WO2007047501A2 - Outer membrane vesicles: novel vaccine for gram-negative biothreat agents - Google Patents

Abstract

An immunogenic composition for the immunization of an individual comprising outer membrane vesicles of Gram-negative bacteria, Francisella or Yersinia spp.

Description

OUTER MEMBRANE VESICLES: NOVEL VACCINE FOR GRAM-NEGATIVE

BIOTHREAT AGENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of earlier-filed U.S. Provisional Application Ser. No. 60/726,238, filed October 14, 2005, which application is incorporated herein by reference in its entirety.

The historical vaccine for tularemia is scarification with Francisella tularensis Live Vaccine Strain (LVS). Heat-killed bacteria are not protective vaccines, although they do generate strong antibody response. (Ellis et al, 2002) Other approaches are now being attempted, including the generation of attenuated strains through rational and directed genetic manipulation of metabolic pathways, DNA vaccines and high throughput cloning and expression systems (reviewed in Isherwood et al, 2005). Currently, there is a great deal of research being performed on tularemia vaccines. Much of this research has been focused on using protein or lipopolysaccharide antigens as subunit vaccines. In general, the use of component vaccines has not resulted in strong protection against challenge with tularemia despite strong induction of antibodies. These and many other findings have led to the concept that an effective tularemia vaccine must induce both a B-cell and a T- cell response. (Isherwood et al, 2005).

Thus, the consensus is that cellular responses, especially CD4+ or CD8+ T- cell response is a fundamental requirement of an effective and protective tularemia vaccine. Vaccination approaches involving the presentation of Francisella protein antigens via recombinant Salmonella (Sjostedt et al, 1992) or the use of immunostimulating complexes (ISCOMs) (Golovliov et al, 1995) have provided some protection against challenge, but not as strong as LVS vaccination alone.

SUMMARY OF THE INVENTION

This invention relates to outer membrane vesicles derived from Francisella and Yersinia spp of bacteria, methods of making such, and pharmaceutical compositions thereof. This invention also relates to broad-spectrum vaccines for the prevention of diseases caused by Francisella and Yersinia spp, especially F. tularensis and Y. pestis. Pathogenic gram-negative microbes produce a wide variety of virulence factors, such as lipopolysacchari.de, toxins, host cell signaling regulators, and other molecules. For some important pathogens knowledge of virulence mechanisms is fairly extensive; for others, such as Francisella tularensis, the causative agent of tularemia, the virulence factors remain largely unidentified. This knowledge gap limits the ability to develop new prophylaxes, therapeutics and vaccines, which have been identified as a current critical need by the US military, NIAID and other government agencies.

Outer membrane vesicles (OMVs) are spherical bi-layered vesicles that are typically 50 to 250 nm in diameter. OVMs are characteristically produced by Gram-negative bacteria (Beveridge, 1999). Examples of bacterial genera that produce OMVs are Escherichia, Serratia, Neisseria, Haemophilus, Salmonella, Shigella, Legionella, Burkholderia, Helicobacter, Proteus, and Pseudomonas. Outer membrane vesicles are constantly being discharged from the bacterial surface during cell growth. A representative model of OMV biogenesis has been proposed by Keuhn and Ketsey (2005) and is shown in FIG. 1.

The composition of outer-membrane vesicles is varied but generally constitutes lipopolysaccharide (LPS), periplasmic proteins, outer membrane proteins (OMPs), phospholipids, DNA, DNA-binding proteins and virulence factors including alkaline phosphatase, phospholipase C (PLC), exotoxin A, DNase, hemolysin, proelastase, protease, and peptidoglycan hydrolase (Beveridge, 1999). Many of these constituents of OMVs can act as strong antigens (Kadurugamuwa & Beveridge, 1996). OMVs have been proposed to play a role in several virulence mechanisms including periplasmic enzyme delivery, DNA transport, bacterial adherence, and evasion of the immune system (Horstman & Kuehn, 2000). One report has indicated that OMVs contain DNA and RNA and may play a role in exchanging genetic material between bacteria (Kolling and Matthews, 1999). Some studies have demonstrated that vesicles released by Neisseria gonorrhoeae and Haemophilus influenza can export DNA from the producing strain and transfer DNA to recipient cells, and Escherichia coli O 157:H7 was shown to release OMVs which contain nucleic acids and Shiga toxins (Kolling and Matthews, 1999).

The pathogenicity of Gram-negative bacteria depends on their ability to secrete many virulence factors into the environment around the targeted tissue (e.g., hemolysin, aerolysin, verotoxin, etc.), in order to promote bacterial adhesion and invasion (Beveridge, 1999). Once free of the pathogen, these factors are diluted by diffusion as well as inactivated by external host constituents (e.g., complement, hydrolytic enzymes, antibodies, and other serum constituents).

It has been reported that OMVs may have direct effects on other bacteria (Li et al, 1998). OMVs have also been suggested to contribute to antibiotic resistance in Gram-negative bacteria by the transfer of β-lactamase activity from a β- lactamase positive cell to an otherwise β-lactamase negative neighbor cell (Li et al., 1988). OMV-mediated delivery of Peptidoglycan Hydrolase (PGase) has been proposed as a virulence mechanism in Pseudomonas, effective against gram- positive organisms directly, and against gram-negative organisms following fusion of OMV with the outer-membrane and release of PGase into the periplasm (Kadurugamuwa & Beveridge, 1996). The potential for Francisella to directly affect other bacteria via OMVs may be critical in situations such as multi-agent infection or for its survival in the environment, perhaps in in the formation of biofilms.

