of Western blot analysis of candidate anthrax vaccine candidates selected according to one embodiment of the present invention.
5 is a graph of anthrax infection survival of immunized guinea pigs using a pharmaceutical composition according to one embodiment of the present invention.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains.

It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to these embodiments and examples and drawings.

Anthrax is a gram-positive spore-forming bacterium that causes anthrax. In 2001, biochemical terrorism using anthrax spores as weapons focused attention on more effective measures against the disease. After entering the host, anthrax sprouts germinate in the macrophage and transfer the bacteria to the local lymph nodes. When they are released from macrophages, extracellular bacilli rapidly proliferate, secrete a high concentration of exotoxin, and spread throughout the body along the bloodstream. The exotoxin is composed of three distinct proteins and is secreted separately as non-toxic monomers: protective antigen (PA), edema factor (EF) and lethal factor (LF). When PA binds to LF or EF, a lethal toxin or edible toxin is formed, respectively. The combination of these factors causes large-scale edema and organ failure. Since PA induces an immune response and protects these anthrax toxins, PA is used as the target antigen of an existing anthrax vaccine.

The first human anthrax vaccine AVA (anthrax vaccine adsorbed, also known as Biothrax) in the United States consists of a PA-containing bacterial filtrate and an aluminum adjuvant. The vaccine is effective, but is also limited by a long and complex dosing schedule (6 consecutive immunizations and 1 additional immunization per year for the first 18 months) and side effects. Side effects are thought to be due to small amounts of EF and LF in the vaccine. Given these two factors - dosage regimen and reactogenicity - the development of improved anthrax vaccines is essential. Second-generation vaccines have been clinically tested in the United States and Korea, and consist of recombinant PA (rPA) and aluminum immunoadjuvants. In some studies, rPA-containing vaccines have shown a protective effect against malignant anthrax spores in rabbits and macaques, but antibodies produced by this vaccine first neutralize the effects of toxins. Then, in order to maintain a sufficient level of defensive antibody, a booster shot is required for drug efficacy every year. Moreover, because rPA is the primary antigen of this vaccine, immunized individuals are vulnerable to attack by genetically modified anthrax (modified PA). Given these problems, strategies are needed to develop more effective vaccines that can be applied to modified anthrax. These strategies include novel immunoadjuvants, various routes of administration, or multivalent vaccine components that target different toxic factors at the outset of infection. Indeed, increased survival rates during co-immunization of rPA and inactivated spores indicate that additional vaccine components can provide enhanced immunity against anthrax.

An immuno-proteomic approach to analyzing and identifying disease-causing microbial immunogens uses 2DE, Western blot of convalescent sera, and MS. Binding with reverse vaccinology using microbial genomic sequences accelerates the development of new vaccine candidates using this technology. In the case of anthrax, several immunogens have been identified that are reactive against AVA-immunized human sera, human serum of patients surviving skin anthrax, or over-immunized animal sera.

To identify new anthrax-vaccine candidates, the proteomics of malignant cells of malignant anthrax H9401 (B. anthracis H9401) were explored using sera from human patients with convalescent guinea pig (GP) or anthrax. Guinea pig serum was isolated from animals that survived repeated infections of malignant spores. Serum was also obtained from five patients with skin and / or digestive anthracosis that developed in 1995 and 2000 in Korea. Thirty four immunogens were identified, six of which were expressed using recombinant techniques and tested in vivo as vaccine candidates. In particular, immunization by rGBAA_0345 or rGBAA_3990 partially protected guinea pigs from malignant spore infections and showed significantly improved survival rates when co-immunized with rGBAA_0345 and rPA.

The first aspect of the present application provides a pharmaceutical composition for the prevention or treatment of anthrax, comprising a protein encoded by a nucleotide sequence comprising the sequence of SEQ ID No. 1 or SEQ ID No. 2, and a pharmaceutically acceptable carrier. have. For example, the pharmaceutical composition may include, but is not limited to, a vaccine composition. The sequence disclosed in Sequence Listing 1 may be the nucleotide sequence of rGBAA_0345, and the sequence disclosed in Sequence Listing 2 may be the nucleotide sequence of rGBAA_3990. The sequence disclosed in Sequence Listing 4 may be the amino acid sequence of rGBAA_0345, and the sequence disclosed in Sequence Listing 5 may be the amino acid sequence of rGBAA_3990.

According to one embodiment of the present invention, the pharmaceutical composition may further include, but is not limited to, anthrax protective antigen (PA). For example, the protective antigen may comprise, but not be limited to, a recombinant protective antigen (rPA). For example, the protective antigen may have an amino acid sequence including the sequence of SEQ ID NO: 3, but the present invention is not limited thereto.

The pharmaceutical composition of the present invention may be administered in unit dose or multiple dose depending on the pharmaceutically effective dose, and may be administered through any conventional route as long as it can reach the target tissue. Methods of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, nasal, pulmonary and rectal administration and the like. However, since the protein is digested during oral administration, effective drugs with the composition must be coated or formulated to prevent degradation by gastric acid in order to be administered orally. In addition, the pharmaceutical composition may be administered using any device capable of delivering the active substance to the target cell.

