WO2008073169A2

WO2008073169A2 - Methods, compositions and kits comprising attenuated anthrax vaccines and methods of delivery

Info

Publication number: WO2008073169A2
Application number: PCT/US2007/018983
Authority: WO
Inventors: Hao Shen; Lauren Zenewicz
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2006-08-31
Filing date: 2007-08-29
Publication date: 2008-06-19
Also published as: WO2008073169A3

Abstract

The present invention includes an attenuated Bacillus anthracis vaccine strain comprising a deletion in a nucleic acid encoding lethal factor and a deletion in a nucleic acid encoding PI-PLC or an attenuated Bacillus anthracis vaccine strain comprising a deletion in a nucleic acid encoding lethal factor and a deletion in edema factor. The present invention further comprises a method of immunizing a mammal against a Bacillus anthracis infection comprising administering the spores of an attenuated Bacillus anthracis vaccine strain intranasally.

Description

TITLE OF THE INVENTION

METHODS, COMPOSITIONS AND KITS COMPRISING ATTENUATED ANTHRAX VACCINES AND METHODS OF DELIVERY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was supported in part by funds obtained from the U.S. Government (National Institutes of Health Grant Number AI45025), and the U.S.

Government may therefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

Bacillus anthracis is the gram-positive, spore-forming bacterium that is the etiological agent of anthrax. Bacillus anthracis spores are widely disseminated in the environment and can remain dormant in the soil for a long time (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826). The spores do not divide, have no measurable metabolism, and are resistant to desiccation, heat, UV light, gamma radiation, and many disinfectants. When the spores receive the proper signals from the environment indicating favorable growth conditions (such as inside a mammalian host), they germinate into vegetative bacilli, which replicate and produce toxins and other virulence factors needed to establish an infection (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826). Bacillus anthracis is one of the top bioterror concerns because of several unique attributes of this organism. It is highly lethal for humans; its spores can be produced quickly and weaponized easily. Dried anthrax spores are very stable and can be dispersed by simply releasing them into the air or hiding them in the simplest of delivery vehicles, e.g. a posted letter (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826; Leppla, et al., 2002, J. Clin. Invest., 110: 141-142), as dramatically illustrated by the criminal dispersal of spores through the U.S. mail system in the autumn of 2001 (Dewan, et al., 2002, Emerg. Infect. Dis. 8: 1066-107). The current anthrax vaccine is not suitable for mass immunization and antibiotic treatments often fail when pulmonary anthrax quickly progresses to the systemic stage of infection (Leppla, et al., 2002, J. Clin. Invest., 110: 141-142). Therefore, a better understanding of Bacillus anthracis pathogenesis and the host immune response, particularly at the early lung stage of pulmonary infection, is urgently needed in order to develop better treatments and vaccines against this deadly pathogen and bioterror agent.

Anthrax presents itself in three forms of infection: cutaneous, gastrointestinal, and pulmonary (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826). The cutaneous form was common in the western world for many centuries especially among those who handled livestock (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826; Baillie, et al., 2001, J. Appl. Microbiol., 91 : 609-613). It is acquired from infected animals or their products and is characterized by an edematous, inflamed, but painless cutaneous carbuncle covered by a black eschar (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826; Hanna, 1998, Curr. Top. Microbiol. Immunol., 225: 13-35). This form has accounted for the majority of human anthrax but is treatable with current antibiotic regimens. In untreated cases, approximately 20% progress to fatal septicemia (Hanna, 1998, Curr. Top. Microbiol. Immunol., 225: 13- 35). Gastrointestinal anthrax is rare but serious, and is the result of ingestion of undercooked, or spore-laden meat from anthrax-infected animals (Baillie, et al., 2001, J. Appl. Microbiol., 91 : 609-613). The most deadly form of anthrax is the pulmonary form which begins with inhalation of Bacillus anthracis spores into the lung. This form was known as wool-sorters disease as it was commonly found among textile workers who breathed spores from contaminated wool and hides of domestic sheep and cattle (Hanna, 1998, Curr. Top. Microbiol. Immunol., 225: 13-35). Improvements in animal management have greatly reduced the threat of contracting anthrax from these sources. However, inhalational anthrax poses the most serious bioterror threat because of the ease by which spores can be dispersed into the air for transmission. It is almost always fatal when it becomes systemic and because of its rapid progression, antibiotic treatments often fail.

In the inhalation form, spores are initially taken up by alveolar macrophages in the lung and subsequent trafficking of these infected macrophages to the lymph nodes aids bacterial dissemination into circulation (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826). In order for the disease to progress, Bacillus anthracis must avoid killing by macrophages but the exact means by which Bacillus anthracis survives and escapes from macrophages are unknown (Hanna, 1998, Curr. Top. Microbiol. Immunol., 225: 13-35; Hanna, et al., 1993, Proc. Nat'l Acad. Sci. USA, 90: 10198-10201). Recent in vitro work has demonstrated that Bacillus anthracis spores germinate inside the macrophages and vegetative bacilli are capable of escaping from the phagosome, a process that was first described and best studied in another human pathogen, Listeria monocytogenes (Guidi-Rontani, et al., 1999, MoI. Microbiol., 31 : 9-17; Gaillard, et al., 1987, Infect. Immun., 55: 2822-2829; Glomski, et al., 2003, Infect. Immun., 71 : 6754-6765). Whether bacilli multiply within macrophages before they are released remains a point of disagreement in the literature. After release from the macrophage, vegetative cells grow and reach titers approaching 10 bacteria per ml of blood, leading to respiratory failure, septicemia, shock and death (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826).

The pathogenesis of Bacillus anthracis is critically dependent on virulence factors encoded on two large plasmids, pXOl and pXO2. The 181 kb pXOl plasmid contains 143 open reading frames (ORF) and encodes anthrax toxins and an important transcriptional regulator of virulence gene expression, AtxX (Okinaka, et al, 1999, J. Bacterid., 181 : 6509-6515; Uchida, et al., 1993, J. Bacteriol., 175: 5329-5338; Koehler, et al., 1994, J. Bacteriol., 176: 586-595). The other plasmid, pXO2 (96 kb), encodes the genes necessary to synthesize a poly D-glutamic acid capsule (Avakyan, et al., 1965, J. Bacteriol., 90: 1082-1095; Uchida, et al., 1985, J. Gen. Microbiol., 131 : (Pt 2) 363-7; Green, et al., 1985, Infect. Immun. 49: 291-297; Mock and Fouet, 2001, Annu. Rev. Microbiol. 55: 647- 671; Zwartouw and Smith, 1956, Biochem. J. 63: 437-442; Okinaka, et al., 1999, J. Appl. Microbiol. 87: 261-262). The capsule inhibits phagocytosis, an important virulence factor, and Bacillus anthracis lacking the capsule are severely attenuated in vivo (Mock and Fouet, 2001, Annu. Rev. Microbiol. 55: 647-671). A strain lacking the pXO2 was developed by Sterne as an attenuated live vaccine and is currently approved for use in livestock.

The anthrax toxins comprise three components: lethal factor (LF), edema factor (EF), and protective antigen (PA), so named because of their respective abilities to induce lethality, edema, and a protective antibody response, respectively (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826; Brassier and Mock, 2001, Toxicon 39: 1747-55). The three components combine to form two different A1/B7 toxins, lethal toxin (LT) and edema toxin (ET). LT comprises LF and PA while ET comprises EF and PA; PA facilitates entry of LF and EF into the host cell by binding to one of two different cellular receptors forming a heptamer (Bradley, et al., 2001, Nature 414: 225-229; Milne, et al., 1994, J. Biol. Chem. 269: 20607-20612) which is then cleaved by a host furin or a furin-like protease allowing LF and/or EF to bind (Klimpel, et al., 1992, Proc. Nat'l. Acad. Sd. USA 89: 10277-10281). Oligomerization of the PA-LF/EF complex triggers receptor-mediated endocytosis (Beauregard, et al., 2000, Cell Microbiol. 2: 251-258). At the low pH of a vacuole, the complex undergoes a conformational change and converts from a prepore to a pore allowing the LF/EF to reach the host cell cytosol (Blaustein, et al., 1989, Proc. Nat'l. Acad. Sci. USA 86: 2209-2213). LF is a zinc metalloprotease and has long been known to be cytotoxic to cells (Smith, et al, 1955, Br. J. Exp. Pathol. 36: 460-472; Thome, et al., 1960, J. Bacterid., 79: 450-455; Klimpel, 1994, MoI. Microbiol., 13: 1093). EF is an adenylate cyclase and its activity leads to the increase of intracellular cAMP and is responsible for inducing the edema that often accompanies infection (Dixon, et al., 1999, N. Eng. J. Med., 341 : 815-826; Leppla, 1982, Proc. Natl. Acad. Sci. USA, 79: 3162-3166). LT is deadly by itself while ET is not lethal in animal models, but contributes to the morbidity of anthrax.

Recent research has demonstrated that LT disrupts MAPK signaling pathways by cleaving the MAPK kinases responsible for p38 MAPK activation (Park, et al., 2002, Science 297: 2048-2051; Duesbery, et al., 1998, Science 280: 734-737). Thus, LT can interfere with the activation and function of various immune cells (Agrawal, et al., 2003, Nature 424: 329-34). LT is also known to induce macrophage apoptosis, eliminating the cells important for bacterial clearance (Park, et al., 2002, Science 297: 2048-2051; Friedlander, 1986, J. Biol. Chem. 261 : 7123-7126; Popov, et al., 2002, Biochem. Biophys. Res. Commun. 293: 349-355). Further, LT inhibits activation of dendritic cells and reduces their ability to prime antigen-specific T cells 4. Additional studies have demonstrated that LT also interferes with TCR signaling and directly inhibits T cell activation (Paccani, et al., 2005, J. Exp. Med. 201: 325-331). While these in vitro studies indicate that LT functions to down-modulate the host immune response, the in vivo relevance of these findings remains to be examined in the context of Bacillus anthracis infection.

In addition to virulence factors encoded on the plasmids, recently completed genome sequence has revealed that the Bacillus anthracis chromosome encodes proteins homologous to many known virulence factors in other pathogenic bacteria (Read, et al., 2003, Nature 423: 81-86). One of these putative virulence factors, PI-PLC is expressed by all pathogenic Bacillus species, B. cereus, B. thuringiensis, and Bacillus anthracis, and share have >94% amino acid identity (Read, et al., 2003, Nature 423: 81-86; Volwerk, et al., 1989, FEMS Microbiol Lett 52: 237-241 ; Klichko, et al., 2003, Biochem. Biophys. Res. Commun. 303: 855-862). PI-PLC plays an important role in B. cereus pathogenesis as B. cereus deficient in this enzyme are less able to cause disease (Callegan, et al., 2002, Infect. Immun. 70: 5381-5389). The PI-PLC genes in B. cereus are regulated by a pleiotropic regulator, PIcR (Read, et al., 2003, Nature 423: 81-86; Okstad, et al., 1999, Microbiology 145: (Pt 11) 3129-3138). Although the PIcR protein is inactive in Bacillus anthracis as a result of truncation (Read, et al., 2003, Nature 423: 81-86), recent results indicate that Bacillus anthracis PI-PLC is expressed but regulated by an anaerobic adaptation system (Milne, 1994, J. Biol. Chem. 269: 20607-20612). Bacterial PI-PLCs cleave PI, but not the multi- phosphorylated forms of PI such as PI-4,5-P2 (PIP2), which are the preferred substrates of mammalian PI-PLCs. In addition to PI cleavage activity, Bacillus PI-PLC also cleaves glycosyl Pi-anchored proteins from the surface of eukaryotic cells. In fact, bacterial PI-PLC and host GPI-anchored proteins were first discovered as a result of investigations on the alkaline phosphatasemia produced during experimental infections of animals with Bacillus anthracis (Ohyabu, 1978, Arch. Biochem. Biophys. 190: 1-7). This led to the discovery of a family of proteins from mammalian cells that are linked to membranes by PI anchors, and to the identification of Bacillus anthracis PI-PLCs that cleave these anchors (Low, et al., 1973, FEBS Lett. 34: 1-4; Low, et al., 1977, Biochem. J. 167: 281-284; Ikezawa, et al., 1989, Toxicon 27: 637-645; Kominami, et al., 1985, Biochem. J. 227: 183-189). GPI- anchored proteins participate in a variety of host cell functions, particularly in host cell signaling (Grakoui, et al., 1999, Science 285: 221-227; Ilangumaran et al., 1997, Biochem. Biophys. Acta. 1328: 227-236; Lafont, et al., 2004, Curr. Opin. Microbiol. 7: 4-10 (2004). Despite the fact that GPI-anchored proteins were discovered through the studies of Bacillus anthracis, the biological significance of cleaving GPI-anchored proteins by Bacillus PI-PLC remains unknown.

Both Bacillus anthracis LT and PI-PLC interfere with dendritic cell activation and function. Thus, like many pathogenic bacteria, Bacillus anthracis employs multiple virulence factors to down-modulate the host response. Collectively these virulence factors have been shown to have a profound impact on the ability of the immune response to control infection in other bacterial systems. It remains to be determined to what extent Bacillus anthracis LT and PI-PLC contribute to the down-modulation of the host immune response in vivo. Although tremendous progress has been made on understanding the mechanism of LT action, most of these studies involve in vitro analysis and surprisingly little has been done in vivo. While the in vitro studies are very useful to provide testable hypotheses, the next critical step is to test these hypotheses in vivo in the context of Bacillus anthracis infection.

There exists a long felt need to provide a safe and effective vaccine to prevent Bacillus anthracis infection, and a means of delivering the vaccine that is inexpensive, rapid, and confers a strong protective immune response. The present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

The present invention includes an attenuated Bacillus anthracis vaccine selected from the group consisting of a Bacillus anthracis vaccine comprising a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding lethal factor and a deletion in the nucleic acid encoding PI-PLC; a Bacillus anthracis vaccine comprising a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding lethal factor and a deletion in the nucleic acid encoding the edema factor; a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding lethal factor; and a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding PI-PLC.

In one aspect of the present invention, the deletion in the nucleic acid encoding lethal factor comprises a deletion of the nucleic acid encoding amino acids 237- 502.

In another aspect of the present invention, the deletion in the nucleic acid encoding lethal factor is an in-frame deletion.

In yet another aspect of the present invention, the deletion in the nucleic acid encoding PI-PLC is an in- frame deletion of the entire open reading frame.

In still another aspect of he present invention, the deletion in the nucleic acid encoding the edema factor is an in-frame deletion of amino acids 359-601.

In still another aspect of the present invention, the vaccine further comprise a pharmaceutically acceptable carrier. In one aspect of the present invention, the vaccine is a Bacillus anthracis spore. In another aspect of the present invention, the vaccine is a vegetative Bacillus anthracis.

The present invention includes a method of immunizing a mammal against a Bacillus anthracis infection, said method comprising administering said mammal an effective amount of the vaccine.

In one aspect of the present invention, the vaccine is a Bacillus anthracis spore.

In another aspect of the present invention, the vaccine is administered intranasally. In yet another aspect of the present invention, the vaccine further comprises a pharmaceutically acceptable carrier.

In still another aspect of the present invention, the mammal is a human.

The present invention includes a method of immunizing a mammal against a Bacillus anthracis infection, said method comprising administering to said mammal an attenuated Bacillus anthracis Sterne vaccine strain, wherein said administration is intranasal administration.

In one aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain is a spore.

In another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding lethal factor.

In yet another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC.

In still another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC and a deletion in a nucleic acid encoding lethal factor.

In still another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding edema factor and a deletion in a nucleic acid encoding lethal factor.