Bacterial OMVs are known to interact with both eukaryotic cells and other bacteria via surface-expressed factors and mediate delivery of virulence factors (Ketsy and Kuehn, 2004). For example, heat-labile enteroroxin (LT) associated with LPS on the surface of enterotoxigenic E. coli (ETEC) vesicles triggers internalization via caveolae and delivers catalytically active LT, which intoxicates the eukaryotic cell (Horstman &. Kuehn, 2000). Helicobacter pylon is a Gram-negative bacterium associated with stomach ulcers, and has been found to produce OMVs which induce a significant increase in the expression of IL-8 (neutrophil-activating chemokine), and thus H. pylon OMVs have been suggested to play a role in promoting host responses via low-grade gastritis which could support the bacterium residing in the stomach (Ismail et al, 2003). Bacteroides gingiυalis OMVs adhere to the epithelial cell of the gums and act as an intermediate for the attachment of bacteria to teeth and gums (Grenier & Mayrand, 1987). OMVs have even been demonstrated to have insecticidal activity (Khandelwal & Banerjee- Bhatnagar, 2003)

The bacterial genus Francisella was previously classified within the genera Pasteurella and Brucella, but was assigned to a new genus in 1947, due to biochemical properties (Oyston et al., 2004). The taxonomy of the genus Francisella is uncertain and in transition. Based on fatty acid composition and 16S rDNA sequence, there are two species within the genus Francisella, F. tularensis and F. philomiragia The subspecies of F. tularensis, F. tularensis subspecies tularensis (also called F. tularensis Group A or neoartica), F. tularensis subspecies holarctica (also called F. tularensis Group B), and F. tularensis subspecies novicida differ in their geographic distribution (Titball et al., 1993, 2003). Variant strains of easily grown organisms designated as FxI and Fx2 have also been isolated from humans. In this scheme, F. novicida is considered to be a subspecies of F. tularensis. However, there is an alternative and older system of nomenclature involving the assignment of strains into one of two biovars, type A and type B, which broadly correspond with F. tularensis subsp. tularensis and subsp. holoarctica. Most of the research into the biology of F. tularensis has been performed using the live vaccine strain (FtLVS), derived from a F. tularensis subsp. holoartica, and is attenuated in humans, while virulent for mice.

F. tularensis is a small, Gram-negative, non-motile, encapsulated, nonsporulating coccobacillus (0.2 x 0.7μm). It is a zoonotic, facultative intracellular pathogen that causes tularemia in humans (Ellis et al, 2002). F. tularensis belongs to the genus Francisella, the Francisellacae family and is part of the γ-subclass of Proteobacteria (Ellis et al, 2002). The natural reservoir of Francisella tularensis may be lagomorphs (rabbits, etc.) and it has recently been found inside a water dwelling amoeba, Acanthamoeba castellanii (Abd et al, 2003). Recently, F. tularensis has been observed to create cell-to-cell connections (pili) (Gil et al, 2004) and to secrete extracellular material that engulfs surrounding bacteria, forming a structure suggestive of a biofilm.

F. tularensis is an unusual pathogen in that it is very efficiently taken up into phagocytic cells, such as macrophages (Fortier et al, 1995, Sjostedt et al, 1996, Elkins et al, 2002), yet does not cause a large activation of the antimicrobial activity of the phagocytic cell. The bacteria enter the host-cell via a phagosome, escape into the cytosol and replicate rapidly until cell-death is induced (Golovliov et al, 2003). The mode of entry of the bacterium into mammalian cells is unknown.

Virulence factors of F. tularensis axe poorly described, despite many years of study (Titball et al, 2003). Virulence factors of bacteria are functionally defined as those proteins which render the bacteria pathogenic, or enable it to be established in the host. The currently recognized virulence factors for Francisella include the 23 kDa protein, also called iglC (Golovliov et al, 1997), and the 17 kDa protein (Sjostedt et al, 1991). The precise function of these proteins is still not known, despite many years of study. Two plasmids, pOMl (Pomerantsev et al, 2001a) and pNFLIO (Pomerantsev et al, 2001b), have been isolated from low virulence strains of Francisella, and so probably do not encode for virulence factors, and no toxins have been reported for Francisella species. Two additional virulence determinants or factors have been identified in F. tularensis - a capsule and lipopolysaccharide (LPS) (Ellis et al, 2002). Capsule produced by F. tularensis has been shown to be essential for serum resistance, but is not required for survival following phagocytosis by polymorphonuclear leukocytes (Sorokin et al, 1996). LPS of F. tularensis activates complement (Phillips et al., 2004) but is much less endotoxic than E. coli LPS. While the precise contribution to virulence of these two factors (LPS and capsule) is still the subject of intense research, it is clear that they are not the only contributors to the virulence or pathogenesis of this organism (Ellis et al, 2002). In this regard, Golovliov and co- workers demonstrate, via electron microscope (EM) images, the uptake and metabolism of Francisella tularensis Live Vaccine Strain (LVS) within a phagosome of monocytic cells (Golovliov et al, 2003).

Another prominent Gram negative bacteria belong to the genus Yersinia. Members of Yersinia, which belong to the family of enterobacteriaceae, are facultative anaerobes. Some members of this genus, such as Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis, are pathogenic in humans. Natural reservoirs of Yersinia bacteria are rodents and sometimes (less frequently) other mammals. Infection may occur either through blood (e.g. as in the case of Y. pestis) or in an alimentary fashion, through consumption of products (esp. vegetables, milk-derived products and meat) contaminated with infected urine or feces. Speculations exist as to whether or not certain Yersinia can also be spread via protozoonotic mechanisms, since Yersinia are known to be facultative intracellular parasites; studies and discussions of the possibility of amoeba-vectored (through the cyst form of the protozoan) Yersinia propagation and proliferation are now in progress.