The pharmaceutical composition may be administered according to a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose of the composition will depend on the severity of the disease, the activity of the drug, the age, body weight, health status, sex, sensitivity to the drug, route of administration, factors including co-administered drugs and other factors well known in the medical arts And the ordinarily skilled physician will readily be able to determine dosages effective for the desired treatment or prophylaxis. The pharmaceutical composition may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. The administration can be in single or multiple doses.

According to one embodiment of the invention, the pharmaceutical composition comprises about 0.36 to 1 part by weight of the anthrax protective antigen relative to 1 part by weight of the protein encoded by the nucleotide sequence comprising the sequence of SEQ ID No. 1 or SEQ ID No. 2, But may not be limited thereto.

For example, the pharmaceutical composition may comprise about 0.05 to 5 parts by weight of the anthrax protective antigen, about 0.1 to 5 parts by weight of the anthrax protective antigen, and about 0.05 to 5 parts by weight of the anthrax protective antigen per 1 part by weight of the protein encoded by the nucleotide sequence comprising the sequence of SEQ ID No. 1 or SEQ ID No. 2 About 0.05 to about 1 part by weight, about 0.05 to about 0.5 part by weight, about 0.05 to about 0.1 part by weight, about 0.05 to about 0.5 part by weight, about 0.5 to about 5 parts by weight, about 1 to about 5 parts by weight, about 3 to about 5 parts by weight, about 0.05 to about 3 parts by weight, , Or about 0.1 to 0.5 parts by weight, or about 0.36 part by weight, but may not be limited thereto.

According to one embodiment of the present invention, the pharmaceutical composition may further include, but is not limited to, an immunosuppressant.

According to one embodiment of the invention, the adjuvant may include, but is not limited to, aluminum hydroxide. For example, the immunoadjuvant may include, but is not limited to, an alhydrogel.

According to one embodiment of the invention, the pharmaceutical composition may be one which induces the production of an antibody against anthrax in vivo. For example, the pharmaceutical composition may be, but not limited to, inducing the production of an antibody that binds to anthrax spores, cell membranes and / or cytoplasm in vivo.

(A) an anthrax alkylhydroperoxide reductase subunit C protein or an anthrax maroon CoA-acyl transfer protein acyltransferase protein; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating anthrax can be provided. For example, the pharmaceutical composition may include, but is not limited to, a vaccine composition.

According to one embodiment of the present invention, the anthrax alkylhydroperoxidase reductase subunit C protein or anthrax maroonyl CoA-acyl transfer protein acyl transferase protein is an alkylhydroperoxide reductase subunit C protein of anthrax H9401, Aconyl transferase protein acyl transferase protein, but may not be limited thereto.

According to one embodiment of the present invention, the pharmaceutical composition may further include, but is not limited to, anthrax protective antigen (PA). For example, the protective antigen may comprise, but not be limited to, a recombinant protective antigen (rPA).

According to one embodiment of the invention, the pharmaceutical composition may further include, but is not limited to, an immunosuppressant.

According to one embodiment of the invention, the adjuvant may include, but is not limited to, aluminum hydroxide. For example, the immunoadjuvant may include, but is not limited to, an alhydrogel.

According to one embodiment of the invention, the pharmaceutical composition may be one which induces the production of an antibody against anthrax in vivo. For example, the pharmaceutical composition may be, but not limited to, inducing the production of an antibody that binds to anthrax spores, cell membranes and / or cytoplasm in vivo.

The third aspect of the present invention provides a method of preventing or treating anthrax, comprising administering the pharmaceutical composition according to the first or second aspect of the present invention to an animal other than a human.

The pharmaceutical compositions herein may be formulated for human or veterinary use, including, but not limited to, pharmaceutically acceptable carriers for administration. Upon oral administration, for example, capsules or tablets; A powder or granule; As a separate unit such as a solution, a syrup or a suspension (in an aqueous or non-aqueous liquid; an edible foam or a whip formulation, or an emulsion) Compositions may be presented, but are not limited thereto.

For example, upon parental administration, the pharmaceutical composition comprises aqueous and non-aqueous sterile injectable solutions, including antioxidants, buffers, bacteriostats, and solutes that allow the blood of the intended recipient to become isotonic And aqueous and non-aqueous sterile suspensions may include, but are not limited to, suspending agents and thickening agents. Additives useful for injectable use include, but are not limited to, water, alcohols, polyols, glycerin and vegetable oils, and the like. The composition may be presented in unit dose (single dose) or multi-dose (multiple dose) containers. For example, it can be presented in sealed ampoules and vials and stored immediately prior to use under freeze-drying conditions requiring only the addition of sterile liquid carriers, such as the water required to make the injections, , But may not be limited thereto. The instant injectable solutions and suspensions may be prepared from, but not limited to, sterile powders, granules and tablets.

According to one embodiment of the invention, the administration may be, but not limited to, administration via intramuscular injection.

For example, administration of the pharmaceutical composition can induce the production of antibodies to anthrax in vivo. For example, the pharmaceutical composition may be administered to induce the production of an antibody that binds to anthrax spores, cell membranes and / or cytoplasm in vivo, but may not be limited thereto.

The first- and second-generation anthrax vaccines used PA as the primary or single immunogen, but the response clearly targets anthrax toxicosis in the final stages of bacterial infection, where the liver balance of anthrax is secreted by anthrax toxin. Thus, it is important to identify novel antigens that either initiate an immune response in the early stages of anthrax infection (e. G., Germination of spores) or increase the bacterial clearance activity of host immune cells.

