Anthrax compositions and methods of use and production

Abstract

Compositions and methods effective for eliciting an immune response for preventing or reducing infection or improving clinical outcomes caused by Bacillus anthracis are provided. The compositions include a naturally occurring or synthetic protein, peptide, or protein fragment containing all or an active portion of an antigenic epitope associated with anthrax toxin proteins optionally combined with a pharmaceutically acceptable carrier. The preferred antigenic epitopes correspond to immunogenic regions of protective antigen, lethal factor or edema factor, either individually or in combination. In addition, methods and compositions containing antibodies for reducing the effects of anthrax toxins are described. The methods involve administering to a human or animal the compositions described herein in a dosage sufficient to elicit an immune response or treat the anthrax infection.

Description

CLAIM OF PRIORITY AND CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 60/799,819, filed May 12, 2006 and is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to the field of immunology and more particularly relates to methods and compositions for treating, preventing or reducing anthrax infection in humans or animals.

BACKGROUND OF THE INVENTION

[0003] Anthrax is a disease caused by the sporulating bacteria Bacillus anthracis. Humans working with animal products are at risk for contracting anthrax, particularly individuals such as veterinarians, laboratory technicians, ranchers and employees working with the skin or hair of animals. Areas such as Iran, Turkey, Iraq, Pakistan, and sub-Saharan Africa are hyperendemic for anthrax, although the organism can be found in most areas of the world. General worldwide populations are vulnerable to B. anthracis infection as a consequence of acts of bioterror, whereas military troops are vulnerable to infection as a consequence of acts of bioterror and/or war.

[0004] Anthrax manifests disease in three different ways: inhalation anthrax disease, gastrointestinal anthrax disease, and cutaneous anthrax disease. Inhalation anthrax disease is caused by inhaling spores. Gastrointestinal anthrax is caused by ingesting spores in contaminated meat, and cutaneous anthrax disease occurs when spores contact an open wound. Untreated inhalation or gastrointestinal anthrax has a case fatality rate of essentially 100 percent while cutaneous anthrax has a case fatality rate of up to 25 percent.

[0005] For persons infected with anthrax, treatment success is limited by several factors, such as the increased incidence of antibiotic resistance, the overwhelming septic responses, which can occur before antibiotics could be effective, and treatment delays based upon decreased suspicion and poor methods of early detection that all can lessen the chance of survival. It is known that early treatment of anthrax with antibiotics is essential to reduce mortality, although not completely effective with inhalation infections. Delays in treatment profoundly decrease survival rates. Early treatment, however, is difficult because initial symptoms of the infection resemble common, non-fatal infections. For example, the inhalation of anthrax spores may initially present symptoms resembling those of the common cold. In addition, symptoms of anthrax infection, depending on how the bacterium is contracted, may take seven to sixty days to appear. Moreover, even prompt, effective antibiotic treatment does not ameliorate the effects of circulating toxins, which can remain in the blood at high levels and continue to mediate their pathogenic effects.

[0006] The pathogenicity of Bacillus anthracis is expressed in two ways: a toxic effect made evident by the appearance of edema, and a so-called lethal toxic effect that may lead to the death of the subject infected. These effects are attributed to the presence of toxins produced by a combination of three proteins that represent the toxin system: protective antigen (PA), lethal factor (LF) and edema factor (EF). In both humans and mammals, toxins increase in the body even during early stages of infection when the host appears asymptomatic. This explains why delays in treatment can be fatal. Thus, there is not only a critical need for improved anthrax intervention therapies, including treatments that interfere with the deleterious actions of the toxins, but also a critical need for point-of-care, rapid, and extremely sensitive diagnostic tests to establish the presence of anthrax early in the infection.

[0007] Passive immunization, an effort to neutralize toxins with antibodies, usually polyclonal antibodies, has been used as a therapeutic intervention for a variety of bacterial infections (Keller and Stiehm, 2000). A major limitation of using polyclonal antisera in patients is the possibility of "serum sickness" due to a patient's immune response to proteins derived from a different species. In addition, higher affinity antibodies are more effective for toxin neutralization, but there is no general way to enhance intentionally the affinity of polyclonal sera or even monoclonal antibodies derived from hybridomas. In addition, traditional approaches using pooled donor intravenous immunoglobulins (IVIG) is not effective as normal donors do not have effective humoral immunity against Bacillus anthracis.

[0008] Although vaccines against Bacillus anthracis are currently available, they are not optimal. Even the most recent commercially available vaccines require multiple primary injections, yearly boosters, and fail to provide proven long-term effectiveness against inhalation attacks and adverse events. In addition, currently available vaccines exhibit severe adverse effects.

[0009] The mechanism for anthrax toxicity is as follows. The 83 kDa form of protective antigen (PA83) is secreted from rapidly growing Bacillus anthracis cells and binds to a specific host cell surface receptor, such as TEM8 or CMG2. (H. M. Scobie and J. A. Young, Curr. Opin. Micro. 8, 106-112 (2005)). Subsequent cleavage by membrane-bound furin, and/or a furin-like protease, possibly PACE4, releases an amino terminal 20 kDa fragment, resulting in receptor-bound PA63. PA63 subunits then oligomerize into heptameric rings, which in turn create binding sites for the lethal factor and edema factor components of Bacillus anthracis toxins. Endocytosis of the receptor/toxin complex into acidic endosomes elicits a conformational change in PA63, whereby the A subunits (LF or EF) of the toxin are released into the endosome. Lysosomal acidification and subsequent receptor release facilitate irreversible membrane insertion of the oligomeric PA63 pore. The pore permits transport of LF and/or EF into the cytoplasm where they elicit their respective toxicities.

[0010] EF is a calcium/calmodulin-dependent adenylate cyclase that is toxic to most cell types and causes local inflammation and edema, but is not usually lethal. Edema toxin, composed of protective antigen and edema factor is been recently shown to cause tissue lesion and death in a mouse model (see "Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice" by Firoved, A. M., G. F. Miller, M. Moayeri, R. Kakkar, Y. Shen, J. F. Wiggins, E. M. McNally, W. J. Tang, and S. H. Leppla. Am J Pathol 167:1309-1320 (2005)).

[0011] LF is a cell-type specific metalloprotease that cleaves MAP-kinase-kinases and several peptide hormones. Lethal toxin, formed by the combination of protective antigen and lethal factor is a zinc dependent protease that results in lysis of macrophages. Lethal factor is the major virulence factor associated with anthrax toxicity and is thought to be responsible for systemic shock and death. Neither of the toxin A subunits are pathogenic in the absence of cytoplasmic delivery by PA or mechanical means (See, "Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process" by A. M. Friedlander J. Biol. Chem. 261, 7123 (1986)).

[0012] The crystal structures of PA83 and heptameric PA63 have been resolved (See, e.g., "Crystal-structure of the anthrax toxin protective antigen" by C. Petosa et al., Nature. 385, 833 (1997)). These structural data support the experimental data (See, e.g., "Characterization of lethal factor-binding and cell-receptor binding domains of protective antigen of Bacillus anthracis using monoclonal-antibodies" by S. F. Little et al., Microbiology-UK. 142, 707 (1996) and "The carboxyl-terminal end of protective antigen is required for receptor-binding and anthrax toxin activity" by Y. Singh et al., J. Biol. Chem. 266,15493 (1991)) that indicate that domain 4, the carboxy-terminus of PA63, is responsible for receptor-mediated uptake of the toxin complex.

[0013] Bacillus anthracis has been used for over sixty years as a biological weapon and is a likely agent for a large-scale attack based upon the relative ease of obtaining and growing the bacterium, as well as the stability of the spores. Weaponized anthrax was first developed in 1941 by the British government, which tested the release of anthrax spores on an island near Scotland (Mourez, M. Rev Physiol Biochem Pharmacol 152:135-164 (2004) and Greenfield, R. A et al. Am J Med Sci 323:299-315 (2002)). While no individuals were infected during this test, the island remained a biohazard for forty-five years until seawater and formaldehyde was used to sterilize the soil. Since that time, however, there have been accidental and deliberate releases of militarized anthrax resulting in human infections and deaths. An accidental release of anthrax from a USSR military facility in 1979 caused 68 deaths and resulted in infection of cattle up to 31 miles away. And in 2001, the deliberate spread of anthrax through infected letters in the US resulted in 22 cases of anthrax (11 cutaneous and 11 inhalation), with five deaths (see Guarner, J. et al. Am J Pathol 163:701-709 (2003) and Quinn, C. P. et al. J Infect Dis 190:1228-1236 (2004)). These incidents highlight the need for a safe, effective vaccine that provides protection from an aerosolized release of Bacillus anthracis spores and potential directed immunotherapeutics for early intervention.

[0014] The current US vaccine (anthrax vaccine absorbed, AVA) is an alhydrogel absorbed cell-free filtrate of the attenuated V770-NP1-R strain, a non-encapsulated bovine isolate. This vaccination is administered at 0, 2, 4 weeks and again at 6, 12, and 18 months with yearly boosters recommended. The primary data on this vaccine has been obtained from animal studies and indicates that, while AVA does not protect mice from lethal challenge with fully virulent strains of B. anthracis, vaccinated mice are protected against challenge with nonencapsulated strains. These models have also demonstrated that passive transfer of antibodies against the major toxin proteins (PA, LF, and EF) can provide protection against challenge with attenuated strains (see Little, S. F. et al. Infect Immun 65:5171-5175 (1997); Little, S. F. et al. Infect Immun 56:1807-1813 (1988); Price, B. M et al. Infect Immun 69:4509-4515 (2001) and Welkos, S., et al. Microbiology 147:1677-1685 (2001)). Antibodies to PA, LF, and EF have also been detected in serum samples of individuals diagnosed with clinical anthrax, but vaccination results in primarily anti-protective antigen antibodies. However, several studies using mice vaccinated with mutant strains of Bacillus anthracis have shown significant contributions of LF and EF to protection (see Mohamed, N. et al. Infect Immun 72:3276-3283 (2004) and Little, S. F. et al. Biochem Biophys Res Commun 199:676-682 (1994)). Thus enhancing the levels of antibodies against these proteins might enhance protection. Accordingly, because the current vaccine is not optimal, new and improved preventative compositions and methods are necessary.

[0015] What are needed are methods and compositions that can prevent, inhibit and diminish the symptoms of Bacillus anthracis infection. The compositions should be able to overcome the activity of anthrax toxins, namely protective antigen, lethal factor and edema factor. Preferably the compositions should be able to induce an immune response in an animal or human. Also needed are compositions that can be used to produce neutralizing antibodies directed to components of the anthrax toxins, such as lethal factor and edema factor. Furthermore, what are needed are methods and compositions that can be used for immunotherapy, specifically directed to Bacillus anthracis infection. Finally, the methods and compositions for preventing, inhibiting and diminishing the symptoms of Bacillus anthracis infection should preferably be non-toxic and produce few side effects.

SUMMARY OF THE INVENTION

[0016] Methods and compositions for treating, preventing, inhibiting and diminishing the symptoms of Bacillus anthracis infection are provided. The compositions described herein uniquely overcome the activity of anthrax toxins, namely protective antigen (PA), lethal factor (LF) and edema factor (EF). More particularly, the compositions include immunogenic compositions containing peptides, or epitopes corresponding to antigenic regions of Bacillus anthracis, or active fragments thereof, optionally combined with delivery vehicles or carrier polypeptide(s), including PA. In certain embodiments, the compositions include multiple epitopes corresponding to one or more antigenic regions associated with anthrax toxins. In a further embodiment, the antigenic regions associated with anthrax toxins are peptides or peptide fragments derived from PA, LF or EF that elicit an immunogenic response when administered to an animal or human. In another embodiment, the composition contains multiple peptides or fragments thereof, derived from PA, LF and/or EF that elicit an immune response when administered to an animal or human. The multiple peptides can be separate or combined, such as in a fusion protein. Though not wishing to be bound by the following theory, the compositions described herein may elicit a humoral immune response that results in prevention or reduction of Bacillus anthracis toxicity and infection.

[0017] The compositions provided herein contain an immunogenic anthrax peptide having a molecular weight less than 20 kDa and are therefore smaller than previously described antigenic domains contemplated for use in anthrax vaccines. The peptide is preferably 40 amino acids in length or less. The peptide is optionally coupled to a carrier, such as but not limited to, a protective antigen protein. Preferred peptides for PA are set forth in Tables 4 and 6. Preferred peptides for LF are set forth in Tables 1-3 and 5. Preferred peptides for EF are set forth in Table 7.

[0018] Preferably, the compositions described herein are useful in a method for inducing an immune response to anthrax, such as for treating, protecting against or vaccinating a human or animal against Bacillus anthracis the causative agent associated with anthrax.

[0019] Certain embodiments of the compositions described herein further contain delivery or carrier vehicles such as liposomes or vesicles having epitopes, epitope fragments, synthetic peptides or conservatively modified peptide fragments presented on their external surfaces. In an alternative embodiment, a desired epitope or immunogenic peptide fragment thereof, may be partially or totally encapsulated with a carrier such as a liposome. In another alternative embodiment, the epitopes or immunogenic fragment thereof or peptide may be transported to desired sites by delivery mechanisms employing the use of colloidal metals such as colloidal gold. The above described compositions are useful as vaccines to improve immunity against certain anthrax toxins, which otherwise would not be effectively targeted by the immune system.

[0020] The compositions are further useful in therapeutic regimens for reducing the debilitating effects of anthrax. Though not wishing to be bound by the following theory, it is thought that the resulting circulating antibodies bind anthrax toxins and thereby prevent the dissemination of Bacillus anthracis infection, reduce existing infection, or diminish the effects of infection.

[0021] The compositions also include isolated and recombinant antibodies specific for Bacillus anthracis antigenic epitopes. Isolated antibodies are produced by and purified from humans or animals with strong primed immune responses and injected into humans or animals with weak, non-functional or non-primed immune systems in need of such circulating antibodies. The antibodies are either produced by administering one or more of the peptides described herein to an animal and then collecting, purifying and modifying the antibodies as needed or by affinity purifying protective, anti-peptide responses from animals immunized with proteins/protein fragments/peptides, vaccinated individuals or survivors of anthrax infection. These affinity purified antibodies can be sequenced and produced recombinantly for use as immunotherapeutics. The antibodies may be monoclonal or polyclonal antibodies. The antibodies are immunoreactive with a peptide of an anthrax protein having a molecular weight less than 20 kDa, preferably 40 amino acids in length or less, that induces an immune response to anthrax when administered to an animal. Thus, anthrax infection is reduced or inhibited either by active immunization of an individual using compositions containing unique epitopes or peptides associated with Bacillus anthracis toxins, or by passive immunization via administering an antibody or a group of antibodies specific for Bacillus anthracis toxins. The antibody may be modified to reduce adverse effects when administered to a patient. Humanized antibodies are ideal for this use.

[0022] Alternatively, the antibodies or peptides are used as reagents in a method, such as an immunoassay. The antibody or peptide reagent is combined with a sample from a potentially infected animal or human, and the formation of an antibody-antigen complex is detected. The immunoassay is useful for detecting anthrax infection in an animal or human, either by detecting circulating antibodies or by detecting anthrax proteins. Alternatively, the immunoassay is useful for detecting specific protective immunity to anthrax infection in an animal or human. If predetermined anti-anthrax antibody titers are detected, then the animal should be protected against anthrax challenge, and subsequent vaccinations or boosting may be unnecessary.

[0023] Accordingly, it is an object of the present invention to provide methods and compositions for preventing and reducing the effects of infection caused by Bacillus anthracis.

[0024] It is another object of the present invention to provide methods and compositions for preventing, reducing and treating the occurrence or spread of anthrax.

[0025] It is a further object of the present invention to provide methods and compositions for preventing and reducing the effects of infection caused by Bacillus anthracis in a human or animal having such an infection by eliciting an active humoral and/or cellular response in the host.

[0026] It is yet another object of the present invention to provide methods and compositions for vaccinating a human or animal against Bacillus anthracis infection.

[0027] It is yet another object of the present invention to provide methods and compositions for passively immunizing a human or animal against Bacillus anthracis infection.

[0028] Another object of the present invention is to provide compositions containing unique epitopes associated with Bacillus anthracis toxins that are antigenic and elicit an immune response against Bacillus anthracis in humans or animals.

[0029] Another object of the present invention is to provide compositions containing unique epitopes associated with Bacillus anthracis toxins that are antigenic and elicit an immune response against Bacillus anthracis in humans or animals wherein the toxin is protective antigen, lethal factor, or edema factor, individually or in combination.

[0030] Yet another object of the present invention is to provide Bacillus anthracis epitopes or peptide fragments modified with antigenic moieties to increase an individual's response to Bacillus anthracis and methods of use thereof.

[0031] Another object of the present invention is to provide antibodies useful for passively immunizing a human or animal against anthrax.

[0032] These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.

[0033] The compositions and method described herein may be understood more readily by reference to the following detailed description of specific embodiments included herein. Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 provides a schematic representation of binding and processing of Bacillus anthracis virulence factors.

[0035] FIG. 2 is a flow chart providing study details for generation of anti-LF and anti-PA antisera containing neutralizing antibodies in mice.

[0036] FIG. 3 is a graph of endpoint titer, providing results of anti-LF and anti-PA IgG antibody titers in immunized A/J mice.

[0037] FIG. 4 is a flow chart providing study design for lethal toxin challenge.

[0038] FIG. 5 provides a schematic representation of a solid phase ELISA technique for detection of antibodies specific for sequential overlapping peptide sequences.

[0039] FIG. 6 provides data of sequential mouse B cell epitopes of lethal factor.

[0040] FIG. 7 provides data of sequential mouse B cell epitopes of protective antigen.

[0041] FIG. 8 provides a design study of strain background versus MHC in B cell epitope selection.

[0042] FIG. 9 provides data of lethal factor epitopes in A/J, B6 and B6.H2.sup.k mice.

[0043] FIG. 10 provides data of protective antigen epitopes in A/J, B6 and B6.H2.sup.k mice.

[0044] FIGS. 11A-D provide data on anti-Bacillus anthracis antibodies following AVA vaccination.

[0045] FIG. 12 provides data of sequential human B cell protective antigen antibodies.

[0046] FIG. 13 provides a table of human protective antigen humoral epitopes.

[0047] FIG. 14 provides data of sequential human B cell lethal factor humoral epitopes.

[0048] FIG. 15 provides a table of human lethal factor humoral epitopes.

[0049] FIG. 16 a flow chart providing study details for generation of anti-Edema Factor (EF) antisera containing neutralizing antibodies in mice.

[0050] FIG. 17 provides results of anti-EF IgG antibody titers in immunized A/J mice.

[0051] FIGS. 18A and B provide results of lethal edema toxin challenge in EF-immunized A/J mice.