In one aspect of the present invention, the mammal is a human. The present invention includes a method eliciting an immune response against a Bacillus anthracis infection, said method comprising administering to said mammal an attenuated Bacillus anthracis Steme vaccine strain, wherein said administration is intranasal administration.

In one aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain is a spore. In another aspect of the present invention, the attenuated Bacillus anthracis

Sterne vaccine comprises a deletion in a nucleic acid encoding lethal factor.

In still another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding edema factor and a deletion in a nucleic acid encoding lethal factor. In one aspect of the present invention, the mammal is a human.

The present invention includes a method of inducing a protective immune response in a mammal, wherein said protective immune response is against a Bacillus anthracis infection, said method comprising administering to said mammal an attenuated Bacillus anthracis Sterne vaccine strain, wherein said administration is intranasal administration.

In another aspect of the present invention, the protective immune response comprises the production of an antibody that specifically binds Bacillus anthracis, or a portion thereof.

In yet another aspect of the present invention, the protective immune response comprises the proliferation of a CD4⁺ T cell that specifically recognizes Bacillus anthracis, or a portion thereof.

In still another aspect of the present invention, the protective immune response comprises the activation of a Bacillus anthracis-specific antigen presenting cell. In one aspect of the present invention, the antigen presenting cell is a dendritic cell.

In another aspect of the present invention, the dendritic cell expresses CD86.

In still another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding lethal factor.

In one aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC.

In another aspect of the present invention, the attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC and a deletion in a nucleic acid encoding lethal factor.

In one aspect of the present invention, the mammal is a human. The present invention includes a kit for immunizing a mammal against a

Bacillus anthracis infection, said kit comprising an immunogenic amount of an attenuated Bacillus anthracis vaccine strain, said kit further comprising an applicator and an instructional material for the use of said kit.

In one aspect of the present invention, the attenuated Bacillus anthracis vaccine strain is a spore.

In another aspect of the present invention, the applicator is an intranasal applicator.

BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 is a graph depicting that Bacillus PI-PLC inhibits dendritic cells activation by TLR ligands. Dendritic cells were incubated with B. thuringienesis PI-PLC (hollow bars) or were left untreated (filled bars) and then stimulated with LPS, poly I:C, or peptidoglycan or with CpG DNA. Dendritic cell activation was determined by measuring up-regulation of CD86 on the cell surface using flow cytometry.

Figure 2, comprising Figures 2A through 2C, is a series of images depicting the specific inhibition of dendritic cell activation by Bacillus PI-PLC. Figure 2A is a graph depicting dendritic cells incubated with B. thuringienesis PI-PLC (hollow bars) or untreated (filled bars), then stimulated with the indicated dose of poly I:C. Dendritic cells were stained for CD86, CD80, and MHCII. Bars represent an increase in the percentage of the dendritic cell population expressing these surface molecules compared with non-poly I:C-, non-PI- PLC-stimulated dendritic cells. Figure 2B depicts flow cytometry results from the dendritic cells depicted in Figure 2A after staining with annexin V and 7-AAD. The number represents the percentage of total cells in each quadrant: live (annexin V^", 7-AAD^"), apoptotic (annexin V⁺, 7-AAD^"), and necrotic/late apoptotic (annexin V⁺, 7-AAD⁺) cells. Figure 2C is a graph depicting dendritic cells that were incubated with dilutions of Bacillus PI-PLC and stimulated with poly I:C. Dendritic cell activation was determined by measuring surface up- regulation of CD86.

Figure 3, comprising Figures 3A through 3E, is a series of images depicting that Bacillus, but not Listeria, PI-PLC inhibits activation of dendritic cells. Figure 3 A is a graph illustrating the activities of B. thuringienesis and L. monocytogenes PI-PLC on the cleavage of PI. Figure 3B is an image depicting flow cytometry measurements of B. thuringienesis and L. monocytogenes PI-PLC cleavage of GPI-anchored proteins. T cells were treated with Bacillus PI-PLC (dashed line) or Listeria PI-PLC (solid line) or were left untreated (shaded region). Surface expression of the GPI-anchored protein Thyl was detected by mAb staining, followed by FACS. Figure 3 C is an image depicting flow cytometry measurements of dendritic cell activation measured by CD86 up-regulation. Dendritic cells were incubated with B. thuringienesis or L. monocytogenes PI-PLC or were left untreated, then stimulated with the indicated dose of poly I:C. Dendritic cells were stained for CDl Ic and CD86. Shaded regions are the controls without poly LC stimulation; black lines are dendritic cells stimulated with poly LC. Numbers below plots indicate the percentage ± SD of cells that are CD86^hlgh (as gated in histograms) when stimulated with or without poly LC. Figures 3D and 3E are flow cytometry results and a graph, respectively, depicting dendritic cell activation measured by TNF-α production. Dendritic cells were treated as described in Figure 3C, except stimulation was in the presence of GolgiStop for intracellular TNF-α staining (Figure 3D). The numbers indicate the percentages of CDl Ic⁺ cells that produce TNF-α. TNF-α in the cell supernatants was measured by ELISA after poly I:C stimulation (Figure 3E). Bars represent the mean ± SD of three samples. Figure 4, comprising Figures 4A and 4 B, is a series of images depicting that

Bacillus PI-PLC treatment inhibits MAPK pathway signaling. Figure 4A is a series of western blots depicting dendritic cells treated with B. thuringienesis PI-PLC or left untreated and then stimulated with the indicated concentration of poly I:C. Cell lysates were then probed with Abs specific to p38 or ERK (total p38 and total ERK) or specific to the phosphorylated forms of p38 or ERK (phos-p38 and phos-ERK). Figure 4B is a series of graphs quantifying the bands in Figure 4A.

Figure 5, comprising Figures 5 A through 5F, is a series of images depicting that L. monocytogenes expressing Bacillus anthracis PI-PLC induces suboptimal T cell priming. Figure 5A is a graph illustrating PI cleavage activity in the supernatants of cultures of the parental L. monocytogenes strain (Lm), the strain lacking Listeria PI-PLC (Lm

ΔplcA), or the strain expressing Bacillus anthracis PI-PLC (Lm ΔplcA: Ba PI-PLC) relative to the parental Lm strain. Figure 5B is a histogram depicting T cells treated with concentrated supernatants from cultures of Lm (gray line) or Lm ΔplcA: Ba PI-PLC (dashed line) or untreated (black line). Surface expression of the GPI-anchored protein Thyl was detected by mAb staining, followed by FACS. Figures 5C and 5D are a series of graphs depicting bacterial loads in the spleen (5C) and liver (5D) of C57BL/6 mice infected with Lm (hollow bars) or Lm ΔplcA: Ba PI-PLC (shaded bars). Figure 5E is an image depicting an intracellular cytokine staining assay for LLO 19₀-2₀1 -specific CD4 T cells in the spleen of mice after infection with Lm or Lm ΔplcA: Ba PI-PLC. The number indicates the mean percentage of CD4 T cells specific to LLOi₉o_₂₀i. Figure 5F is an graph depicting the total numbers of LLOi ₉₀-₂₀1 -specific cells per spleen after infection (mean ± SD; three or four mice per group).

Figure 6, comprising Figures 6A through 6H, is a series of images depicting that spores persist in mice that were intranasally immunized with Bacillus anthracis Sterne vaccine strain spores. Figures 6A and 6B illustrate bacterial loads in the lungs (6A) and spleens (6B) of immunized mice at different days post-immunization (p.i.). Each data point represents one mouse, bars represent the mean, and dashed lines indicate the limits of detection. Figures 6C through 6E are a series of H&E stained histology images illustrating that peripheral mononuclear cells (PMN) and macrophages infiltrate into the lungs of immunized mice. Figure 6C depicts naϊve lungs, and Figures 6D and 6E depict immunized lungs. Figure 6F is a series of dot plots of lung cells from naϊve or immunized mice demonstrating PMN (GR-I⁺, CDl lb+) or macrophage (GR-I^', CDl lb+) populations. The number indicates the percent of live cells that fall within that gate. Figures 6G and 6H are graphs illustrating the kinetics of PMN and macrophage infiltration into the lungs of immunized mice. Figure 7, comprising Figures 7A through 7F, is a series of images demonstrating that immunization with attenuated Bacillus anthracis spores activates antigen presenting cells (APCs). Figure 7A is an image depicting dot plots of APCs in mice after intranasal Bacillus anthracis immunization with Bacillus anthracis spores (with and without ampicillin), B. cereus spores, or Bacillus anthracis vegetative bacilli. The number represents the percent of cells in that quadrant. Unactivated cells (CD86^", MHC IF) are in the bottom left quadrant, activated (CD86⁺, MHC H⁺) are in the top right quadrant. Figure 7B is a graph illustrating the mean ± SD of colony forming units (CFU) recovered per lung of mice (n=3) depicted in Figure 7 A. The dashed line indicates the limit of detection. Figure 7C is an image of protein cytokine array membranes depicting the cytokines from dendritic cells infected (right panel) or uninfected (middle panel) with the Sterne strain and then stimulated. Key to cytokines on left panel. +, positive control; -, negative control; A, GCSF; B, GM- CSF; C, IL-2; D, IL-3; E, IL-4; F, IL-5; G, IL-6; H, IL-9; I, IL-IO; J, IL-12; K, IL-12p70; L, IL-13; M, IL-17; N, IFNγ; O, MCP-I ; P, MCP-5; Q, RANTES; R, SCF; S, sTNFRI; T, TNFα; U, thrombopoietin; V, VEGF; unmarked, blank. Figure 7D is a graph illustrating IL- 12 measured by ELISA in the cell supernatants of dendritic cells infected with vegetative

Bacillus anthracis bacilli and stimulated. Bars represent mean ± SD of three samples. Figure 7E is an series of histograms depicting the activation of dendritic cells in the lung following intranasal administration of an attenuated Bacillus anthracis vaccine strain. Dendritic cell activation in the lungs of resistant mice immunized with Bacillus anthracis spores (with and without antibiotics) and B. cereus spores is depicted. The number represents the percent of cells in that quadrant. Unactivated cells (CD86^", MHC IF) are in the bottom left quadrant, activated (CD86⁺, MHC H⁺) are in the top right quadrant. Figure 7F depicts the detection of IL- 12 by intracellular cytokine staining in dendritic cells stimulated with Bacillus anthracis. Figure 8, comprising Figures 8A through 8E, is a series of images illustrating that T cells infiltrate the lungs of immunized mice and are activated. Figures 8A through 8D are histology images of lung sections from naive (8A) and infected (8B-8D) mice stained with anti-CD3 Ab and hematoxylin. Figure 8E is a series of histograms depicting surface expression of CD44 and CD62L on CD4⁺ or CD8⁺ cells from the lungs of naϊve or immunized mice. Numbers represent the percentage(mean ± SD) of CD4⁺ or CD8⁺ cells within the activated cell gate. Figure 9, comprising Figures 9A through 9D, is a series of graphs depicting that Bacillus anthracis intranasal immunization generates Bacillus anthracis-specific CD4 and CD8 T cell responses. Figures 9A and 9B depict IFNγ levels from splenocytes from naϊve mice cultured with heat-killed Bacillus anthracis (9A) or recombinant protective antigen protein (9B) (open bars) or left unstimulated (shaded bars). Figures 9C and 9D depict IFNγ levels from splenocytes from immunized mice administered either CD4 (9C) or CD8 (9D) depleting Ab or left untreated.

Figure 10 is a graph depicting that Bacillus anthracis T cell immunity is long- lasting and generates recall responses to protective antigen in naϊve mice (1°), immunized mice (M), or post-challenge immunized mice (2°). Figure 11 is a series of graphs illustrating that intranasal Bacillus anthracis immunization generates Bacillus anthracis-specific IgG antibody titers as detected by ELISA to protective antigen or lethal factor in the serum of either naϊve or immunized mice. Figure 12, comprising Figures 12A through 12K, is a series of images depicting that PMN are essential for controlling early bacterial growth in the lung. Figure 12A through 12C are a series of graphs depicting survival (12A), spore and bacterial presence in lungs (12B) and spleens (12C) in PMN-depleted and control mice immunized intranasally with Sterne spores. Each symbol represents one mouse; bar indicates the mean. The dashed line is the limit of detection. Figures 12D through 12F are a series of histology images depicting Gram stain of lung from control (12D) and PMN-depleted (12E- 12F) mice. Figure 12G is a graph depicting the survival of C57BL/6 (dotted line) and AJJCr (solid line) mice infected intranasally with Sterne spores. Figure 12H is a graph illustrating bacteria recovery from the lungs (left panel) or spleens (right panel) of A/JCr mice infected intranasally with Sterne spores and administered ampicillin (shaded bar) or no ampicillin. Figures 121 is a graph illustrating the percentage ± SD of GR-I + cells in the lungs of A/JCr mice treated with MIP -2, as determined by FACS. Figures 12J and 12K are a series of graphs depicting mortality of A/JCr mice intranasally administered MIP-2 (triangles) or PBS (squares) one day before (12J) or after (12K) infection with B. anthracic spores.

Figure 13 is a graph depicting that intranasal immunization with the Sterne strain spores provides protective immunity and increases survival of A/JCr mice intranasally immunized with Bacillus anthracis Sterne strain spores and then intranasally challenged with spores (triangles). Age-matched naϊve mice challenged with spores are depicted by squares.

Figure 14, comprising Figures 14A through 14C, is a series of images depicting the construction of a derivative of Sterne strain (SdL) with an unmarked, in-frame deletion of LF. Figure 14A is a schematic diagram of the LF loci in the Sterne and SdL mutant. Figure 14B is an image of a gel depicting PCR confirmation of the LF depletion in the SdL mutant. PCR with primer 1/3 amplified 1.0-kb and 0.7-kb from Sterne and SdL, respectively. PCR with primer 2/3 (deleted region) amplified 0.4-kb DNA fragment in Sterne but no product in SdL. Figure 14 C is a graph depicting LT cytotoxicity of supernatants from Sterne and the SdL mutant in J774 cells administered the indicated concentrations of Sterne and SdL supernatants measured by an MTT cytotoxicity assay. Figure 15, comprising Figures 15A and 15B, are a series of images depicting that the SdL mutant is highly attenuated as measured by survival in A/JCr mice and GR-I- depleted C57BL/6 mice intranasally infected with spores of Sterne (solid line) or SdL (dotted line) strains.

Figure 16 is an image depicting dendritic cell activation and lung titers as measured by CD86 expression on CDl Ic⁺ cells from lungs following intranasal spore administration of attenuated Bacillus anthracis vaccine strains Sterne and SdL to A/JCr mice. Mice immunized with spores of Sterne strain succumbed to infection by day 7.

Figure 17 is a series of images depicting FACS analysis of activation of antigen-specific T cells by peptide-pulsed dendritic cells in vitro. (+) indicates with OVA_2S7. ₂(_A peptide, (-) indicates without the peptide. Figure 18, comprising Figures 18A through 18C, is a series of images depicting the increased adaptive immune responses following immunization with SdL in AJ]CT mice immunized with spores of Sterne and SdL strains. Figure 18A depicts total T cell activation assessed by FACS analysis of CD44 expression (mice immunized with Sterne Spores succumbed to infection). Figures 18B and 18C are a series of graphs depicting antigen-specific T cell and antibody response as measured by ELISA.