Among the pathogenic species, Y. pestis is best-known as an etiologic agent of bubonic plague. The type species, Y. pestis, is a nonmotile, non- spore-forming coccobacillus (0.5 to 0.8 mm in diameter and 1 to 3 mm long) that exhibits bipolar staining with Giemsa, Wright's, or Wayson staining. The organism grows at temperatures from 4 up to 408⁰C (optimum at 28 to 308⁰C); the optimum pH for growth ranges between 7.2 to 7.6; however, extremes of pH 5 to 9.6 are tolerated. Y. pestis has typical cell wall and whole-cell lipid compositions and an enterobacterial antigen, in common with other enteric bacteria. Its lipopolysaccharide is characterized as rough, possessing core components but lacking extended O-group side chains; while there is no true capsule, a carbohydrate-protein envelope, termed capsular antigen or fraction 1 (Fl), forms during growth above 338⁰C. This facultative anaerobe possesses a constitutive glyoxylate bypass and unregulated L-serine deaminase expression but lacks detectable adenine deaminase, aspartase, glucose 6-phosphate dehydrogenase, ornithine decarboxylase, and urease activities, as well as a possible lesion in a- ketoglutarate dehydrogenase. At all temperatures, Y. pestis has nutritional requirements for L-isoleucine, L-valine, L-methionine, L-phenylalanine, and glycine (or L- threonine); these auxotrophies, some of which are capable of reversion, are due to cryptic genes. At 378C, the organism has additional requirements for biotin, thiamine, pantothenate, and glutamic acid. These metabolic requirements preclude a saprophytic existence; Y. pestis is an obligate parasite. Diagnostic tests characterize Y. pestis as positive by an o-nitrophenyl-b-D-galactopyranoside (ONPG) test without acid production from lactose.

Three biotypes (or biovars) of Y. pestis are recognized on the basis of conversion of nitrate to nitrite and fermentation of glycerol. Biotype antiqua is positive for both characteristics, orientalis forms nitrite but does not ferment glycerol, and mediaevalis ferments glycerol but does not form nitrite from nitrate. Strains of the three biotypes exhibit no difference in their virulence or pathology in animals or humans. DNA macrorestriction patterns of the three biotypes determined by pulsed-field gel electrophoresis support this division. However, individual isolates within a strain demonstrate pulsed-field gel electrophoresis heterogeneity, suggesting that relatively frequent spontaneous DNA rearrangements are occurring, possibly due to insertion sequences within the genome. Ribotyping identified 16 patterns that can be organized into the three classical biotypes. Two ribotypes (B and O) comprise the majority of strains examined and may be responsible for all three plague pandemics.

Yersinia enterocolitica and bacteria that resemble it are ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. They comprise a biochemically heterogeneous group that can grow at refrigeration temperatures (a strong argument for use of cold enrichment). Based on their biochemical heterogeneity and DNA relatedness, members of this group were separated into four species: Y. enterocolitica, Y. intermedia, Y. fredeήksenii, and Y. kristensenii (Bercovier et al., 1980). Through additional revisions, the genus Yersinia has grown to include eleven species. Of these, Y. enterocolitica is most important as a cause of foodborne illness.

The association of human illness with consumption of Y. enterocolitica- contaminated food, animal wastes, and unchlorinated water is well documented (Auliso et al., 1982, 1983). Refrigerated foods are potential vehicles because contamination is possible at the manufacturing site or in the home. This organism may survive and grow during refrigerated storage.

Yersinia spp. that cause human yersiniosis carry a plasmid (41-48 Mdal) (Zink et al., 1980) that is associated with a number of traits related to virulence: autoagglutination in certain media at 35-37⁰C; inhibition of growth in calcium- deficient media and binding of crystal violet dye at 35-37°C; increased resistance to normal human sera; production of a series of outer membrane proteins at 35-37⁰C; ability to produce conjunctivitis in guinea pig or mouse (Sereny test); and lethality in adult and suckling mice by intraperitoneal (i.p.) injection of live organisms. The plasmid associated with virulence can be detected by gel electrophoresis or DNA colony hybridization. Recent evidence, however, indicates that presence of plasmid alone is not sufficient for the full expression of virulence in Yersinia. The intensity of some plasmid-mediated virulence properties such as mouse lethality and conjunctivitis is variable, depending on the genes carried on the bacterial chromosome and the serogroup, suggesting that chromosomal genes also contribute to Yersinia virulence.

Virulent strains of Yersinia invade mammalian cells such as HeLa cells in tissue culture (Lee et al., 1977). However, strains that have lost other virulent properties retain HeLa invasiveness, because the invasive phenotype for mammalian cells is encoded by chromosomal loci (Miller et al., 1988, 1989).

Y. pseudotuberculosis is less ubiquitous than Y. enterocolitica, and although frequently associated with animals, has only rarely been isolated from soil, water, and foods. Among Y. pseudotuberculosis strains there is little or no variation in biochemical reactions, except with the sugars melibiose, raffinose, and salicin. Serologically (based on a heat-stable somatic antigen), the Y. pseudotuberculosis strains are classified into six groups, each serogroup containing pathogenic strains. It has been reported that serogroup III strains harbor a 42-Mdal plasmid as do serogroup II strains that are lethal to adult mice. The association of yersiniosis in humans with the presence of a 42-Mdal plasmid in Y. pseudotuberculosis has been established (Schiemann et al., 1992).

The present invention relates to compositions, such as vaccines, and their use to elicit immune responses against Francisella and Yersinia spp, especially protective immune responses. OMVs have the ability to present antigenic molecules in a native form while activating both T and B cell responses, and thus provide a solution to the problem of a non-living vaccine for tularemia. In addition, OMV vaccines are known to be safe and effective in humans. Thus, the invention relates to an immunogenic composition for the immunization of an individual comprising outer membrane vesicles of Gram- negative bacteria, Francisella or Yersinia spp, e.g., F. tularensis or F. philomiragia, e.g., F. tularensis subspecies tularensis (Group A), F. tularensis subspecies holarctica (Group B), or F. tularensis subspecies novicida. It also relates to a method for preparing native outer membrane vesicles from Francisella, comprising, (i) shearing Francisella cells;

(ii) separating cells and cellular debris from native outer membrane vesicles; (iii) passing said native outer membrane vesicles through an ion exchange matrix; and

(iv) ultrafiltering said native outer membrane vesicles; and to a vaccine for protection against infection with Gram negative bacteria, Francisella or Yersinia spp, comprising outer membrane vesicles of said bacteria in an amount effective to elicit protective antibodies in an animal to said Gram negative bacteria, and a pharmaceutically acceptable carrier, e.g., wherein said Francisella spp is F. tularensis or F. philomiragia, e.g., wherein said F. tularensis is F. tularensis subspecies tularensis (Group A), F. tularensis subspecies holarctica (Group B), or F. tularensis subspecies novicida or wherein said Yersinia spp is Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis.