[0052] FIG. 19A and B provide data of sequential mouse B cell epitopes of EF.

[0053] FIG. 20 provides data showing that peptide-binding antibodies purified from PA- and LF-immunized mice can neutralize the action of lethal toxin in vitro.

[0054] FIG. 21 provides graphs showing IgG anti-PA peptide concentration and percent viability.

[0055] FIGS. 22A-G are graphs of data showing confirmation of peptide epitopes.

[0056] FIG. 23A-C provide data showing that some peptide-binding antibodies from human AVA vaccines can neutralize the action of lethal toxin in vitro.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0057] Immunogenic anthrax peptide compositions and methods for inducing an immune response to anthrax in a human or animal are described herein. Anti-anthrax antibodies, methods of making the antibodies, and methods for treating anthrax infection using the antibodies are also described. In addition, reagents and assays for the detection of anthrax infection are described.

[0058] The anthrax peptides provided herein are immunogenic and have a molecular weight less than 20 kDa. The peptides are portions or fragments of the immunologically important anthrax proteins known as protective antigen (PA) protein, lethal factor (LF) protein, and edema factor (EF) protein, or combinations thereof. Preferably, the amino acid sequence of each anthrax peptide is less than 40 amino acids. More preferably, the amino acid sequence is greater than six amino acids and less than 40 amino acids. Alternatively, the amino acid sequence is greater than six amino acids and less than 20 amino acids in length. In a preferred embodiment, the amino acid sequence of the anthrax peptide has a length between 12 and 18 amino acids. Alternatively, the preferred length is 8 to 10 amino acids in length. In one embodiment, the consecutive amino acid sequence of the anthrax peptide is 12 amino acids in length. Selective and representative anthrax peptide amino acid sequences are provided herein.

[0059] The antibodies provided herein are immunoreactive with anthrax proteins, particularly PA, LF or EF and are prepared by administering the anthrax peptides described herein to an animal and collecting or purifying a biological fluid from the animal containing antibodies specific for the anthrax peptides. The antibodies are administered to an animal, such as a human patient infected with anthrax, as an immunotherapy to treat the disease.

[0060] The anthrax peptides and antibodies described herein are also useful as reagents in immunoassays for the detection or diagnosis of anthrax infection.

[0061] Nucleic acid sequences that encode the anthrax peptides described herein are also contemplated within the scope of the invention. These nucleic acid sequences are useful for production of the anthrax peptides. The nucleic acid sequences are also useful as a DNA vaccine and can be inserted into a vector to transfect animals or humans to introduce the antigenic epitope or peptide into the system, thereby causing the body to mount an immune response to the immunogenic peptide.

Definitions

[0062] The terms "a", "an" and "the" as used herein are defined to mean one or more and include the plural unless the context is inappropriate.

[0063] The terms "antibody" or "antibodies" as used herein include monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, simianized, and humanized antibodies as well as Fab fragments, including the products of an Fab immunoglobulin expression library.

[0064] The term "antigen" refers to an entity or fragment thereof which can induce an immune response in a animal. The term includes immunogens and regions responsible for antigenicity or antigenic determinants.

[0065] As employed herein, the phrase "biological activity" refers to the functionality, reactivity, and specificity of compounds that are derived from biological systems or those compounds that are reactive to them, or other compounds that mimic the functionality, reactivity, and specificity of these compounds. Examples of suitable biologically active compounds include enzymes, antibodies, antigens and proteins.

[0066] The phrases "biologically pure" or "isolated" refer to material substantially or essentially free from components that normally accompany it as found in its native state. Thus, the peptides described herein do not contain materials normally associated with their in situ environment. Typically, the isolated, antiproliferative peptides described herein are at least about 80% pure, usually at least about 90% pure, and preferably at least about 95% pure as measured by band intensity on a silver stained gel.

[0067] Protein purity or homogeneity may be indicated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.

[0068] Preferably, the epitopes and peptides of the present invention are relatively short in length (i.e., less than about 40 amino acids). Such short amino acid sequences can be synthesized using standard chemical peptide synthesis techniques.

[0069] Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the immunogenic peptides described herein. Techniques for solid phase synthesis are known to those skilled in the art.

[0070] Alternatively, the antigenic peptides described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide and, if required, renaturing the peptide. Techniques sufficient to guide one of skill through such procedures are found in the literature.

[0071] Once expressed, recombinant peptides can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of about 50 to 95% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.

[0072] One of skill in the art will recognize that after chemical synthesis, biological expression or purification, the immunogenic peptides may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is often necessary to denature and reduce the immunogenic peptide and then to cause the peptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art.

[0073] The term "bodily fluid," as used herein, includes, but is not limited to, saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, synovial fluid, blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, seminal fluid, mammary secretions, and vitreal fluid, and nasal secretions.

[0074] The term "carrier" as used herein means a structure in which a immunogen or a immunogenic peptide fragment of Bacillus anthracis toxin can be incorporated into or can be associated with, thereby presenting or exposing the immunogen or part of the immunogenic peptide to the immune system of a human or animal and rendering the immunogenic composition antigenic for Bacillus anthracis. The term "carrier" further comprises methods of delivery wherein immunogenic peptides or peptide fragment compositions may be transported to desired sites by delivery mechanisms. In addition, the term "carrier" further includes vaccine delivery mechanisms known to those skilled in the art including, but not limited to, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetanus toxoid, and polypeptides derived from B. anthracis, including protective antigen (PA), particularly recombinant PA, and other adjuvants. It is also to be understood that the antigenic epitope compositions provided herein can further include adjuvants, preservatives, diluents, emulsifiers, stabilizers, and other components that are known and used in vaccines. Any adjuvant system known in the art can be used in the compositions described herein. Such adjuvants include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersed .beta.-(1,4) linked acetylated mannan, polyoxyethylene-polyoxypropylene copolymer adjuvants, modified lipid adjuvants, saponin derivative adjuvants, large polymeric anions such as dextran sulfate, and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate.

[0075] Carrier proteins, or carriers, that can be used in the antigenic peptide compositions provided herein include, but are not limited to, maltose binding protein "MBP"; bovine serum albumin "BSA"; keyhole lympet hemocyanin "KLH";

[0076] ovalbumin; flagellin; thyroglobulin; serum albumin of any species; gamma globulin of any species; syngeneic cells; syngeneic cells bearing Ia antigens; polymers of D- and/or L- amino acids ; tetanus toxoid, and polypeptides derived from B. anthracis itself, particularly recombinant PA. The carrier is conjugated or otherwise covalently or non-covalently bound to the anthrax peptide.

[0077] The phrase "consisting essentially of" is used herein to exclude any elements that would substantially alter the essential properties of the peptides to which the phrase refers. Thus, the description of a peptide "consisting essentially of . . . " excludes any amino acid substitutions, additions, deletions or modifications that would substantially alter the biological activity of that peptide.

[0078] As used herein, the term "edema factor" or "EF" refers to wild-type full-length edema factor of Bacillus anthracis (See SEQ ID NO:3 and Genbank accession number: AAA79215; Robertson D L, Tippetts M T, Leppla S H. Gene. 1988 Dec. 20; 73(2):363-71).

[0079] The term "effective amount" refers to the amount of epitope or immunogenic peptide which, when administered to a human or animal, elicits an immune response, prevents, reduces or lessens Bacillus anthracis infection, causes a reduction in reactivity or inhibits the spread and proliferation of Bacillus anthracis disease. The effective amount is readily determined by one of skill in the art following routine procedures.

[0080] For example, immunogenic epitope compositions may be administered intramuscularly, parenterally, subcutaneously, via inhalation as an aerosol or orally in a range of approximately 1.0 .mu.g to 5.0 mg per patient, though this range is not intended to be limiting. The actual amount of epitope or immunogenic peptide composition required to elicit an immune response will vary for each individual patient depending on the immunogenicity of the epitope administered and on the immune response of the individual. Consequently, the specific amount administered to an individual will be determined by routine experimentation and based upon the training and experience of one skilled in the art.

[0081] The term "epitope" as used herein means a part of a macromolecule recognized by the immune system such as a three-dimensional structural feature that binds to an antibody. Alternatively, the term "epitope" refers to a linear epitope determined by an amino acid sequence.

[0082] Also as used herein, the term "immunogenic" refers to substances that elicit or enhance the production of antibodies, T-cells and other reactive immune cells directed against Bacillus anthracis and contribute to an immune response in a human or animal. An immune response occurs when an animal or individual produces sufficient antibodies, T-cells and other reactive immune cells against compositions of the present invention to prevent, lessen or alleviate the effect of Bacillus anthracis infection.

[0083] As used herein, the term "lethal factor" or "LF" refers to wild-type full-length lethal factor of Bacillus anthracis (See SEQ ID NO:2 and Genbank accession number: AAM26117; Read T D, Salzberg S L, Pop M, Shumway M, Umayam L, Jiang L, Holtzapple E, Busch J D, Smith K L, Schupp J M, Solomon D, Keim P, Fraser C M. Science. 2002 Jun. 14; 296(5575):2028-33)).

[0084] As used herein, the term "protective antigen" or "PA" refers to the wild-type full-length protective antigen of Bacillus anthracis (See SEQ ID NO:1 and Genbank accession number: AAA22637; Welkos S L, Lowe J R, Eden-McCutchan F, Vodkin M, Leppla S H, Schmidt J J. Gene. 1988 Sep. 30; 69(2):287-300)).

[0085] The term "peptides," are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term "amino terminus" (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" (abbreviated C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide.

[0086] Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the peptide. Thus, when one amino acid is said to "follow" another, that amino acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid.

[0087] The term "residue" refers to an amino acid (D or L) or an amino acid mimetic incorporated in an oligopeptide by an amide bond or amide bond mimetic. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

[0088] Furthermore, one of skill will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

[0089] 1) Alanine (A), Serine (S), Threonine (T);

[0090] 2) Aspartic acid (D), Glutamic acid (E);

[0091] 3) Asparagine (N), Glutamine (Q);

[0092] 4) Arginine (R), Lysine (K), Histidine (H);

[0093] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and;

[0094] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Anthrax Peptide Compositions

[0095] The anthrax peptides provided herein are immunogenic portions or fragments of PA, LF, or EF Bacillus anthracis toxin proteins, and the peptides have a molecular weight less than 20 kDa. Each peptide contains at least one epitope of an anthrax toxin protein as described in more detail below. Preferably, the amino acid sequence of each anthrax peptide is less than 40 amino acids. In one embodiment, the amino acid sequence of the anthrax peptide is greater than six amino acids and less than 40 amino acids or 20 amino acids in length. In a preferred embodiment, the amino acid sequence of the anthrax peptide is greater than six amino acids and less than 20 amino acids in length. Most preferably, the amino acid sequence of the anthrax peptide has a length between 12 and 18 amino acids. Ideally, the anthrax peptide has a sequence between eight and 10 amino acids. Alternatively, the anthrax peptide is 12 amino acids in length.

[0096] Combinations of anthrax peptides from the same or different anthrax toxin proteins, fusion proteins containing the anthrax peptides, anthrax peptides coupled to a carrier, and conservatively modified peptides are also included within the scope of the peptides described herein. In addition, the peptides may be naturally occurring, recombinant, modified or synthetic.

[0097] Depending on their intended use, the peptides are attached to or admixed with adjuvants, excipients, preservatives, delivery vehicles, other antigenic moieties and the like to produce an immunogenic composition such as an anthrax vaccine composition. The anthrax peptides can also be modified so as to increase their antigenicity. Examples of antigenic moieties and adjuvants include, but are not limited to, lipophilic muramyl dipeptide derivatives, nonionic block polymers, aluminum hydroxide or aluminum phosphate adjuvant, and mixtures thereof.

[0098] As mentioned above, each peptide contains at least one epitope of an anthrax toxin protein. An epitope is that part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. An epitope is a single antigenic site on a protein against which an antibody reacts. Although usually epitopes are thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes. Most B cell epitopes can be thought of as three-dimensional surface features of an antigen molecule; these features fit precisely and thus bind to antibodies. The part of an antibody that recognizes the epitope is called a paratope. Sequential epitopes can be recognized by antibodies and can either be found on the surface of the molecule or partially on the surface of the molecule. The methodology used herein constructs sequential epitopes, which are synthesized in extremely high concentrations/densities on the solid phase surfaces. This approach allows the detection of antibodies which bind to a sequential sequence, and at times, even identifies two regions of binding which come together on the surface of the molecule leading to one functional epitope.

[0099] As used herein, an epitope is defined as containing a region of immunogenicity associated with at least one of the following: protective antigen, lethal factor or edema factor. In one embodiment, the epitope contains a range of consecutive amino acids that are immunogenic to protective antigen, lethal factor or edema factor. Preferably, the composition described herein contains one or more epitopes from each of these three Bacillus anthracis toxin proteins. More preferably, the composition contains one or more epitopes from each domain of each toxin protein, PA, LF and EF, of the Bacillus anthracis toxin system.

[0100] The immunogenic peptides provided herein are peptides, portions or fragments of full-length anthrax proteins. Full-length recombinant protective antigen, lethal factor and edema factor proteins are produced using a recombinant vector such as standard bacterial or Baculoviral gene expression systems. Full-length proteins can be cleaved into individual domains or digested using various methods. In addition, the amino acid sequences and domains of full length protective antigen, lethal factor and edema factor are well known in the art (see also SEQ ID NO: 1-3 and Genbank Accession Numbers AA22637, AAM26117, and AA79215, respectively). In a preferred embodiment, the composition contains one or more immunogenic peptides from each of these Bacillus anthracis toxin proteins.

[0101] In a preferred embodiment, the composition contains one or more immunogenic peptides from each Bacillus anthracis toxin protein.

[0102] In one embodiment, the peptides in the anthrax peptide composition are preferably epitopes (a) 1-9 PA (Table 6); 1-10 LF (Table 5) and 1-13 EF (Table 7) and fragments thereof for human (see FIGS. 12-15) and (b) 1-27 LF (Table 1); 1-28 LF (Table 2); 1-27 LF (Table 3); 1-25 PA (Table 4); 1-17 EF (Table 8) and 1-9 EF (Table 9) and fragments thereof for mouse. (see FIGS. 6, 7 and 19).

[0103] In another embodiment, the peptides are those of Tables 1-9. Preferred anthrax peptides from protective antigen include the peptide sequences of Tables 4 and 6-7.

[0104] Preferred anthrax peptides from lethal factor include the peptide sequences of Tables 1-3 and 5.

[0105] Preferred anthrax peptides from edema factor include the peptide sequences of Tables 8-10.

[0106] Preferred anthrax peptides from protective antigen and lethal factor include the peptide sequences referred to in FIGS. 13 and 15, respectively.

[0107] Highly preferred anthrax peptides are set forth in the Tables as described below:

Table 1: Mouse Summary Lethal Factor

[0108] Table 1 discloses immunogenic decapeptides and corresponding composite immunogenic peptides for mouse lethal factor. A total of 392 reactive amino acids were identified out of 810 amino acids that comprise LF. A peptide was considered reactive if the peptide was observed to have reactivity greater than or equal to 0.3 as determined by a normalized OD measurement. An extended epitope, consisting of adjacent overlapping peptides was required to contain at least one peptide demonstrating an OD of 0.4 or greater. A single reactive peptide that did not demonstrate an adjacent reactive peptide was not considered to constitute an epitope unless the OD exceeded 1.0. In total, 27 epitopes of lethal factor in the immunized murine system were identified based on this classification.