Figure 19, comprising Figure 19A through 19B, is a series of images depicting the increased innate response in the lung following INSI with PI-PLC mutant. Figure 19A depicts day 3 after immunization with the PI-PLC mutant (SdP), the LF mutant (SdL), or the LF/EF double mutant (SdLE), infiltration into the lung by GRl⁺ cells including PMN (CDl Ib⁺GRl⁺) and macrophages (CDl Ib⁺GRl^") was examined by FACS. Figure 19B depicts day 3 after immunization with the PI-PLC mutant (SdP), the LF mutant (SdL), or the LF/EF double mutant (SdLE). SSC stands for side scatter.

DETAILED DESCRIPTION OF THE INVENTION

Direct comparison of anthrax vaccines in animal models has demonstrated that the live attenuated Sterne vaccine confers better protection than the acellular vaccine (Turnbull, et al., 1986, Infect. Immun., 52: 356-363; Welkos and Friedlander, 1988, Microb. Pathog., 5: 127-139). However, the Sterne vaccine is not approved for human use in the US due to safety concerns. Thus, further attenuation of Sterne strain is needed but substantial attenuation frequently results in loss of immunogenicity, a paradox that often impedes live vaccine development. The present invention provides attenuated Bacillus anthracis vaccine strains that improve upon the Sterne vaccine strain. Further, because of the aforementioned paradox, the present invention additionally provides methods for administering an attenuated Bacillus anthracis vaccine strain that stimulates the proliferation and activation of antigen presenting cells, thus leading to a robust humoral and cellular immune response, including a memory T cell response, from a live Bacillus anthracis vaccine strain.

Definitions As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one

(i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, to "alleviate" a disease means reducing the severity of one or more symptoms of the disease.

As used herein, "amino acids" are represented by the full name thereof, by the three-letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D

Glutamic Acid GIu E

Lysine Lys K

Arginine Arg R

Histidine His H Tyrosine Tyr Y

Cysteine Cys C

Asparagine Asn N

Glutamine GIn Q

Serine Ser S Threonine Thr T

Glycine GIy G

Alanine Ala A

Valine VaI V

Leucine Leu L Isoleucine He I

Methionine Met M

Proline Pro P

Phenylalanine Phe F

Tryptophan Tip W "Antisense"refers particularly to the nucleic acid sequence of the non- coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

By the term "applicator", as the term is used herein, is meant any device including, but not limited to, a hypodermic syringe, a pipette, a nasal sprayer, and the like, for administering the compounds and compositions of the invention.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated, then the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

By the term "effective amount", as used herein, is meant an amount that when administered to a mammal, causes a detectable level of immune response compared to the immune response detected in the absence of the compound. Immune response can be readily assessed by a plethora of art-recognized methods.

The skilled artisan would understand that the amount of the compound or composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

As used herein, "eliciting an immune response" or "elicits an immune response" comprises causing an adaptive and/or innate immune response to be detectably elevated after contact with and/or administration of an antigen to a mammal or cell, wherein the detectable elevation is greater than in a similar or identical mammal or cell not contacted with and/or administered an antigen, or wherein the detectable elevation is greater than in the same mammal or cell before being contacted with and/or administered an antigen.

"Instructional material, "as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and/or compound of the invention in the kit for effecting alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue or a mammal, including as disclosed elsewhere herein. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

By "complementary to a portion or all of the nucleic acid encoding" a protein of the invention, is meant a sequence of nucleic acid which does not encode a protein. Rather, the sequence which is being expressed in the cells is identical to the non-coding strand of the nucleic acid encoding the protein and thus, does not encode the protein.

The terms "complementary" and "antisense" as used herein, are not entirely synonymous. "Antisense" refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. "Complementary" as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e. g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e. g., A:T and G:C nucleotide pairs). As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

A "coding region" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g. , naked or contained in liposomes) and viruses (e.g., retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. A first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent one another or if the two regions are separated by no more than about 1000 nucleotide residues, and preferably no more than about 100 nucleotide residues.

As used herein, the term "fragment" as applied to a nucleic acid, may ordinarily be at least about 18 nucleotides in length, preferably, at least about 24 nucleotides, more typically, from about 24 to about 50 nucleotides, preferably, at least about 50 to about 100 nucleotides, even more preferably, at least about 100 nucleotides to about 200 nucleotides, yet even more preferably, at least about 200 to about 300, even more preferably, at least about 300 nucleotides to about 400 nucleotides, yet even more preferably, at least about 400 to about 500, and most preferably, the nucleic acid fragment will be greater than about 500 nucleotides in length.

As applied to a protein, a "fragment" of a protein is about 6 amino acids in length. More preferably, the fragment of a protein is about 8 amino acids, even more preferably, at least about 10, yet more preferably, at least about 15, even more preferably, at least about 20, yet more preferably, at least about 30, even more preferably, about 40, and more preferably, at least about 50, more preferably, at least about 60, yet more preferably, at least about 70; even more preferably, at least about 80, and more preferably, at least about 100 amino acids in length.

A "genomic DNA" is a DNA strand which has a nucleotide sequence homologous with a gene as it exists in the natural host. By way of example, a fragment of a chromosome is a genomic DNA.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e. g., between two nucleic acid molecules, e. g., two DNA molecules or two RNA molecules, or between two polypeptide molecules.

When a subunit position in both of the two molecules is occupied by the same monomelic subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are completely or 100% homologous at that position. The percent homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% identical, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 5'-ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

In addition, when the terms "homology" or "identity" are used herein to refer to the nucleic acids and proteins, it should be construed to be applied to homology or identity at both the nucleic acid and the amino acid sequence levels.

A first oligonucleotide anneals with a second oligonucleotide with "high stringency" or "under high stringency conditions" if the two oligonucleotides anneal under conditions whereby only oligonucleotides which are at least about 60%, more preferably at least about 65%, even more preferably at least about 70%, yet more preferably at least about 80%, and preferably at least about 90% or, more preferably, at least about 95% complementary anneal with one another. The stringency of conditions used to anneal two oligonucleotides is a function of, among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the oligonucleotides, the G-C content of the oligonucleotides, and the expected degree of non-homology between the two oligonucleotides, if known. Methods of adjusting the stringency of annealing conditions are known (see, e.g., Sambrook et al., 2001, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. MoI. Biol. 215: 403- 410), and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) world wide web government site of the National Library of Medicine as part of the National Institutes of Health. BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward= 1 ; expectation value 10.0 ; and word size= 11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI- Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules and relationships between molecules which share a common pattern. When using BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used. See the publicly available government web site of National Center for Biotechnology

Information (NCBI) of the National Library of Medicine at the National Institutes of Health.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted. An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

By describing two polynucleotides as "operably linked" it is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region. Preferably, when the nucleic acid encoding the desired protein further comprises a promoter/regulatory sequence, the promoter/regulatory sequence is positioned at the 5' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell. Together, the nucleic acid encoding the desired protein and its promoter/regulatory sequence comprise a "transgene." As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

A "polyadenylation sequence" is a polynucleotide sequence which directs the addition of a poly A tail onto a transcribed messenger RNA sequence. A "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.

The term "nucleic acid" typically refers to large polynucleotides.

The term "oligonucleotide" typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G,C) in which "U" replaces "T."

Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5 'to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences. A "portion" of a polynucleotide means at least at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.

"Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications.

A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

"Probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

"Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc. ) as well.

A "recombinant polypeptide" is one which is produced upon expression of a recombinant polynucleotide.

"Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

The term "protein" typically refers to large polypeptides.

The term "peptide" typically refers to short polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the term "reporter gene" means a gene, the expression of which can be detected using a known method. By way of example, the Escherichia coli lacZ gene may be used as a reporter gene in a medium because expression of the lacZ gene can be detected using known methods by adding a chromogenic substrate such as O-nitrophenyl/3- galactoside to the medium (Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC, p. 574).

A "restriction site" is a portion of a double-stranded nucleic acid which is recognized by a restriction endonuclease.

As used herein, the term "transgene" means an exogenous nucleic acid sequence which exogenous nucleic acid is encoded by a transgenic cell or mammal. A "recombinant cell" is a cell that comprises a transgene. Such a cell may be a eukaryotic cell or a prokaryotic cell. Also, the transgenic cell encompasses, but is not limited to, an embryonic stem cell comprising the transgene, a cell obtained from a chimeric mammal derived from a transgenic ES cell where the cell comprises the transgene, a cell obtained from a transgenic mammal, or fetal or placental tissue thereof, and a prokaryotic cell comprising the transgene.

By the term "exogenous nucleic acid" is meant that the nucleic acid has been introduced into a cell or an animal using technology which has been developed for the purpose of facilitating the introduction of a nucleic acid into a cell or an animal.

By "tag" polypeptide is meant any protein which, when linked by a peptide bond to a protein of interest, may be used to localize the protein, to purify it from a cell extract, to immobilize it for use in binding assays, or to otherwise study its biological properties and/or function. As used herein, the term "transgenic mammal" means a mammal, the germ cells of which comprise an exogenous nucleic acid.

As used herein, to "treat" means reducing the frequency with which symptoms of a disease (i.e., Bacillus anthracis infection, sequelae following infection, and the like) are experienced by a patient.

By the term "vector" as used herein, is meant any plasmid or virus encoding an exogenous nucleic acid. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like. The vector may be a viral vector which is suitable as a delivery vehicle for delivery of a nucleic acid that encodes a protein and/or antibody of the invention, to the patient, or the vector may be a non-viral vector which is suitable for the same purpose.

Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12744-12746). Examples of viral vectors include, but are not limited to, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, and the like (Cranage et al., 1986, EMBO J. 5: 3057-3063; International Patent Application No. WO 94/17810, published August 18,1994 ; International Patent Application No. WO 94/23744, published October 27,1994). Examples of non- viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.

A "therapeutic" treatment is a treatment administered to a patient who exhibits signs of pathology for the purpose of diminishing or eliminating those signs and/or decreasing or diminishing the frequency, duration and intensity of the signs. By the term "specifically binds," as used herein, is meant an antibody which recognizes and binds with a protein present in a sample, but which antibody does not substantially recognize or bind other molecules in the sample.

As used herein "LF" or "lethal factor" refers to a Bacillus anthracis lethal factor. As used herein, a "PI-PLC" or a "phosphatidylinositol-specific phospholipase" refers to a Bacillus anthracis PI-PLC, unless otherwise designated. By the term "vaccine" as used herein, is meant a composition, preferably an attenuated whole cell bacteria or a spore, which serves to protect an animal against a disease and/or to treat an animal already infected compared with an otherwise identical animal to which the vaccine is not administered or compared with the animal prior to the administration of the vaccine.

As used herein, the term "immunizing a mammal against a Bacillus anthracis infection" means administering to the mammal a composition, preferably an attenuated whole cell bacteria or spore thereof, which elicits an immune response in the mammal, which immune response provides protection to the mammal against anthrax disease when compared with an otherwise identical mammal to which the composition is not administered or to the mammal prior to such administration.

Description

The present invention encompasses an attenuated Bacillus anthracis vaccine strain. As demonstrated by the data disclosed herein, attenuated mutants of the whole cell Bacillus anthracis Sterne strain that comprise a deletion in the lethal factor gene and/or a deletion in PI-PLC, and/or a deletion in the edema fact or gene result in an attenuated Bacillus anthracis vaccine strain that, when administered to a mammal, preferably a human, result in the activation of antigen presenting cells, and develop robust, Bacillus anthracis specific humoral and cellular immune responses.

The present invention further encompasses a method of eliciting an immune response against Bacillus anthracis by administering a Bacillus anthracis Sterne strain bacteria that has been attenuated according to the present invention. This is because, as demonstrated by the data disclosed herein, attenuated whole cell Bacillus anthracis Sterne strain comprising a deletion in the lethal factor gene and/or a deletion in PI-PLC and/or a deletion in the edema factor gene result in an attenuated Bacillus anthracis vaccine strain that, when administered to a mammal, preferably a human, result in the activation of antigen presenting cells, and develop Bacillus anthracis specific humoral and cellular immune responses. As disclosed elsewhere herein, the deletion of nucleotides 707 to 1505 in the nucleic acid encoding lethal factor (SEQ ID NO.: 1) corresponds to an in- frame deletion of amino acids 237 to 502 of the LF protein (SEQ ID NO.: 2). However, the present invention is not limited to the deletions of lethal factor described herein, but include any deletion that would result in a non-functioning lethal factor protein. Such deletions include, but are not limited to, deletions in the enzymatic site(s) of lethal factor, truncations of the gene encoding lethal factor, deletion or otherwise disruption of the lethal factor promoter/regulatory element, and the like.

As disclosed elsewhere herein, the entire open reading frame was deleted from the nucleic acid sequence encoding PI-PLC (SEQ ID NO.: 3). In addition, the deletion of the nucleic acid sequence encoding PI-PLC is not limited to the deletion described herein, but rather includes any deletion that would result in a non- functioning PI-PLC protein (SEQ ID NO.: 4) or a PI-PLC with diminished function. Such deletions include deletions in the nucleic acid encoding PI-PLC, or deletions in the promoter/regulatory element driving expression of PI-PLC, and the like.

As disclosed elsewhere herein, the deletion of nucleotides 1059 to 1778 in the nucleic acid encoding edema factor (SEQ ID NO.: 5) corresponds to an in-frame deletion of amino acids 359-601 of the EF protein (SEQ ID NO.: 6). In addition, the deletion of the nucleic acid encoding the edema factor is not limited to the deletion described herein, but rather includes any deletion that would result in a non-functioning edema factor or an edema factor with diminished function. Such deletions include deletions in the nucleic acid encoding edema factor, or deletions in the promoter/regulatory element driving expression of edema factor, and the like.

The vaccine of the invention is not limited to deletion mutants of Bacillus anthracis or the Sterne vaccine strain. That is, the present invention can further comprise mutations in a protein which give rise to different lengths and can comprise insertion, deletion or point mutations. An insertion mutation is one where additional base pairs are inserted into a nucleic acid encoding the protein, such as, but not limited to, a mutation that causes a frameshift mutation and/or a stop codon such that the nucleic acid is not translated past the novel premature stop codon. A deletion mutation is one where base pairs have been removed from a nucleic acid molecule. A point mutation is one where a single base pair alteration has been made in a nucleic acid molecule. Each of these mutations is designed such that creation of any one of them in a nucleic acid molecule effects an alteration in the nature of any polypeptide expressed by that nucleic acid, which alteration results in a protein that function as would the wild type protein, or does so to a lesser extent, compared with the wild type full-length protein which is not truncated or modified.

Methods for determining if lethal factor, PI-PLC or edema factor are non- functioning or have diminished function compared to the wild type protein are disclosed elsewhere herein and include, for example, enhanced dendritic cell function, GPI-anchored protein cleavage, MTT macrophage function assays, and the like.

As demonstrated by the data disclosed herein, the present vaccine is effective when administered as a spore. Methods for inducing Bacillus anthracis bacteria to form spores are known in the art, and are disclosed elsewhere herein. However, for other methods of administration, use of the present attenuated Bacillus anthracis vaccine strain as a vegetative bacilli are also useful. Methods for propagating and maintaining Bacillus anthracis in a vegetative state are known in the art and are described elsewhere herein.

The present invention further comprises a method for immunizing an animal, preferably a mammal, even more preferably a human, against a Bacillus anthracis infection. This is because, as demonstrated by the data disclosed herein, administration of attenuated Bacillus anthracis vaccine strain spores intranasally results in an immune response characterized by, among other things, activation of dendritic cells, the proliferation of Bacillus anthracis T cells, and the production of Bacillus anthracis-specific antibodies. Thus, the present, invention includes a method for immunizing a mammal against a Bacillus anthracis infection.