The invention also relates to isolated outer membrane vesicles from a strain of Francisella or Yersinia, e.g., from a Francisella strain which expresses iron uptake proteins.

It also relates to a method of preventing infection by Francisella or Yersinia Gram-negative bacteria in an animal comprising administering a vaccine of the invention, e.g., wherein said animal is a human.

It also relates to a method for reducing Francisella infection symptoms, comprising administering to a patient in need of such treatment an effective amount of antibodies against native outer membrane vesicles of Francisella in a pharmaceutically acceptable excipient, or a method of eliciting an immune response against Francisella comprising administering outer membrane vesicles from Francisella.

Membrane vesicles, also known as blebs, are little bud-like protrusions formed in the cell wall, outer membrane, cytoplasmic, and/ or plasma membrane of a microorganism. When cultured under selected conditions the membrane vesicles break away from the whole cell into the medium. The membrane vesicles are generally spherical, possess a bilayer, and have a diameter of about 10 to 200 nm, preferably 50-150 nm, most preferably 80 to 100 nm.

The membrane vesicles may be natural membrane vesicles of a microorganism which produces membrane vesicles. Natural membrane vesicles contain outer membrane and periplasm components. Natural membrane vesicles can be produced without exposing the microorganism to a surface-active agent. Thus, this invention in one aspect relates to compositions free of surface active agents. Treatment with a surface active agent produces membrane vesicles which are larger than the natural vesicles. These large membrane vesicles typically contain outer membrane, cytoplasmic membrane or plasma membrane components, and cytoplasm. Membrane vesicles produced by treatment with surface-active agents also include natural membrane vesicles. The membrane vesicles used in the vaccine, methods, and compositions of the invention include both natural membrane vesicles and the larger membrane vesicles.

By way of analogy, natural membrane vesicles of Pseudomonas aeruginosa contain mainly B-band LPS, mature periplasmic enzymes and secretory enzymes which are in transit. Secretory enzymes may be mature enzymes or proenzymes; the latter being activated once they are liberated from the cell surface. The antimicrobial agent gentamicin increases the incidence of membrane vesicles and frequently results in membrane vesicles which contain outer membrane, cytoplasmic membrane and/or plasma membrane components. Both types of membrane vesicles can be enriched with peptidoglycan-hydrolysing enzymes (i.e., autolysins) .

While we do not wish to be bound by any particular models or theory, a proposed model for the formation of membrane vesicles is set out in schematic form in FIG. 1. Suitable microorganisms for producing the membrane vesicles are of Francisela and Yeήsinia spp of bacteria.

In accordance with preferred embodiments of the invention, the microorganism is selected from the bacterial strains F. tularensis, F. phϊlomiragia, Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis.

This invention in one aspect provides isolated or biologically pure (e.g., substantially free of toxic components) outer membrane vesicles. "Isolated" in general refers to the vesicles in a state other than the natural state.

Another aspect of the instant invention relates to a method for the production and purification of OVMs derived from Francisella or Yersinia spp. Suitable microorganisms which may be used to prepare outer membrane vesicles are described above. The strains of the microorganism used to prepare the membrane vesicles may be reference strains which may be obtained from research institutes working in the field, or from public depositories such as the American Type Culture Collection (Bethesda, MD). The microorganism strains may also be obtained from animals, preferably humans suffering from naturally occurring infections.

The microorganisms are grown under suitable conditions that permit natural membrane vesicles to be formed. Suitable growth conditions will be selected having regard to the type of microorganism, and the desired characteristics of the membrane vesicles. Generally, growth mediums suitable for culturing the microorganisms so that they produce membrane vesicles contain a nitrogen source and a carbon source.

Suitable nitrogen sources are nitrogen salts. The initial concentration of the nitrogen source is related to the temperature of the fermentation during the growth phase. There should be enough nitrogen source present to provide a final cell mass of a least about 0.5-1.0 g/1. A useful range of initial nitrogen concentration is selected so that less than 0.1 g/1 remains at the conclusion of the growth phase.

As carbon source, sugars such as glucose (or crude glucose such as dextrose), sucrose, fructose, erythrose, mannose, xylose, and ribose, or mixtures of these sugars may be used. Commercial sources of these sugars can conveniently be used. Such sources include liquid sucrose, high fructose corn syrup and dextrose corn syrup. Other carbon sources can be used in combination with these sugars such as mannitol and other sugar derivatives.

The medium preferably includes other components useful in fermentation processes. For example, the medium may include a source of magnesium such as magnesium sulfate, a source of phosphate such as K2HPO4, a source of iron such as iron sulfate, and a source of zinc such as zinc sulfate. Useful concentration ranges of magnesium, phosphate, iron and zinc are 2-5 mM, 0.5-5.0 mM, 2-5 mM, 1-5 mM, 0.5 mM and 0.5-5.0 mM, respectively.

The medium may also contain components which support the production of specific enzymes. For example, choline (2-hydroxy methyl-trimethyl ammonium chloride salt) may be added to the medium to support the production of phospholipase C, or chelating compounds such as transferrin to support siderophore production. By way of example, bacterial culture media and conditions are provided in Example 1 of the specification.

Commercially available media, which favor the production of membrane vesicles, for example, Mueller-Hinton broth, or Trypticase soy broth, Brain-Heart Infusion, Chamberlain's medium (CM), and blood agar, may also be used.