TABLE-US-00001 TABLE 1 No. on Sequence Pin # Composite Sequence Length Structure GAGGHGDVGM 17 GAGGHGDVGMHVKEKE 16 (SEQ ID NO: 4) (SEQID NO: 92) GGHGDVGMHV 18 (SEQ ID NO: 5) HGDVGMHVKE 19 (SEQ ID NO: 6) DVGMHVKEKE 20 (SEQ ID NO: 7) KEKNKDENKR 24 KEKNKDENKRKD 12 (SEQ ID NO: 8) (SEQ ID NO: 93) KNKDENKRKD 25 (SEQ ID NO: 9) RNKTQEEHLK 31 RNKTQEEHLKEIMKHI 16 (SEQ ID NO: 10) (SEQID NO: 94) KTQEEHLKEI 32 (SEQ ID NO: 11) QEEHLKEIMK 33 (SEQ ID NO: 12) EHLKEIMKHI 34 (SEQ ID NO: 13) EKVPSDVLEM 49 EKVPSDVLEMYKAIGGKI 18 1 (SEQ ID NO: 14) (SEQID NO: 95) VPSDVLEMYK (SEQ ID NO: 15) 50 SDVLEMYKAI (SEQ ID NO: 16) 51 VLEMYKAIGG (SEQ ID NO: 17) 52 EMYKAIGGKI (SEQ ID NO: 18) 53 SEDKKKIKDI 66 SEDKKKIKDIYGKDALLHEH 20 2 (SEQ ID NO: 19) (SEQ ID NO: 96) DKKKIKDIYG 67 (SEQ ID NO: 20) KKIKDIYGKD 68 (SEQ ID NO: 21) IKDIYGKDAL 69 (SEQ ID NO: 22) DIYGKDALLH 70 (SEQ ID NO: 23) YGKDALLHEH 71 (SEQ ID NO: 24) LHEHYVYAKE 74 LHEHYVYAKEGY 12 (SEQ ID NO: 25) (SEQ ID NO: 97) EHYVYAKEGY 75 (SEQ ID NO: 26) SNEVQEVFAK 122 SNEVQEVFAKAF 12 (SEQ ID NO: 27) (SEQ ID NO: 98) EVQEVFAKAF 123 (SEQ ID NO: 28) QHRDVLQLYA 131 QHRDVLQLYAPEAFNYMDKFNEQEINL 34 3 (SEQ ID NO: 29) SLEELKD RDVLQLYAPE 132 (SEQ ID NO: 30) VLQLYAPEAF 133 (SEQ ID NO: 31) QLYAPEAFNY 134 (SEQ ID NO: 32) YAPEAFNYMD 135 (SEQ ID NO: 33) PEAFNYMDKF 136 (SEQ ID NO: 34) AFNYMDKFNE 137 SPLIT INTO (SEQ ID NO: 35) 26 AND 16 NYMDKFNEQE 138 (SEQ ID NO: 36) MDKFNEQEIN 139 (SEQ ID NO: 37) KFNEQEINLS 140 (SEQ ID NO: 38) NEQEINLSLE 141 (SEQ ID NO: 39) QEINLSLEEL 142 (SEQ ID NO: 40) INLSLEELKD 143 (SEQ ID NO: 41) LEELKDQRML 145 LEELKDQRMLAR 12 (SEQ ID NO: 42) (SEQ ID NO: 100) ELKDQRMLAR 146 (SEQ ID NO: 43) MLARYEKWEK 149 MLARYEKWEKIKQH 14 (SEQ ID NO: 44) (SEQID NO: 101) ARYEKWEKIK 150 (SEQ ID NO: 45) YEKWEKIKQH 151 (SEQ ID NO: 46) LLKKLQIPIE 163 LLKKLQIPIE 10 4 (SEQ ID NO: 47) (SEQ ID NO: 47) SLSQEEKELL 172 SLSQEEKELLKRIQ 14 5 (SEQ ID NO: 48) (SEQ ID NO: 102) SQEEKELLKR 173 (SEQ ID NO: 49) EEKELLKRIQ 174 (SEQ ID NO: 50) DFLSTEEKEF 181 DFLSTEEKEFLKKLQIDI 18 6 (SEQ ID NO: 51) (SEQ ID NO: 103) LSTEEKEFLK 182 (SEQ ID NO: 52) TEEKEFLKKL 183 (SEQ ID NO: 53) EKEFLKKLQI 184 (SEQ ID NO: 54) EFLKKLQIDI 185 (SEQ ID NO: 55) SLSEEEKELL 191 SLSEEEKELLNRIQ 14 7 (SEQ ID NO: 56) (SEQ ID NO: 104) SEEEKELLNR 192 (SEQ ID NO: 57) EEKELLNRIQ 193 (SEQ ID NO: 58) LSEKEKEFLK 201 LSEKEKEFLKKLKLDI 16 8 (SEQ ID NO: 59) (SEQID NO: 105) EKEKEFLKKL 202 (SEQ ID NO: 60) EKEFLKKLKL 203 (SEQ ID NO: 61) EFLKKLKLDI 204 (SEQ ID NO: 62) QPYDINQRLQ 209 QPYDINQRLQDT 12 9 (SEQ ID NO: 63) (SEQID NO: 106) YDINQRLQDT 210 LIDSPSINLDVRKQ (SEQ ID NO: 64) LIDSPSINLD 216 (SEQ ID NO: 65) (SEQ ID NO: 107) 14 10 DSPSINLDVR 217 (SEQ ID NO: 66) PSINLDVRKQ 218 (SEQ ID NO: 67) NRGIFNEFKK 249 NRGIFNEFKK 10 (SEQ ID NO: 68) (SEQID NO: 68) KYSISSNYMI 255 KYSISSNYMIVD 12 (SEQ ID NO: 69) (SEQID NO: 108) SISSNYMIVD 256 (SEQ ID NO: 70) LIKKVTNYLV 324 LIKKVTNYLVDG 12 (SEQ ID NO: 71) (SEQ ID NO: 109) KKVTNYLVDG 325 (SEQ ID NO: 72) LVDGNGRFVF 328 LVDGNGRFVFTDITLP 16 11 (SEQ ID NO: 73) (SEQ ID NO: 110) DGNGRFVFTD 329 (SEQ ID NO: 74) NGRFVFTDIT 330 (SEQ ID NO: 75) RFVFTDITLP 331 (SEQ ID NO: 76) QYTHQDEIYE 338 QYTHQDEIYEQV 12 12 (SEQ ID NO: 77) (SEQ ID NO: 111) THQDEIYEQV 339 (SEQ ID NO: 78) VPESRSILLH 347 VPESRSILLHGP 12 (SEQ ID NO: 79) (SEQ ID NO: 112) ESRSILLHGP 348 (SEQ ID NO: 80) DSEGFIHEFG 357 DSEGFIHEFGHAVD 14 (SEQ ID NO: 81) (SEQ ID NO: 113) EGFIHEFGHA 358 (SEQ ID NO: 82) FIHEFGHAVD 359 (SEQ ID NO: 83) NSKKFIDIFK 372 NSKKFIDIFKEE 12 (SEQ ID NO: 84) (SEQ ID NO: 114) KKFIDIFKEE 373

(SEQ ID NO: 85) DHAERLKVQK 391 DHAERLKVQKNAPKTF 16 (SEQ ID NO: 86) (SEQ ID NO: 115) AERLKVQKNA 392 (SEQ ID NO: 87) RLKVQKNAPK 393 (SEQ ID NO: 88) KVQKNAPKTF 394 (SEQ ID NO: 89) PKTFQFINDQ 397 PKTFQFINDQIK 12 (SEQ ID NO: 90) (SEQ ID NO: 116) TFQFINDQIK 398 (SEQ ID NO: 91)

Table 2: Extended Mouse Summary Lethal Factor

[0109] Table 2 discloses immunogenic decapeptides and corresponding composite immunogenic peptides for mouse lethal factor wherein the peptides and composite sequences were considered reactive if the peptide reactivity was greater than 0.2 as determined by optical density measurement. Each extended epitope (denoted as composite sequence) was required to contain at least one decapeptide demonstrating an optical density (O.D.) of greater than or equal to 0.4. In this instance, 440 reactive amino acid were identified out of a total of 810 amino acids comprising LF. In total, 28 epitopes were identified based on this classification.

TABLE-US-00002 TABLE 2 Pin Sequence # Composite Sequence Length VQGAGGHGDV 16 VQGAGGHGDVGMHVKEKE 18 (SEQ ID NO: 117) (SEQ ID NO: 139) GAGGHGDVGM 17 (SEQ ID NO: 4) GGHGDVGMHV 18 (SEQ ID NO: 5) HGDVGMHVKE 19 (SEQ ID NO: 6) DVGMHVKEKE 20 (SEQ ID NO: 7) KEKEKNKDEN 23 KEKEKNKDENKRKD 14 (SEQ ID NO: 118) (SEQ ID NO: 140) KEKNKDENKR 24 (SEQ ID NO: 8) KNKDENKRKD 25 (SEQ ID NO: 9) KRKDEERNKT 28 KRKDEERNKTQE 12 (SEQ ID NO: 119) (SEQ ID NO: 141) KDEERNKTQE 29 (SEQ ID NO: 120) RNKTQEEHLK 31 RNKTQEEHLKEIMKHI 16 (SEQ ID NO: 10) (SEQ ID NO: 94) KTQEEHLKEI 32 (SEQ ID NO: 11) QEEHLKEIMK 33 (SEQ ID NO: 12) EHLKEIMKHI 34 (SEQ ID NO: 13) EKVPSDVLEM 49 EKVPSDVLEMYKAIGGKI 18 (SEQ ID NO: 14) (SEQ ID NO: 95) VPSDVLEMYK 50 (SEQ ID NO: 15) SDVLEMYKAI 51 (SEQ ID NO: 16) VLEMYKAIGG 52 (SEQ ID NO: 17) EMYKAIGGKI 53 (SEQ ID NO: 18) ISLEALSEDK 63 ISLEALSEDKKKIKDIYGK 26 (SEQ ID NO: 121) DALLHEH (SEQ ID NO: 142) LEALSEDKKK 64 (SEQ ID NO: 122) ALSEDKKKIK 65 (SEQ ID NO: 123) SEDKKKIKDI 66 (SEQ ID NO: 19) DKKKIKDIYG 67 (SEQ ID NO: 20) KKIKDIYGKD 68 (SEQ ID NO: 21) IKDIYGKDAL 69 (SEQ ID NO: 22) DIYGKDALLH 70 (SEQ ID NO: 23) YGKDALLHEH 71 (SEQ ID NO: 24) LHEHYVYAKE 74 LHEHYVYAKEGY 12 (SEQ ID NO: 25) (SEQ ID NO: 97) EHYVYAKEGY 75 (SEQ ID NO: 26) DFSVEFLEQN 117 not included (SEQ ID NO: 124) SNEVQEVFAK 122 SNEVQEVFAKAF 12 (SEQ ID NO: 27) (SEQ ID NO: 98) EVQEVFAKAF 123 (SEQ ID NO: 28) QHRDVLQLYA 131 QHRDVLQLYAPEAFNYMD 34 (SEQ ID NO: 29) KFNEQEINLSLEELKD (SEQ ID NO: 99) RDVLQLYAPE 132 (SEQ ID NO: 30) VLQLYAPEAF 133 (SEQ ID NO: 31) QLYAPEAFNY 134 (SEQ ID NO: 32) YAPEAFNYMD 135 (SEQ ID NO: 33) PEAFNYMDKF 136 (SEQ ID NO: 34) AFNYMDKFNE 137 SPLIT INTO 26 AND 16 (SEQ ID NO: 35) NYMDKFNEQE 138 (SEQ ID NO: 36) MDKFNEQEIN 139 (SEQ ID NO: 37 KFNEQEINLS 140 (SEQ ID NO: 38) NEQEINLSLE 141 (SEQ ID NO: 39) QEINLSLEEL 142 (SEQ ID NO: 40) INLSLEELKD 143 (SEQ ID NO: 41) LEELKDQRML 145 LEELKDQRMLAR 12 (SEQ ID NO: 42) (SEQ ID NO: 100) ELKDQRMLAR 146 (SEQ ID NO: 43) MLARYEKWEK 149 MLARYEKWEKIKQH 14 (SEQ ID NO: 44) (SEQ ID NO: 101) ARYEKWEKIK 150 (SEQ ID NO: 45) YEKWEKIKQH 151 (SEQ ID NO: 46) SEEGRGLLKK 160 not included (SEQ ID NO: 274) LLKKLQIPIE 163 LLKKLQIPIEPK 12 (SEQ ID NO: 47) (SEQ ID NO: 144) KKLQIPIEPK 164 (SEQ ID NO: 125) SLSQEEKELL 172 SLSQEEKELLKRIQ 14 (SEQ ID NO: 48) (SEQ ID NO: 102) SQEEKELLKR 173 (SEQ ID NO: 49) EEKELLKRIQ 174 (SEQ ID NO: 50) DFLSTEEKEF 181 DFLSTEEKEFLKKLQIDI 18 (SEQ ID NO: 51) (SEQ ID NO: 103) LSTEEKEFLK 182 (SEQ ID NO: 52) TEEKEFLKKL 183 (SEQ ID NO: 53) EKEFLKKLQI 184 (SEQ ID NO: 54) EFLKKLQIDI 185 (SEQ ID NO: 55) SLSEEEKELL 191 SLSEEEKELLNRIQ 14 (SEQ ID NO: 56) (SEQ ID NO: 104) SEEEKELLNR 192 (SEQ ID NO: 57) EEKELLNRIQ 193 (SEQ ID NO: 58) LSEKEKEFLK 201 LSEKEKEFLKKLKLDIQP 18 (SEQ ID NO: 59) (SEQ ID NO: 143) EKEKEFLKKL 202 (SEQ ID NO: 60) EKEFKKLKL 203 (SEQ ID NO: 61) EFLKKLKLDI 204 (SEQ ID NO: 62) LKKLKLDIQP 205 (SEQ ID NO: 126) DIQPYDINQR 208 DIQPYDINQRLQDTGG 16 (SEQ ID NO: 127) (SEQ ID NO: 145) QPYDINQRLQ 209 (SEQ ID NO: 63) YDINQRLQDT 210 (SEQ ID NO: 64) INQRLQDTGG 211 (SEQ ID NO: 128) LIDSPSINLD 216 LIDSPSINLDVRKQYKRD 18 (SEQ ID NO: 65) (SEQ ID NO: 146) DSPSINLDVR 217 (SEQ ID NO: 66) PSINLDVRKQ 218 (SEQ ID NO: 67) INLDVRKQYK 219 (SEQ ID NO: 129) LDVRKQYKRD 220 (SEQ ID NO: 130) NRGIFNEFKK 249 NRGIFNEFKKNF 12 (SEQ ID NO: 68) (SEQ ID NO: 147) GIFNEFKKNF 250 (SEQ ID NO: 131) KYSISSNYMI 255 KYSISSNYMIVD 12 (SEQ ID NO: 69) (SEQ ID NO: 108) SISSNYMIVD 256 (SEQ ID NO: 70) LIKKVTNYLV 324 LIKKVTNYLVDG 12 (SEQ ID NO: 71) (SEQ ID NO: 109) KKVTNYLVDG 325 (SEQ ID NO: 72) LVDGNGRFVF 328 LVDGNGRFVFTDITLP 16 (SEQ ID NO: 73) (SEQ ID NO: 110) DGNGRFVFTD 329 (SEQ ID NO: 74) NGRFVFTDIT 330 (SEQ ID NO: 75) RFVFTDITLP 331 (SEQ ID NO: 76) NIAEQYTHQD 336 not included (SEQ ID NO: 132) QYTHQDEIYE 338 QYTHQDEIYEQVHSKG 16 (SEQ ID NO: 77) (SEQ ID NO: 148) THQDEIYEQV 339 (SEQ ID NO: 78) QDEIYEQVHS 340 (SEQ ID NO: 133) EIYEQVHSKG 341 (SEQ ID NO: 134) VPESRSILLH 347 VPESRSILLHGPSKGV 16 (SEQ ID NO: 79) (SEQ ID NO: 149) ESRSILLHGP 348 (SEQ ID NO: 80) RSILLHGPSK 349 (SEQ ID NO: 135) ILLHGPSKGV 350 (SEQ ID NO: 136) DSEGFIHEFG 357 DSEGFIHEFGHAVD 14 (SEQ ID NO: 81) (SEQ ID NO: 113) EGFIHEFGHA 358 (SEQ ID NO: 82) FIHEFGHAVD 359 (SEQ ID NO: 83) NSKKFIDIFK 372 NSKKFIDIFKEE 12 (SEQ ID NO: 84) (SEQ ID NO: 114) KKFIDIFKEE 373 (SEQ ID NO: 85) DHAERLKVQK 391 DHAERLKVQKNAPKTFQF 18 (SEQ ID NO: 86) (SEQ ID NO: 150) AERLKVQKNA 392 (SEQ ID NO: 87) RLKVQKNAPK 393 (SEQ ID NO: 88) KVQKNAPKTF 394 (SEQ ID NO: 89) QKNAPKTFQF 395

(SEQ ID NO: 137) PKTFQFINDQ 397 PKTFQFINDQIKFI 14 (SEQ ID NO: 90) (SEQ ID NO: 151) TFQFINDQIK 398 (SEQ ID NO: 91) QFINDQIKFI 399 (SEQ ID NO: 138)

Table 3: Additional Mouse Lethal Factor Data

[0110] Table 3 discloses immunogenic decapeptides and corresponding composite immunogenic peptides for mouse lethal factor. A total of 392 reactive amino acids were identified out of 810 amino acids that comprise LF. A peptide was considered reactive if the peptide was observed to have reactivity greater than or equal to 0.2 as determined by a normalized OD measurement. An extended epitope, consisting of adjacent overlapping peptides was required to contain at least one peptide demonstrating an OD of 0.4 or greater. A single reactive peptide that did not demonstrate an adjacent reactive peptide was not considered to constitute an epitope unless the OD exceeded 1.0. In total, 27 epitopes of lethal factor in the immunized murine system were identified based on this classification.

TABLE-US-00003 TABLE 3 Epitope number Pin # Sequence 1 17-20 GAGGHGDVGMHVKEKE (SEQ ID NO: 92) 2 24-25 KEKNKDENKRKD (SEQ ID NO: 93) 3 31-34 RNKTQEEHLKEIMKHI (SEQ ID NO: 94) 4 49-53 EKVPSDVLEMYKAIGGKI (SEQ ID NO: 95) 5 66-71 SEDKKKIKDIYGKDALLHEH (SEQ ID NO: 96) 6 74-75 LHEHYVYAKEGY (SEQ ID NO: 97) 7 122-123 SNEVQEVFAKAF (SEQ ID NO: 98) 8 131-143 QHRDVLQLYAPEAFNYMDKFNEQEINLSLEE LKD (SEQ ID NO: 99) 9 145-146 LEELKDQRMLAR (SEQ ID NO: 100) 10 149-151 MLARYEKWEKIKQH (SEQ ID NO: 101) 11 163 LLKKLQIPIE (SEQ ID NO: 47) 12 172-174 SLSQEEKELLKRIQ (SEQ ID NO: 102) 13 181-185 DFLSTEEKEFLKKLQIDI (SEQ ID NO: 103) 14 191-193 SLSEEEKELLNRIQ (SEQ ID NO: 104) 15 201-204 LSEKEKEFLKKLKLDI (SEQ ID NO: 105) 16 209-210 QPYDINQRLQDT (SEQ ID NO: 106) 17 216-218 LIDSPSINLDVRKQ (SEQ ID NO: 107) 18 249 NRGIFNEFKK (SEQ ID NO: 68) 19 255-256 KYSISSNYMIVD (SEQ ID NO: 108) 20 324-325 LIKKVTNYLVDG (SEQ ID NO: 109) 21 328-331 LVDGNGRFVFTDITLP (SEQ ID NO: 110) 22 338-339 QYTHQDEIYEQV (SEQ ID NO: 111) 23 347-348 VPESRSILLHGP (SEQ ID NO: 112) 24 357-359 DSEGFIHEFGHAVD (SEQ ID NO: 113) 25 372-373 NSKKFIDIFKEE (SEQ ID NO: 114) 26 391-394 DHAERLKVQKNAPKTF (SEQ ID NO: 115) 27 397-398 PKTFQFINDQIK (SEQ ID NO: 116)

Table 4: Mouse PA Compiled

[0111] Table 4 discloses immunogenic decapeptides and corresponding epitopes for mouse protective antigen wherein the peptides and composite sequences were considered reactive if the peptide reactivity was greater than 0.2 as determined by optical density measurement and containing at least one decapeptide with reactivity of 0.4 or greater. In this instance, 25 epitopes were identified based on this classification.