In one embodiment of the present method, the attenuated Bacillus anthracis vaccine strain is a derivative of the Sterne strain. Preferably, the attenuated Bacillus anthracis vaccine strain is a deletion mutant of the Sterne strain, including a lethal factor deletion mutant and/or a PI-PLC deletion mutant, and/or an edema factor deletion mutant. Preferably, the attenuated Bacillus anthracis vaccine strain is delivered intranasally as a spore.

The term "intranasal delivery" or "intranasal administration" as used herein refers to a systemic form of administration of an active ingredient, whereby a therapeutically effective amount of the active ingredient, for example an attenuated Bacillus anthracis vaccine strain, is propelled or otherwise introduced into the nasal passages of an animal such that it contacts the nasal mucosa, from which it is absorbed into the systemic circulation. The vaccine formulations of this invention are made by conventional techniques for spore formation, described elsewhere herein. Further, as described below, methods for making a vaccine, such as a spore-based vaccine, into a suitable form for intranasal delivery, are known in the art. For a discussion of the state of the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.,

18th Edition, 1975.

As is known in the art, spores are tolerant to extreme environmental conditions and are stable for long periods of time. Thus, the attenuated Bacillus anthracis vaccine strain spores of the present invention are highly suitable for storage in the form of an intranasal preparation, including a powder, or a solution or suspension of spores. The attenuated Bacillus anthracis vaccine spores of the present invention can be delivered with a pharmaceutically acceptable carrier it increase stability or enhance delivery to a mammal. Such carriers used in the intranasal administration of a vaccine include, for example, a high molecular weight polysaccharide, for example dextran, and optionally an absorption enhancer, for example sodium glycocholate.

If the spores of the present attenuated Bacillus anthracis vaccine strain are too large for nasal administration, they must be processed further. Methods for decreasing the particle size of spores, or for disseminating clumped spores for intranasal administration, include pulverization. Suitable methods for pulverization include cutting, chopping, crushing, grinding, milling, micronization, screening, trituration, and the like.

The resulting spore particles are then size classified. Suitable size classification methods include screening, sieving and the like. Spore particles of the preferred size in the vaccine of this invention are less than about 100 microns in diameter, preferably between about 60 microns and about 100 microns in diameter, and are obtained by passing the spores through a #200 standard mesh.

The present invention further comprises a method of inducing a protective immune response in a mammal. As used herein, a protective immune response means an immune response whereby the severity of the resulting disease absent a protective immune response would be worse than with a protective immune response. The present invention comprises administering an attenuated Bacillus anthracis vaccine strain to a mammal, preferably a human, to generate a protective immune response.

Preferably, the protective immune response is generated by administering an attenuated Bacillus anthracis vaccine strain intranasally in the form of a spore. This is because, as demonstrated by the data disclosed herein, mammal susceptible to anthrax developed a protective immune response, including activated antigen presenting cells and humoral and cellular immune responses, when administered spores of an attenuated Bacillus anthracis vaccine strain intranasally. Further, as demonstrated by the data disclosed herein, humoral immune responses generated in response to administration of the vaccine of the present invention include the production of IgG antibodies that specifically bind Bacillus anthracis, or a portion thereof, such as a Bacillus anthracis antigen.

Methods for determining if an animal is immunized, or if an animal has developed a protective immune response are known in the art and are described elsewhere herein, and include an examination of dendritic cell activation, antihody production, and T cell memory. A protective immune response to Bacillus anthracis is also one which affords protection to the animal from lethal challenge with wild type Bacillus anthracis. Protection against lethal challenge with wild type Bacillus anthracis is typically assessed by first immunizing a series of animals with the present vaccines to generate serum capable of neutralizing Bacillus anthracis infectivity in a standard neutralization assay. The animals are then inoculated with a serial dilutions of wild type Bacillus anthracis, which dilutions contain sufficient Bacillus anthracis to kill non-immunized animals. The death rate of the animals is quantitated and is compared to the level of the Bacillus anthracis neutralizing immune response in each of the animals. Protection from lethal challenge has been effected when non-immunized animals die and immunized animals do not die as a result of infection with Bacillus anthracis.

As the data presented herein establish, the attenuated Bacillus anthracis vaccine strains of the invention, and/or combinations thereof, protected an animal against infection by Bacillus anthracis better than the Sterne vaccine, which is the only whole cell vaccine approved in any part of the world, although not in the United States. Thus, the attenuated Bacillus anthracis vaccine of the invention is capable of protecting an animal against anthrax to a level that surpasses the currently available whole cell vaccine. Table 1. Attenuated Bacillus anthracis vaccines

The attenuated Bacillus anthracis vaccine of the invention may be formulated to be suspended in a pharmaceutically acceptable carrier suitable for use in animals and in particular, humans. Such formulations include the use of adjuvants such asmuramyl dipeptide derivatives (MDP) or analogs which are described in U.S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and, 4,406,890. Other adjuvants which are useful include alum (Pierce Chemical Co. ), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL- 12. Other components may include a polyoxypropylene-polyoxyethylene block polymer (Pluronic), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Patent No. 4,606, 918). The attenuated Bacillus anthracis vaccine of the invention can also be encapsulated into liposomes for administration to the animal. See for example, U.S. Patent Nos. 4,053,585; 4,261,975 and 4,406,890. The attenuated Bacillus anthracis vaccine (e.g., Sterne vaccine strain spore,

SdL, SdP, SdLP, and/or SdLE spores) of the invention is administered to a human by any suitable route of administration, for example, subcutaneously, intramuscularly, orally, intravenously, intradermally, intranasally or intravaginally. However, as demonstrated by the data disclosed herein, the preferred means of administering the vaccine of the present invention is intranasal administration.

The attenuated Bacillus anthracis vaccine, preferably in a spore form, is first suspended in a pharmaceutically acceptable carrier which is suitable for the chosen route of administration and which will be readily apparent to those skilled in the art of vaccine preparation and administration. The dose of vaccine to be used may vary dependent upon any number of factors including the age of the individual and the route of administration. Typically, the subunit vaccine is administered in a range of 0.5 mg to 50 mg of protein per dose. Approximately 1-10 doses are administered to the individual at intervals ranging from once per week, to once per month, to once per year, to once every few years.

The attenuated Bacillus anthracis vaccine of the invention can be formulated and administered to a mammal for treatment and/or prevention of anthrax infection as now described.

The invention encompasses the preparation and use of pharmaceutical compositions comprising an attenuated Bacillus anthracis vaccine useful for treatment, more preferably, prevention of anthrax infection as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, as a combination of at least one active ingredient (e.g., an immunogenic dose of an attenuated Bacillus anthracis vaccine) in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional (active and/or inactive) ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-or multi-dose unit. Although the descriptions of pharmaceutical compositions provided herein are principally directed to attenuated Bacillus anthracis vaccines which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration, but preferably for intranasal administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one- half or one-third of such a dosage. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject immunized and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include antibiotics and/or adjuvants, and the like. Antibiotics for use in the methods of the present invention include, but are not limited to, ciprofloxacin and beta-lactam antibiotics such as penicillin and ampicillin. Controlled-or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology. A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder- dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65° F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken, i. e., by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1 % (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein. Typically dosages of the compound of the invention which may be administered to an animal, preferably a human, range in amount from 1 mg to about 100 g per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.

The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being immunized against, the type and age of the animal, etc. A compound used to treat anthrax infection may be co-administered with the immunogenic dose of an attenuated Bacillus anthracis vaccine vaccine of the invention. Alternatively, the compound (s) may be administered an hour, a day, a week, a month, or even more, in advance of the immunogenic dose (s) of an attenuated Bacillus anthracis vaccine vaccine, or any permutation thereof. The vaccine of the invention is useful for prevention of anthrax disease in an animal, preferably a human. However, the vaccine is also useful as a therapeutic agent for treatment of ongoing anthrax infection in order to boost the immune response in the animal. Thus the invention contemplates both prophylactic and therapeutic uses for the attenuated Bacillus anthracis vaccine of the invention. It should be appreciated that the attenuated Bacillus anthracis vaccine of the invention can be combined with other subunit vaccines, such as attenuated Bacillus anthracis vaccines comprising other truncated Bacillus anthracis proteins, each of which may be generated and used according to published protocols and the procedures described herein.

Kits The invention includes various kits which comprise an attenuated Bacillus anthracis vaccine of the invention, an applicator, and instructional materials which describe use of the kit to perform the methods of the invention. Although exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the invention.

In one aspect, the invention includes a kit for treating an anthrax infection in a human. The kit is used pursuant to the methods disclosed in the invention. Briefly, the kit is used to administer an attenuated Bacillus anthracis vaccine strain, preferably a spore, to a mammal (e.g., a human) having an anthrax infection, or at risk of contracting an anthrax infection. This is because, as more fully disclosed elsewhere herein, the data disclosed herein demonstrate that an attenuated Bacillus anthracis vaccine strain of the invention when administered to an animal in an art-recognized model of anthrax infection, elicits an immune response that protects the otherwise susceptible animal from anthrax infection and resulting mortality. The kit further comprises an applicator useful for administering an attenuated

Bacillus anthracis vaccine strain to the animal. The particular applicator included in the kit will depend on, e.g., the method used to administer an attenuated Bacillus anthracis vaccine strain, as well as the animal to which the attenuated Bacillus anthracis vaccine strain is to be administered, and such applicators are well-known in the art and may include, among other things, a pipette, a syringe, a dropper, and the like. Preferably, the applicator is an applicator for administering spores intranasally. Moreover, the kit comprises an instructional material for the use of the kit. These instructions simply embody the disclosure provided herein.

The kit can further include a pharmaceutically-acceptable carrier. The composition is provided in an appropriate amount as set forth elsewhere herein. Further, the route of administration and the frequency of administration are as previously set forth elsewhere herein.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLE 1 : Bacillus anthracis PI-PLC Inhibition of Dendritic Cell Activation The complete genome sequence has revealed that Bacillus anthracis encodes proteins homologous to known virulence factors of Lm (Read et al., 2003, Nature, 423, 81- 86). In a transient intracellular step during the initial lung stage of pulmonary anthrax, Bacillus anthracis survives phagocytic killing by escaping to the cytosol and hijacking macrophages for dissemination into bloodstream, leading to systemic infection (Dixon et al., 2000, Cell Microbiol., 2: 453-463). This intracellular step was first described and is best studied in Listeria monocytogenes and indicates that the cellular immune response in the lung may play a role in the initial control of pulmonary anthrax. The present Example discloses the immune modulating function of Bacillus anthracis, particularly that Bacillus PI- PLC inhibits dendritic cell activation and the T cell response.

Bacillus anthracis PI-PLC Inhibits Dendritic Cell Activation and the T cell Response

GPI anchored proteins participate in a variety of host cell functions, particularly in mediating host cell signaling. There are many GPI anchored proteins but only a limited number have been identified with known functions; most notable are an LPS receptor (CD14) and FcγRIII (CDl 6) expressed by antigen-presenting cells. The results disclosed herein demonstrate that treatment of dendritic cells with purified Bacillus PI-PLC affects the ability of dendritic cells to respond to toll-like receptor (TLR) ligand stimulation as measured by reduced co-stimulatory and MHC molecule upregulation, TNFα production and MAPK signaling. Conversely, dendritic cell functions were not affected by Listeria PI- PLC, which differs from Bacillus PI-PLC in its inability to efficiently cleave GPI anchored proteins. Infection of mice with Listeria monocytogenes expressing Bacillus anthracis PI- PLC resulted in reduced CD4 T cell priming. These data demonstrate that Bacillus anthracis PI-PLC downmodulates dendritic cell function, possibly by removing GPI anchored proteins important for dendritic cell activation (Zenewicz, et al., 2005, J. Immunol., 174: 8011-8016).

PI-PLC Treatment and TLR Ligand Activation of Dendritic Cells Dendritic cells were cultured from the bone marrow of C57BL/6 mice as described in, for example, (Lutz et al., 1999, J. Immunol. Methods 223: 77-92). Dendritic cells were left untreated or treated for 1 hour with 6 μg/ml purified recombinant Bacillus or Listeria PI-PLC unless otherwise indicated. Polyinosine-polycytidylic acid (poly I: C), LPS, peptidogylcan (Sigma-Aldrich, St. Louis, MO), or CpG DNA (5'-

TCCATGACGTTCCTGATGCT-3'; SEQ ID NO:1) was then added at the indicated concentration for the indicated time. Recombinant B. thuringienesis and L. monocytogenes PI-PLC were provided by M. F. Roberts (Boston College, Chestnut Hill, MA). They were expressed in Escherichia coli and purified as described in Feng et al. (J. Biol. Chem., 277: 19867-19875). B. thuringienesis PI-PLC shares high homology with PI-PLC from other Bacillus species. Between the mature PI-PLC of B. thuringienesis and Bacillus anthracis there are only two amino acid differences, one of which is a conservative substitution. These two residues are not important for substrate binding or catalysis (Gassier et al., 1997, Biochemistry, 36:12802-12813).

Dendritic Cell Viability and Surface Marker Expression

Annexin V and 7-aminoactinomycin D (7-AAD) staining were performed according to manufacturer's directions (BD Pharmingen, Franklin Lakes, NJ). For surface staining of dendritic cells, cells were stained with mAb anti-CD 1 Ic (clone HL3), anti-CD86 (clone GLl), anti-CD80 (clone 16-10A1), and anti-MHC II (clone 2G9) in 1% BSA/PBS with FcR block. All mAb for FACS were purchased from BD Pharmingen. After several washes in 1% BSA/PBS, cells were fixed with 2% paraformaldehyde and analyzed by FACS.

TNF-α production by Dendritic Cells For intracellular staining to analyze TNF-α production, dendritic cells were incubated for 5 hours at 37°C with 5% CO₂ in the presence of GolgiStop (BD Pharmingen). Dendritic cells were surface stained as described herein with anti-CD 1 Ic, and then, according to the manufacturer's protocol, dendritic cells were permeabilized with Cytofix/Cytoperm solution, stained with anti-TNFα mAb (clone MP6-XT22), fixed, and analyzed by FACS. TNF-α in dendritic cell supernatants was measured by ELISA according to the manufacturer's directions (R&D Systems, Minneapolis, MN). Analysis of p38 and ERK phosphorylation

Dendritic cells were pretreated with 6 μg/ml PI-PLC for 1 hour, then stimulated with the indicated concentration of poly I:C for 60 minutes. Na₃VO₄ (1 mM) was added to inhibit phosphatases. Cells were lysed in sample buffer with 100 mM DTT, and lysates were separated on a 4-15% gradient gel. A Western blot was performed using one of the following Abs: rabbit polyclonal anti-ERK (p44/42 MAPK), anti-p38 MAPK, or Abs that recognize the phosphorylated forms of these proteins (Cell Signaling Technology, Danvers, MA). Blots were developed by enhanced chemiluminescensce (ECL), and bands were quantitated using Image Gauge (version 4.01 ; Fuji, Valhalla, NY).

Construction of recombinant L. monocytogenes strains expressing Bacillus anthracis PI-PLC

Recombinant L. monocytogenes was constructed using the highly attenuated ΔdalΔdat mutant as the parental strain (Lm) (Thompson, et al., 1998, Infect. Immun., 66: 3552-3561). An L. monocytogenes strain with an in-frame deletion of plcA (Lm ΔplcA) was constructed on the ΔdalΔdat background using alleic exchange methods described in Camilli et al., (1993, MoI. Microbiol., 8: 143-157). The gene encoding Bacillus anthracis PI-PLC was then integrated into the chromosome of the Lm ΔplcA strain, generating a strain with the Bacillus anthracis PI-PLC gene under control of the Listeria plcA promoter and signal sequence (Lm ΔplcA: Ba PI-PLC).