Here we used an immunopathologic approach to identify 34 anthrax proteins that are immunoreactive to the serum of convalescent guinea pigs or human anthrax patients. Based on Western blot immunoreactivity (intensity and xenogeneic recognition), recombinant versions of the six immunogens were expressed and tested in an anthrax infection guinea pig model. Among these proteins, immunization of rGBAA_0345 provided the most effective defense against anthrax H9401 spores. rGBAA_0345 corresponds to the alkylhydroperoxidase reductase subunit C (Ahpc), which reduces the organic hydroperoxides to the corresponding alcohols. In Mycobacterium leprae, AhpC binds to a soluble cytoplasmic protein but is also found in the cell membrane part. AhpC appears to promote the survival of leprosy bacteria when macrophages produce high levels of reactive oxygen during the immune response. AhpC from Salmonella typhimurium protects both bacteria and human cells from reactive nitrogen intermediates. Helicobacter pylori infection causes immunogenicity to AhpC, and the level of antibody to AhpC has a positive correlation with the progression of gastric ulcer. A recent paper describes rAhpC as a potential candidate for Helicobacter pylori infection. Homologs of GBAA_0345 are expressed in the cell membrane part of anthrax, secretory and spores. Immunogenicity of anthrax infections in guinea pigs, rabbits, and humans has also been reported. GBAA_0345 is also reported to be present in the cytoplasmic part, but not in the culture medium of anthrax. Although GBAA_0345 is predicted to lack the signal peptide for extracellular secretion, the enzyme may bind to the spore bark of the parental cell during spore formation, which may lead to antibody-response in anthrax spore-infected hosts. In our experiments, GBAA_0345 was expressed in a nutrient cell-type cytoplasm of anthrax, and in a nutrient cell protein (VCP) fraction containing both cell membrane portion and spore (see FIG. Moreover, in this study, the inventors have for the first time reported an increase in protection by co-administration of rGBAA_0345 and rPA against completely malignant anthrax spore infection for the first time. As a result, guinea pigs immunized with rGBAA_0345 or rGBAA_0345-rPA were partially or almost completely defensively protected from anthrax exposure, respectively. Antibodies to rGBAA_0345 bind to both spores and bacilli, which may produce antibodies that bind to the native GBAA_0345 protein present in spore or trophoblastic cells and that high levels of reactive oxygen Resulting in the elimination of bacteria by phagocytic action promoted by the antibody.

This greatly enhances the host's resistance to anthrax infection. As a first step in analyzing the safety of this vaccine, we monitored body weight during vaccination. There was no significant difference in body weight change between the rGBAA_0345 group and the PBS control group. Finally, since the immunoreactivity of rGBAA_0345 was detected in the sera of both guinea pigs and human patients at the recovery phase of the anthrax, it is presumed that the immunization associated with rGBAA_0345 effectively induced a humoral immune response.

Another immunogen that provides partial protection against anthrax infection is GBAA_3990. This protein corresponds to the FabD, maronyl CoA-acyl transfer protein acyl transferase, which promotes acyl transfer of malonate (from maronyl-CoA) to acyl transfer protein during fatty acid biosynthesis. In Mycobacterium tuberculosis, the homologue of this protein makes the cell wall of mycobacteria relatively impermeable and confer intrinsic resistance to host macrophages. Although the molecular basis for the partial protection provided by the rGBAA_3990 vaccination was not disclosed, an increase in survival rate in combination with rPA shows the potential of rGBAA_3990 as a candidate vaccine candidate. Two components of the outer membrane (exosporium, BclA and BxpB) have been reported to increase the effect of rPA on co-immunity, although they do not provide protection by itself for malignant-spore infection.

Over the past several decades, a variety of anthrax vaccines have been identified through an immuno-proteomic approach. Immunizations of previously identified immunogen DNA vaccines or genetically engineered spores failed to defend guinea pigs from anthrax infections. On the other hand, our vaccination strategy provided partial or almost complete defense, respectively, when guinea pigs were vaccinated with GBAA_0345 or GBAA_0345-rPA. These differences appear to be due to a number of factors, including different means of vaccination, different forms of immunogen, and different immune adjuvants. We administered the recombinant-protein vaccine through intramuscular injection. This labor-intensive approach may not be appropriate for testing a large number of identified immunogens, but it is the most accurate way to assess the potential of a vaccine. Moreover, co-immunization with low doses of rPA (e.g., to provide ~ 28% of the defense) provides another important means of assessing the usefulness of immunogens, and multi-valent vaccination will be more effective in future prevention of anthrax . ≪ / RTI >

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

[Example]

1. Bacterial culture and protein extraction

Complete malignant, somatic and anthrax anthrax H9401 were used in the paper immuno-proteomics analysis. One colony of Anthrax H9401 was inoculated into 200 mL of medium (0.8% w / v nutrient broth containing 0.3 w / v yeast extract, 0.5% w / v glucose, and 0.9% NaHCO ₃ ) And cultured for 24 hours. Bacteria were then collected and lysed for analysis. Nutrient cells were collected by centrifugation at 10,000 xg for 15 min. The bacterial pellet was washed with 50 mM Tris-HCl (pH 7.5) containing 1 M PMSF and then with 2 DE sample buffer (7 M urea, 2 M thiourea, 4% w / v 3- [3-cholamidopropyl] -dimethylammonium] 1-propanesulfonate (CHAPS), 0.5% v / v ampholyte, 100 mM DTT, 120 mM Tris-HCl (pH 7.5), and 1x protease inhibitor. Bacterial cells were lysed by sonication and treated with DNase I (Invitrogen, Grand Island, NY, USA). Soluble proteins were collected by centrifugation and concentrated by precipitation with trichloroacetic acid (Sigma, St. Louis, Mo., USA).