TABLE-US-00004 TABLE 4 No. on Epitope Pin Structure Number Number Sequence 1 1 27-30 FSDLNFQAPMWTSST (SEQ ID NO: 152) 2 34-39 STTGDLSIPSSELENIPSEN (SEQ ID NO: 153) 3 75-77 YQRENPTEKGLDFK (SEQ ID NO: 154) 4 82-85 LYWTDSQNKKEVISSD (SEQ ID NO: 155) 2 5 93-99 LKQKSSNSRKKRSTSAGPTV PD (SEQ ID NO: 156) 6 101-102 GPTVPDRDNDGI (SEQ ID NO: 157) 7 110-111 EGYTVDVKNKRT (SEQ ID NO: 158) 8 119-124 SNIHEKKGLTKYKSSPEKWS (SEQ ID NO: 158) 3 9 129-135 TASDPYSDFEKVTGRIDKNV SP (SEQ ID NO: 159) 10 139 SPEARHPLVA (SEQ ID NO: 160) 11 143-144 VAAYPIVHVDME (SEQ ID NO: 161) 4 12 147-151 VDMENIILSKNEDQSTQN (SEQ ID NO: 162) 5 13 168-172 NAEVHASFFDIGGSVSAG (SEQ ID NO: 163) 14 199 NTGTAPIYNV (SEQ ID NO: 164) 15 213-215 AKENQLSQILAPNN (SEQ ID NO: 165) 16 276 TLKEALKIAF (SEQ ID NO: 166) 6 17 287-290 GKDITEFDFNFDQQTS (SEQ ID NO: 167) 18 295 QNIKNQLAEL (SEQ ID NO: 168) 19 304-305 LDKIKLNAKMNI (SEQ ID NO: 169) 20 312-314 KRFHYDRNNIAVGA (SEQ ID NO: 169) 7 21 317-323 AVGADESVVKEAHREVINSS TE (SEQ ID NO: 170) 8 22 327 TEGLLLNIDK (SEQ ID NO: 171) 23 329-331 LLNIDKDIRKILSG (SEQ ID NO: 172) 24 357-359 KLPLYISNPNYKVN (SEQ ID NO: 173) 25 366-377 TKENTIINPSEN (SEQ ID NO: 174)

[0112] Table 5. Human Lethal Factor Compiled

[0113] Table 5 discloses immunogenic decapeptides and corresponding epitopes for human lethal factor wherein the peptides and composite sequences were considered reactive if the response was greater than the average plus 2 standard deviations above controls and more than 50% of the individuals were positive for a given region. In this instance, 10 epitopes were identified based on this classification.

TABLE-US-00005 TABLE 5 Epitope Number Pin Number Sequence 1 3-6 KEFIKVISMSCLVTAI (SEQ ID NO: 175) 2 13-14 PVFIPLVQGAGG (SEQ ID NO: 176) 3 66-68 SEDKKKIKDIYGKD (SEQ ID NO: 177) 4 146-147 ELKDQRMLARYE (SEQ ID NO: 178) 5 294-296 KIDTKIQEAQLNIN (SEQ ID NO: 179) 6 298-300 AQLNINQEWNKALG (SEQ ID NO: 180) 7 310-312 NVHNRYASNIVESA (SEQ ID NO: 181) 8 333-334 TDITLPNIAEQY (SEQ ID NO: 182) 9 339-343 THQDEIYEQVHSKGLYVP (SEQ ID NO: 183) 10 389-393 MHSTDHAERLKVQKNAPK (SEQ ID NO: 184)

Table 6. Protective Antigen Human Epitopes Newly Identified

[0114] Table 6 discloses immunogenic decapeptides and corresponding epitopes for human protective antigen wherein the peptides and composite sequences were considered reactive if the peptide response was greater than the average plus 1.5 standard deviations above controls and more than 30% of the individuals were positive for a given region In this instance, nine epitopes were identified based on this classification.

TABLE-US-00006 Epitope Number Pin Number Sequence 1 14-16 IQAEVKQENRLLNE (SEQ ID NO: 185) 2 43-44 ENQTFQSAIWSG (SEQ ID NO: 186) 3 49-50 FIKVKKSDEYTF (SEQ ID NO: 187) 4 64-66 NKASNKIRLEKG (SEQ ID NO: 188) 5 77-78 NPTEKGLDFKLY (SEQ ID NO: 189) 6 111-112 YTVDVKNKRT (SEQ ID NO: 190) 7 132-133 SDFEKVTGRIDK (SEQ ID NO: 191) 8 180-181 STVAIDHSLSLA (SEQ ID NO: 192) 9 218-219 APNNYYPSKNLA (SEQ ID NO: 193)

Table 7. Epitopes Within the Protective Antigen Protein as Recognized by AVA Vaccinated Individual Sera and Stratified by Neutralization.

[0115] Table 7 discloses immunogenic decapeptides and corresponding humoral epitopes for human protective antigen when individuals were stratified based upon ability of sera to inhibit macrophage killing in an in vitro protection assay. Epitopes were considered reactive if the peptide response was greater than the average plus 2 standard deviations above controls and more than 30% of the inviduals were positive for a given response. Using this methodology, we were able to identify 1 unique epitope within the low responders, 7 unique epitopes within the medium responders, and 5 unique epitopes within the high responders for a total of 13 epitopes.

TABLE-US-00007 Epitope A.A. No..sup.a No..sup.b Sequence Domain Function 1 95-106 SGFIKVKKSDEY I PA20 (SEQ ID NO: 194) 2 131-144 NSNKIRLEKGRLYQ I PA20 (SEQ ID NO: 195) 3 153-172 NPTEKGLDFKLYWTDSQNK I PA20 K (SEQ ID NO: 196) 4 181-200 QLPELKQKSSNSRKKRSTSA I/I' Furin Cleavage (SEQ ID NO: 197) 5 221-234 YTVDVKNKRTFLSP I' Ligand binding (SEQ ID NO: 198) 6 261-275 PYSDFEKVTGRIDKNV I' Ligand binding (SEQ ID NO: 199) 7 313-326 SETRTISKNTSTSR II Translocation (SEQ ID NO: 200) 8 3450362 IGGSVSAGFSNSNSSTVA II Translocation (SEQ ID NO: 201) 9 389-398 LNANIRYVNT II Translocation (SEQ ID NO: 202) 10 435-448 APNNYYPSKNLAPI II Translocation (SEQ ID NO: 203) 11 585-596 QQTSQNIKNQLA III Heptamerization (SEQ ID NO: 204) 12 657-666 LLNIDKDIRK IV Receptor binding (SEQ ID NO: 205) 13 753-762 ILIFSKKGYE IV Receptor binding (SEQ ID NO: 206) .sup.aEpitope number as defined in FIG. 23 .sup.bPubmed accession number AAA 22637

Table 8. Edema Factor Human Compiled

[0116] Table 8 discloses immunogenic decapeptides and corresponding epitopes for human edema factor wherein the peptides and composite sequences were considered reactive if the peptide response was greater than the average plus 2 standard deviations above controls and more than 60% of the individuals were positive for a given region. In this instance, 17 epitopes were identified based on this classification.

TABLE-US-00008 TABLE 8 Epitope Number Pin Number Sequence 1 3-4 KFIPNKFSIISF (SEQ ID NO: 207) 2 70-71 SRFVFEKKRETP (SEQ ID NO: 208) 3 192-193 GNLENKKSITEH (SEQ ID NO: 209) 4 200-201 GKIPLKLDHLRI (SEQ ID NO: 210) 5 208-209 ENGIIKGKKEI (SEQ ID NO: 211) 6 233-237 VLGEKFNWRNIEVMAKNV (SEQ ID NO: 212) 7 239-243 VMAKNVEGVLKPLTADYD (SEQ ID NO: 213) 8 252-255 TEIKKQIPTKRMDKW (SEQ ID NO: 214) 9 261-264 PNSLEKQKGVTNLLIK (SEQ ID NO: 215) 10 266-267 TNLLIKYGIERK (SEQ ID NO: 216) 11 274-280 KTLSNWQKQMLDRLNEAVKYT (SEQ ID NO: 217) 12 333-335 FIKNLSSIRRSSNV (SEQ ID NO: 218) 13 358 FSQEKKRKIS (SEQ ID NO: 219) 14 373-376 APEYKNYFQYLKERIT (SEQ ID NO: 220) 15 381-383 NQVQLLTHQKSNI (SEQ ID NO: 221) 16 385-386 HQKSNIEFKLLY (SEQ ID NO: 222) 17 394-395 ENETDNFEVFQK (SEQ ID NO: 223)

Table 9. Mouse Summary Edema Factor

[0117] Table 9 discloses immunogenic decapeptides and corresponding composite immunogenic peptides for mouse Edema Factor wherein the peptides and composite sequences were considered reactive if the peptide reactivity was greater than 0.16 as determined by optical density measurement. Each extended epitope (denoted as composite sequence was required to contain at least one decapeptide demonstrating an optical density (O.D.) of greater than or equal to 0.3. In this instance, 118 reactive amino acids were identified out of a total of 800 amino acids comprising EF. In total, 9 epitopes were identified based on this classification.

TABLE-US-00009 TABLE 9 Pin Composite Epitope Sequence # Sequence Length Number knsmnsrgek 63 KNSMNSRGEKVPFASR 18 1 (SEQ ID NO: 224) FV (SEQ ID NO: 256) smnsrgekvp 64 (SEQ ID NO: 225) nsrgekvpfa 65 (SEQ ID NO: 226) rgekvpfasr 66 (SEQ ID NO: 227) ekvpfasrfv 67 (SEQ ID NO: 228) yainseqske 80 YAINSEQSKEVY 12 2 (SEQ ID NO: 229) (SEQ ID NO: 257) inseqskevy 81 (SEQ ID NO: 230) ssdlifsqkf 103 SSDLLFSQKFKE 12 3 (SEQ ID NO: 240) (SEQ ID NO: 258) dllfsqkfke 104 (SEQ ID NO: 241) lelyapdmfe 129 LELYAPDMFEYM 12 4 (SEQ ID NO: 242) (SEQ ID NO: 259) Lyapdmfeym 130 (SEQ ID NO: 243) egeigkiplk 198 EGEIGKIPLKLD 12 5 (SEQ ID NO: 244) (SEQ ID NO: 260) eigkiplkld 199 (SEQ ID NO: 245) dydifalaps 246 DYDLFALAPSLT 12 6 (SEQ ID NO: 246) (SEQ ID NO: 261) difalapsit 247 (SEQ ID NO: 247) nwqkqmldrl 276 NWQKQMLDRLNEAV 14 7 (SEQ ID NO: 248) (SEQ ID NO: 262) qkqmldrlne 277 (SEQ ID NO: 249) qmldrlneav 278 (SEQ ID NO: 250) neavkytgyt 281 NEAVKYTGYTGG 12 8 (SEQ ID NO: 251) (SEQ ID NO: 263) avkytgytgg 282 (SEQ ID NO: 252) qdneefpekd 291 QDNEEFPEKDNEIF 14 9 (SEQ ID NO: 253) (SEQ ID NO: 264) neefpekdne 292 (SEQ ID NO: 254) efpekdneif 293 (SEQ ID NO: 255)

Table 10: Additional Mouse Edema Factor Data

[0118] Table 10 discloses immunogenic decapeptides and corresponding epitopes for mouse edema factor wherein the peptides and composite sequences were considered reactive if the peptide reactivity was greater than 0.16 as determined by optical density measurement and containing at least one decapeptide with reactivity of 0.3 or greater. In this instance, 9 epitopes were identified based on this classification.

TABLE-US-00010 TABLE 10 Epitope Number on Pin Amino acid structure Numbers # AAA79215 AA Sequence 1 63-67 125-142 KNSMNSRGEKVPFASRFV (SEQ ID NO: 256) 2 80-81 159-170 YAINSEQSKEVY (SEQ ID NO: 257) 3 103-104 205-216 SSDLLFSQKFKE (SEQ ID NO: 258) 4 129-130 257-268 LELYAPDMFEYM (SEQ ID NO: 259) 5 198-199 395-406 EGEIGKI PLKLD (SEQ ID NO: 260) 6 246-247 491-502 DYDLFALAPSLT (SEQ ID NO: 261) 7 276-278 551-564 NWQKQMLDRLNEAV (SEQ ID NO: 262) 8 281-282 561-572 NEAVKYTGYTGG (SEQ ID NO: 263) 9 291-293 581-594 QDNEEFPEKDNEIF (SEQ ID NO: 264)

Anthrax Peptide Formulations

[0119] One or more of the anthrax peptides can be prepared in a physiologically acceptable formulation, such as in a pharmaceutically acceptable carrier, using known techniques. For example, the peptide is combined with a pharmaceutically acceptable excipient to form a therapeutic composition.

[0120] Alternatively, the gene or nucleic acid sequence encoding the peptide may be delivered in a vector for continuous administration using gene therapy techniques. The compositions provided herein may be administered in the form of a solid, liquid or aerosol. Examples of solid compositions include pills, creams, and implantable dosage units. Pills may be administered orally. Therapeutic creams may be administered topically. Implantable dosage units may be administered locally, or may be implanted for systematic release of the therapeutic composition, for example, subcutaneously. Examples of liquid compositions include formulations adapted for injection intramuscularly, subcutaneously, intravenously, intra-arterially, and formulations for topical and intraocular administration. Examples of aerosol formulations include inhaler formulations for administration to the lungs.

[0121] The compositions may be administered by standard routes of administration. In general, the composition may be administered by topical, oral, rectal, nasal or parenteral (for example, intravenous, subcutaneous, or intramuscular) routes. In addition, the composition may be incorporated into a sustained release matrix or matrices such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.

[0122] A sustained release matrix, as used herein, is a matrix made of materials, usually polymers that are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained release matrix desirably is chosen by biocompatible materials such as liposomes, polylactides (polylactide acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).

[0123] Alternatively, the immunogenic anthrax peptide can be inserted into a carrier, such as a membranous carrier, so as to present on the carrier surface immunogenic regions of protective antigen, lethal factor and/or edema factor. Protective antigen, lethal factor and edema factor are all immunogenic because they are recognized by the immune system as "non-self." However, inserting less than whole protective antigen, lethal factor and edema factor toxin onto the surface of liposomes may alter the presentation of the epitope to the immune system, and in some cases, may render the epitope less immunogenic.

[0124] Immunogenic liposomes may be made by reconstituting liposomes in the presence of purified or partially purified protective antigen, lethal factor and edema factor. Additionally, protective antigen, lethal factor and/or edema factor peptides may be reconstituted into liposomes. Liposomes that can be used in the compositions include those known to one skilled in the art. Any of the standard lipids useful for making liposomes may be used. Standard bilayer and multi-layer liposomes may be used to make compositions of the present invention.

[0125] The dosage of the composition will depend on the progression of infection, the particular composition used, and other clinical factors such as weight and condition of the patient, and the route of administration.

Anti-Anthrax Antibody Compositions

[0126] Anthrax peptides having the characteristics set forth above are used for the production of both monoclonal and polyclonal antibodies reactive with anthrax proteins. The compositions containing the immunogenic peptides produce monoclonal or polyclonal antibodies specifically immunoreactive with protective antigen, lethal factor and/or edema factor. Antibodies are made by methods well known to those of ordinary skill in the art.

[0127] The immunogenic anthrax peptide compositions and anti-LF, -EF and -PA antibodies are administered to a human or animal by any appropriate means, preferably by injection. For example, immunogenic peptides of protective antigen, lethal factor and edema factor are administered by subcutaneous or intramuscular injection. Whether internally produced or provided from external sources, the circulating anti-LF, -PA or -EF antibodies bind to appropriate receptors and reduce or inactivate Bacillus anthracis' ability to stimulate infection and disease.

[0128] The preferred antibody is a monoclonal antibody, due to its higher specificity for analyte. Monoclonal antibodies are generated by methods well known to those skilled in the art. The preferred method is a modified version of the method of Kearney, et al., J. Immunol. 123:1548-1558 (1979). Briefly, animals such as mice or rabbits are inoculated with the immunogen in adjuvant, and spleen cells are harvested and mixed with a myeloma cell line, such as P3X63Ag8,653. The cells are induced to fuse by the addition of polyethylene glycol. Hybridomas are chemically selected by plating the cells in a selection medium containing hypoxanthine, aminopterin and thymidine (HAT). Hybridomas are subsequently screened for the ability to produce anti-anthrax monoclonal antibodies. Hybridomas producing antibodies are cloned, expanded and stored frozen for future production.

[0129] The anti-anthrax antibodies described herein are useful both as therapeutic compositions for the treatment of anthrax infection and as reagents in immunoassays for the detection or diagnosis of anthrax infection. The antibodies can be modified according to their designed use.

[0130] Anti-anthrax antibodies for use as a therapeutic treatment to be administered to an animal or human patient infected with anthrax may be modified so that the antibody is more easily tolerated and has fewer adverse side effect. For example, the antibodies may be modified to become hybrid, chimeric or altered antibodies as known to those skilled in the art and described in U.S. Pat. No. 6,331,415. Preferably, the antibodies are "humanized" in accordance with methods known to those skilled in the art. The humanized antibodies, such as humanized monoclonal antibodies, are produced from immunogenic sequences of patients that target key epitopes of protective antigen, lethal factor and/or edema factor. The immunogenic sequences can be used to prevent toxin formation in those individuals already infected with anthrax. These anti-anthrax antibodies are also administered to individuals to passively immunize them against Bacillus anthracis and thereby prevent the initiation of infection, reduce reactivity or inhibit the proliferation of Bacillus anthracis. When administered to individuals, the anti-LF, -EF and -PA antibodies bind to Bacillus anthracis, thereby reducing the effective circulating concentration of Bacillus anthracis toxins. Consequently, Bacillus anthracis toxin-dependent infection is prevented, reduced or inhibited.

[0131] If the antibody is intended for use an a reagent in an immunoassay for the detection of anthrax toxin, such as for the detection of anthrax infection in a patient, the antibody may be modified so that it can be directly or indirectly detection. For example, the antibody may be labeled directly with a detectable label for identification and quantitation of anthrax protein. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances including colored particles such as colloidal gold and latex beads.

[0132] Alternatively, the antibody may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin, such as protein A or G or second antibodies. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art.

Diseases and Conditions to be Treated

[0133] The anthrax peptide compositions described herein are useful for treating, preventing, lessening or alleviating disease, symptoms and complications of Bacillus anthracis infection. The compositions described herein are particularly useful as a prophylactic against the causative agent of anthrax and are especially useful as a vaccine against Bacillus anthracis. The compositions are especially useful for treating or preventing Bacillus anthracis by producing neutralizing antibodies directed against the toxin components protective antigen, lethal factor and edema factor. The anti-anthrax antibody compositions described herein are useful for providing an immunotherapeutic agent for the treatment of anthrax infection.

[0134] Administration of the compositions provided herein to a human or animal infected with Bacillus anthracis is useful for reducing or alleviating the symptoms and immunogenic reaction of the infection.

[0135] Administration of the compositions provided herein to a human or animal at risk of being exposed to Bacillus anthracis is useful for preventing, reducing or alleviating the symptoms and immunogenic reaction of the infection. Compositions containing immunogenic peptides of protective antigen, lethal factor and/or edema factor are administered to a human or animal to induce immunity to Bacillus anthracis. The immunized human or animal develops circulating antibodies against Bacillus anthracis which bind to corresponding regions of protective antigen, lethal factor and edema factor, thereby reducing or inactivating its ability to stimulate Bacillus anthracis infection.

Antibodies and Immunoassay Detection Methods

[0136] Anthrax peptides, antibodies, methods, and kits for the detection of Bacillus anthracis proteins in a sample are provided. The anthrax peptides described above are useful for detecting anti-anthrax antibodies in a biological sample from a patient. In addition, the anthrax peptides are useful for the production of anti-anthrax antibodies for the detection of anthrax toxin in a biological sample.