PI-PLC activity

PI-PLC activity on PI was determined by measuring the cleavage of L-3- phosphatidyl-[2-³H]inositol by the purified enzymes or supernatants of overnight cultures as described in Goldfine and Knob, (1992, Infect. Immun., 60: 4059-4067). Activity on GPI- anchored proteins was determined by incubating either enzymes or concentrated supernatants from overnight bacterial cultures with T cells for 1 hour at 37⁰C. Cells were then surface stained, as described above, with mAb to the GPI-anchored protein Thyl (clone 53-2.1) and analyzed by FACS.

Analysis of T cell response C57BL/6 mice were intravenously infected with 2 X 10⁸ CFU of Lm or Lm ΔplcA:Ba PI-PLC in PBS. Twenty milligrams of D-alanine was injected with the bacteria to allow transient bacterial growth in vivo. Bacterial loads in the spleen and liver were determined by plating serial dilutes of organ homogenates on brain heart infusion agar with 100 μg/ml D-alanine. Splenocytes from day 7 postinfection mice were stimulated with or without listeriolysin

(LLOi₉₀_₂o₁) peptide, and intracellular IFN-γ staining was performed as described in Zenewicz, et al., (2002, J. Immunol., 169: 5805-5812).

Bacillus PI-PLC Inhibits Dendritic Cell Activation by TLR Ligands The pathogenic Bacillus species, B. cereus, B. thuringiensis, and Bacillus anthracis, all express PI-PLCs that have >94% amino acid identity (Read et al., 2003, Nature 423: 81-86; Volwerk, et al., 1989, FEMS Microbiol. Lett., 52: 237-241; Klichko, et al., 2003, Biochem. Biophys. Res. Commun., 303: 855-862). To investigate whether Bacillus PI-PLC modulates dendritic cell function, dendritic cells were treated with or without Bacillus PI- PLC for 1 hour, then stimulated them for 18 hours with TLR ligands, including LPS (TLR4), poly I:C (TLR3), peptidogylcan (TLR2), and CpG DNA (TLR9). Dendritic cell activation was examined by staining for surface expression of CD86, which is up-regulated upon TLR stimulation.

Bacillus PI-PLC treatment greatly reduced the percentage of dendritic cells that expressed high levels of CD86 upon TLR stimulation, and this was true for all four TLR ligands tested (Figure 1). Thus, Bacillus PI-PLC reduced the ability of dendritic cells to become activated and up-regulate the costimulatory molecule CD86 in response to various TLR ligands.

To further investigate the inhibitory effect of Bacillus PI-PLC on Dendritic cell activation, the following parameters were examined: 1) the surface expression of several surface markers (CD80, CD86, and MHC class II) upon stimulation with various concentrations of poly I:C, 2) the effect of Bacillus PI-PLC on the viability of dendritic cells, and 3) the potency of PI-PLC in inhibiting dendritic cell activation. Bacillus PI-PLC inhibited dendritic cell activation over a range of poly I:C concentrations (0.1-10 μg/ml), resulting in reduced up-regulation of CD80, CD86, MHC class II surface expression compared with non-PI-PLC-treated dendritic cells (Figure 2). This phenotype cannot be attributed to increased cell death, because dendritic cells treated with or without PI-PLC had similar annexin V and 7-AAD staining profiles (Figure 2).

Titration of Bacillus PI-PLC resulted in a dose-dependent inhibition of the activation of dendritic cells (Figure 2). Inhibitory effects were observed at a concentration as low as 1 ng/ml. Thus, Bacillus PI-PLC reduced the ability of dendritic cells to become activated and up-regulate the surface expression of costimulatory and Ag presentation molecules in response to TLR stimulation.

Bacillus PI-PLC, but not Listeria PI-PLC, Inhibits Dendritic Cell activation To determine whether the inhibitory effect on dendritic cell activation is unique to Bacillus PI-PLC or is shared by other bacterial PI-PLCs, treatment with Bacillus and Listeria PI-PLC on dendritic cell activation was compared. Listeria and Bacillus PI-PLC had comparable activity in the cleavage of PI, but only Bacillus PI-PLC had the ability to cleave GPI-anchored proteins (Figure 3). No defect was observed in CD86 up-regulation when cells were treated with Listeria PI-PLC before poly I:C stimulation (Figure 3).

In addition to increased surface expression of costimulatory and Ag presentation molecules, dendritic cell activation is characterized by cytokine production. Therefore, the ability of Bacillus or Listeria PI-PLC to affect the production of TNF-α in dendritic cells was also examined. Non-PI-PLC-treated dendritic cells produced TNF-α in a dose-dependent manner in response to poly I:C stimulation (Figure 3). Treatment of dendritic cells with Bacillus PI-PLC inhibited the ability of dendritic cells to produce TNF-α in response to poly I:C stimulation (Figure 3). At 1 μg/ml poly I:C, only 5% of Bacillus PI- PLC-treated cells produced TNF-α compared with 20% of dendritic cells producing TNF-α in the control group lacking Bacillus PI-PLC treatment. This defect in cytokine production by Bacillus PI-PLC-treated dendritic cells was also evident when measured by TNF-α secretion into the cell supernatant after overnight stimulation (Figure 3). In contrast, Listeria PI-PLC-treated dendritic cells produced TNF-α at comparable levels to non-PI-PLC-treated cells (Figure 3). Thus, Bacillus, but not Listeria, PI-PLC affects the ability of dendritic cells to respond to TLR ligands. Because the major difference between Bacillus and Listeria PI- PLC is the inability of Listeria PI-PLC to cleave GPI-anchored proteins, these data indicate that modulation of dendritic cell function by Bacillus PI-PLC may be due to its ability to cleave GPI-anchored proteins from the dendritic cell surface that are important for activation.

Bacillus PI-PLC Treatment Inhibits MAPK Pathway Signaling TLR interaction with their ligands leads to activation of the MAPK signaling pathway, which, in turn, results in activation of transcription factors responsible for up- regulation of costimulatory molecules and cytokines (Alexopoulou, et al., 2001, Nature 413: 732-738; Akira, et al., 2004, Nat. Rev. Immunol., 4: 499-511). To further examine the effect of Bacillus PI-PLC on dendritic cell activation, activation of the MAPK signaling pathway was analyzed by measuring phosphorylation of the MAPKs ERK (p44/42) and p38.

Compared with untreated dendritic cells, dendritic cells treated with Bacillus PI-PLC had less phosphorylated ERK or p38 in response to poly I: C stimulation (Figure 4). These data demonstrate that Bacillus PI-PLC down-modulates MAPK pathway signaling in dendritic cells. Previous studies have demonstrated that lethal toxin also blocks MAPK signaling (Park, et al., 2002, Science 297: 2048-2051; Agrawal, et al., 2003, Nature 424: 329-334). Thus, Bacillus anthracis has multiple virulence factors that interfere with this important signaling pathway central to the initiation of an effective immune response.

L. monocytogenes Expressing Bacillus anthracis PI-PLC Induces Suboptimal T cell Priming in vivo

Because Bacillus PI-PLC treatment of dendritic cells before activation results in reduced surface expression of costimulatory molecules and cytokine production, Bacillus

PI-PLC may down-modulate the cellular immune response by interfering with dendritic cell function. In order to examine whether Bacillus PI-PLC has an effect on the T cell response, the affect of expression of Bacillus anthracis PI-PLC in L. monocytogenes on the T cell response was investigated using the well-defined murine model of listeriosis that allows for quantitative analysis of Ag-specific T cell responses in vivo.

A recombinant L. monocytogenes strain was constructed that has its PI-PLC gene (plcA) replaced by the Bacillus anthracis PI-PLC gene. A highly attenuated ΔdalΔdat mutant of L. monocytogenes was used as the parental strain. The ΔdalΔdat strain has deletions of two genes encoding the enzymes D-alanine racemase (dal) and D-amino acid aminotransferase (dat) that are required for synthesizing D-alanine, an essential component of the cell wall. Because D-alanine cannot be obtained from the environment or host cells, these bacteria can grow only when D-alanine is provided and thus are highly attenuated in vivo (U.S. Patent 6,635,749). L. monocytogenes strains expressing endogenous PI-PLC or Bacillus

PI-PLC had similar activity on the cleavage of PI (Figure 5), as expected because Listeria and Bacillus PI-PLC have comparable PLC activity on PI (Goldfine, et al., 1992, Infect. Immun., 70: 4059-4067). In contrast, only supernatants from L. monocytogenes expressing Bacillus anthracis PI-PLC had significant GPI anchor cleavage activity, as measured by their ability to cleave the GPI-anchored protein Thyl from the surface of T cells (Figure 5). This demonstrates that the recombinant L. monocytogenes strain indeed expresses the Bacillus PI- PLC with a strong activity for GPI anchor cleavage.

To investigate whether Bacillus anthracis PI-PLC modulated the immune response, C57BL/6 mice were infected with L. monocytogenes expressing endogenous PI- PLC or Bacillus anthracis PI-PLC. Similar bacterial loads between the two strains were recovered from the spleens and livers of mice at 12 and 24 hours postinfection (Figure 5). By 48 hours, postinfection, both strains were cleared from the spleens and livers of infected mice. On day 7 after infection, the Ag-specifϊc CD4 T cell response was analyzed by intracellular cytokine staining after stimulation with LLOl₉o_₂oi, an I-A^b-restricted epitope derived from the L. monocytogenes protein LLO (Geginat, et al., 2001, J. Immunol., 166: 1877-1884). A greater percentage of CD4 T cells were specific to the LLOi₉o-₂oi epitope in mice infected with the parental strain of L. monocytogenes than in mice infected with L. monocytogenes expressing Bacillus anthracis PI-PLC (Figure 5). When the total number of LLO i9o-2oi -specific CD4 T cells per spleen was calculated, mice infected with the parent L. monocytogenes strain had ~3-fold more LLO 19₀-2₀1 -specific CD4 T cells than mice infected with L. monocytogenes expressing Bacillus anthracis PI-PLC (Figure 5). These in vivo results demonstrate that Bacillus anthracis PI-PLC expression results in a reduced Ag- specific CD4 T cell response to infection.

The data disclosed herein demonstrate that Bacillus but not Listeria PI-PLC inhibits the activation of dendritic cells by TLR ligands. A major difference between Bacillus and Listeria PI-PLC is the inability of Listeria PI-PLC to cleave GPI-anchored proteins. These results further indicate that Bacillus PI-PLC can inhibit dendritic cell activation by cleaving GPI-anchored proteins important in TLR signaling. Our results also demonstrate that expression of Bacillus anthracis PI-PLC in Listeria monocytogenes results in reduced T cell response in vivo.

EXAMPLE 2: Intranasal immunization with spores of attenuated Bacillus anthracis activate antigen presenting cells in lung.

Even though the most deadly form of anthrax is inhalational infection, little is known about the early immune response in the lung that may play a role in slowing the disease progression. Targeting the early lung stage of infection and mobilizing the innate and adaptive cellular immune response in the lung can block or reduce bacterial dissemination into the blood, thus preventing systemic infection, or at least providing more time and a better chance for antibiotic or antibody therapies to work.

The current vaccine approved for Bacillus anthracis infections in humans is an acellular vaccine (AVA) that is administered subcutaneously via needle injection. This vaccine requires repeated immunizations and frequent boosts for induction of protective immunity, making it impractical for mass immunization. A live attenuated vaccine (Sterne) provides better protective immunity than the acellular AVA vaccine in animal models and is currently used in livestock by a single subcutaneous injection of bacterial spores. The following Example demonstrates the use of the Sterne vaccine in a vaccine platform based on intranasal spore immunization that is easy to use and targets the lung, the initial site of pulmonary anthrax infection. The data disclosed herein demonstrates that intranasal immunization with Sterne spores is particularly effective in activating antigen-presenting cells (APCs) in the lung and stimulating them to produce cytokines, such as IL- 12, IL-6 and RANTES, that are conducive to the induction of a ThI response. Following a single immunization, spores persist for greater than two weeks in the lung and provide prolonged antigenic stimulation for the induction of a strong immune response. Further, as demonstrated by the data herein, immunized mice had high levels of antigen-specific antibodies and long-lasting memory T cells that mounted a protective recall response upon challenge. These results demonstrate that simple intranasal immunization with spores of an attenuated strain offer a vaccination strategy that is suitable for mass immunization against pulmonary anthrax.

Bacteria and Mouse Strains Bacillus anthracis Sterne vaccine strain 7702 (pXOl+, pXO2-) was provided by Rick Rest, Drexel University School of Medicine, Philadelphia, PA (Sterne, 1939, Onderstepoort J. Vet. Sci. A. Ind., 13: 307-312). B. cereus ATCC 6464 was provided by Howard Goldfine, University of Pennsylvania. C57BL/6 and A/JCr mice (National Cancer Institute; Frederick, MD) were immunized at 6-8 weeks old. All mice were housed in insulator cages.

Spore Preparation

Spores were prepared as described in Guidi-Rontani, et al., 1999, MoI.

Microbiol., 31 : 9-17). Briefly, an overnight bacterial culture was diluted 1 : 100 into phage assay medium and incubated at 3O⁰C with aeration for 3 days. Spores were harvested by centrifugation, washed with PBS, and then heat-treated at 65°C for 30 minutes to kill remaining vegetative bacilli. Spores were extensively washed with phosphate buffered saline

(PBS), resuspended in PBS, and plated to determine CFU/ml. Spores were maintained at

4°C until use.

Immunization

Forty microliters of spores in PBS (2x10⁶ CFU for C57BL/6 mice and as indicated for A/JCr mice) were applied to the nasal flares of the mice after light anesthesia by halothane. For antibiotic treated mice, mice were given drinking water containing 2 mg/ml ampicillin (Sigma; St. Louis, MO) at the time of immunization. As indicated, in vivo depletion of CD4 and/or CD8 T cells was performed 24 hours before harvesting the spleens.

Mice were injected intraperitoneally (i.p.) with 400 μg anti-CD4 (clone GKl .5) (BioSource;

Carmillo, CA) or 250 μg anti-CD8 (clone H-35), a gift from Phil Scott, University of

Pennsylvania. Depletion in the spleen and lung was greater than 99% as verified by flow cytometry. Cell Preparation

Spleens and lungs were aseptically removed from sacrificed mice, placed into cold RPMI 1640 and passed through a wire mesh screen. Lungs were treated with 1 mg/ml collagenase (Sigma) for 1 hour. Red blood cells were lysed with 0.83% ammonium chloride. Splenocytes or lung cells were resuspended in complete RPMI 1640 medium containing 5% FCS, 200 μM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

Bacterial Recovery

Spleen and lung homogenates were serially diluted in sterile PBS and the samples were halved. One dilution was plated directly onto brain heart infusion agar and the other was first heat-treated for 30 minutes at 65⁰C before plating. CFU after heat- treatment represented the number of spores and CFU from untreated homogenate minus heat-treated CFU represented number of vegetative bacilli. All bacterial loads presented in the Figures represent total CFU/lung since no statistically significant difference was detected between non- and heat-treated samples.