The microorganisms can be cultured in two stages. The first stage is carried out at a temperature sufficient to promote the growth phase of the microorganism. After rollover into the stationary phase, the temperature of the growth medium may be reduced to a temperature which promotes production of membrane vesicles. For example, the temperature may be reduced to 20 to 25°C, preferably room temperature.

The final medium is subjected to a variety of steps to recover the desired membrane vesicles. For example, the membrane vesicles may be isolated by precipitation, filtration, and/or differential centrifugation.

Formation of membrane vesicles may optionally be induced using surface- active agents. The release of membrane vesicles typically increases several fold after the microorganism is exposed to an agent. Suitable surface agents include surface- active antimicrobial agents such as polymyxin, atypical metal ions, and EDTA. Preferably, the surface-active agent is an antimicrobial surface-active agent, most preferably an aminoglycoside. Examples of suitable aminoglycosides include gentamicin, hygromycin, tobramycin, amakacin, kanamycin, neomycin, paromomycin, and streptomycin. The method for inducing formation of the membrane vesicles is generally as described above. The microorganism is cultured using the above described conditions, and the surface-active agent is added after the first stage, i.e., after early stationary growth phase. The concentration of antimicrobial agent that is added is about four times the minimal inhibitory concentration (MIC).

By way of example, F. tularensis can be induced to release membrane vesicles into the medium on exposure of the organism to gentamicin. In particular, F. tularensis strains are grown in BHI broth to the early stationary phase at 37⁰C.

The antigens associated with the surface of membrane vesicles may be identified using conventional methods. For example, Western immunoblots of solubilized components of the membrane vesicles can be prepared and specific antigens can be identified using antibodies specific for the antigen (e.g., antibodies specific for LPS, pilin, flagellin etc.). LPS can also be identified using immunogold electron microscopic detection. Enzymes contained in the membrane vesicles may be identified using conventional enzyme assays. For example, phospholipase C activity may be determined using the synthetic substrate p-nitrophenyl phosphorylcholine (Sigma) as described by Berka et al. (Infect. Immun. 34: 1071-1074, 1981); protease may be determined by the assay described by Howe and Iglewski (Infect. Immun. 43: 1058- 1063, 1984) using Hide powder azure (Sigma); alkaline phosphatase may be assayed using p-nitrophenyl phosphate (pNPP) (Sigma) as described in Tan, A. S. P. and E. A. Worobec (FEMS Microbial. Letts. 106:281-286, 1993); elastase may be determined using elastin Congo red (Sigma) as a substrate in an assay based on the method of Kessler and Safrin (Kessler, E., and M. Safrin, J. Bacteriol. 170:5241-5247, 1988); and hemolysin activity may be measured as described in Bergmann et al. (Infect. Immun. 57:2187-2195, 1989). Peptidoglycan hydrolases may be determined using SDS-PAGE zymogram systems as outlined in Bernadsky, G., et al. (J. Bacteriol. 176:5225-5232, 1994). Immunogold electron microscopic detection may also be used to identify enzymes contained in a membrane vesicle.

Methods of obtaining the outer membrane vesicles from the bacterial preparations are, e.g., shown in the examples.

The invention also provides a vesicle preparation. For administration to a patient, the vesicles are preferably formulated as immunogenic compositions, and more preferably as compositions suitable for use as a vaccine in humans (e.g. children or adults). Vaccines of the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection), but will typically be prophylactic.

The composition of the invention is preferably sterile, or preferably pyrogen- free. The composition of the invention generally has a pH of between 6.0 and 7.0, more preferably to between 6.3 and 6.9 e.g. 6.6+/-0.2. The composition is preferably buffered at this pH. Other components suitable for human administration are disclosed in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

The compositions, e.g., vaccines of the invention are applicable for administration to animals, including mammals, avian species, and fish; preferably humans and various other mammals, including bovines, equines, and swine.

Immunogenic compositions comprise an immunologically effective amount of antigen, as well as any other compatible components, as needed. By "immunologically effective amount" is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group or individual to be treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g., including booster doses). The vaccine may be administered in conjunction with other immunoregulatory agents.

The antigen compositions or individual antigens to be administered are provided in a pharmaceutically acceptable solution such as an aqueous solution, often a saline solution, or they can be provided in powder form. The compositions may also include an adjuvant. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v Tween 80, 0.5% w/v Span 85), Cp G- containing nucleic acid (where the cytosine is ummethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/ CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For experimental animals, one can use Freund's, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP) , N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-(l'-2'-dip- almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic antigen.

Further exemplary adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59.TM. (WO90/ 14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (DM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (2) saponin adjuvants, such as QS21 or Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M- CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O- deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in- water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin MoI Ther 2001 3: 15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al, J. Immunol, 1998, 160, 870-876; Chu et al., J. Exp. Med, 1997, 186, 1623-1631; Lipford et al, Ear. J. Immunol., 1997, 27, 2340-2344; Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg et al, Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840-1845; Cowdery et al, J. Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167, 72-78; Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al, J. Immunol., 1996, 157, 2116-2122; Messina et al, J. Immunol, 1991, 147, 1759-1764; Yi et al, J. Immunol, 1996, 157, 4918-4925; Yi et al, J. Immunol, 1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160, 4755-4761; and Yi et al, J. Immunol, 1998, 160, 5898-5906; International patent applications WO96/02555, WO98/ 16247, WO98/ 18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one CG dinucleotide, where the cytosine is unmethylated; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/ 52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (WOO 1/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO00/62800); (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/ 11241; (13) a saponin (e.g. QS21)+3dMPL+lM2 (optionally+a sterol) e.g. WO98/57659; (14) other substances that act as immuno stimulating agents to enhance the efficacy of the composition. Muramyl peptides include N-acetyl- muramyl-L-threonyl-- D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl- D-isoglutamine (nor-MDP) , N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (l'-2'-di- palmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.

The antigens may be combined with conventional excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antigen in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. The resulting compositions may be in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or the like.