[0137] As mentioned above, anti-anthrax monoclonal and polyclonal antibodies having sensitivity for PA, EF and LF toxins are produced by immunizing an animal with one or more of the anthrax peptides described herein, isolating antibodies that react with the epitopes, and collecting and purifying the antibodies from a biological fluid such as blood in accordance with methods well known to those skilled in the art.

[0138] Immunoassay methods containing the antibodies immunoreactive with the anthrax toxins are useful for the detection of anthrax in a sample such as an animal sample. Immunoassays employing such antibodies are capable of detecting low concentrations of protein in the sample. The antibodies are immunoreactive with one or more epitopes on the PA, EF and LF proteins and react minimally with other proteins that may be present in the sample, thus providing for an accurate determination of the presence of anthrax in a sample, such as a human possibly exposed to anthrax. The preferred antibodies are monoclonal antibodies, produced by hybridomas, due to their high specificity.

[0139] The antibodies are collectively assembled in a kit with conventional immunoassay reagents for detection of anthrax protein. The kit may optionally contain both monoclonal and polyclonal antibodies and a standard for the determination of the presence of anthrax protein in a sample. The kit containing these reagents provides for simple, rapid, on site detection of anthrax protein.

[0140] Immunoassays

[0141] A sensitive immunoassay employing antibodies as reagents, prepared using the anthrax peptides described above, is provided. The immunoassay is useful for detecting the presence or amount of anthrax toxin in a variety of samples, particularly human samples. The sample may be obtained from any source in which the anthrax proteins are accessible to the antibody. For example, the sample may be a biological fluid of an animal suspected of being infected with anthrax, such as a blood or serum sample. The immunoassay is therefore useful for diagnosis or prognosis of an anthrax infection.

[0142] An immunoassay employing the anthrax peptides described herein as reagents is also provided. The peptides are used as reagents to test for specific protective immunity to anthrax in biological samples taken from anthrax vaccinated individuals.

[0143] The peptides bind to the antibodies in the biological sample. Results from this type of immunoassay provide information on the immune responsiveness to anthrax of the vaccinated individual that can be used to decrease the number of boosters needed by that individual or can be used to identify individuals who require alternative protective procedures.

[0144] The reagents may be employed in any heterogeneous or homogeneous sandwich or competitive immunoassay for the detection of anthrax protein. Either the reagent is labeled with a detectable label or is coupled to a solid phase. Methods for coupling reagents to solid phases are well known to those skilled in the art. In accordance with the immunoassay method, the sample containing the analyte is reacted with the reagent for a sufficient amount of time under conditions that promote the binding of antibody to peptide. It will be understood by those skilled in the art that the immunoassay reagents and sample may be reacted in different combinations and orders. A physical means is employed to separate reagents bound to the solid phase from unbound reagents such as filtration of particles, decantation of reaction solutions from coated tubes or wells, magnetic separation, capillary action, and other means known to those skilled in the art. It will also be understood that a separate washing of the solid phase may be included in the method.

[0145] The concentration of anthrax protein, or anti-anthrax antibody, in the sample is determined either by comparing the intensity of the color produced by the sample to a color card or by using a reflectometer.

[0146] The resulting reaction mixture, or combination of reagent and sample, is prepared in a solution that optimizes antibody-analyte binding kinetics. An appropriate solution is an aqueous solution or buffer. The solution is preferably provided under conditions that will promote specific binding, minimize nonspecific binding, solubilize analyte, stabilize and preserve reagent reactivity, and may contain buffers, detergents, solvents, salts, chelators, proteins, polymers, carbohydrates, sugars, and other substances known to those skilled in the art.

[0147] The reaction mixture solution is reacted for a sufficient amount of time to allow the antibody to react and bind to the peptide to form an antibody-analyte complex. The shortest amount of reaction time that results in binding is desired to minimize the time required to complete the assay. An appropriate reaction time period for an immunochromatographic strip test is less than or equal to 20 minutes or between approximately one minute and 20 minutes. A reaction time of less than five minutes is preferred. Most preferably, the reaction time is less than three minutes. By optimizing the reagents, binding may be substantially completed as the reagents are combined.

[0148] The reaction is performed at any temperature at which the reagents do not degrade or become inactivated. A temperature between approximately 4.degree. C. and 37.degree. C. is preferred. The most preferred reaction temperature is ambient or room temperature (approximately 25.degree. C.).

[0149] Immunoassay Kit

[0150] An immunoassay kit for the detection of anthrax protein in a sample contains one or more antibodies prepared using the epitopes described above.

[0151] The kit may additionally contain equipment for obtaining the sample, a vessel for containing the reagents, a timing means, a buffer for diluting the sample, and a colorimeter, reflectometer, or standard against which a color change may be measured.

[0152] In a preferred embodiment, the reagents, including the antibody are dry. Addition of aqueous sample to the strip results in solubilization of the dry reagent, causing it to react.

[0153] An alternative immunoassay kit would be useful for the detection of specific, protective anthrax epitope reactivity. After vaccination, individuals often need to be assessed to determine if protective immunity has been generated (or continues to be generated). This kit contains one or a mixture of the immunogenic peptides described herein coupled to a solid phase system. Dilutions of sera are incubated with these peptides and detection systems used to measure the presence and concentration of protective antibodies.

[0154] The compositions and methods are further illustrated by the following non-limiting examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLE 1

Generation of Mouse Anti-sera Containing Protective Antibodies to Anthrax Lethal Toxin and Protective Antigen

[0155] FIG. 2 shows the study design used to immunize groups of A/J mice with recombinant PA, recombinant LF or with adjuvant alone. The initial immunization was conducted in the presence of Complete Freund's Adjuvant (CFA) on day 0 and boosters were given on days 10, 24 and 38.

[0156] Blood samples for antibody testing and epitope mapping were collected from individual mice four days after each boost and again before toxin challenge, which was conducted 70-80 days after the final boost.

[0157] FIG. 3 demonstrates verification that the immunized mice were producing robust antibody responses to the recombinant LF and PA immunogens. FIG. 3 shows IgG antibody titers to LF and PA as determined by ELISA using commercially available LF and PA preparations.

[0158] LF-immunized mice produced high titers of anti-LF antibodies at day 28, and PA-immunized mice similarly produced high titers of anti-PA antibodies at day 28, while mice immunized with adjuvant alone did not. Samples collected prior to immunization were also negative for these antibodies. The average ODs of 1/100 serum dilutions of each group of sera, also confirm this pattern.

EXAMPLE 2

Lethal Toxin Challenge

[0159] Before conducting epitope mapping, the mice of Example 1 were studied to determine whether they had produced lethal toxin-neutralizing antibodies. To check this, the mice were challenged with a lethal dose of the anthrax lethal toxin, which is co-injected PA plus LF. The dose used was three times the LD50 dose as determined in 6 week old A/J mice (See FIG. 4).

[0160] Only one of seven mice in the control group survived the challenge.

[0161] In contrast nine of ten PA-immunized mice survived the challenged (p=0.009), and seven of eight LF-immunized mice (p=0.007) survived the challenge. These data suggested that the mice had produced toxin-neutralizing antibodies.

EXAMPLE 3

Epitope Mapping

[0162] To map anti-LF, anti-EF and anti-PA antibodies produced in immunized mice sera, sera was screened against a series of solid phase bound 10 mer peptides that overlap by eight amino acids (See FIG. 5). The overlapping decapeptides span the entire length of protective antigen (379 peptides), edema factor (396 peptides) and lethal factor (401 peptides).

[0163] The decapeptides were conveniently synthesized onto polyethylene rods in a 96-well format. In a modified ELISA technique, the pins were lowered into 96-well plates for various incubations and washed in between.

[0164] Each individual decapeptide for each Bacillus anthracia toxin component was tested for immunogenic activity. FIG. 6 demonstrates results obtained in mice for B cell epitopes of lethal factor.

EXAMPLE 4

Epitope Mapping of Lethal Factor

[0165] FIG. 6 demonstrates the mapping results of LF-immunized A/J mice from day 28 bleed to day 108 bleed. It was found that multiple epitopes exist throughout the molecule. Although some epitopes decreased in reactivity by day 108, certain regions of the lethal factor toxin remained elevated or changed in intensity by day 108. Mice immunized with adjuvant alone failed to significantly bind any of the decapeptides. Regions that spanned at least one decapeptide with an O.D. of 1.0 or above were considered epitopes. Subsets of these epitopes are highlighted in FIG. 6 as examples.

[0166] Individual decapeptides observed to be immunogenic above a scale of 0.2 (O.D) were identified as immunogenic peptides. Each extended epitope (denoted as composite sequence) was required to contain at least one decapeptide demonstrating an O.D. of greater than or equal to 0.4.

[0167] A subset of the epitopes highlighted as examples were superimposed onto the crystal structure of lethal factor.

[0168] The epitopes as superimposed onto the crystal structure of FIG. 6 and also identified in Table 1 correspond to the following amino acid locations of wild-type lethal factor (See SEQ ID NO:2). [0169] 1: EKVPSDVLEMYKAIGGKI (SEQ ID NO:95): corresponds to amino acids 94-114 of wild type lethal factor. [0170] 2: SEDKKKIKDIYGKDALLHEH (SEQ ID NO:96): corresponds to amino acids 131-150 of wild type lethal factor. [0171] 3: QHRDVLQLYAPEAFNYMDKFNEGEINLSLEELKD (SEQ ID NO:265): corresponds to amino acids 261-294 of wild type lethal factor. [0172] 4: LLKKLQIPIE (SEQ ID NO:47): corresponds to amino acids 325-334 of wild type lethal factor. [0173] 5: SLSQEEKELLKRIQ (SEQ ID NO:102): corresponds to amino acids 343-356 of wild type lethal factor. [0174] 6: DFLSTEEKEFLKKLQIDIRD (SEQ ID NO:266): corresponds to amino acids 361-378 of wild type lethal factor. [0175] 7: SLSEEEKELLNRIQ (SEQ ID NO:104): corresponds to amino acids 381-394 of wild type lethal factor. [0176] 8: LSEKEKEFLKKLKLDI (SEQ ID NO:105): corresponds to amino acids 401-416 of wild type lethal factor. [0177] 9: GPYDINGRLQDT (SEQ ID NO:267): corresponds to amino acids 417-428 of wild type lethal factor. [0178] 10: LIDSPSINLDVRKQ (SEQ ID NO:107): corresponds to amino acids 431-444 of wild type lethal factor. [0179] 11: LVDGNGRFVFTDITLP (SEQ ID NO:110): corresponds to amino acids 655-670 of wild type lethal factor. [0180] 12: GYTHQDEIYEGV (SEQ ID NO:268): corresponds to amino acids 675-686 of wild type lethal factor.

[0181] Interestingly, the lethal factor epitopes tended to cluster in domain I, the PA binding domain and domain III, which has some residues that contact the MAP-kinase kinase substrate.

[0182] Two epitopes were also observed in domain IV of lethal factor, the catalytic domain.

EXAMPLE 5

Epitope Mapping of Protective Antigen

[0183] FIG. 7 demonstrates the mapping results of PA-immunized A/J mice from day 28 bleed to day 108 bleed. Again it was found that multiple epitopes exist throughout the molecule. PA harbored fewer epitopes than observed for LF, despite high antibody titers.

[0184] Regions that spanned at least one decapeptide with reactivity of 0.4 (0.D) or greater were considered epitopes. Eight prominent epitopes, a subset of the total identified epitopes, were overlaid onto the PA crystal structure as examples. Interestingly one of the epitopes spans the furin cleavage site; whereas two of the epitopes occur in regions of PA that have been implicated in influencing binding to LF and EF.

[0185] Epitope 5 is not resolved in the crystal structure.

[0186] Also shown in FIG. 7 are several unmarked epitopes. In this example, individual decapeptides observed to be immunogenic above a scale of 0.2 and containing at least one decapeptide with reactivity of 0.4 or greater define immunogenic peptides.

[0187] The epitopes as superimposed onto the crystal structure of FIG. 7 and also identified in Table 4 (No. on structure) correspond to the following amino acid locations of wild-type protective antigen (See SEQ ID NO:1). [0188] 1: STTGDLSIPSSELENIDSEN (SEQ ID NO:269): corresponds to amino acids 67-86 of wild-type protective antigen. [0189] 2: LKQKSSNSRKKRSTSAGPTVPD (SEQ ID NO:156): corresponds to amino acids 185-206 of wild-type protective antigen. [0190] 3: TASDPYSDFEKVTGRIDKNVSP (SEQ ED NO:159): corresponds to amino acids 257-278 of wild-type protective antigen. [0191] 4: VDMENIILSKNEDQSTQN (SEQ ID NO:162): corresponds to amino acids 293-310 of wild-type protective antigen. [0192] 5: NAEVHASFEDIGGSVSAG (SEQ ID NO:270): corresponds to amino acids 335-352 of wild-type protective antigen. [0193] 6: GKDITEFDFNFDQQTS (SEQ ID NO:167): corresponds to amino acids 573-588 of wild-type protective antigen. [0194] 7: (SEQ ID NO:170): corresponds to amino acids 633-654 of wild-type protective antigen. [0195] 8: TEGLLLNIDK (SEQ ID NO:171): corresponds to amino acids 653-662 of wild-type protective antigen.

EXAMPLE 6

Strain Background Versus MHC

[0196] In order to broaden the number of identifiable B cell epitopes of PA and LF, epitopes were also mapped in immunized C57BL/6 mice (See FIG. 8).

[0197] In order to assess the relative importance of MHC class II haplotype versus genetic background in epitope selection, responses in C57BL/6 mice congenic for the MHC class II haplotype of A/J mice were also assessed (See FIG. 8).

[0198] FIG. 9 shows the mapping results after LF immunization in Day 28 bleeds.

[0199] Some epitopes were identified as common among the strains, denoted by the "C", while other epitopes depended upon the genetic background, denoted by the "B". In this case, the A/J background was important.

[0200] Interestingly, other epitopes were strongly influenced by MHC class II, and these epitopes are denoted by "M". In those instances, the "k" haplotype was required for this epitope.

[0201] In total, most epitopes were determined by non-MHC background genes, while several epitopes were common between the two strains.

[0202] MHC class II haplotype was found not to be an important determinant of B cell epitope selection.

[0203] FIG. 10 shows the mapping results after PA immunization in Day 28 bleeds.

EXAMPLE 7

Evaluation of Antibodies to Protective Antigen in Vaccinated Humans

[0204] To begin examining the humoral immune response following anthrax vaccination, individuals who had received three or more doses of the anthrax vaccine absorbed (AVA) were recruited and peripheral blood was collected. A cohort of 114 vaccinated individuals as well as 26 unvaccinated controls were recruited into the study. The majority of the individuals, 61.4%, were Caucasian with 23.68% African American, 5.26% Asian, and 9.64% of other nationalities. The average age was 33.83 (.+-.8.6), the average number of vaccinations was 5.12 (.+-.1.47), and the average time since the last vaccination was 2.45 (.+-.1.39) years.

[0205] The sera from these individuals was used in a standard ELISA to detect the level of antibodies against protective antigen, lethal factor, and edema factor. An optical density greater than the average plus twice the standard deviation of the controls was considered positive. The vaccinated individuals had detectable anti-protective antigen antibodies (FIG. 11), while only a subset had detectable anti-lethal factor or anti-edema factor antibodies. Individuals with anti-protective antigen antibodies could be divided into four groups based upon antibody titer (1:10, 1:100, 1:1,000, and 1:10,000). Surprisingly, the level of antigen-specific antibodies did not correlate with age of the vaccinee nor the number of vaccinations (FIG. 11). Additionally, the presence or absence of anti- lethal or edema factor antibodies did not correlate with the level of anti-protective antigen antibodies. However, a significant (p=0.0008) inverse correlation between the years post vaccination and the anti-PA titer was found. Thus, those individuals with the highest titer of anti-protective antigen antibodies were most recently vaccinated (1.0.+-.0.4 years post vaccination, FIG. 11).

EXAMPLE 8

Evaluation of AVA Vaccinated Individual Sera for In Vitro Inhibition of Toxin Macrophage Kills

[0206] Because antibodies that are potentially neutralizing are most interesting, this example focused on the epitopes identified in those individuals who were high responders (FIG. 23 and Table 7) and antibodies in regions with high potential for toxin neutralization. The most prominent epitope identified in the high responders was an epitope, QLPELKQKSSNSRKKRSTSA (SEQ ID NO:197), which was identified as the region containing the furin cleavage site. In fact, five out of six (or 83%) of the high responders had antibodies directed against this antigenic region based upon the solid phase peptide assay. In addition to this epitope within the furin cleavage site, the ability of two other epitopes to mediate protection was analyzed. The first of these was an epitope within the ligand binding region (PYSDFEKVTGRJDKNV, SEQ ID NO:199). The final epitope was identified using solid phase peptide assays on sera from mice immunized with protective antigen (described below) and is within the receptor binding domain of the protective antigen.

[0207] To test for antibodies directed at these three epitopes of interest, peptides covering these regions were synthesized and used in a peptide-specific ELISA. As shown in FIG. 22 (panels A-C), 16 individuals were identified with antibodies directed at the furin cleavage site epitope, 12 individuals with antibodies directed at the ligand binding site epitope, and 18 individuals with antibodies directed at the receptor binding site. Once these unique antigen regions had been identified and confirmed, the ability of antibodies to these epitopes to mediate protection was tested. Thus, peptide-specific antibodies were enriched using column absorption followed by peptide-specific ELISAs and toxin neutralization. As shown in FIG. 22 (panels D-F), in all cases it was possible to significantly enrich and deplete for antibodies specific for the corresponding peptide. These enriched and depleted samples were then used in a standard toxin neutralization assay to test the ability of these peptide-specific antibodies to mediate protection. As shown in FIG. 22 (panel G), antibodies directed against the epitope within the furin cleavage site were able to block toxin activity while antibodies directed at the other two epitopes were less capable. Thus, the furin peptide-specific antibodies generated following AVA vaccination are capable of neutralizing toxin activity.

EXAMPLE 9

Specificity of Protective Antigen Antibodies

[0208] The fine specificity of the protective antigen antibody response was evaluated utilizing overlapping decapeptides synthesized on a solid phase support, as described above for Example 3. Briefly, sera from a vaccinated individual was incubated with overlapping decapeptides of PA, followed by incubation with a human IgG conjugate linked to HRP, and absorbance at 450 nm determined. As shown in FIG. 12, the fine specificity of the protective antigen antibody response following vaccination can be clearly observed. FIG. 12 also presents the most reactive epitopes (epitopes #1-9) superimposed onto the crystal structure of PA. The observation of regions associated with immunogenicity allows for the detection of epitopes and individual decapeptides that are immunogenic peptide fragments.