Cytokine ELISAs

For IFNγ ELISAs, high-binding ELISA plates were coated with 5 μg/ml purified anti-IFNγ Ab (BD Pharmingen; San Diego, CA) in PBS overnight. Plates were then washed with 0.05% Tween/PBS and blocked with PBS/1 % BSA/5% sucrose for 1 hour. Supernatants from cell cultures were diluted in 1% BS A/PBS and incubated for 2 hours at room temperature. Plates were then washed and incubated with 1 μg/ml biotinylated anti- IFNγ Ab (BD Pharmingen) in 1% BSA/PBS for 2 hours at room temperature, followed by another wash and incubation with streptavidin conjugated to horse-radish peroxidase (HRP). After washing, plates were then developed with tetramethylbenzidine (TMB)

(Sigma), the reaction was quenched with 2 N H₂SO₄ and the plates were read on a spectrophotometer at 450 nm. IFNγ concentration was determined by a standard curve with 2-fold diluted purified IFNγ samples run in parallel. IL-12 in dendritic cell supernatants was measured by ELISA according to the manufacturer's directions (R&D systems; Minneapolis, MN). Lethal Factor and Protective Antigen Ab Titer ELISAs

To measure Ab in the serum, plates were coated with 2.5 μg/ml purified protective antigen or lethal factor (List Biological Laboratories, Inc; Campbell, CA) in PBS overnight. Plates were washed and blocked as described herein. Serum samples were serially diluted in 3-fold dilutions in 1 % BSA/PBS and incubated for 1.5 hour at 37⁰C. Plates were washed and incubated with anti-mouse IgG conjugated to HRP at 37°C for 1.5 hours. After a final washing, plates were developed and assayed as described above. Titer was defined as the greatest dilution of serum with a positive result above background.

In vitro Dendritic Cell Stimulation and Cytokine Arrays

Dendritic cells were cultured from the bone marrow of C57BL/6 mice. Dendritic cells were treated with Bacillus anthracis vegetative bacilli (MOI = 1) for 2 hours, antibiotics were added (50 μg/ml gentamycin, 10 μg/ml tetracycline, 100 U/ml penicillin, and 100 μg/ml streptomycin) and the cells were stimulated for another 16 hours. Cell supernatants from unstimulated and stimulated dendritic cells were then hybridized to RayBio mouse cytokine arrays (RayBiotech, Inc.; Norcross, GA) according to the manufacturer's directions.

Histology Lungs were fixed in formalin for 24 hours, embedded in paraffin, and sectioned into 6 μm thick sections. Sections were stained with 1 :150 of anti-CD3 mAb (clone CD3-12) (Novocastra Laboratories; Newcastle Upon Tyne, UK) according to manufacturer's directions and then counterstained with hematoxylin.

Surface Stain

All mAb were purchased from BD Pharmingen unless otherwise specified. To examine T cells, cells were stained with mAb anti-CD8 (clone 53-6.7), and anti-CD4 (clone RM4-5), anti-CD44 (clone IM7), and anti-CD62L (clone MEL- 14) and to examine APCs, cells were stained with mAb anti-CDl Ic (clone HL3), anti-CD86 (clone GLl) and anti-MHCII (clone 2G9) in 1% BSA/PBS. After several washes in 1% BSA/PBS, cells were fixed with 2% paraformaldehyde. Cells were analyzed with a FACS Calibur (Becton- Dickinson) and data were analyzed using Flow-Jo, version 3.7 (TreeStar, Inc., Ashland, OR).

Spores Persist in the Lungs of Immunized Mice To evaluate the potential of intranasal spore immunization as a vaccine platform C57BL/6 mice were intranasally administered 2xlO⁶ spores of the attenuated Sterne vaccine strain of Bacillus anthracis. Immunized mice did not exhibit any signs of morbidity throughout the study (over 200 days post-immunization (p.i.)). For several weeks p.i. spores could be recovered from the lungs of immunized mice (Figure 6). In addition, a low number of bacteria were found in the spleens of immunized mice (Figure 6). Thus, intranasal immunization with the Sterne vaccine strain delivers spores to the lung, where they persist for several weeks, as well as disseminate to the spleen in small numbers, without causing observable adverse effects to the host.

To examine the early innate response in the lung, C57BL/6 mice were inoculated intranasally with 2x10⁶ spores of the Sterne strain. There was a rapid infiltration of peripheral mononuclear cells (PMN) within 12 hours, as visualized in H&E staining of lung sections (Figure 6) and by FACS analysis of lung lymphocytes (Figure 6). Kinetic analysis demonstrated that PMN (CDl lb+/GR-l+) infiltration peaked at 12-24 hours p.i., followed by infiltration of macrophages that peak at 72 hours (Figure 6). Thus, intranasal vaccination with attenuated Bacillus anthracis induces a robust innate response in the lung that is characterized by rapid PMN infiltration, followed by recruitment of inflammatory macrophages.

APC Activation Following Intranasal Spore Immunization APCs link innate and adaptive immunity by recognition of unique components of pathogens. This in turn leads to activation of the APC, resulting in upregulation of costimulatory and antigen presentation molecules, both of which enhance the APCs ability to stimulate T cells (Medzhitov, 2001, Nat. Rev. Immunol., 1 : 135-145). As a starting point to characterize the immune response to intranasal spore immunization, the activation of CDl Ic⁺ cells (comprising both dendritic cells and alveolar macrophages) was examined in the lung following immunization of C57BL/6 mice. On day three p.i., expression of the co-stimulatory molecule CD86 and MHC class II on CDl lc+ cells from the lungs of naive and immunized mice were compared. APCs from the lungs of immunized mice had increased levels of both CD86 and MHC class II molecules on their surface compared to APCs from non-immunized control mice (Figure 7).

To determine if APC activation was induced by spores per se or vegetative bacilli following spore immunization, bacterial growth was inhibited by treating immunized mice with the antibiotic ampicillin, which inhibits the growth of vegetative bacilli, but has no effect on dormant spores. APCs from the lungs of immunized mice treated with antibiotics had reduced levels of activation compared to those from immunized mice without antibiotic treatment (Figure 7). Thus, spores themselves are not effective in inducing APC activation, but spore germination and subsequent growth of vegetative bacilli are important for inducing APC activation.

Since germination of spores into vegetative bacilli is important for APC activation, the immune response induced by intranasal administration of vegetative bacilli instead of spores was examined. Surprisingly, little APC activation was found in the lungs of mice intranasally administered 2x10 CFU of vegetative bacilli (Figure 7). The lack of APC activation was not due to the failure of vegetative bacilli to reach the lungs since virtually all of the administered bacteria could be recovered from the lungs five minutes after immunization. However, the vegetative bacilli were quickly cleared and these mice had no detectable bacteria in their lungs three days after administration (Figure 7). Therefore, although bacterial growth after spore immunization is needed to induce APC activation, intranasal administration of vegetative bacilli cannot drive an immune response, likely due to their rapid clearance from the lung. Together these results demonstrate that survival and germination of spores are critical for the induction of an immune response in the lung.

In order to determine if immunization with spores of other Bacillus species was also effective in stimulating a robust response in the lung, C57BL/6 mice were intranasally administered spores of the closely related pathogen Bacillus cereus that causes gastrointestinal but not pulmonary infection. APCs from the lungs of B. cereus immunized mice were less activated than those found in Bacillus anthracis immunized mice, although equal numbers of CFU were recovered (Figure 7). These results indicate that a species- specific attribute(s) of Bacillus anthracis associated with its ability to initiate pulmonary infection may contribute to efficient activation of APC in the lung.

Bacillus anthracis Stimulated Dendritic cells Secrete ThI Inducing Cytokines In addition to increased surface expression of co-stimulatory and antigen presentation molecules, dendritic cells also express different cytokines when they become activated. The amount and types of cytokines secreted by dendritic cells influence the type of resulting immune response. Therefore, the cytokine profile produced by dendritic cells in response to the Sterne strain of Bacillus anthracis was examined. Dendritic cells were stimulated in vitro with vegetative bacilli of the Sterne strain and the cytokines secreted into the supernatant were identified using a cytokine array blot. Among 22 different cytokines that can be detected by the cytokine array blot, IL-6, RANTES, and IL- 12 were detected in the supernatant of stimulated dendritic cells (Figure 7). RANTES recruits macrophages to the site of infection (Zou, et al., 2000, J. Immunol., 165: 4388-4396) and IL-6 inhibits suppression of regulatory (CD4+, CD25+) T cells (Pasare and Medzhitov, 2003, Science 299: 1033-1036). IL-12 is critical for induction of a ThI response (Hsieh, et al., 1993, Science 260: 547-549; Seder, et al., 1993, Proc. Nat'l Acad. Sci. USA, 90: 10188-10192). Therefore IL-12 was quantified by ELISA and found to be secreted by dendritic cells in a Bacillus anthracis dose dependent manner (Figure 7). Thus, dendritic cells activated by the Sterne vaccine express cytokines conducive to a ThI response.

T cell Responses Following Intranasal Spore Immunization

Intranasal immunization with spores is particularly effective at activating

APCs in the lung and Bacillus anthracis stimulation of dendritic cells results in secretion of ThI inducing cytokines. Therefore, intranasal immunization may generate a Bacillus anthracis-specific T cell response.

As a first step in examining the T cell response, whether intranasal spore immunization led to T cell infiltration and activation was examined. Few CD3+ cells were observed in the lungs of non-immunized mice (Figure 8). However, in the lungs of immunized mice seven days p.i., CD3+ cells were numerous and localized around the alveoli epithelium (Figure 8). Comparison of the expression of surface activation markers on CD4 or CD8 T cells from the lungs of naive and immunized mice revealed that T cells from immunized mice had an activated phenotype. Both CD4 and CD8 T cells from immunized mice expressed greater levels of CD44 and reduced levels of CD62L compared to T cells from naϊve mice (Figure 8). Thus, in response to intranasal spore immunization, T cells infiltrate into the lungs and these cells have an activated phenotype.

Antigen-specific T cell responses to Bacillus anthracis have not been well characterized. A Bacillus anthracis-specific T cell response may contribute to protective immunity conferred by vaccination. Since T cells infiltrate into the lungs of Bacillus anthracis immunized mice and are activated, the Bacillus anthracis-specific T cell response in immunized mice was examined. Seven days p.i., splenocytes from immunized mice were restimulated with either heat-killed vegetative bacilli, protective antigen protein, or left unstimulated. Only upon stimulation did cells produce IFNγ (Figure 9). Splenocytes from non-immunized mice incubated with heat-killed bacteria or protective antigen did not produce IFNγ. No detectable IL-4 was produced by any of the stimulated cells. Thus, intranasal spore immunization generates a Bacillus anthracis-specific T cell response.

Both CD4 and CD8 T cells can produce IFNγ. To determine the contribution of CD4 and CD8 T cells to the IFNγ response a CD4 or CD8 depleting antibody was administered to immunized mice one day prior to harvesting splenocytes. T cell depletion was greater than 99% effective as measured by flow cytometry. Splenocytes of immunized mice depleted of CD4+ cells produced significantly less IFNγ upon stimulation with heat- killed bacteria than non-depleted mice (Figure 9). A significant reduction was also observed in CD8 depleted mice compared to non-depleted mice (Figure 9), although the reduction was not as great as that seen with CD4 depletion. Thus, intranasal Bacillus anthracis immunization generates a ThI response, consisting of both CD4 and CD8 Bacillus anthracis- specific T cells.

Bacillus anthracis Intranasal Immunization Generates Memory T cells

The generation of long-lasting antigen-specific T cells is paramount to vaccine design. To examine if intranasal immunization with Bacillus anthracis spores induces persisting memory T cells, the T cell response was examined four months p.i. Splenocytes from immunized mice produced IFNγ when stimulated with protective antigen (Figure 10). These amounts were slightly lower than the levels produced by splenocytes of day seven p.i. mice (Figure 10). Upon secondary immunization with another intranasal administration of Bacillus anthracis spores, IFNγ levels produced upon stimulation with protective antigen were significantly greater than those secreted from cells from either day seven post primary immunization or memory mice (Figure 10). Intranasal immunization with live Bacillus anthracis spores of the Sterne vaccine strain therefore induces long-lasting Bacillus anthracis- specific T cells that are able to recall upon re-immunization.

Intranasal Immunization Generates Bacillus anthracis-specific Antibodies Antibodies are the gold standard for evaluating Bacillus anthracis immunity.

Bacillus anthracis-specific antibody titers often correlate with protection (Friedlander, et al., 2002, Curr. Top. Microbiol., 271 : 33-60). Subcutaneous injection of Sterne strain spores is well known to induce anti-Bacillus anthracis antibodies. To examine if intranasal immunization with the Sterne strain also generates an antibody response, the serum of immunized mice was examined for antibodies against the lethal toxin components, protective antigen and lethal factor. Immunized mice, unlike naive mice, had both protective antigen- and lethal factor-specific IgG in their serum (Figure 11). Like other vaccination regimens, intranasal immunization with spores generates a Bacillus anthracis-specific antibody response.

Susceptible Mouse Models for Study of Immunosuppression

As disclosed elsewhere herein, there were few vegetative bacilli detectable following intranasal administration of attenuated Bacillus anthracis to C57BL/6 mice. Since virulence factors are expressed mostly by vegetative bacilli, one would not expect to observe much immunosuppression in resistant C57BL/6 mice. To overcome this limitation, susceptible mice were employed in which vegetative bacilli are not rapidly cleared but have a chance to replicate and express virulence factors.

GrI -depleted mice are highly susceptible to INSI with Sterne. The rapid infiltration of PMN suggests that PMN play a role in the early control of bacterial growth in the lung following intranasal administration of attenuated Bacillus anthracis. Therefore, PMN were depleted in vivo using the mAb against GR-I (clone RB6-8C5), which specifically depletes neutrophils (Hestdal, et al., 1991, J. Immunol., 147: 22-28; Czuprynski, et al., 1994, Infect. Immun., 62: 5161-5163). Mice depleted of PMNs were highly susceptible, and succumbed to infection within 24 hours. PMN-depleted mice had high levels of vegetative bacteria while vegetative bacilli were not detectable in the control group (Figure 12), although there were similar numbers of spores in the lungs between the two groups. Gram staining of lung tissue also revealed extensive vegetative bacteria in the PMN- depleted lungs but not in the control group (Figure 12). In addition to uncontrolled bacterial growth in the lung, PMN-depleted mice also had significantly more vegetative bacteria in their spleens compared to control mice (Figure 12). Thus, depletion of PMN allows vegetative bacteria to grow in the lung and further disseminate, leading to host mortality in C57BL/6 mice which are otherwise resistant and rapidly clear vegetative bacteria of the noncapsulated Sterne strain.

A/JCr mice are more susceptible to the Sterne strain following i.p. or i.v. injection (Welkos, et al., 1986, Infect. Immun., 51 : 795-800). It was not known if A/JCr mice are also susceptible to Sterne strain following intranasal infection. The susceptibility of A/JCr mice was measured by intranasal vaccination by administering 2x10⁶ Sterne spores. Unlike C57BL/6 mice that exhibit no signs of morbidity and no detectable vegetative bacilli (Figure 12), A/JCr mice became moribund and had high loads of vegetative bacilli (Figure 12). Antibiotic treatment at the time of immunization prevented death and these mice had reduced levels of vegetative bacteria in the lung and spleen (Figure 12). These results demonstrate that, like PMN-depleted B6 mice, lethality in A/JCr mice is due to uncontrolled bacterial growth in the lung, leading to systemic dissemination.

A/JCr mice are known to have a defect in complement function although the precise genetic defect responsible for susceptibility of A/JCr mice to B. anthacis remains unknown. While complement by itself is not very effective in killing gram-positive bacteria, complement activation can enhance PMN response. Since PMN play an important role in the early control of vegetative growth in resistant B6 mice, susceptibility in A/JCr mice might be due, at least in part, to an inadequate PMN response. This was examined by intranasally treating A/JCr mice with the potent PMN attractant MIP-2 (Wolpe, et al., 1989, Proc. Nat'l Acad. Sci. USA, 86: 612-616), resulting in rapid recruitment of GR-1+ cells into the lungs (Figure 12). When infected with 2x10⁶ spores (-10 X LD₅₀) one day post MIP2 -treatment, a greater percentage of MIP-2 treated mice survived infection compared to control mice (50% versus 20%). For mice that did succumb to infection, MIP-2 treated mice survived longer (mean survival time of 6.2 versus 4.7 days; Fig 12). Further, MIP2-treatment one day after spore-exposure also improved the outcome of infection, with only 20% of MIP-2- treated mice succumbing to infection compared to 50% of untreated control mice. Thus, enhanced recruitment of PMN to the lung increased resistance of A/JCr mice, indicating an inadequate PMN response in susceptible A/JCr mice and providing additional evidence supporting the role of PMN in the early control of non-capsulated bacilli in the lung following intranasal administration of attenuated Bacillus anthracis.