2. Serum preparation

Six week old female Hartley SPF guinea pigs (Samtaco, Osan, Korea) were infected through intramuscular injection (i.m.) by spores of the complete malignant species B. anthracis H9401. The dose ranged from 25 to 200 spores. After 14 days, the animals were reinfected by the same method. After another 13 days, serum was collected from surviving animals and pooled for immuno-proteomic analysis. Pooled samples of normal human serum were obtained from Innovative Research Inc. (Peary Court Novi, MI, USA). Serum from five patients infected with anthrax was also used. These patients were infected with anthrax in Seoul or Changning, Korea. In Seoul, patients were ingested raw meat from cattle infected with anthrax and were diagnosed with anthrax anemia (one patient died). The patient was contacted with contaminated beef and had an ankylosing lesion in his hand. They were diagnosed with skin anthrax. One of these patients had lesions in the palate, which represent the oral pharyngeal form of anthrax anthracnose. Serum was collected within one week from the onset of these patients. The patient's serum was pooled for immuno-proteomic analysis. Animal Testing Protocol (NIH-08-09) was approved by the National Institutes of Health's Animal Experimental Ethics Committee to reduce animal suffering. Breeding and management of animals has been subject to all relevant guidelines and requirements of the National Institutes of Health's Animal Experimental Ethics Committee. Animals were raised in specific pathogen free (SPF) and infectious laboratories, and guinea pigs moved to the BL3 facility at the National Institutes of Health. Experiments related to human serum were carried out in accordance with the provisions of the Institutional Ethics Review Board of the National Institute of Health.

3. Electrophoresis and western blot

For 2DE analysis, protein pellets were resuspended in 7 M urea, 2 M thiourea, 2% CHAPS, 1% w / v DTT, and 1% v / v malate (pH range 4-10) It was destroyed. The protein (400 ㎍) was applied to a 23-cm IPG strip (nonlinear, pH gradient 4-10, Genomine, Pohang, Korea) equilibrated with the same solution. For the IEF, a voltage of 150 to 2500 V was applied linearly for the first 3 hours to allow the sample to enter. The voltage was then maintained at 3500 V, for a total of 98 kVh. In subsequent IEFs, each strip was run 2 DE using an ISO-DALT 2 DE system (Amersham Biosciences, San Francisco, Calif., USA). One gel was stained with CBB and the proteins of the other gels were transferred to a PVDF membrane under an electric field (230 mA) in a Hoefer-DALT Western Vertical buffer circulation chamber with Tris-glycine buffer (25 mM Tris, 192 mM glycine, 20% v / v methanol; pH 8.0) for 16-20 hours. After blocking the PVDF membrane with 10% w / v skim milk in a Tris-buffered saline containing 0.05% w / v Tween 20, each of the pooled sera obtained from the anthrax patient or normal human is applied to the membrane (10 [mu] g / mL, respectively). Western blotting was also performed separately for the guinea pig serum or the normal guinea pig serum at the recovery period. Specific binding was detected using a HRP-conjugated human secondary IgG (Southern Biotech, Birmingham, AL, USA) and chemiluminescent-producing ECL reagent (Amersham Biosciences, San Francisco, Calif. Respectively. CBB-stained protein spots and western blot signals were compared, and immunoreactive protein spots were selected. The results of VCP samples isolated by 2DE and stained with CBB are shown in Figure 1A. In Western blot analysis, the results of 2DE gel proteins were transferred to PVDF membrane followed by analysis of the serum of the recovered GP (FIG. 1B) and the sera of the anthrax patient (FIG. 1C) as probes are shown in FIGS. 1B and 1C . 25 to 14 immunoreactive spots were identified using guinea pigs and human probes, respectively. In order to identify these proteins, gel fragments containing separate protein spots were cut out, separated using trypsin, and then purified using Ettan MALDI TOF Pro MS (MALDI-TOF-MS) (Amersham Biosciences, San Francisco, CA, USA). The mass spectrometry results of GBAA_0345 and GBAA_0399 are shown in FIG. 2A and FIG. 2B, respectively. The results of protein identification by MALDI-TOF-MS are shown in SWISS-PROT (http://expasy.org/tools/tagdent.html) and NCBI (http://blast.ncbi.nlm.nih.gov) databases with The ProFound program was developed by Rockefeller University (http://prowl.rockefeller.edu/prowl-cgi/profound.exe). Of these, the information of GBAA_0345 and GBAA_3990 is shown in Table 1 below.