[0209] FIG. 23 presents the fine specificity of the response stratified by ability of the vaccinated individual sera to inhibit macrophase killing (surrogate in vitro model of neutralization). Additional and unique areas of humoral epitope specificity were determined by this method.

EXAMPLE 10

Evaluation of Antibodies to Lethal Factor in Vaccinated Humans

[0210] As discussed above, for examination of humoral and cellular immunity following anthrax vaccination, sera from individuals who had received three or more doses of the anthrax vaccine absorbed (AVA), as well as unvaccinated controls matched by age, sex, and race, were analyzed. As seen in FIG. 11, the majority of individuals tested had received the recommended six vaccinations, and the average time since the last vaccination was 2.33 (.+-.1.9) years. The sera were tested by ELISA to detect the level of antibodies against lethal factor. An optical density greater than the average plus twice the standard deviation of the controls was considered positive.

[0211] All of the vaccinated individuals (n=29) had detectable anti-protective antigen antibodies (FIG. 11). However, only a subset of these vaccinated individuals had detectable antibodies to lethal factor (n=9) or edema factor (n=10).

[0212] The fine specificity of the lethal factor antibody response was evaluated utilizing overlapping decapeptides synthesized on a solid phase support, as described above for Example 3. Briefly, sera from a vaccinated individual was incubated with overlapping decapeptides of LF, followed by incubation with a human IgG conjugate linked to HRP, and absorbance at 450 nm determined. As shown in FIG. 14, the fine specificity of the lethal factor antibody response following vaccination can be clearly observed. FIG. 14 also presents the most reactive epitopes (epitopes #1-10) superimposed onto the crystal structure of LF. The observation of regions associated with immunogenicity allows for the detection of epitopes and individual decapeptides that are immunogenic peptide fragments.

EXAMPLE 11

Generation of Anti-Sera Containing Protective Antibodies to Anthrax Edema Factor

[0213] FIG. 16 shows the study design used to immunize groups of A/J mice with recombinant EF or with adjuvant alone. The initial immunization was conducted in the presence of CFA on day 0 and boosters were given on days 10, 24 and 38.

[0214] Blood samples for antibody testing and epitope mapping were collected from individual mice 4-5 days after each boost and again prior to edema toxin challenge, which was 129 days after the final boost.

[0215] FIG. 17 demonstrates verification that the immunized mice were producing robust antibody responses to the recombinant EF. FIG. 17 shows IgG antibody titers to EF as determined by ELISA using a commercially available EF preparation.

[0216] All EF-immunized mice produced high titers of anti-EF antibodies at days 28, 42 and 171.

EXAMPLE 12

Edema Toxin Challenge

[0217] The mice of Example 11 were studied to determine whether they had produced edema toxin-neutralizing antibodies. To check this, the LD50 of edema toxin was first determined in unimmunized six week old A/J mice (see FIG. 18A). The mice of Example 11 were then challenged with a lethal dose of the anthrax edema toxin, which is co-injected PA plus EF. The dose used was four times the LD50 dose determined in A/J mice.

[0218] Fifty percent of the EF-immunized mice survived the lethal edema toxin challenge, while 0% of the control mice that had been immunized with adjuvant alone survived the challenge (see FIG. 18B). These data suggested that the EF-immunized mice of Example 11 had produced edema toxin-neutralizing antibodies.

EXAMPLE 13

Epitope Mapping of Edema Factor

[0219] FIG. 19 demonstrates the mapping results of EF-immunized A/J mice from day 15 and day 28 bleeds. It was found that the response initiated with a single epitope on day 15 then expanded to include multiple epitopes by day 28. Mice immunized with adjuvant alone failed to significantly bind any of the decapeptides. Regions that spanned at least one decapeptide with an O.D. of 0.3 and above were considered epitopes. These epitopes are highlighted in FIG. 19 and superimposed onto the known EF crystal structure as examples from this particular mouse strain.

[0220] The epitopes, as superimposed onto the crystal structure of FIG. 19 and also identified in Table 9, correspond to the following amino acid locations of wild-type edema factor (See SEQ ID NO:3). [0221] 1: KNSMNSRGEKVPFASRFV (SEQ ID NO:256): corresponds to amino acids 125-142 of wild type edema factor. [0222] 2: YAINSEQSKEVY (SEQ ID NO:257): corresponds to amino acids 159-170 of wild type edema factor. [0223] 3: SSDLLFSQKFKE (SEQ ID NO:258): corresponds to amino acids 205-216 of wild type edema factor. [0224] 4. LELYAPDMFEYM (SEQ ID NO:259): corresponds to amino acids 257-268 of wild type edema factor. [0225] 5. EGEIGKIPLKLD (SEQ ID NO:260): corresponds to amino acids 395-406 of wild type edema factor. [0226] 6. DYDLFALAPSLT (SEQ ID NO:261): corresponds to amino acids 491-502 of wild type edema factor. [0227] 7. NWQKQMLDRLNEAV (SEQ ID NO:262): corresponds to amino acids 551-564 of wild type edema factor. [0228] 8. NEAVKYTGYTGG (SEQ ID NO:263): corresponds to amino acids 561-572 of wild type edema factor. [0229] 9. QDNEEFPEKDNEIF (SEQ ID NO:264): corresponds to amino acids 581-594 of wild type edema factor.

EXAMPLE 14

Identification of Neutralizing Peptide Epitopes of PA and LF Using Murine Immune Serum

[0230] Immune sera from LF- and PA-immunized A/J mice were subjected to affinity chromatography using multiple antigenic peptide columns constructed from the following epitopes which are also disclosed in FIGS. 6 and 7, respectively, and Tables 1-4: PA 164-177, RKKRSTSAGPTVPD (SEQ ID NO: 271); PA 230-243, SDPYSDFEKVTGRIDK (SEQ ID NO: 272); and LF 232-247, VLQLYAPEAFNYMDKF (SEQ ID NO: 273). The numbers here refer to amino acids in SEQ ID NO:1 for PA and SEQ ID NO:2 for LF. Individual column fractions were concentrated to the original serum volume and titrated into a standard Lethal Toxin-mediated macrophage cell death assay. As shown in FIG. 20, all three preparations of the thus-purified antibodies demonstrated a capacity for neutralizing the ability of Lethal Toxin to kill a mouse macrophage cell line, as indicated by increased % cell viability. The concentration of total IgG in each retained (i.e. peptide-binding) fraction was quantitated and is indicated in FIG. 20.

[0231] All scientific articles, publications, abstracts, patents and patent applications mentioned above are hereby incorporated by reference in their entirety.

[0232] While this invention has been described in specific detail with reference to the disclosed embodiments, it will be understood that many variations and modifications may be effected within the spirit and scope of the invention.

Sequence CWU 1

1

2671764PRTBacillus anthracis 1Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser Thr Ile1 5 10 15Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu Val Lys 20 25 30Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser Gln Gly Leu 35 40 45Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro Met Val Val 50 55 60Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu65 70 75 80Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile Trp Ser Gly 85 90 95Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala Thr Ser Ala 100 105 110Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val Ile Asn Lys 115 120 125Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg Leu Tyr Gln 130 135 140Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp145 150 155 160Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu Val Ile Ser 165 170 175Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser 180 185 190Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp Arg Asp 195 200 205Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp 210 215 220Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His225 230 235 240Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser 245 250 255Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile 260 265 270Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val Ala Ala Tyr 275 280 285Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser Lys Asn Glu 290 295 300Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr Ile Ser Lys305 310 315 320Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His Gly Asn Ala 325 330 335Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser Ala Gly 340 345 350Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser Leu Ser 355 360 365Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu Asn Thr Ala 370 375 380Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr385 390 395 400Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu Gly Lys 405 410 415Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln 420 425 430Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile 435 440 445Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn 450 455 460Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp465 470 475 480Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly 485 490 495Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro Gln 500 505 510Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn 515 520 525Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu Glu 530 535 540Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe545 550 555 560Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile 565 570 575Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys 580 585 590Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val Leu Asp 595 600 605Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys Arg 610 615 620Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser Val625 630 635 640Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr Glu Gly Leu 645 650 655Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile 660 665 670Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile Asn Asp Arg 675 680 685Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe 690 695 700Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn705 710 715 720Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr Ile 725 730 735Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys 740 745 750Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 755 7602809PRTBacillus anthracis 2Met Asn Ile Lys Lys Glu Phe Ile Lys Val Ile Ser Met Ser Cys Leu1 5 10 15Val Thr Ala Ile Thr Leu Ser Gly Pro Val Phe Ile Pro Leu Val Gln 20 25 30Gly Ala Gly Gly His Gly Asp Val Gly Met His Val Lys Glu Lys Glu 35 40 45Lys Asn Lys Asp Glu Asn Lys Arg Lys Asp Glu Glu Arg Asn Lys Thr 50 55 60Gln Glu Glu His Leu Lys Glu Ile Met Lys His Ile Val Lys Ile Glu65 70 75 80Val Lys Gly Glu Glu Ala Val Lys Lys Glu Ala Ala Glu Lys Leu Leu 85 90 95Glu Lys Val Pro Ser Asp Val Leu Glu Met Tyr Lys Ala Ile Gly Gly 100 105 110Lys Ile Tyr Ile Val Asp Gly Asp Ile Thr Lys His Ile Ser Leu Glu 115 120 125Ala Leu Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly Lys Asp 130 135 140Ala Leu Leu His Glu His Tyr Val Tyr Ala Lys Glu Gly Tyr Glu Pro145 150 155 160Val Leu Val Ile Gln Ser Ser Glu Asp Tyr Val Glu Asn Thr Glu Lys 165 170 175Ala Leu Asn Val Tyr Tyr Glu Ile Gly Lys Ile Leu Ser Arg Asp Ile 180 185 190Leu Ser Lys Ile Asn Gln Pro Tyr Gln Lys Phe Leu Asp Val Leu Asn 195 200 205Thr Ile Lys Asn Ala Ser Asp Ser Asp Gly Gln Asp Leu Leu Phe Thr 210 215 220Asn Gln Leu Lys Glu His Pro Thr Asp Phe Ser Val Glu Phe Leu Glu225 230 235 240Gln Asn Ser Asn Glu Val Gln Glu Val Phe Ala Lys Ala Phe Ala Tyr 245 250 255Tyr Ile Glu Pro Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu 260 265 270Ala Phe Asn Tyr Met Asp Lys Phe Asn Glu Gln Glu Ile Asn Leu Ser 275 280 285Leu Glu Glu Leu Lys Asp Gln Arg Met Leu Ala Arg Tyr Glu Lys Trp 290 295 300Glu Lys Ile Lys Gln His Tyr Gln His Trp Ser Asp Ser Leu Ser Glu305 310 315 320Glu Gly Arg Gly Leu Leu Lys Lys Leu Gln Ile Pro Ile Glu Pro Lys 325 330 335Lys Asp Asp Ile Ile His Ser Leu Ser Gln Glu Glu Lys Glu Leu Leu 340 345 350Lys Arg Ile Gln Ile Asp Ser Ser Asp Phe Leu Ser Thr Glu Glu Lys 355 360 365Glu Phe Leu Lys Lys Leu Gln Ile Asp Ile Arg Asp Ser Leu Ser Glu 370 375 380Glu Glu Lys Glu Leu Leu Asn Arg Ile Gln Val Asp Ser Ser Asn Pro385 390 395 400Leu Ser Glu Lys Glu Lys Glu Phe Leu Lys Lys Leu Lys Leu Asp Ile 405 410 415Gln Pro Tyr Asp Ile Asn Gln Arg Leu Gln Asp Thr Gly Gly Leu Ile 420 425 430Asp Ser Pro Ser Ile Asn Leu Asp Val Arg Lys Gln Tyr Lys Arg Asp 435 440 445Ile Gln Asn Ile Asp Ala Leu Leu His Gln Ser Ile Gly Ser Thr Leu 450 455 460Tyr Asn Lys Ile Tyr Leu Tyr Glu Asn Met Asn Ile Asn Asn Leu Thr465 470 475 480Ala Thr Leu Gly Ala Asp Leu Val Asp Ser Thr Asp Asn Thr Lys Ile 485 490 495Asn Arg Gly Ile Phe Asn Glu Phe Lys Lys Asn Phe Lys Tyr Ser Ile 500 505 510Ser Ser Asn Tyr Met Ile Val Asp Ile Asn Glu Arg Pro Ala Leu Asp 515 520 525Asn Glu Arg Leu Lys Trp Arg Ile Gln Leu Ser Pro Asp Thr Arg Ala 530 535 540Gly Tyr Leu Glu Asn Gly Lys Leu Ile Leu Gln Arg Asn Ile Gly Leu545 550 555 560Glu Ile Lys Asp Val Gln Ile Ile Lys Gln Ser Glu Lys Glu Tyr Ile 565 570 575Arg Ile Asp Ala Lys Val Val Pro Lys Ser Lys Ile Asp Thr Lys Ile 580 585 590Gln Glu Ala Gln Leu Asn Ile Asn Gln Glu Trp Asn Lys Ala Leu Gly 595 600 605Leu Pro Lys Tyr Thr Lys Leu Ile Thr Phe Asn Val His Asn Arg Tyr 610 615 620Ala Ser Asn Ile Val Glu Ser Ala Tyr Leu Ile Leu Asn Glu Trp Lys625 630 635 640Asn Asn Ile Gln Ser Asp Leu Ile Lys Lys Val Thr Asn Tyr Leu Val 645 650 655Asp Gly Asn Gly Arg Phe Val Phe Thr Asp Ile Thr Leu Pro Asn Ile 660 665 670Ala Glu Gln Tyr Thr His Gln Asp Glu Ile Tyr Glu Gln Val His Ser 675 680 685Lys Gly Leu Tyr Val Pro Glu Ser Arg Ser Ile Leu Leu His Gly Pro 690 695 700Ser Lys Gly Val Glu Leu Arg Asn Asp Ser Glu Gly Phe Ile His Glu705 710 715 720Phe Gly His Ala Val Asp Asp Tyr Ala Gly Tyr Leu Leu Asp Lys Asn 725 730 735Gln Ser Asp Leu Val Thr Asn Ser Lys Lys Phe Ile Asp Ile Phe Lys 740 745 750Glu Glu Gly Ser Asn Leu Thr Ser Tyr Gly Arg Thr Asn Glu Ala Glu 755 760 765Phe Phe Ala Glu Ala Phe Arg Leu Met His Ser Thr Asp His Ala Glu 770 775 780Arg Leu Lys Val Gln Lys Asn Ala Pro Lys Thr Phe Gln Phe Ile Asn785 790 795 800Asp Gln Ile Lys Phe Ile Ile Asn Ser 8053800PRTBacillus anthracis 3Met Thr Arg Asn Lys Phe Ile Pro Asn Lys Phe Ser Ile Ile Ser Phe1 5 10 15Ser Val Leu Leu Phe Ala Ile Ser Ser Ser Gln Ala Ile Glu Val Asn 20 25 30Ala Met Asn Glu His Tyr Thr Glu Ser Asp Ile Lys Arg Asn His Lys 35 40 45Thr Glu Lys Asn Lys Thr Glu Lys Glu Lys Phe Lys Asp Ser Ile Asn 50 55 60Asn Leu Val Lys Thr Glu Phe Thr Asn Glu Thr Leu Asp Lys Ile Gln65 70 75 80Gln Thr Gln Asp Leu Leu Lys Lys Ile Pro Lys Asp Val Leu Glu Ile 85 90 95Tyr Ser Glu Leu Gly Gly Glu Ile Tyr Phe Thr Asp Ile Asp Leu Val 100 105 110Glu His Lys Glu Leu Gln Asp Leu Ser Glu Glu Glu Lys Asn Ser Met 115 120 125Asn Ser Arg Gly Glu Lys Val Pro Phe Ala Ser Arg Phe Val Phe Glu 130 135 140Lys Lys Arg Glu Thr Pro Lys Leu Ile Ile Asn Ile Lys Asp Tyr Ala145 150 155 160Ile Asn Ser Glu Gln Ser Lys Glu Val Tyr Tyr Glu Ile Gly Lys Gly 165 170 175Ile Ser Leu Asp Ile Ile Ser Lys Asp Lys Ser Leu Asp Pro Glu Phe 180 185 190Leu Asn Leu Ile Lys Ser Leu Ser Asp Asp Ser Asp Ser Ser Asp Leu 195 200 205Leu Phe Ser Gln Lys Phe Lys Glu Lys Leu Glu Leu Asn Asn Lys Ser 210 215 220Ile Asp Ile Asn Phe Ile Lys Glu Asn Leu Thr Glu Phe Gln His Ala225 230 235 240Phe Ser Leu Ala Phe Ser Tyr Tyr Phe Ala Pro Asp His Arg Thr Val 245 250 255Leu Glu Leu Tyr Ala Pro Asp Met Phe Glu Tyr Met Asn Lys Leu Glu 260 265 270Lys Gly Gly Phe Glu Lys Ile Ser Glu Ser Leu Lys Lys Glu Gly Val 275 280 285Glu Lys Asp Arg Ile Asp Val Leu Lys Gly Glu Lys Ala Leu Lys Ala 290 295 300Ser Gly Leu Val Pro Glu His Ala Asp Ala Phe Lys Lys Ile Ala Arg305 310 315 320Glu Leu Asn Thr Tyr Ile Leu Phe Arg Pro Val Asn Lys Leu Ala Thr 325 330 335Asn Leu Ile Lys Ser Gly Val Ala Thr Lys Gly Leu Asn Glu His Gly 340 345 350Lys Ser Ser Asp Trp Gly Pro Val Ala Gly Tyr Ile Pro Phe Asp Gln 355 360 365Asp Leu Ser Lys Lys His Gly Gln Gln Leu Ala Val Glu Lys Gly Asn 370 375 380Leu Glu Asn Lys Lys Ser Ile Thr Glu His Glu Gly Glu Ile Gly Lys385 390 395 400Ile Pro Leu Lys Leu Asp His Leu Arg Ile Glu Glu Leu Lys Glu Asn 405 410 415Gly Ile Ile Leu Lys Gly Lys Lys Glu Ile Asp Asn Gly Lys Lys Tyr 420 425 430Tyr Leu Leu Glu Ser Asn Asn Gln Val Tyr Glu Phe Arg Ile Ser Asp 435 440 445Glu Asn Asn Glu Val Gln Tyr Lys Thr Lys Glu Gly Lys Ile Thr Val 450 455 460Leu Gly Glu Lys Phe Asn Trp Arg Asn Ile Glu Val Met Ala Lys Asn465 470 475 480Val Glu Gly Val Leu Lys Pro Leu Thr Ala Asp Tyr Asp Leu Phe Ala 485 490 495Leu Ala Pro Ser Leu Thr Glu Ile Lys Lys Gln Ile Pro Thr Lys Arg 500 505 510Met Asp Lys Val Val Asn Thr Pro Asn Ser Leu Glu Lys Gln Lys Gly 515 520 525Val Thr Asn Leu Leu Ile Lys Tyr Gly Ile Glu Arg Lys Pro Asp Ser 530 535 540Thr Lys Gly Thr Leu Ser Asn Trp Gln Lys Gln Met Leu Asp Arg Leu545 550 555 560Asn Glu Ala Val Lys Tyr Thr Gly Tyr Thr Gly Gly Asp Val Val Asn 565 570 575His Gly Thr Glu Gln Asp Asn Glu Glu Phe Pro Glu Lys Asp Asn Glu 580 585 590Ile Phe Ile Ile Asn Pro Glu Gly Glu Phe Ile Leu Thr Lys Asn Trp 595 600 605Glu Met Thr Gly Arg Phe Ile Glu Lys Asn Ile Thr Gly Lys Asp Tyr 610 615 620Leu Tyr Tyr Phe Asn Arg Ser Tyr Asn Lys Ile Ala Pro Gly Asn Lys625 630 635 640Ala Tyr Ile Glu Trp Thr Asp Pro Ile Thr Lys Ala Lys Ile Asn Thr 645 650 655Ile Pro Thr Ser Ala Glu Phe Ile Lys Asn Leu Ser Ser Ile Arg Arg 660 665 670Ser Ser Asn Val Gly Val Tyr Lys Asp Ser Gly Asp Lys Asp Glu Phe 675 680 685Ala Lys Lys Glu Ser Val Lys Lys Ile Ala Gly Tyr Leu Ser Asp Tyr 690 695 700Tyr Asn Ser Ala Asn His Ile Phe Ser Gln Glu Lys Lys Arg Lys Ile705 710 715 720Ser Ile Phe Arg Gly Ile Gln Ala Tyr Asn Glu Ile Glu Asn Val Leu 725 730 735Lys Ser Lys Gln Ile Ala Pro Glu Tyr Lys Asn Tyr Phe Gln Tyr Leu 740 745 750Lys Glu Arg Ile Thr Asn Gln Val Gln Leu Leu Leu Thr His Gln Lys 755 760 765Ser Asn Ile Glu Phe Lys Leu Leu Tyr Lys Gln Leu Asn Phe Thr Glu 770 775 780Asn Glu Thr Asp Asn Phe Glu Val Phe Gln Lys Ile Ile Asp Glu Lys785 790 795 800410PRTBacillus anthracis 4Gly Ala Gly Gly His Gly Asp Val Gly Met1 5 10510PRTBacillus anthracis 5Gly Gly His Gly Asp Val Gly Met His Val1 5 10610PRTBacillus anthracis 6His Gly Asp Val Gly Met His Val Lys Glu1 5 10710PRTBacillus anthracis 7Asp Val Gly Met His Val Lys Glu Lys Glu1 5 10810PRTBacillus anthracis 8Lys Glu Lys Asn Lys Asp Glu Asn Lys Arg1 5 10910PRTBacillus anthracis 9Lys Asn Lys Asp Glu Asn Lys Arg Lys Asp1 5 101010PRTBacillus anthracis 10Arg Asn Lys