Intranasal Vaccination Provides Protection in a Sterne Strain Susceptible Mouse Strain

The present data demonstrates that intranasal immunization with live spores of the Sterne vaccine strain generates an adaptive immune response, resulting in anti-Bacillus anthracis serum antibodies and a Bacillus anthracis-specific T cell response. To determine if intranasal spore immunization can provide protection against pulmonary anthrax infection, a mouse strain that is known to be susceptible to infection with the Sterne vaccine strain was used (Welkos, et al, 1986, Infect. Immun., 51 : 795-800; Pickering, et al., 2004, Infect. Immun., 72: 6382-6389). Unlike C57BL/6 mice, intranasal administration of A/JCr mice with 2x10⁶ spores results in mortality. Immunization with a lower dose of spores (1x10⁵ CFU) generated Bacillus anthracis-specific antibody and T cell responses, similar to that observed for C57BL/6 mice. When vaccinated (2 months p.i.) and naive control mice were challenged with a lethal dose of spores, vaccinated A/JCr mice survived infection whereas naive mice succumbed to disease two days post-infection (Figure 13). Thus, intranasal immunization with spores of a Bacillus anthracis strain provides protection against lethal pulmonary Bacillus anthracis infection.

The data disclosed herein demonstrate that following intranasal administration of attenuated Bacillus anthracis, there is little bacterial replication but a strong innate response in the lung characterized by rapid recruitment of APCs and macrophages, followed by activation of dendritic cells and a robust antigen-specific antibody and T cell response, including a T cell memory response.

EXAMPLE 3: Efficacy of attenuated Bacillus anthracis vaccine comprising in- frame deletions of PI-PLC, Lethal Toxin, and/or Edema Toxin

As demonstrated by the data disclosed herein, TLRs play a critical role in host recognition of microbes, leading to activation of various innate immune effectors, including macrophages and dendritic cells. The present data demonstrates that PI-PLC inhibits dendritic cell activation by TLR ligands and that Bacillus anthracis PI-PLC functions as a virulence factor to downmodulate the immune response. In addition, LT is known to inhibit dendritic cell and T cell activation by interfering with MAPK signaling pathways. The present Example discloses the role of BaPI-PLC and LT in down-modulation of the innate response in vivo in the context of bacterial infection, and the role of PI-PLC and LT in an attenuated Bacillus anthracis vaccine.

PI-PLC Assays

Hydrolysis of PI is measured by incubation of culture supernatants or protein preparations with [3^H-inositol] PI and detergent in buffer as described elsewhere herein. Cleavage of the GPI-anchored proteins is determined by FACS analysis of surface expression of CD90, a GPI-anchored protein expressed on T cells and dendritic cell, following incubation with serial dilutions of purified enzymes or supernatants from overnight bacterial cultures for 1 hour at 37°C. hi addition, cleavage of the bulk of GPI-anchored proteins on dendritic cells is measured by staining with proaerolysin (Sotgia, et al., 2002, MoI. Cell. Biol., 22: 3905-3926). In this assay, cells are fixed for 30 minutes in PBS containing 2% paraformaldehyde and rinsed with PBS. The cells are then labeled with 10^"8 M proaerolysin. Cells are then washed repeatedly in PBS and incubated with an anti-aerolysin MAb from Protox Biotech (Victoria, BC Canada). The bound primary Ab is visualized with FITC- labeled goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA).

B. cereus PI-PLC and Bacillus anthracis PI-PLC Site-directed Mutagenesis Mutant constructs are generated by PCR-mediated sequence overlap extension using Pfx high fidelity DNA polymerase (Invitrogen, La Jolla, CA), resulting in in-frame gene replacements. Single amino acid changes are incorporated into the oligonucleotide primers for PCR. DNA sequences of mutant constructs are confirmed by automated cycle sequencing.

Overexpression and Purification of Mutant Proteins

Overexpression of the recombinant protein is induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) at mid-log phase as described in Feng et al. (2002, J. Biol. Chem., 277: 19867-19875). Protein is purified by chromatography on

Q-sepharose and phenyl-sepharose (Feng et al., 2002, J. Biol. Chem., 277: 19867-19875). Activity is monitored by assay for PI hydrolysis. Inactive protein purification is monitored by SDS-PAGE of column fractions and by Western blotting using an antibody against Bacillus PI-PLC. To exclude the possibility of residual LPS in protein preparations, the concentrated stock solution of purified PI-PLC is passed over three LPS-removing columns (DETOXI-GEL™ system), Pierce Biotechnology, Rockford, IL). The lack of LPS contamination is confirmed with the Limulus amebocyte lysate assay (Sigma).

Protein Stability Protein thermal stability is measured using circular dichroism by Tm measurements in standard buffers (Feng et al., 2002, J. Biol. Chem., 277: 19867-19875), in guanidinium-HCl (Elwell and Schellman, 1975, Biochem. Biophys. Acta., 386: 309-323) or urea solutions (O'Neil, 2005, Science 250: 646-651). A Jasco J-810 instrument (Jasco, Easton, MD) equipped with an automatic titrator and an 8-cell temperature-controlled sample changer is used for dichroism measurements.

Dendritic Cell Activation/Inhibition Assay

Dendritic cells are cultured from the bone marrow of C57BL/6 mice as described elsewhere herein and in Lutz, et al. (1999, J. Immunol. Methods 223: 77-92). Dendritic cells are left untreated or treated for 1 hour with varying concentration of PI-PLC to determine the amount and time required to inhibit dendritic cell activation. Dendritic cell activation is performed with poly I:C, LPS, peptidogylcan or CpG DNA (5' TCCATGACGTTCCTGATGCT 3'; SEQ ID NO:7) and activation is measured by surface staining of activation markers, intracellular staining and ELISA for cytokines, and western blot for p38 and ERK phosphorylation, as disclosed herein and in Wolpe, et al. (1989, Proc. Nat'l. Acad. Sci. USA, 86: 612-616). Briefly, for surface staining of dendritic cells, cells are stained with mAb that specifically binds to CDl Ic, CD86, CD80, and MHC II in 1% BSA/PBS with Fc receptor block. Dendritic cell viability is evaluated by Annexin-V and 7- AAD staining (BD Pharmingen, Franklin Lakes, NJ). For intracellular staining to analyze TNFα production, dendritic cells are incubated for 5 hours at 37⁰C with 5% CO₂ in the presence of GolgiStop (BD Pharmingen), surface stained with anti-CD 1 Ic, and then permeabilized with Cytofix/Cytoperm solution, stained with anti-TNF mAb (clone MP6- XT22). TNFα in dendritic cell supematants is measured by ELISA (R&D systems; Minneapolis, MN). For analysis of p38 and ERK phosphorylation, dendritic cells are pretreated with PI-PLC for 1 hour and then stimulated with poly I:C for 60 minutes. One mM Na₃VO₄ is added to inhibit phosphatases. Cells are lysed in sample buffer with 100 mM DTT and lysates are separated on a 4-15% gradient gel. A western blot is performed using one of the following Ab: rabbit polyclonal anti-ERK (p44/42 MAP kinase), anti-p38 MAP kinase or an Ab that recognizes the phosphorylated forms of these proteins (Cell Signaling Technology; Beverly, MA).

Immunization

Spores are prepared as described herein and in Guidi-Rontani, et al. (1999, MoI. Microbiol., 31 : 9-17). Briefly, an overnight bacterial culture is diluted 1 :100 into phage assay medium and incubated at 30°C with aeration for 3 days. Spores are harvested by centrifugation, washed with PBS, and then heat-treated at 65⁰C for 30 minutes to kill remaining vegetative bacilli before plating to determine CFU/ml. Forty microliters of spore suspension are applied to the nasal flares of a mammal with or without light anesthesia.

LDsn and Bacterial Virulence LD₅₀ is determined by infecting groups (5 per group) of mammals with 10- fold serial dilutions of bacteria and monitoring survival daily. For bacterial titres, spleen and lung homogenates are serially diluted. Half of each dilution is plated directly and the other half is heat-treated for 30 minutes at 65°C to kill vegetative bacilli and titer the number of spores. The number of vegetative bacilli are calculated by subtracting CFU after heat- treatment from CFU without heat-treatment.

Histology

Lungs are fixed, embedded in paraffin and sectioned. Sections are stained with mAbs against CD3, CDl 1C, GR-I and CDl Ib and then counterstained with hematoxylin. Gram-stain is performed to visualize vegetative bacilli in the lung.

Surface Stain

Lymphocyte suspensions are stained with mAbs against CDl Ib and Ly6C/G to assess neutrophil and macrophage recruitment, with mAbs against CDl Ic and CD86 and MHCII to measure dendritic cell activation, and with mAb to CD8, CD4, CD44, and CD62L to visualize T cell activation.

Bronchoalveolar lavage (BAL^")

Fifteen milliliters of HBSS/5mM EDTA is instilled in 1 ml aliquots into the lungs of euthanised/anesthetized mammals to collect lavage fluid. Lavaged cells are pooled, fractionated by centrifugation over a Ficoll-Hypaque gradient, counted and resuspended in RPMI medium for FACS or for in vitro culture to measure cytokine production. For culture, cells are seeded at 1x10⁶ cells/ml into 48 well plates and stimulated with HKBa or PA protein. Supernatants are harvested at specific timepoints for ELISA.

Cytokine Quantitation by ELISA and Intracellular Staining

Dilutions of cell-free supernatants or BAL fluids (50 μl) are added to plates coated with Ab against various cytokines, followed by standard ELISA assays. The concentrations of cytokines are derived from the linear portion of the standard curve for each respective cytokine. For intracellular cytokine staining, dendritic cells isolated from the lung and BAL of infected mammals are incubated with heat-killed baccilli at an MOI of 10 for 5 days. After incubation, cells are stained first for surface expression of CDl Ic, CDl Ib and GR-I, permeabilized and then stained for IFNγ, TNFα, and/or IL-2 using the Cytofix/Cytoperm Kit (BD Pharmingen).

Antigen Specific T cell Responses 5 x 10⁶ lymphocytes from spleen or lung are added to a 48 well plate and then stimulated with PA protein or heat-killed bacteria at an MOI of 10. Supernatants are assessed for IFNγ, IL-4 and IL-IO. ELISPOT and intracellular cytokine staining are performed to quantitate antigen specific T cells, using overlapping PA peptides (Bei Resources, Manassas, VA) and dendritic cells pulsed with HKBa.

Measurement of Ab Titers

To measure Ab in the serum, plates are coated with HKBa, PA or LF protein (List Biological Laboratories, CA) in PBS. Serially diluted serum samples are added. After incubation and washing, biotinylated anti -mouse IgG Ab against various IgG isotypes (IgGl, IgG2a, IgG2b and IgG3) are added. Titer is defined as the greatest dilution of serum with an OD₄₅₀ twice that of control. Neutralizing toxin Ab is measured by the ability of serum from immune mice to protect LT sensitive J774 macrophages from purified LT. 5 x 10⁴ J774 MΦacrophages are added to 96 well plates and incubated at 37° for 12 hours. LT is added to cultures in the presence or absence of serial two fold dilutions of immune or control serum and incubated for 12 hours. MTT is added to wells and viable macrophages are assessed by colorimetric assay.

Construction of Attenuated Bacillus anthracis Vaccine Strains

Attentuated Bacillus anthracis strains are constructed with an in-frame deletion of PI-PLC, LT, and ET singly and in combination. These vaccines are administered to a mammal to examine the innate and the specific immune response to an attenuated Bacillus anthracis vaccine.

Mutations in the Bacillus anthracis genome are introduced using techniques adapted from similar Gram-positive bacteria, such as L. monocytogenes (Shen et al., 1998, Cell, 92: 535-545; Foulds, et al., 2002, J. Immunol., 168, 1528-1532; San Mateo, et al., 2002, J. Immunol., 169: 5202-5208; Shen, et al., 1995, Proc. Nat'l Acad. Sci. USA 92: 3987-3991). Defined, unmarked mutations are introduced into the Bacillus anthracis gemome, in contrast with other Bacillus anthracis mutants, which have been made using site-directed insertional mutagenesis with antibiotics-resistance markers. Insertional mutations often have a polar effect on downstream gene expression, and the presence of antibiotic-resistance is unacceptable in attenuated live vaccines because of the risk associated with introducing antibiotic-resistance into the environment and clinical setting. To overcome these limitations, attenuated Bacillus anthracis mutants are constructed using a two-step allelic exchange system that allows the introduction of site-directed, unmarked deletions/mutations in the Bacillus anthracis genome. An allelic exchange system was used to construct a derivative of Sterne

(named "SdL") with an in-frame deletion of LF (deleted of codons 237-502 (amino acids 331-418), Figure 14). The genotype of the deletion in the SdL strain was been verified by PCR (Fig. 14) and is further confirmed by Southern blot. The phenotype of this strain was tested for the lack of LT cytotoxicity in the culture supernatant using the standard MTT cytotoxicity assay on J774 macrophages, described herein and in Hering, et al., (2004,

Biologicals 32: 17-27) and is further confirmed by western blot using commercially available anti-LF antibodies.

Using the same approach, a derivative of Sterne strain with an in- frame deletion of PI-PLC (named "SdP"; deletion of the entire ORF) was also constructed. The genotype of PI-PLC deletion was confirmed by PCR as was done with the SdL mutant and is further verified by Southern blot. The phenotype of the SdP strain is assessed by Western blot and by assaying PI- and GPI-cleavage activities as described elsewhere herein to confirm the absence of a functional PI-PLC protein.

A double attenuated Bacillus anthracis mutant is constructed by introducing a PI-PLC deletion into the SdL mutant. The resultant strain has a deletion in both the LF and PI-PLC gene (SdLP). The genotype and phenotype of both LF and PI-PLC deletions in the SdLP strain is confirmed by PCR, Southern Blot, western blot, and by assessing PI and GPI cleavage activity as described above for the SdL and SdP strains.

A double attenuated Bacillus anthracis mutant is also constructed by introducing an in-frame deletion of EF into the SdL mutant (LF). The resultant strain has a deletion in both the LF and EF gene (SdLE). The genotype of both LF and EF deletions in the SdLE strain been confirmed by PCR.

Virulence As disclosed elsewhere herein, PI-PLC and LT are virulence factors involved in immune down-modulation, and therefore, inactivation of these factors results in attenuation of virulence. An LT mutant of the Sterne strain has reduced virulence in a subcutaneous infection model (LD₅₀ raised from 10⁶ for wild type to >10⁹ for an LF deletion strain; Pezard, et al., 1995, Infect. Immun. 63: 1369-1372; Pezard, et al., 1991, Infect. Immun., 59: 3472-3477).