4. Expression of DNA constructs and recombinant proteins

Of the identified immunogens, the coding sequences of GBAA_0345, GBAA_2267, GBAA_3338, GBAA_3609, GBAA_3990, and GBAA_4182 (gene positions of B. anthracis Ames Ancestor) were PCR-amplified from the genomic DNA of anthrax H9501. The primers used in this reaction included a restriction enzyme recognition site. PCT was performed in a DNA thermal cycler (Applied Biosystems, Carlsbad, Calif., USA) using a PCR reagent kit (Qiagen, Hilden, Germany). PCR was carried out for 30 cycles at 94 ° C for 60 seconds, 60 ° C for 60 seconds, and 72 ° C for 90 seconds. The PCR products were cloned into TA-vector (Promega, Madison, Wis., USA) and transformed into E. coli DH10B competent cells. After white / blue selection, the PCR products were subcloned into pET19b (+), pET28a (+), or pET30a (+) expression vector (Novagen, Madison, Wis., USA) containing the N-terminal His6- tag, respectively. Each construct was identified by DNA sequencing and transformed into competent E. coli BL21 (DE3) pLysS (GBAA_2267) or E. coli BL21 (DE3) (all other samples) (Novagen, Madison, _{600 nm} = 0.6, and protein expression was induced under 1 mM IPTG. After dissolving the cell pellet, each recombinant protein was purified by affinity chromatography using Ni-NTA sepharose column (Qiagen) The purified proteins were stored at -70 ° C until use.

5. Guinea pig immunization and bio-defense analysis

Five-week-old female Hartley guinea pigs (Samtaco, Osan, Korea) were randomized into groups and immunized via intramuscular injection on days 1 and 28 using varying amounts of rPA and / or recombinant antigens. The antigens were dissolved in 500 [mu] l PBS containing 250 [mu] g alhydrogel immunoadjuvant (Brenntag Biosector, Frederikssund, Denmark). PBS containing 250 μg of the alhydrogel was used as a negative control. Anesthetized guinea pigs developed hemorrhage through the perforation of the heart 14 days after the second immunization. To determine the protective activity against each antigen, the immunized guinea pigs were infected with anthrax H9401 10 LD ₅₀ after 28 days of final immunization. Animals surviving 14 days later were considered as survivors.

6. Statistical Analysis

A log-rank test was used to measure the differences between sample groups. For survival comparisons, Kaplan-Meier analysis was performed. A probability value of p <0.05 was considered statistically significant.

7. Immunoreactivity of anthrax H9401 protein

Guinea pigs were repeatedly infected with spores of anthrax H9401, a malignant species. Serum isolated from live animals was used as a probe for subsequent Western blot analysis. Three different protein extracts (spore lysate, secreted protein (SP), and nutrient cell protein (VCP)) were prepared from anthrax H9401 and SDS-PAGE and western blotting were performed. The VCP samples showed the strongest immunoreactivity against the guinea pig serum of the recovery period and exhibited two major bands of relative molecular masses (Mr) ~ 22 and 80 kDa and three minor bands of ~ 36, 50 and 57 kDa, respectively (See FIG. 3). In contrast to the previous results of Chitlaru et al., Where 100 ug of SP was used for 2DE and western blot analysis, the SP samples of this experiment did not show many bands that were recognizable in the western blot, Is less. Since SP immunoreactive proteins have been extensively studied by the above group, the present inventors have focused on performing 2DE and Western blot analysis using the antigen-rich VCP fraction.

8. Identification of Immunoreactive Proteins

The VCP extract of anthrax H9401 was isolated via 2 DE and the gel was stained with CBB (see Figures 1A-1C). More than 850 protein spots were found here, which were primarily 30 to 100 kDa and had pI values of 4.5 to 7.0. In the western blot, the guinea pig serum-reactive spots were widely located in both pI and Mr, but most of the serum-reactive spots in human anthrax patients were clustered around Mr 40-90 kDa, pI 5-6. 81 immunoreactive spots (23 human sera and 38 guinea pigs) were identified from gel internal trypsin isolation and MALDI-TOF MS. Twenty-two spots (9 human sera and 13 guinea pigs) corresponded to the multiple isoforms of the 34 identified proteins, 5 of which were in serum from both human patients and convalescent guinea pigs. The identified immunogens were identified from six proteins (GBAA_pXO1_0164, GBAA_pXO1_0164, GBAA_pXO1_0172, GBAA_0885, GBAA_0887, GBAA_4035, and GBAA_5580) in the first generation vaccine and nine proteins identified in human skin anthrax (GBAA_pXO1_0164, GBAA_0107, GBAA_0108, GBAA_0309, GBAA_0445, GBAA_3744, GBAA_4182 , GBAA_5369, and GBAA_5547). Among these 14 proteins, nine proteins (GBAA_pXO1_0164, GBAA_pXO1_0172, GBAA_0108, GBAA_0108, GBAA_0885, GBAA_0885, GBAA_0887, GBAA_5369 and GBAA_5580) and three additional proteins (GBAA_0344, GBAA_3338 and GBAA_3609) have been previously reported as animal immunogens . Finally, we have identified 17 novel anthrax vaccines with potential for energy metabolism, protein synthesis, and fatty acid synthesis.