Thr Gln Glu Glu His Leu Lys1 5 101110PRTBacillus anthracis 11Lys Thr Gln Glu Glu His Leu Lys Glu Ile1 5 101210PRTBacillus anthracis 12Gln Glu Glu His Leu Lys Glu Ile Met Lys1 5 101310PRTBacillus anthracis 13Glu His Leu Lys Glu Ile Met Lys His Ile1 5 101410PRTBacillus anthracis 14Glu Lys Val Pro Ser Asp Val Leu Glu Met1 5 101510PRTBacillus anthracis 15Val Pro Ser Asp Val Leu Glu Met Tyr Lys1 5 101610PRTBacillus anthracis 16Ser Asp Val Leu Glu Met Tyr Lys Ala Ile1 5 101710PRTBacillus anthracis 17Val Leu Glu Met Tyr Lys Ala Ile Gly Gly1 5 101810PRTBacillus anthracis 18Glu Met Tyr Lys Ala Ile Gly Gly Lys Ile1 5 101910PRTBacillus anthracis 19Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile1 5 102010PRTBacillus anthracis 20Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly1 5 102110PRTBacillus anthracis 21Lys Lys Ile Lys Asp Ile Tyr Gly Lys Asp1 5 102210PRTBacillus anthracis 22Ile Lys Asp Ile Tyr Gly Lys Asp Ala Leu1 5 102310PRTBacillus anthracis 23Asp Ile Tyr Gly Lys Asp Ala Leu Leu His1 5 102410PRTBacillus anthracis 24Tyr Gly Lys Asp Ala Leu Leu His Glu His1 5 102510PRTBacillus anthracis 25Leu His Glu His Tyr Val Tyr Ala Lys Glu1 5 102610PRTBacillus anthracis 26Glu His Tyr Val Tyr Ala Lys Glu Gly Tyr1 5 102710PRTBacillus anthracis 27Ser Asn Glu Val Gln Glu Val Phe Ala Lys1 5 102810PRTBacillus anthracis 28Glu Val Gln Glu Val Phe Ala Lys Ala Phe1 5 102910PRTBacillus anthracis 29Gln His Arg Asp Val Leu Gln Leu Tyr Ala1 5 103010PRTBacillus anthracis 30Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu1 5 103110PRTBacillus anthracis 31Val Leu Gln Leu Tyr Ala Pro Glu Ala Phe1 5 103210PRTBacillus anthracis 32Gln Leu Tyr Ala Pro Glu Ala Phe Asn Tyr1 5 103310PRTBacillus anthracis 33Tyr Ala Pro Glu Ala Phe Asn Tyr Met Asp1 5 103410PRTBacillus anthracis 34Pro Glu Ala Phe Asn Tyr Met Asp Lys Phe1 5 103510PRTBacillus anthracis 35Ala Phe Asn Tyr Met Asp Lys Phe Asn Glu1 5 103610PRTBacillus anthracis 36Asn Tyr Met Asp Lys Phe Asn Glu Gln Glu1 5 103710PRTBacillus anthracis 37Met Asp Lys Phe Asn Glu Gln Glu Ile Asn1 5 103810PRTBacillus anthracis 38Lys Phe Asn Glu Gln Glu Ile Asn Leu Ser1 5 103910PRTBacillus anthracis 39Asn Glu Gln Glu Ile Asn Leu Ser Leu Glu1 5 104010PRTBacillus anthracis 40Gln Glu Ile Asn Leu Ser Leu Glu Glu Leu1 5 104110PRTBacillus anthracis 41Ile Asn Leu Ser Leu Glu Glu Leu Lys Asp1 5 104210PRTBacillus anthracis 42Leu Glu Glu Leu Lys Asp Gln Arg Met Leu1 5 104310PRTBacillus anthracis 43Glu Leu Lys Asp Gln Arg Met Leu Ala Arg1 5 104410PRTBacillus anthracis 44Met Leu Ala Arg Tyr Glu Lys Trp Glu Lys1 5 104510PRTBacillus anthracis 45Ala Arg Tyr Glu Lys Trp Glu Lys Ile Lys1 5 104610PRTBacillus anthracis 46Tyr Glu Lys Trp Glu Lys Ile Lys Gln His1 5 104710PRTBacillus anthracis 47Leu Leu Lys Lys Leu Gln Ile Pro Ile Glu1 5 104810PRTBacillus anthracis 48Ser Leu Ser Gln Glu Glu Lys Glu Leu Leu1 5 104910PRTBacillus anthracis 49Ser Gln Glu Glu Lys Glu Leu Leu Lys Arg1 5 105010PRTBacillus anthracis 50Glu Glu Lys Glu Leu Leu Lys Arg Ile Gln1 5 105110PRTBacillus anthracis 51Asp Phe Leu Ser Thr Glu Glu Lys Glu Phe1 5 105210PRTBacillus anthracis 52Leu Ser Thr Glu Glu Lys Glu Phe Leu Lys1 5 105310PRTBacillus anthracis 53Thr Glu Glu Lys Glu Phe Leu Lys Lys Leu1 5 105410PRTBacillus anthracis 54Glu Lys Glu Phe Leu Lys Lys Leu Gln Ile1 5 105510PRTBacillus anthracis 55Glu Phe Leu Lys Lys Leu Gln Ile Asp Ile1 5 105610PRTBacillus anthracis 56Ser Leu Ser Glu Glu Glu Lys Glu Leu Leu1 5 105710PRTBacillus anthracis 57Ser Glu Glu Glu Lys Glu Leu Leu Asn Arg1 5 105810PRTBacillus anthracis 58Glu Glu Lys Glu Leu Leu Asn Arg Ile Gln1 5 105910PRTBacillus anthracis 59Leu Ser Glu Lys Glu Lys Glu Phe Leu Lys1 5 106010PRTBacillus anthracis 60Glu Lys Glu Lys Glu Phe Leu Lys Lys Leu1 5 106110PRTBacillus anthracis 61Glu Lys Glu Phe Leu Lys Lys Leu Lys Leu1 5 106210PRTBacillus anthracis 62Glu Phe Leu Lys Lys Leu Lys Leu Asp Ile1 5 106310PRTBacillus anthracis 63Gln Pro Tyr Asp Ile Asn Gln Arg Leu Gln1 5 106410PRTBacillus anthracis 64Tyr Asp Ile Asn Gln Arg Leu Gln Asp Thr1 5 106510PRTBacillus anthracis 65Leu Ile Asp Ser Pro Ser Ile Asn Leu Asp1 5 106610PRTBacillus anthracis 66Asp Ser Pro Ser Ile Asn Leu Asp Val Arg1 5 106710PRTBacillus anthracis 67Pro Ser Ile Asn Leu Asp Val Arg Lys Gln1 5 106810PRTBacillus anthracis 68Asn Arg Gly Ile Phe Asn Glu Phe Lys Lys1 5 106910PRTBacillus anthracis 69Lys Tyr Ser Ile Ser Ser Asn Tyr Met Ile1 5 107010PRTBacillus anthracis 70Ser Ile Ser Ser Asn Tyr Met Ile Val Asp1 5 107110PRTBacillus anthracis 71Leu Ile Lys Lys Val Thr Asn Tyr Leu Val1 5 107210PRTBacillus anthracis 72Lys Lys Val Thr Asn Tyr Leu Val Asp Gly1 5 107310PRTBacillus anthracis 73Leu Val Asp Gly Asn Gly Arg Phe Val Phe1 5 107410PRTBacillus anthracis 74Asp Gly Asn Gly Arg Phe Val Phe Thr Asp1 5 107510PRTBacillus anthracis 75Asn Gly Arg Phe Val Phe Thr Asp Ile Thr1 5 107610PRTBacillus anthracis 76Arg Phe Val Phe Thr Asp Ile Thr Leu Pro1 5 107710PRTBacillus anthracis 77Gln Tyr Thr His Gln Asp Glu Ile Tyr Glu1 5 107810PRTBacillus anthracis 78Thr His Gln Asp Glu Ile Tyr Glu Gln Val1 5 107910PRTBacillus anthracis 79Val Pro Glu Ser Arg Ser Ile Leu Leu His1 5 108010PRTBacillus anthracis 80Glu Ser Arg Ser Ile Leu Leu His Gly Pro1 5 108110PRTBacillus anthracis 81Asp Ser Glu Gly Phe Ile His Glu Phe Gly1 5 108210PRTBacillus anthracis 82Glu Gly Phe Ile His Glu Phe Gly His Ala1 5 108310PRTBacillus anthracis 83Phe Ile His Glu Phe Gly His Ala Val Asp1 5 108410PRTBacillus anthracis 84Asn Ser Lys Lys Phe Ile Asp Ile Phe Lys1 5 108510PRTBacillus anthracis 85Lys Lys Phe Ile Asp Ile Phe Lys Glu Glu1 5 108610PRTBacillus anthracis 86Asp His Ala Glu Arg Leu Lys Val Gln Lys1 5 108710PRTBacillus anthracis 87Ala Glu Arg Leu Lys Val Gln Lys Asn Ala1 5 108810PRTBacillus anthracis 88Arg Leu Lys Val Gln Lys Asn Ala Pro Lys1 5 108910PRTBacillus anthracis 89Lys Val Gln Lys Asn Ala Pro Lys Thr Phe1 5 109010PRTBacillus anthracis 90Pro Lys Thr Phe Gln Phe Ile Asn Asp Gln1 5 109110PRTBacillus anthracis 91Thr Phe Gln Phe Ile Asn Asp Gln Ile Lys1 5 109216PRTBacillus anthracis 92Gly Ala Gly Gly His Gly Asp Val Gly Met His Val Lys Glu Lys Glu1 5 10 159312PRTBacillus anthracis 93Lys Glu Lys Asn Lys Asp Glu Asn Lys Arg Lys Asp1 5 109416PRTBacillus anthracis 94Arg Asn Lys Thr Gln Glu Glu His Leu Lys Glu Ile Met Lys His Ile1 5 10 159518PRTBacillus anthracis 95Glu Lys Val Pro Ser Asp Val Leu Glu Met Tyr Lys Ala Ile Gly Gly1 5 10 15Lys Ile9620PRTBacillus anthracis 96Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly Lys Asp Ala Leu1 5 10 15Leu His Glu His 209712PRTBacillus anthracis 97Leu His Glu His Tyr Val Tyr Ala Lys Glu Gly Tyr1 5 109812PRTBacillus anthracis 98Ser Asn Glu Val Gln Glu Val Phe Ala Lys Ala Phe1 5 109934PRTBacillus anthracis 99Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu Ala Phe Asn Tyr1 5 10 15Met Asp Lys Phe Asn Glu Gln Glu Ile Asn Leu Ser Leu Glu Glu Leu 20 25 30Lys Asp10012PRTBacillus anthracis 100Leu Glu Glu Leu Lys Asp Gln Arg Met Leu Ala Arg1 5 1010114PRTBacillus anthracis 101Met Leu Ala Arg Tyr Glu Lys Trp Glu Lys Ile Lys Gln His1 5 1010214PRTBacillus anthracis 102Ser Leu Ser Gln Glu Glu Lys Glu Leu Leu Lys Arg Ile Gln1 5 1010318PRTBacillus anthracis 103Asp Phe Leu Ser Thr Glu Glu Lys Glu Phe Leu Lys Lys Leu Gln Ile1 5 10 15Asp Ile10414PRTBacillus anthracis 104Ser Leu Ser Glu Glu Glu Lys Glu Leu Leu Asn Arg Ile Gln1 5 1010516PRTBacillus anthracis 105Leu Ser Glu Lys Glu Lys Glu Phe Leu Lys Lys Leu Lys Leu Asp Ile1 5 10 1510612PRTBacillus anthracis 106Gln Pro Tyr Asp Ile Asn Gln Arg Leu Gln Asp Thr1 5 1010714PRTBacillus anthracis 107Leu Ile Asp Ser Pro Ser Ile Asn Leu Asp Val Arg Lys Gln1 5 1010812PRTBacillus anthracis 108Lys Tyr Ser Ile Ser Ser Asn Tyr Met Ile Val Asp1 5 1010912PRTBacillus anthracis 109Leu Ile Lys Lys Val Thr Asn Tyr Leu Val Asp Gly1 5 1011016PRTBacillus anthracis 110Leu Val Asp Gly Asn Gly Arg Phe Val Phe Thr Asp Ile Thr Leu Pro1 5 10 1511112PRTBacillus anthracis 111Gln Tyr Thr His Gln Asp Glu Ile Tyr Glu Gln Val1 5 1011212PRTBacillus anthracis 112Val Pro Glu Ser Arg Ser Ile Leu Leu His Gly Pro1 5 1011314PRTBacillus anthracis 113Asp Ser Glu Gly Phe Ile His Glu Phe Gly His Ala Val Asp1 5 1011412PRTBacillus anthracis 114Asn Ser Lys Lys Phe Ile Asp Ile Phe Lys Glu Glu1 5 1011516PRTBacillus anthracis 115Asp His Ala Glu Arg Leu Lys Val Gln Lys Asn Ala Pro Lys Thr Phe1 5 10 1511612PRTBacillus anthracis 116Pro Lys Thr Phe Gln Phe Ile Asn Asp Gln Ile Lys1 5 1011710PRTBacillus anthracis 117Val Gln Gly Ala Gly Gly His Gly Asp Val1 5 1011810PRTBacillus anthracis 118Lys Glu Lys Glu Lys Asn Lys Asp Glu Asn1 5 1011910PRTBacillus anthracis 119Lys Arg Lys Asp Glu Glu Arg Asn Lys Thr1 5 1012010PRTBacillus anthracis 120Lys Asp Glu Glu Arg Asn Lys Thr Gln Glu1 5 1012110PRTBacillus anthracis 121Ile Ser Leu Glu Ala Leu Ser Glu Asp Lys1 5 1012210PRTBacillus anthracis 122Leu Glu Ala Leu Ser Glu Asp Lys Lys Lys1 5 1012310PRTBacillus anthracis 123Ala Leu Ser Glu Asp Lys Lys Lys Ile Lys1 5 1012410PRTBacillus anthracis 124Asp Phe Ser Val Glu Phe Leu Glu Gln Asn1 5 1012510PRTBacillus anthracis 125Lys Lys Leu Gln Ile Pro Ile Glu Pro Lys1 5 1012610PRTBacillus anthracis 126Leu Lys Lys Leu Lys Leu Asp Ile Gln Pro1 5 1012710PRTBacillus anthracis 127Asp Ile Gln Pro Tyr Asp Ile Asn Gln Arg1 5 1012810PRTBacillus anthracis 128Ile Asn Gln Arg Leu Gln Asp Thr Gly Gly1 5 1012910PRTBacillus anthracis 129Ile Asn Leu Asp Val Arg Lys Gln Tyr Lys1 5 1013010PRTBacillus anthracis 130Leu Asp Val Arg Lys Gln Tyr Lys Arg Asp1 5 1013110PRTBacillus anthracis 131Gly Ile Phe Asn Glu Phe Lys Lys Asn Phe1 5 1013210PRTBacillus anthracis 132Asn Ile Ala Glu Gln Tyr Thr His Gln Asp1 5 1013310PRTBacillus anthracis 133Gln Asp Glu Ile Tyr Glu Gln Val His Ser1 5 1013410PRTBacillus anthracis 134Glu Ile Tyr Glu Gln Val His Ser Lys Gly1 5 1013510PRTBacillus anthracis 135Arg Ser Ile Leu Leu His Gly Pro Ser Lys1 5 1013610PRTBacillus anthracis 136Ile Leu Leu His Gly Pro Ser Lys Gly Val1 5 1013710PRTBacillus anthracis 137Gln Lys Asn Ala Pro Lys Thr Phe Gln Phe1 5 1013810PRTBacillus anthracis 138Gln Phe Ile Asn Asp Gln Ile Lys Phe Ile1 5 1013918PRTBacillus anthracis 139Val Gln Gly Ala Gly Gly His Gly Asp Val Gly Met His Val Lys Glu1 5 10 15Lys Glu14014PRTBacillus anthracis 140Lys Glu Lys Glu Lys Asn Lys Asp Glu Asn Lys Arg Lys Asp1 5 1014112PRTBacillus anthracis 141Lys Arg Lys Asp Glu Glu Arg Asn Lys Thr Gln Glu1 5 1014226PRTBacillus anthracis 142Ile Ser Leu Glu Ala Leu Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile1 5 10 15Tyr Gly Lys Asp Ala Leu Leu His Glu His20 2514318PRTBacillus anthracis 143Leu Ser Glu Lys Glu Lys Glu Phe Leu Lys Lys Leu Lys Leu Asp Ile1 5 10 15Gln Pro14412PRTBacillus anthracis 144Leu Leu Lys Lys Leu Gln Ile Pro Ile Glu Pro Lys1 5 1014516PRTBacillus anthracis 145Asp Ile Gln Pro Tyr Asp Ile Asn Gln Arg Leu Gln Asp Thr Gly Gly1 5 10 1514618PRTBacillus anthracis 146Leu Ile Asp Ser Pro Ser Ile Asn Leu Asp Val Arg Lys Gln Tyr Lys1 5 10 15Arg Asp14712PRTBacillus anthracis 147Asn Arg Gly Ile Phe Asn Glu Phe Lys Lys Asn Phe1 5 1014816PRTBacillus anthracis 148Gln Tyr Thr His Gln Asp Glu Ile Tyr Glu Gln Val His Ser Lys Gly1 5 10 1514916PRTBacillus anthracis 149Val Pro Glu Ser Arg Ser Ile Leu Leu His Gly Pro Ser Lys Gly Val1 5 10 1515018PRTBacillus anthracis 150Asp His Ala Glu Arg Leu Lys Val Gln Lys Asn Ala Pro Lys Thr Phe1 5 10 15Gln Phe15114PRTBacillus anthracis 151Pro Lys Thr Phe Gln Phe Ile Asn Asp Gln Ile Lys Phe Ile1 5 1015216PRTBacillus anthracis 152Phe Ser Asp Leu Asn Phe Gln Ala Pro Met Val Val Thr Ser Ser Thr1 5 10 1515320PRTBacillus anthracis 153Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu Asn Ile1 5 10 15Pro Ser Glu Asn 2015414PRTBacillus anthracis 154Tyr Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp Phe Lys1 5 1015516PRTBacillus anthracis 155Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu Val Ile Ser Ser Asp1 5 10 1515622PRTBacillus anthracis 156Leu Lys Gln Lys Ser Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala1 5 10 15Gly Pro Thr Val Pro Asp 2015712PRTBacillus anthracis 157Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile1 5 1015812PRTBacillus anthracis 158Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg Thr1 5 1015920PRTBacillus anthracis 159Ser Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro1 5 10 15Glu Lys Trp Ser 2016022PRTBacillus anthracis 160Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile1 5 10 15Asp Lys Asn Val Ser Pro 2016110PRTBacillus anthracis 161Ser Pro Glu Ala Arg His Pro Leu Val Ala1 5 1016212PRTBacillus anthracis 162Val Ala Ala Tyr Pro Ile Val His Val Asp Met Glu1 5 1016318PRTBacillus anthracis