The concentration of immunogenic antigens of the invention in the pharmaceutical formulations can vary widely, i.e. from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active vesicles are combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are also described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions include, albeit not exclusively, solutions of the membrane vesicles in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may also be applied to implants such as catheters, pace-makers, etc. which are often sites for colonization of pathogens, and thus sources of infectious diseases.

The vaccine may be stored in a sealed vial, ampule or the like. The present vaccine can generally be administered in the form of a spray for intranasal administration, or by nose drops, inhalants, swabs on tonsils, or a capsule, liquid, suspension or elixirs for oral administration. In the case where the vaccine is in a dried form, the vaccine is preferably dissolved or suspended in sterilized distilled water before administration. Any inert carrier is preferably used, such as saline, phosphate buffered saline, or any such carrier which the vaccine has suitable solubility.

The compositions of the invention can be administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the composition to be administered in which any toxic effects are outweighed by the therapeutic effects of the membrane vesicles.

The composition may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration inhalation, transdermal application, or rectal administration. The pharmaceutical compositions are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the active membrane vesicles or as powders of the vesicles to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids. For local use, those preparations in the form of creams or ointments for topical use, or in the form of sprays are suitable; for inhalant uses, preparations in the form of sprays, for example nose sprays, are suitable.

Generally, the vaccine may be administered orally, subcutaneously, intradermally or intramuscularly, intranasally or orally in a dose effective for the production of neutralizing antibody and resulting in protection from infection or disease. The vaccine may be in the form of single dose preparations or in multi- dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.

The microorganisms which produce membrane vesicles described herein may also be transfected with one or more nucleotide sequences encoding exogenous proteins in order to provide membrane vesicles have exogenous proteins incorporated into the membrane vesicles or associated with their surface. For example, the exogenous proteins include antigens which are associated with infectious diseases caused by infectious agents which do not produce membrane vesicles including viruses such as human immunodeficiency virus (HIV), influenza (nuriminidase/haemagglutinin), adenovirus, Herpes simplex, measles, simian immunodeficiency virus; fungi such as Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatidis, Candida albicans; protozoa such as Leishmania mexicana, Plasmodium falciparum and Taxoplasma gondii; and, gram- positive bacteria such as Streptococcus mutans, and S. pneumoniae (cell wall antigens) . Microorganisms transfected with such antigens may be used to produce membrane vesicles which may be used as vaccines against the infectious agent. The microorganism may also be transfected with a nucleotide sequence encoding an exogenous protein having a known therapeutic or regulatory activity such as hormones preferably insulin, blood clotting factor VIII, growth hormones, hirudin, cytokines such as gamma interferon, tumor necrosis factor, IL-I, IL-2,IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I l, GM-CSF, CSF-I, and G-CSF. Membrane vesicles containing therapeutic or regulatory proteins may be used to deliver the proteins to a host. The microorganisms may also be transfected with proteins which facilitate targeting of a membrane vesicle having the proteins associated with their surfaces to specific target tissues or cells. For example, tumor-associated antigens, CD4 proteins on T-helper cells, and gρl20 in HIV.

Nucleotide sequences encoding exogenous proteins may also be introduced into microorganisms which produce membrane vesicles using methods well known to those skilled in the art. The necessary elements for the transcription and translation of the inserted nucleotide sequences may be selected depending on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. A reporter gene which facilitates the selection of host cells transformed or transfected with a nucleotide acid sequence may also be incorporated in the microorganism. (See, e.g., Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, for transfection/ transformation methods and selection of transcription and translation elements, and reporter genes). Sequences which encode exogenous proteins may generally be obtained from a variety of sources, including for example, depositories which contain plasmids encoding sequences including the American Type Culture Collection (ATCC, Rockville Md.), and the British Biotechnology Limited (Cowley, Oxford England) .

The present invention also can be used to provide a vaccine against an infectious disease caused by an infectious agent, e.g., comprising a carrier strain having a membrane vesicle of a microorganism integrated into the cell surface of the carrier strain, wherein the membrane vesicle has an amount of an antigen associated with its surface which is effective to provide protection against the infectious agent. The term "integrating" or "integrated" used herein refers to the fusion of the cell membrane of the membrane vesicle with the cell surface of the carrier strain, or the adherence of the membrane vesicle to the cell surface of the carrier strain.

"Infectious disease" refers to any disease or condition due to the action of the infectious agents against which this invention is directed. The infectious agent may be a microorganism which produces membrane vesicles, or a microorganism which does not produce membrane vesicle.

Therefore, in an embodiment of the invention, a vaccine against the mentioned infectious diseases is provided which comprises a carrier strain having a membrane vesicle of the microorganism integrated into the cell surface of the carrier strain. The vaccines may be used for the prophylaxis or active immunization and treatment of the infectious diseases.

The membrane vesicles employed in the vaccines of the present invention may be natural membrane vesicles of the microorganism or they may be membrane vesicles produced by treating the microorganism with a surface-active agent. The membrane vesicles are selected so that they have an amount of an antigen (i.e. immunogen) associated with their surfaces which is effective to provide protection against the pathogenic infectious agent/microorganism. For example, for the pathogens listed in Table 1, membrane vesicles may be selected which contain the specific antigens identified in Table 1. In particular, membrane vesicles may be selected for F. tularensis which have endotoxin (A- and B-band lipopolysaccharide) , outer membrane proteins, pilin, and/or flagellin associated with their surfaces. These membrane vesicles may be fused with a carrier strain to provide a vaccine which is useful for protecting against infections caused by F. tularensis.

The carrier strain is selected so that it is incapable of multiplying in vivo. Carrier strains are obtained through selection of variants which occur naturally, or using conventional means known to those skilled in the art. Examples of suitable carrier strains are Shigella species, Salmonella species, preferably S. typhi Ty21a, S. typhimurium, Vibrio species, and Escherichia species.