The data disclosed herein demonstrate that SdL is highly attenuated when administered as an intranasal spore inoculation (Figure 15). While the LD₅₀ of the Sterne strain is ~lxlθ⁶ cfu in naive A/JCr mice, the LD₅₀ of the SdL strain is > 10⁷ cfu. SdL is also highly attenuated in neutrophil (GR- 1+) depleted B6 mice that are extremely susceptible to intranasal infection with the Sterne strain as described elsewhere herein. In addition, the

LD₅₀ following intravenous injection of vegetative SdL into naive B6 mice is > 10⁶ bacteria, while the LD50 for intravenous injection with the Sterne strain is ~10³ cfu.

The virulence of SdL is further characterized by comparing the kinetics of bacterial growth and dissemination into various organs following intranasal spore inoculation. A dose of 1 xl O⁵ spores (0.1 LD₅₀ of Sterne) for both Sterne and SdL strain are used so that a comparison can be made when similar infectious doses are administered. A dose of 1x10⁷ of attenuated Bacillus anthracis vaccine strain SdL strain is used to compare the kinetics of bacterial growth and dissemination to compare when an 0.1 LD₅₀ of each strain is used for infection. Similarly, bacterial growth and dissemination is measured following intravenous infection with vegetative bacteria. This allows a determination of the ability of an attenuated Bacillus anthracis vaccine strain to replicate in vivo once the infection becomes systemic.

As described above with the SdL strain, the virulence of an attenuated Bacillus anthracis vaccine strain, such as SdP, SdL and SdLP, is measured to gain a full assessment of attenuation. The LD₅₀ of the SdP and SdLP strain in A/JCr mice, the kinetics of bacterial growth and dissemination into various organs following intranasal spore inoculation with a sublethal dose (0.1 LD50), the LD₅₀ and bacterial growth and dissemination following intravenous infection and the virulence (LD₅₀, bacterial growth and dissemination) of the attenuated Bacillus anthracis vaccine strain C57BL/6 mice depleted of GR- 1+ cells are all measured. All experiments are carried out as described herein for the Sterne strain and the SdL strain, and the parental Sterne strain is included as a control for side-by-side comparison.

Innate Immunogenicity

To examine the LT-mediated down-modulation of the innate immune response, dendritic cell activation is measured following immunization with the SdL mutant. Dendritic cell activation was not observed in A/JCr mice following intranasal administration with 10⁵ spores (0.1 LD₅₀) of the Sterne strain. The lack of dendritic cell activation was not due to insufficient stimulation since there was bacterial growth, and at the next dose level A/JCr mice succumbed to infection. While intranasal administration with 10⁵ spores of SdL also did not induce dendritic cell activation, due to the attenuation of the SdL strain, immunization with 10⁶ spores (>0.1 LD₅₀ of SdL) was possible without mice succumbing to infection. At a dose greater than 0.1 LD₅₀, there was clear dendritic cell activation as evident by upregulation of CD86 (Figure 16).

The immune reaction in response to administration of an attenuated Bacillus anthracis vaccine strain in vivo is measured by analyzing the recruitment of polymorphonuclear leukocytes (PMNs) and macrophages, activation of dendritic cells, and measuring the inflammatory cytokine response using the methods disclosed herein. A/JCr mice are immunized by intranasal inoculation with spores of the Sterne, SdL, SdP, SdLP, and SdLE strains. Infiltration of PMNs and macrophages into the lung are examined by histology and by FACS analysis at different time-points (12, 24, 48, and 72 hours post infection as described above). Initially, the response is measured by removing the entire lung and isolating total lung lymphocytes for analysis. Further measurements are taken from cells isolated from BAL, parenchymal lung tissue, and draining mediastinal lymph nodes. Isolated cells are stained with mAbs to CDl Ic, GR-I and CDl Ib and examined by FACS analysis to the recruitment of cells of the innate immune system after immunization with SdL, SdP or SdLP strains. The activation of dendritic cells at these time-points is analyzed by FACS analysis of MHC II and CD86 expression on CDl Ic⁺ cells. The lung contains a complex population of myeloid and plasmacytoid dendritic cells, not all of which express CDl Ic (Gonzalez-Juarrero, 2003, J. Immunol., 171 : 3128-3135). Therefore, the subsets of dendritic cell populations in the lung are identified by staining with CDl Ib, GR-I and B220 to examine the dendritic cell subsets in the lung in response to immunization with attenuated Bacillus anthracis vaccine strains.

In addition, cytokine expression by lung dendritic cells is assessed by intracellular cytokine staining of dendritic cells isolated from BAL fluid and from parenchymal lung tissue. Control or dendritic cells stimulated with heat-killed bacteria are assessed for production of the ThI cytokines IL-12 and IFNγ or the Th2 cytokines 11-4, IL-6 and IL-10, to evaluate the cytokine profile in a mammal in response to immunization with an attenuated Bacillus anthracis vaccine strain.

Additionally, supernatants are collected from cultured dendritic cells and cytokine production is assessed with cytokine arrays and ELISA as described above. The inflammatory cytokine response in BAL fluid is examined at different time-points post infection by ELISA analysis for IFNα, TNFα, IL-12 and IFNγ. Together, these studies provide a kinetic and quantitative comparison of the innate immune response in mammals immunized with attenuated Bacillus anthracis vaccine strains.

Adaptive Immunogenicity

Dendritic cells are key antigen-presenting cells for initiating the adaptive immune response. As demonstrated by the data disclosed herein, inhibition of dendritic cell activation by PI-PLC can thus lead to down-modulation of not only innate but also adaptive antigen-specific responses. In order measure the impact of inhibited dendritic cell activation on T cell activation and adaptive immunity, dendritic cells are treated with PI-PLC and activated with LPS as disclosed elsewhere herein. After extensive washing to remove PI- PLC, treated dendritic cells are then pulsed with the antigenic peptides, OVA_2S7-264 and OVA_323-339, and used to stimulate CFSE-labeled CD8 and CD4 T cells from OT-I and OT-II mice (Murphy, et al., 1990, Science 250: 1720-1723, Barnden, et al., 1998, Immunol. Cell Biol. 76: 34-40) which express transgenic TCR specific to the MHC Class I and MHC Class II-restricted epitopes, OVA_257-264 and OVA_323-33Q, respectively.

The ability of dendritic cells to stimulate T cells is quantitatively measured by FACS analysis of T cell CFSE fluorescence intensity, which decreases incrementally as T cells divide (Figure 17). T cell stimulation is further tested using an adoptive transfer model in which recipient mice are injected i.v. with CFSE-labeled OT-I and OT-II cells followed by i.p. immunization with peptide-pulsed dendritic cells.

The direct effect of PI-PLC on T cells is measured by purifying T cells from spleens of C57BL/6 and BALB/c mice using MACS (Miltenyi Biotec, Germany), and then labeling the T cells with CFSE. These cells (lxlO⁶/well in 96-well plates) ared stimulated with plate-bound anti-CD3 and anti-CD28 mAb in the presence of increasing concentrations of PI-PLC. Stimulation with anti-CD3/ anti-CD28 mAb are used instead of dendritic cells to allow assessment of direct effects on T cells without the complication of PI-PLCs effect on dendritic cells. Control groups include no PI-PLC and no anti-CD3/anti-CD28 mAb. Proliferation of T cells is assayed on days 2 and 3 by FACS analysis of CFSE fluorescence. T cell activation is examined by staining for T cell activation phenotypes (CD44^hlgh, CD62L^low, CD69^high, and CD25^high) and by measuring production of cytokines (such as IFNγ, IL-4, and TNF α) by intracellular cytokine staining and ELISA of culture supernatants, as disclosed elsewhere herein. Modulation of the adaptive immune responses in vivo is measured following immunization with attenuated Bacillus anthracis vaccine strains, including the SdL, SdP and SdLP strains. Vaccination with the SdL strain induces a stronger T cell and antibody response than the parental Sterne strain (Figure 18). While activation of total T cells was similar between SdL and Sterne when immunized with 10⁵ spores (this dose represents -0.1 LD₅₀ for Sterne and -0.01 LDs₀ for SdL), there were higher levels of antigen-specific T cells and antibodies in SdL immunized mice. Attenuation of SdL strain allowed immunization with a higher dose,^;and there was increased activation of T cells when A/JCr mice were immunized with 10⁶ spores (-0.1 LD₅₀ of SdL), while mice immunized with 10⁶ Sterne all succumbed to infection. Further, mice immunized with 10⁶ spores of SdL had greatly increased antigen-specific T cell and antibody responses. These results demonstrate that inactivation of LT results in increased immunogenicity while further attenuating the Sterne strain and provide in vivo evidence for the role of attenuated Bacillus anthracis vaccine strains in immunizing against anthrax.

As described with regard to the SdL strain above, A/JCr mice are immunized intranasally with the Sterne, SdP, SdL and SdLP strain at a dose of 10⁵ spores for all strains and a dose of 0.1 LD₅₀ of each respective strain. The total T cell activation and antigen- specific T cell responses on day 7 and T cell memory and antibodies on day 30 post- immunization are measured. Further, spores and vegetative bacilli in the lung and spleen are measured according to the methods disclosed elsewhere herein, permitting correlations between the immune response and bacterial loads in vivo in addition to immunization doses. Uninfected mice are included as controls in these assays.

Tissue sections are prepared to examine lung pathology, infiltration of T cells into the lung tissues (using immunohistology (anti-CD3 mAb)), and the presence of bacteria by gram-staining. Activation of T cells in the lung and spleen is examined by FACS analysis using mAb to CD4 and CD8, and a panel of activation markers (CD44, CDl Ia, CD62L, and CD69). Antigen-specific T cell responses are measured by stimulating lymphocytes isolated from lung, spleen and lymph nodes with HkBa and PA protein. After 24, 48 and 72 hour stimulation, T cell proliferation is measured by ³H-thymidine incorporation (pulsed during the final 24 hour) and supernatants are harvested to measure production of cytokines (IFNγ, IL-4). The contribution of CD4 and CD8 T cells to the IFNγ and IL-4 responses is measured by depleting CD4 and CD8 T cells, as described elsewhere herein.

The antibody response is measured by ELISA using HKBa, PA and LF protein as antigens and by measuring the response by various IgG isotypes (IgGl, IgG2a, IgG2b and IgG3). The titer of toxin-neutralizing Ab is measured by assessing the ability of serum from immune mice to protect LT sensitive J774 macrophages from purified LT using the standard MTT cytotoxicity assay described above.

As described above, A/J mice were immunized with 10⁵ spores of the Sterne, PI-PLC mutant (SdP) as well as the mutant deleted of LF (SdL) and the double mutant (SdLE) deleted of both LF and EF (edema factor). Interestingly, there were similar levels of PMN and macrophages in the lung of Sterne and SdL (LF mutant) immunized mice but substantially more in the SdLE (LF/EF double mutant) and SdP immunized mice. These results indicate that inactivation of PI-PLC leads to an increased early innate response and provides in vivo evidence that PI-PLC down-modulates the immune response (Figure 19).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. An attenuated Bacillus anthracis vaccine selected from the group consisting of (a) a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding lethal factor and a deletion in the nucleic acid encoding PI-PLC; (b) a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding lethal factor and a deletion in the nucleic acid encoding edema factor; (c) a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding lethal factor; and (d) a Bacillus anthracis Sterne strain comprising a deletion in the nucleic acid encoding PI-PLC.

2. The vaccine of claim 1, wherein said deletion in the nucleic acid encoding lethal factor (LF) comprises a deletion of the nucleic acid encoding amino acids 237-502 of the LF protein.

3. The vaccine of claim 1, wherein said deletion in the nucleic acid encoding lethal factor is an in-frame deletion.

4. The vaccine of claim 1, wherein said deletion in the nucleic acid encoding PI-PLC comprises a deletion of the nucleic acid corresponding to the entire open reading frame of the PI-PLC protein.

5. The vaccine of claim 1, wherein said deletion in the nucleic acid encoding edema factor (EF) comprises a deletion of the nucleic acid encoding amino acids 359-601 of the EF protein.

6. The vaccine of claim 1, wherein said vaccine further comprise a pharmaceutically acceptable carrier.

7. The vaccine of claim 1, wherein said vaccine is a Bacillus anthracis spore.

8. The vaccine of claim 1, wherein said vaccine is a vegetative Bacillus anthracis.

9. A method of immunizing a mammal against a Bacillus anthracis infection, said method comprising administering said mammal an effective amount of the vaccine of claim 1.

10. The method of claim 9, wherein said vaccine is a Bacillus anthracis spore.

11. The method of claim 9, wherein said vaccine is administered intranasally.

12. The method of claim 9, wherein said vaccine further comprises a pharmaceutically acceptable carrier.

13. The method of claim 9, wherein said mammal is a human.

14. A method of immunizing a mammal against a Bacillus anthracis infection, said method comprising administering to said mammal an attenuated Bacillus anthracis Sterne vaccine strain, wherein said administration is intranasal administration.

15. The method of claim 14, wherein said attenuated Bacillus anthracis Sterne vaccine strain is a spore.

16. The method of claim 14, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding lethal factor.

17. The method of claim 14, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC.

18. The method of claim 14, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC and a deletion in a nucleic acid encoding lethal factor.

19. The method of claim 14, wherein said attenuated Bacillus anthracis Steme vaccine strain comprises a deletion in a nucleic acid encoding edema factor and a deletion in a nucleic acid encoding lethal factor.

20. The method of claim 14, wherein said mammal is a human.

21. A method eliciting an immune response against a Bacillus anthracis infection, said method comprising administering to said mammal an attenuated Bacillus anthracis Sterne vaccine strain, wherein said administration is intranasal administration.

22. The method of claim 21, wherein said attenuated Bacillus anthracis Sterne vaccine strain is a spore.

23. The method of claim 21, wherein said attenuated Bacillus anthracis Steme vaccine comprises a deletion in a nucleic acid encoding lethal factor.

24. The method of claim 21, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC.

25. The method of claim 21, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC and a deletion in a nucleic acid encoding lethal factor.

26. The method of claim 21, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding edema factor and a deletion in a nucleic acid encoding lethal factor.

27. The method of claim 21, wherein said mammal is a human.

28. A method of inducing a protective immune response in a mammal, wherein said protective immune response is against a Bacillus anthracis infection, said method comprising administering to said mammal an attenuated Bacillus anthracis Sterne vaccine strain, wherein said administration is intranasal administration.

29. The method of claim 28, wherein said attenuated Bacillus anthracis Sterne vaccine strain is a spore.

30. The method of claim 28, wherein said protective immune response comprises the production of an antibody that specifically binds Bacillus anthracis, or a portion thereof.

31. The method of claim 28, wherein said protective immune response comprises the proliferation of a CD4⁺ T cell that specifically recognizes Bacillus anthracis, or a portion thereof.

32. The method of claim 28, wherein said protective immune response comprises the activation of a Bacillus anthracis-specific antigen presenting cell.

33. The method of claim 32, wherein said antigen presenting cell is a dendritic cell.

34. The method of claim 33, wherein said dendritic cell expresses CD86.

35. The method of claim 28, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding lethal factor.

36. The method of claim 28, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC.

37. The method of claim 28, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding PI-PLC and a deletion in a nucleic acid encoding lethal factor.

38. The method of claim 28, wherein said attenuated Bacillus anthracis Sterne vaccine strain comprises a deletion in a nucleic acid encoding edema factor and a deletion in a nucleic acid encoding lethal factor.

39. The method of claim 28, wherein said mammal is a human.

40. A kit for immunizing a mammal against a Bacillus anthracis infection, said kit comprising an immunogenic amount of an attenuated Bacillus anthracis vaccine strain, said kit further comprising an applicator and an instructional material for the use of said kit.

41. The kit of claim 40, wherein said attenuated Bacillus anthracis vaccine strain is a spore.

42. The kit of claim 40, wherein said applicator is an intranasal applicator.