163Val Asp Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr1 5 10 15Gln Asn16418PRTBacillus anthracis 164Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser1 5 10 15Ala Gly16510PRTBacillus anthracis 165Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val1 5 1016614PRTBacillus anthracis 166Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn1 5 1016710PRTBacillus anthracis 167Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe1 5 1016816PRTBacillus anthracis 168Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser1 5 10 1516910PRTBacillus anthracis 169Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu1 5 1017012PRTBacillus anthracis 170Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile1 5 1017114PRTBacillus anthracis 171Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala1 5 1017222PRTBacillus anthracis 172Ala Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His Arg Glu Val1 5 10 15Ile Asn Ser Ser Thr Glu 2017310PRTBacillus anthracis 173Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys1 5 1017414PRTBacillus anthracis 174Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly1 5 1017514PRTBacillus anthracis 175Lys Leu Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val Asn1 5 1017612PRTBacillus anthracis 176Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn1 5 1017716PRTBacillus anthracis 177Lys Glu Phe Ile Lys Val Ile Ser Met Ser Cys Leu Val Thr Ala Ile1 5 10 1517812PRTBacillus anthracis 178Pro Val Phe Ile Pro Leu Val Gln Gly Ala Gly Gly1 5 1017914PRTBacillus anthracis 179Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly Lys Asp1 5 1018012PRTBacillus anthracis 180Glu Leu Lys Asp Gln Arg Met Leu Ala Arg Tyr Glu1 5 1018114PRTBacillus anthracis 181Lys Ile Asp Thr Lys Ile Gln Glu Ala Gln Leu Asn Ile Asn1 5 1018214PRTBacillus anthracis 182Ala Gln Leu Asn Ile Asn Gln Glu Trp Asn Lys Ala Leu Gly1 5 1018314PRTBacillus anthracis 183Asn Val His Asn Arg Tyr Ala Ser Asn Ile Val Glu Ser Ala1 5 1018412PRTBacillus anthracis 184Thr Asp Ile Thr Leu Pro Asn Ile Ala Glu Gln Tyr1 5 1018518PRTBacillus anthracis 185Thr His Gln Asp Glu Ile Tyr Glu Gln Val His Ser Lys Gly Leu Tyr1 5 10 15Val Pro18618PRTBacillus anthracis 186Met His Ser Thr Asp His Ala Glu Arg Leu Lys Val Gln Lys Asn Ala1 5 10 15Pro Lys18714PRTBacillus anthracis 187Ile Gln Ala Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu1 5 1018812PRTBacillus anthracis 188Glu Asn Gln Thr Phe Gln Ser Ala Ile Trp Ser Gly1 5 1018912PRTBacillus anthracis 189Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe1 5 1019012PRTBacillus anthracis 190Asn Lys Ala Ser Asn Lys Ile Arg Leu Glu Lys Gly1 5 1019112PRTBacillus anthracis 191Asn Pro Thr Glu Lys Gly Leu Asp Phe Lys Leu Tyr1 5 1019210PRTBacillus anthracis 192Tyr Thr Val Asp Val Lys Asn Lys Arg Thr1 5 1019312PRTBacillus anthracis 193Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys1 5 1019412PRTBacillus anthracis 194Ser Thr Val Ala Ile Asp His Ser Leu Ser Leu Ala1 5 1019512PRTBacillus anthracis 195Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala1 5 1019612PRTBacillus anthracis 196Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr1 5 1019714PRTBacillus anthracis 197Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg Leu Tyr Gln1 5 1019820PRTBacillus anthracis 198Asn Pro Thr Glu Lys Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser1 5 10 15Gln Asn Lys Lys 2019920PRTBacillus anthracis 199Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser Arg Lys Lys Arg1 5 10 15Ser Thr Ser Ala 2020014PRTBacillus anthracis 200Tyr Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro1 5 1020116PRTBacillus anthracis 201Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val1 5 10 1520214PRTBacillus anthracis 202Ser Glu Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg1 5 1020318PRTBacillus anthracis 203Ile Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr1 5 10 15Val Ala20410PRTBacillus anthracis 204Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr1 5 1020514PRTBacillus anthracis 205Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile1 5 1020612PRTBacillus anthracis 206Gln Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala1 5 1020710PRTBacillus anthracis 207Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys1 5 1020810PRTBacillus anthracis 208Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu1 5 1020912PRTBacillus anthracis 209Lys Phe Ile Pro Asn Lys Phe Ser Ile Ile Ser Phe1 5 1021012PRTBacillus anthracis 210Ser Arg Phe Val Phe Glu Lys Lys Arg Glu Thr Pro1 5 1021112PRTBacillus anthracis 211Gly Asn Leu Glu Asn Lys Lys Ser Ile Thr Glu His1 5 1021212PRTBacillus anthracis 212Gly Lys Ile Pro Leu Lys Leu Asp His Leu Arg Ile1 5 1021311PRTBacillus anthracis 213Glu Asn Gly Ile Ile Lys Gly Lys Lys Glu Ile1 5 1021418PRTBacillus anthracis 214Val Leu Gly Glu Lys Phe Asn Trp Arg Asn Ile Glu Val Met Ala Lys1 5 10 15Asn Val21518PRTBacillus anthracis 215Val Met Ala Lys Asn Val Glu Gly Val Leu Lys Pro Leu Thr Ala Asp1 5 10 15Tyr Asp21616PRTBacillus anthracis 216Thr Glu Ile Lys Lys Gln Ile Pro Thr Lys Arg Met Asp Lys Val Val1 5 10 1521716PRTBacillus anthracis 217Pro Asn Ser Leu Glu Lys Gln Lys Gly Val Thr Asn Leu Leu Ile Lys1 5 10 1521812PRTBacillus anthracis 218Thr Asn Leu Leu Ile Lys Tyr Gly Ile Glu Arg Lys1 5 1021921PRTBacillus anthracis 219Lys Thr Leu Ser Asn Trp Gln Lys Gln Met Leu Asp Arg Leu Asn Glu1 5 10 15Ala Val Lys Tyr Thr 2022014PRTBacillus anthracis 220Phe Ile Lys Asn Leu Ser Ser Ile Arg Arg Ser Ser Asn Val1 5 1022110PRTBacillus anthracis 221Phe Ser Gln Glu Lys Lys Arg Lys Ile Ser1 5 1022216PRTBacillus anthracis 222Ala Pro Glu Tyr Lys Asn Tyr Phe Gln Tyr Leu Lys Glu Arg Ile Thr1 5 10 1522313PRTBacillus anthracis 223Asn Gln Val Gln Leu Leu Thr His Gln Lys Ser Asn Ile1 5 1022412PRTBacillus anthracis 224His Gln Lys Ser Asn Ile Glu Phe Lys Leu Leu Tyr1 5 1022512PRTBacillus anthracis 225Glu Asn Glu Thr Asp Asn Phe Glu Val Phe Gln Lys1 5 1022610PRTBacillus anthracis 226Lys Asn Ser Met Asn Ser Arg Gly Glu Lys1 5 1022710PRTBacillus anthracis 227Ser Met Asn Ser Arg Gly Glu Lys Val Pro1 5 1022810PRTBacillus anthracis 228Asn Ser Arg Gly Glu Lys Val Pro Phe Ala1 5 1022910PRTBacillus anthracis 229Arg Gly Glu Lys Val Pro Phe Ala Ser Arg1 5 1023010PRTBacillus anthracis 230Glu Lys Val Pro Phe Ala Ser Arg Phe Val1 5 1023110PRTBacillus anthracis 231Tyr Ala Ile Asn Ser Glu Gln Ser Lys Glu1 5 1023210PRTBacillus anthracis 232Ile Asn Ser Glu Gln Ser Lys Glu Val Tyr1 5 1023310PRTBacillus anthracis 233Ser Ser Asp Leu Leu Phe Ser Gln Lys Phe1 5 1023410PRTBacillus anthracis 234Asp Leu Leu Phe Ser Gln Lys Phe Lys Glu1 5 1023510PRTBacillus anthracis 235Leu Glu Leu Tyr Ala Pro Asp Met Phe Glu1 5 1023610PRTBacillus anthracis 236Leu Tyr Ala Pro Asp Met Phe Glu Tyr Met1 5 1023710PRTBacillus anthracis 237Glu Gly Glu Ile Gly Lys Ile Pro Leu Lys1 5 1023810PRTBacillus anthracis 238Glu Ile Gly Lys Ile Pro Leu Lys Leu Asp1 5 1023910PRTBacillus anthracis 239Asp Tyr Asp Leu Phe Ala Leu Ala Pro Ser1 5 1024010PRTBacillus anthracis 240Asp Leu Phe Ala Leu Ala Pro Ser Leu Thr1 5 1024110PRTBacillus anthracis 241Asn Trp Gln Lys Gln Met Leu Asp Arg Leu1 5 1024210PRTBacillus anthracis 242Gln Lys Gln Met Leu Asp Arg Leu Asn Glu1 5 1024310PRTBacillus anthracis 243Gln Met Leu Asp Arg Leu Asn Glu Ala Val1 5 1024410PRTBacillus anthracis 244Asn Glu Ala Val Lys Tyr Thr Gly Tyr Thr1 5 1024510PRTBacillus anthracis 245Ala Val Lys Tyr Thr Gly Tyr Thr Gly Gly1 5 1024610PRTBacillus anthracis 246Gln Asp Asn Glu Glu Phe Pro Glu Lys Asp1 5 1024710PRTBacillus anthracis 247Asn Glu Glu Phe Pro Glu Lys Asp Asn Glu1 5 1024810PRTBacillus anthracis 248Glu Phe Pro Glu Lys Asp Asn Glu Ile Phe1 5 1024918PRTBacillus anthracis 249Lys Asn Ser Met Asn Ser Arg Gly Glu Lys Val Pro Phe Ala Ser Arg1 5 10 15Phe Val25012PRTBacillus anthracis 250Tyr Ala Ile Asn Ser Glu Gln Ser Lys Glu Val Tyr1 5 1025112PRTBacillus anthracis 251Ser Ser Asp Leu Leu Phe Ser Gln Lys Phe Lys Glu1 5 1025212PRTBacillus anthracis 252Leu Glu Leu Tyr Ala Pro Asp Met Phe Glu Tyr Met1 5 1025312PRTBacillus anthracis 253Glu Gly Glu Ile Gly Lys Ile Pro Leu Lys Leu Asp1 5 1025412PRTBacillus anthracis 254Asp Tyr Asp Leu Phe Ala Leu Ala Pro Ser Leu Thr1 5 1025514PRTBacillus anthracis 255Asn Trp Gln Lys Gln Met Leu Asp Arg Leu Asn Glu Ala Val1 5 1025612PRTBacillus anthracis 256Asn Glu Ala Val Lys Tyr Thr Gly Tyr Thr Gly Gly1 5 1025714PRTBacillus anthracis 257Gln Asp Asn Glu Glu Phe Pro Glu Lys Asp Asn Glu Ile Phe1 5 1025834PRTBacillus anthracis 258Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu Ala Phe Asn Tyr1 5 10 15Met Asp Lys Phe Asn Glu Gly Glu Ile Asn Leu Ser Leu Glu Glu Leu 20 25 30Lys Asp25920PRTBacillus anthracis 259Asp Phe Leu Ser Thr Glu Glu Lys Glu Phe Leu Lys Lys Leu Gln Ile1 5 10 15Asp Ile Arg Asp 2026012PRTBacillus anthracis 260Gly Pro Tyr Asp Ile Asn Gly Arg Leu Gln Asp Thr1 5 1026112PRTBacillus anthracis 261Gly Tyr Thr His Gln Asp Glu Ile Tyr Glu Gly Val1 5 1026220PRTBacillus anthracis 262Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu Asn Ile1 5 10 15Asp Ser Glu Asn 2026318PRTBacillus anthracis 263Asn Ala Glu Val His Ala Ser Phe Glu Asp Ile Gly Gly Ser Val Ser1 5 10 15Ala Gly26414PRTBacillus anthracis 264Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp1 5 1026516PRTBacillus anthracis 265Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys1 5 10 1526616PRTBacillus anthracis 266Val Leu Gln Leu Tyr Ala Pro Glu Ala Phe Asn Tyr Met Asp Lys Phe1 5 10 1526710PRTBacillus anthracis 267Ser Glu Glu Gly Arg Gly Leu Leu Lys Lys1 5 10

Claims

1. A composition comprising an immunogenic peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa and induces an immune response to anthrax when administered to an animal.

2. The composition of claim 1, wherein the anthrax protein is a protective antigen, lethal factor or edema factor protein.

3. The composition of claim 1, wherein the peptide has less than 40 amino acids.

4. The composition of claim 1, wherein the peptide is coupled to a carrier.

5. The composition of claim 4, wherein the carrier is a protective antigen protein.

6. The composition of claim 1, comprising two or more of the immunogenic peptides.

7. The composition of claim 1, wherein the immunogenic peptide is a fusion protein of two or more immunogenic peptides of an anthrax protein, wherein each peptide has a molecular weight less than 20 kDa and induces an immune response when administered to an animal.

8. The composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of anthrax protective antigen amino acid sequences set forth in Table 4 (SEQ ID NOS: 152-174) and Table 6 (SEQ ID NOS: 185-193).

9. The composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of anthrax lethal factor protein amino acid sequences set forth in Tables 1-3 (SEQ ID NOS: 92-116) and 5 (SEQ ID NOS: 175-184).

10. The composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of anthrax edema factor protein amino acid sequences set forth in Table 7 (SEQ ID NOS: 194-206).

11. A method of inducing an immune response to anthrax comprising administering to an animal an effective amount of a composition comprising an immunogenic peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa.

12. The method of claim 11, wherein the animal is infected with anthrax and the induced immune response is effective for treating the anthrax infection in the animal.

13. The method of claim 11, wherein the induced immune response is effective for protecting against anthrax infection in the animal.

14. A composition comprising an antibody immunoreactive with a peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa and induces an immune response to anthrax when administered to an animal. 25

15. The composition of claim 14, wherein the antibody is a humanized antibody.

16. A method of treating an animal infected with anthrax comprising administering to an animal an effective amount of a composition comprising an antibody immunoreactive with a peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa.

17. A method of producing an antibody to anthrax comprising administering to an animal an effective amount of a composition comprising an immunogenic peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa.

18. The method of claim 17, wherein the antibody is a monoclonal or polyclonal antibody.

19. A method for detecting anthrax infection in an animal comprising combining a sample from the animal with an antibody immunoreactive with a peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa and induces an immune response to anthrax when administered to an animal, and detecting a complex formed between the antibody and an anthrax protein in the sample, wherein detection of a complex indicates anthrax infection in the animal.

20. A method for detecting anthrax infection in an animal comprising combining a sample from the animal with an immunoreactive peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 lcDa and induces an immune response to anthrax when administered to an animal, and detecting a complex formed between an antibody in the sample and the immunoreactive peptide, wherein detection of a complex indicates anthrax infection in the animal.

21. A method for detecting specific protective immunity to anthrax infection in an animal comprising combining a sample from the animal with an immunoreactive peptide of an anthrax protein, wherein the peptide has a molecular weight less than 20 kDa and induces an immune response to anthrax when administered to an animal, and detecting a complex formed between an antibody in the sample and the immunoreactive peptide, wherein detection of a predetermined amount of complex indicates specific protective immunity to anthrax infection in the animal.