A membrane vesicle may be integrated into the cell surface of a carrier strain by contacting the membrane vesicle with the carrier strain. By way of example, exponential growth phase cultures of the carrier strain (e.g., S. typhimurium aro A, and S. typhiTy21a) in a suspension are incubated with membrane vesicles from, for example, F. tularenis or Y. pestis.

The vaccine may be a multivalent vaccine and additionally contain immunogens related to other infectious diseases in a prophylactically or therapeutically effective manner. Multivalent vaccines against infectious diseases caused by different infectious agents may contain a carrier strain having membrane vesicles integrated into the cell surface of the carrier strain, wherein the membrane vesicles have amounts of antigens associated with their surfaces which are effective to provide protection against the infectious agents.

A multivalent vaccine may comprise at least two carrier strains each having membrane vesicles with different immunogens associated with different infectious agents. In an embodiment of the invention a multivalent vaccine is provided comprising at least two carrier strains each having membrane vesicles of different pathogenic microorganisms integrated into the cell surface of the carrier strain For example, a multivalent vaccine may contain a carrier strain having a selected membrane vesicle of different subspecies of F. tularenis or Y. pestis integrated into its cell surface.

A multivalent vaccine may contain a carrier strain having at least two membrane vesicles having different immunogens associated with different infectious agents. In an embodiment of the invention, a multivalent vaccine is provided comprising a carrier strain and membrane vesicles from at least two different microorganisms integrated into the cell surface of the carrier strain. Thus, a carrier strain may contain immunogens relating to more than one pathogenic microorganism. For example, a carrier strain may be contacted with a selected membrane vesicle obtained from another different strain or species of bacteria, and a membrane vesicle obtained from F. tularenis or Y. pestis using the methods described herein, to produce a carrier strain having immunogens from both bacteria associated with the cell surface.

The present invention also contemplates a pharmaceutical composition comprising a membrane vesicle of the invention containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity in an amount effective to have a bactericidal effect on gram-negative and/ or gram-positive bacterial pathogens, and a pharmaceutically acceptable vehicle or diluent.

The invention also contemplates a method of treating an infectious disease caused by a gram-negative and/ or gram-positive bacterial pathogen comprising administering an amount of a membrane vesicle containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity, effective to have a bactericidal effect on the gram-negative and/ or gram-positive bacterial pathogen.

Membrane vesicles containing enzymes with peptidoglycan hydrolase, lipase, and proteolytic activity may be selected using conventional enzyme assays.

The present invention also relates to a composition comprising the outer membrane vesicles of the invention containing a therapeutic agent in an amount which is effective to introduce the therapeutic agent into a host. The invention also relates to a method for administering a therapeutic agent to a host comprising administering to the host a suspension of the therapeutic agent encapsulated in a membrane vesicle of the invention.

Therapeutic agents may be encapsulated in the outer membrane vesicles by culturing the microorganisms capable of producing membrane vesicles in the presence of the therapeutic agents. The therapeutic agents may also be produced by the microorganism by transforming the microorganism with a gene which expresses the therapeutic agent preferably in the periplasmic space.

Any of a wide variety of therapeutic agents may be encapsulated in the membrane vesicles described herein. Among these may be mentioned antimicrobial agents, metabolic regulators, immune modulators, antiproliferative agents, chemotherapeutics, etc. For example, the invention is well suited for antimicrobial agents, such as polymyxin, and aminoglycosides including gentamicin, hygromycin, tobramycin, amakacin, kanamycin, neomycin, paromomycin, streptomycin; and antiviral agents such as interferon, interleukins, and octreotide.

The membrane vesicles may also have carbohydrate, proteins, glycoproteins or glycolipids associated with their surfaces which will target the therapeutic agent to the tissue where it is most needed. Alternatively, specific adhesins such as bacterial fimbriae can be incorporated in the surface of the membrane vesicles. This will enable targeting to only the tissues at risk while reducing the exposure of other tissues to toxic side effects of the drug. Slow sustained release of therapeutic agents from vesicles will also prolong the residence time of the therapeutic agent in areas where it is most needed.

In one embodiment of the invention, a composition is provided comprising membrane vesicles of a microorganism containing an antimicrobial agent, in an amount which is effective to introduce the antimicrobial agent into a host. The invention also relates to a method for administering an antimicrobial agent into a host comprising administering to the host a membrane vesicle of a microorganism containing the antimicrobial agent. Membrane vesicles containing antimicrobial agents for use in these compositions and methods may be prepared using the methods described herein. For example, membrane vesicles containing antimicrobial agents may be prepared by exposing a microorganism which is capable of producing membrane vesicles (for example, the microorganisms, preferably F. tularenis or Y. pestis) to an antimicrobial agent. Preferably the antimicrobial agent is polymyxin, or an aminoglycoside such as gentamicin, hygromycin, tobramycin, amakacin, kanamycin, neomycin, paromomycin, and streptomycin.

The invention also relates to a method of inserting nucleic acid molecules into a target cell which comprises encapsulating the nucleic acid in a membrane vesicle of the invention, and bringing the membrane vesicle in contact with the target cell whereby the nucleic acid molecule is inserted into the cell. Nucleic acid molecules which may be encapsulated in a membrane vesicle may be from eucaryotic or prokaryotic cells and they may be endogenous or exogenous to a microorganism that produces membrane vesicles. Examples of nucleic acid molecules which may be encapsulated in a membrane vesicle are nucleic acid molecules encoding (a) mammalian proteins such as hormones preferably insulin, blood clotting factor VIII, growth hormones, hirudin, cytokines, and a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR); (b) viral antigens such as HIV glycoprotein, hepatitis B surface antigens, influenza antigens; fungal antigens for example from Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatidis, Candida albicans;, and (c) protozoal antigens for example from Leishmania mexicana, Plasmodium falciparum and Taxoplasma gondii. "Target cells" as used herein refers to a cell of a living organism, plant, animal, or microbe. The cell may be unicellular such as a microorganism or it may be multicellular including animals such as humans. Membrane vesicles containing nucleic acid molecules may be prepared by the methods described herein preferably using surface-active agents. For example, treatment of a microorganism which produces membrane vesicles (which has or has not been transfected with an exogenous nucleic acid molecule) , with a surface- active agent such as gentamicin will produce membrane vesicles incorporating DNA. The encapsulated nucleic acid molecule can be inserted into a target cell by contacting the membrane vesicle containing the nucleic acid molecule with the surface of the target cell. For microorganisms the contact is with the cell wall, and for animal cells it is with the membrane. Cells associated with multi-cellular organisms may be contacted in vivo or in vitro. The nucleic acid molecule passes into the target cells when the membrane vesicle contacts the target cell, and it is taken up by the target cell through fusion of the membrane vesicle with the cell wall or membrane, or by endocytosis. Conventional techniques are used to contact the membrane vesicles with the target cells. For example, if the contact is to be effected in vitro, the cells and membrane vesicles are admixed. In vivo the membrane vesicles may be injected intravenously or given orally into the host organism in combination with a pharmaceutically acceptable carrier.