9. Selection and expression of vaccine candidates

From the list of immunogens, eight proteins were selected for further analysis as the most likely vaccine target. Three proteins (GBAA_0309, GBAA_3338, and GBAA_4182) were selected that are jointly identified in both guinea pigs and human sera, since proteins with immunological reactivity to both guinea pigs and humans were considered to be one of the most important factors for screening vaccine targets . Although the infection history of humans and guinea pigs is different, it has been speculated that their common immunogen may be a universal target for anthrax. Five other proteins (GBAA_0067, GBAA_0345, and GBAA_3990 from guinea pig serum; GBAA_2267 and GBAA_3609 from human serum) were selected according to the signal intensity of Western blot to CBB staining by densitometer. Recombinant forms of the six targets, except GBAA_0067 and GBAA_0309, of eight targets were successfully expressed using the E. coli pET system. Five proteins were produced and secreted in soluble form, but rGBAA_3990 was found in the inclusion body. All six proteins were purified. The results of purification and western blot analysis of rGBAA_0345 and rGBAA_3990 are shown in FIG.

10. Protective Effects of Candidate Proteins in Guinea Pigs

In each of the purified recombinant proteins, in vivo defense analysis using guinea pigs was performed. The animals were immunized twice with recombinant proteins at intervals of 4 weeks, and alhydrogel (250 μg) was used as an adjuvant. The guinea pigs were also immunized with either rPA (5 [mu] g) or PBS vehicle as positive control and negative control, respectively. Four weeks after the second immunization, the animals were infected with 10 LD ₅₀ of the malignant anthrax H9401 spore and monitored for survival for 14 days. All PBS-injected animals died, but animals receiving rPA survived. rGBAA_0345 or rGBAA_3990 showed 33% and 17% survival rates, respectively (see Table 2, below). Other recombinant proteins showed no protective effect against anthrax infection.

The two recombinant proteins showing the protective effect were then tested in combination with rPA. Guinea pigs were immunized with 1.8 μg of rPA, which showed suboptimal survival when used with 5 μg recombinant protein and 250 μg alhydrogel. The group immunized with rPA and rGBAA_0345 (ie, rGBAA_0345-rPA) significantly improved after infection with 10 LD ₅₀ malignant anthrax H9401 spores compared to rPA-immunized group with 28% (5/18) survival The survival rate was 94% (17/18; p = 0.0001). On the other hand, the survival rate of the rGBAA_3990-rPA group was 65% (13/20; p = 0.0166). For analysis of immunogenicity, some blood was separated after 2 weeks of the second immunization. Each of the sera of the same group was pooled and analyzed by ELISA to determine specific IgG that was inverse to each recombinant antigen. The endpoint reversal of the anti-rPA antibody in the bivalent vaccination group was higher than in the monovalent rPA group, which means that the immunogenicity of rPA was not inhibited by the presence of other antigens. In particular, the divalent vaccine composed of rGBAA_0345 and rPA gave significantly improved resistance to the malignant anthrax H9401 spore.

Figure 5 is the survival curves of vaccinated guinea pigs following malignant anthrax H9401 spore infection. Hartey guinea pigs (n = 18-20 per group) were immunized via intramuscular injection using the protein or protein combination. PBS containing 250 [mu] g alhydrogel was used as a control. After the final immunization, the guinea pigs were infected by intramuscular injection of 10 LD ₅₀ of anthrax H9401 spores. Survival of guinea pigs was observed for 14 days and survival curves were obtained by Kaplan-Meier analysis (* p < 0.05, *** p < 0.0005).

RGBAA_0345 was not detected in the 2DE assay when human serum was used as a probe in Western blotting (see FIG. To confirm the antigenicity of rGBAA_0345 in human infection, western blot analysis was performed on VCP and rGBAA_0345 using sera from anthrax patients as probes. No native GBAA_0345 was detected in the VCP sample, but a rGBAA_0345 specific signal was detected. Serum from anthrax patients includes antibodies to GBAA_0345. When normal serum was used as a probe, no signal was detected in the VCP or rGBAA_0345 samples.

11. Localization of GBAA_0345

In a conventional study, GBAA_0345 was found in the membrane portion of anthrax, secretome and spore protein bodies. Antibodies specific to rGBAA_0345 were prepared to confirm the ubiquitination of GBAA_0345 protein. Using this reagent, GBAA_0345 was detected in both spores and bacillus of anthrax using a confocal laser scanning microscope and a transmission electron microscope, and no specific binding was observed in the normal serum. This means that immunization with rGBAA_0345 can produce antibodies that bind to both spore and trophic cells, thus reducing the risk of anthrax infection by phagocytes.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention .

<110> KOREA CENTER FOR DISEASE CONTROL AND PREVENTION GREEN CROSS CORPORATION <120> PHAMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF ANTHRAX <130> 0000 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 564 <212> DNA <213> Bacillus anthracis <400> 1 atgttattaa tcggcacaga agtaaaaccg tttaaagcta atgcttacca taatggagaa 60 tttatccaag ttactgacga aagtttaaaa ggaaaatgga gtgtagtttg tttctaccca 120 gctgacttca cattcgtttg cccaactgaa cttgaagact tacaaaacca atatgcaact 180 cttaaagagt taggcgttga agtatactct gtatctacag acactcactt cactcacaaa 240 gcatggcatg atagctcaga aactatcggt aaaatcgagt acatcatgat tggtgaccca 300 actcgcacaa tcactacaaa cttcaacgtt ttaatggaag aagaaggtct tgctgctcgt 360 ggtacattca tcatcgatcc agacggtgtt atccaatcta tggaaatcaa tgctgacggt 420 atcggccgtg acgcaagcat tcttgttaac aaaattaaag cagctcaata cgtacgtaac 480 aacccaggtg aagtttgccc agctaaatgg caagagggtt ctgctacact taaaccaagc 540 cttgaccttg taggcaaaat ctaa 564 <210> 2 <211> 945 <212> DNA <213> Bacillus anthracis <400> 2 atgggaaaac tagcatttct ttttccgggc caaggttcac aggcagttgg aatgggtaaa 60 cagttagcag agaataataa agaggttgca aatgtatttg caaaagcaga tgaagtgttg 120 caagattcac tttcagaagt gatttttgaa gggtcgcagg aaaagcttac gttaacgaca 180 aatgcgcaac cggctttatt aacaacgagt tttgctattt tgacagcttt aaaagagtat 240 gatattacac cagactttgt cgcagggcat agtttaggtg agtatagcgc tcttgtagca 300 gcgggtgcat taacttttga ggatgcagta tatgctgtaa ggaaacgtgg ggaatatatg 360 gaagaagctg ttcctggcgg agaaggtgca atggcggcta ttttaggtgc tgatccacat 420 atgctgaaac gagttacaga agaggtaaca ggtgaagggt atgcagtaca aatcgctaat 480 atgaatagta cgaagcaaat tgtaatttct ggaacaaagc aaggggtcga gattgcttct 540 caaagagcga aagaaaatgg tgcaaagaga gcgattccgc ttaaagtaag tggaccgttc 600 cactctgcgc ttatgaagcc agcagcggag aaattccaaa gtgtcttaaa tgaaattaca 660 attcaagata caaatattcc ggtaatcgca aatgtaacag cagacgttat tacaagtggt 720 gcgcatattc aagaaaagct aatcgaacag ctctactcgc ctgtcttatg gtacccatcc 780 attataatata tggtgaatca aggggtcgat acatttattg aaattggacc tgggaaagta 840 cttgctggac ttatgaagtc aattgattca tcagtgaaag catacgcgat atacgatgaa 900 gagacattaa aagataccat ttcaaatttg agaggagaaa actga 945 <210> 3 <211> 764 <212> PRT <213> Bacillus anthracis <400> 3 Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser Thr Ile   1 5 10 15 Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu Val Lys              20 25 30 Gln Glu Asn Arg Leu Leu Asn Glu Ser Ser Ser Ser Ser Gln Gly Leu          35 40 45 Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro Met Val Val      50 55 60 Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu  65 70 75 80 Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile Trp Ser Gly                  85 90 95 Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala Thr Ser Ala             100 105 110 Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val Ile Asn Lys         115 120 125 Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg Leu Tyr Gln     130 135 140 Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp 145 150 155 160 Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu Val Ile Ser                 165 170 175 Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser             180 185 190 Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp Arg Asp         195 200 205 Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp     210 215 220 Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His 225 230 235 240 Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser                 245 250 255 Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile             260 265 270 Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val Ala Ala Tyr         275 280 285 Pro Ile Val His Val Asp Met Glu Asn Ile Leu Ser Lys Asn Glu     290 295 300 Asp Gln Ser Thr Gln Asn Thr Asp Ser Gln Thr Arg Thr Ile Ser Lys 305 310 315 320 Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His Gly Asn Ala                 325 330 335 Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser Ala Gly             340 345 350 Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser Leu Ser         355 360 365 Leu Ala Gly Glu Arg Thr Ala Glu Thr Met Gly Leu Asn Thr Ala     370 375 380 Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr 385 390 395 400 Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu Gly Lys                 405 410 415 Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln             420 425 430 Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile         435 440 445 Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn     450 455 460 Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp 465 470 475 480 Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly                 485 490 495 Arg Val Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro Gln             500 505 510 Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn         515 520 525 Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu Glu     530 535 540 Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe 545 550 555 560 Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile                 565 570 575 Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys             580 585 590 Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val Leu Asp         595 600 605 Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys Arg     610 615 620 Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser Val 625 630 635 640 Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu Gly Leu                 645 650 655 Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile             660 665 670 Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn Asp Arg         675 680 685 Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe     690 695 700 Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn 705 710 715 720 Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr Ile                 725 730 735 Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys             740 745 750 Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly         755 760 <210> 4 <211> 187 <212> PRT <213> Bacillus anthracis <400> 4 Met Leu Leu Ile Gly Thr Glu Val Lys Pro Phe Lys Ala Asn Ala Tyr   1 5 10 15 His Asn Gly Glu Phe Ile Gln Val Thr Asp Glu Ser Leu Lys Gly Lys              20 25 30 Trp Ser Val Val Cys Phe Tyr Pro Ala Asp Phe Thr Phe Val Cys Pro          35 40 45 Thr Glu Leu Glu Asp Leu Gln Asn Gln Tyr Ala Thr Leu Lys Glu Leu      50 55 60 Gly Val Glu Val Tyr Ser Val Ser Thr Asp Thr His Phe Thr His Lys  65 70 75 80 Ala Trp His Asp Ser Ser Glu Thr Ile Gly Lys Ile Glu Tyr Ile Met                  85 90 95 Ile Gly Asp Pro Thr Arg Thr Ile Thr Thr Asn Phe Asn Val Leu Met             100 105 110 Glu Glu Glu Gly Leu Ala Ala Arg Gly Thr Phe Ile Ile Asp Pro Asp         115 120 125 Gly Val Ile Gln Ser Met Glu Ile Asn Ala Asp Gly Ile Gly Arg Asp     130 135 140 Ala Ser Ile Leu Val Asn Lys Ile Lys Ala Ala Gln Tyr Val Arg Asn 145 150 155 160 Asn Pro Gly Glu Val Cys Pro Ala Lys Trp Gln Glu Gly Ser Ala Thr                 165 170 175 Leu Lys Pro Ser Leu Asp Leu Val Gly Lys Ile             180 185 <210> 5 <211> 314 <212> PRT <213> Bacillus anthracis <400> 5 Met Gly Lys Leu Ala Phe Leu Phe Pro Gly Gln Gly Ser Gln Ala Val   1 5 10 15 Gly Met Gly Lys Gln Leu Ala Glu Asn Asn Lys Glu Val Ala Asn Val              20 25 30 Phe Ala Lys Ala Asp Glu Val Leu Gln Asp Ser Leu Ser Glu Val Ile          35 40 45 Phe Glu Gly Ser Gln Glu Lys Leu Thr Leu Thr Thr Asn Ala Gln Pro      50 55 60 Ala Leu Leu Thr Thr Ser Phe Ala Ile Leu Thr Ala Leu Lys Glu Tyr  65 70 75 80 Asp Ile Thr Pro Asp Phe Val Ala Gly His Ser Leu Gly Glu Tyr Ser                  85 90 95 Ala Leu Val Ala Gla Ala Leu Thr Phe Glu Asp Ala Val Tyr Ala             100 105 110 Val Arg Lys Arg Gly Glu Tyr Met Glu Glu Ala Val Pro Gly Gly Glu         115 120 125 Gly Ala Met Ala Ale Ile Leu Gly Ala Asp Pro His Met Leu Lys Arg     130 135 140 Val Thr Glu Glu Val Thr Gly Glu Gly Tyr Ala Val Gln Ile Ala Asn 145 150 155 160 Met Asn Ser Thr Lys Gln Ile Val Ile Ser Gly Thr Lys Gln Gly Val                 165 170 175 Glu Ile Ala Ser Gln Arg Ala Lys Glu Asn Gly Ala Lys Arg Ala Ile             180 185 190 Pro Leu Lys Val Ser Gly Pro Phe His Ser Ala Leu Met Lys Pro Ala         195 200 205 Ala Glu Lys Phe Gln Ser Val Leu Asn Glu Ile Thr Ile Gln Asp Thr     210 215 220 Asn Ile Pro Ile Val Ale Asn Val Thr Ala Asp Val Ile Thr Ser Gly 225 230 235 240 Ala His Ile Gln Glu Lys Leu Ile Glu Gln Leu Tyr Ser Pro Val Leu                 245 250 255 Trp Tyr Pro Ser Ile Glu Tyr Met Val Asn Gln Gly Val Asp Thr Phe             260 265 270 Ile Glu Ile Gly Pro Gly Lys Val Leu Ala Gly Leu Met Lys Ser Ile         275 280 285 Asp Ser Ser Val Lys Ala Tyr Ala Ile Tyr Asp Glu Glu Thr Leu Lys     290 295 300 Asp Thr Ile Ser Asn Leu Arg Gly Glu Asn 305 310

Claims (15)

A protein encoded by a nucleotide sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2;
Anthrax protective antigen (PA) having an amino acid sequence comprising the sequence of Sequence Listing 3; And
A pharmaceutical composition for the treatment of anthrax, comprising a pharmaceutically acceptable carrier,
Wherein the anthrax protective antigen is 0.36 to 1 part by weight based on 1 part by weight of the protein encoded by the nucleotide sequence comprising the sequence of SEQ ID No. 1 or SEQ ID No. 2. delete delete The method according to claim 1,
Wherein the composition further comprises an adjuvant. 5. The method of claim 4,
Wherein the adjuvant comprises aluminum hydroxide. The method according to claim 1,
A pharmaceutical composition for inducing the production of an antibody against anthrax in vivo. (a) anthrax alkylhydroperoxidase reductase subunit C protein or anthrax marronyl CoA-acyl transfer protein acyl transferase protein;
(b) anthrax protective antigen (PA) having an amino acid sequence comprising the sequence of Sequence Listing 3; And
(c) a pharmaceutically acceptable carrier
/ RTI &gt;
Wherein the anthrax protective antigen is 0.05 to 5 parts by weight based on 1 part by weight of the anthrax alkylhydroperoxide reductase subunit C protein or anthrax maroonyl CoA-acyl transfer protein acyl transferase protein. Composition. 8. The method of claim 7,
The anthrax alkylhydroperoxidase reductase subunit C protein or anthrax maroonyl CoA-acyl transfer protein acyl transferase protein is an alkylhydroperoxide reductase subunit C protein of anthrax H9401 or an anthrax maroon CoA-acyl transfer protein acyl transferase Lt; RTI ID = 0.0 &gt; anthracycline &lt; / RTI &gt; protein. delete delete 8. The method of claim 7,
Wherein the composition further comprises an adjuvant. 12. The method of claim 11,
Wherein the adjuvant comprises aluminum hydroxide. 8. The method of claim 7,
A pharmaceutical composition for inducing the production of an antibody against anthrax in vivo. A method for treating anthrax, comprising administering a pharmaceutical composition according to any one of claims 1, 4 to 8, 11 to 13 to an animal other than a human. 15. The method of claim 14,
Wherein said administration is by intramuscular injection.

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