It will also be appreciated that the membrane vesicles described herein may be used to isolate products produced by genetic engineering techniques. For example, a host microorganism which produces membrane vesicles may be transformed with a recombinant vector having a gene encoding a desired gene product and having the necessary transcription and translation elements required for the gene product to be expressed in the host cell, and preferably transported to the periplasmic space. The microorganism expressing the gene product may be cultured under suitable conditions to produce natural membrane vesicles, or the microorganism may be induced to produce membrane vesicles after exposure to a surface-active agent. Membrane vesicles containing the gene product may be isolated and the gene product can be removed from the membrane vesicles. Products (e.g. cell surface antigens and enzymes) which are endogenous to a microorganism which produces membrane vesicles may also be isolated from membrane vesicles in a similar fashion.

In one embodiment, the above-said method comprises growing bacteria in 0.1% L-cysteine HCl (Fisher Scientific) brain heart infusion broth (C-BHI) (Becton- Dickinson) for 24-48 hr at 37⁰C with shaking at 200 rpm. Chamberlain's medium (CM) is a chemically defined medium that was used to grow Francisella spp., at 37°C for 1-2 days shaking at 200 rpm. Bacterial species are streaked onto Chocolate Agar II (Becton-Dickinson) plates and incubated at 37⁰C for 1-2 days depending on species. Isolated colonies are used to observe bacteria and identify the presence of OMVs by AFM and TEM. Plates are wrapped with parafilm and then stored at 4°C until needed. For enrichment and isolation of OMV, frozen stock of F. novicida, F. philomiragia, and FtLVS are transferred to 100 mL of C-BHI. Cultures are grown for 1-2 days to determine the time at which OMVs were produced. Broth cultures are centrifuged at 10,000 x g for 15 min, and supernatants are harvested. Supernatants are sequentially passed through a 0.45μm-, 0.22μm-, and O. lOμm - filtration system. Filtered supernatants are subjected to ultracentrifugation for 2 hrs at 150,000 x g (Ti-70 rotor, Beckman Instruments, Inc.). The supernatant is removed and the pellet, which consists of OMVs is resuspended in IX phosphate buffered saline (PBS) and stored at -80⁰C. After determining the protein concentration of each sample, the volume is adjusted so that each sample is normalized with an equivalent concentration of protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views, and wherein:

FIG. 1. Proposed Model of vesicle biogenesis from Keuhn and Kesty (2005). OMVs are proteoliposom.es consisting of outer membrane phospholipids and LPS, a subset of outer membrane proteins (OMPs), and periplasmic proteins. Proteins such as labile enterotoxin (LT) (red) that adhere to the external surface of the bacteria are associated with the external surface of vesicles. Proteins and lipids of the inner membrane and cytosolic content are excluded from OMVs. Vesicles are likely to bud at sites where the links between the peptidoglycan and OM are infrequent, absent, or broken. (LPS) Lipopolysaccharide; (Pp) periplasm; (OM) outer membrane; (PG) peptidoglycan; (IM) inner membrane; (Cyt) cytosol (Diagram and caption is from Keuhn et al. (2005). FIG. 2. Shows the growth curve of F. novicida (F. noυicida). The growth curve was generated to determine when F. novicida enters stationary phase and used to determine the production of outer membrane vesicles (OMV).

FIG. 3. Shows a graph of Francisella outer membrane vesicles (OMV) protein concentration versus time.

FIG. 4. Atomic Force Microscopy (AFM) image of Francisella tularensis noυicida (formerly F. novicida) bacteria. a) Atomic Force Microscopy (AFM) image of OMVs prepared from Francisella tularensis novicida (formerly F. novicida). b) AFM image of OMVs prepared from Francisella philomiragia. Note: Different scale of X axis between A and B.

FIG. 5. Characterization of OMVs through AFM.

(a) F. novicida OMV: F. novicida OMVs are domed shaped to an irregular flattened form due to this method. The average diameter of OMVs isolated from F. novicida was 47 nm. (Bar = 500 nm)

(b) F. novicida bacterium with OMVs. F. noυicida bacterium isolated from a 48 hr chocolate agar plate. OMVs are being produced on the surface of the bacterium (short black arrows) and a pilus like structure is protruding from the central right portion of the cell (long black arrow), which is highly encapsulated. Bar = 500 nm.

(c) F. novicida bacterium. F. novicida bacterium from a 24hr liquid broth culture has a cocobacillus shape, and has capsule (Bar = lμm).

FIG. 6. Characterization of OMVs through negative staining - TEM.

(a) Isolated OMVs of F. novicida and

(b) OMVs isolated from F. philomiragia were fixed and then negatively stained and the viewed under TEM. Evident are round spherical bilayered vesicles. Bars = 500 nm

(c) Fixed F. novicida bacterium with and one OMV protruding from the surface of the bacteria. Bar = lμm. FIG. 7. Acid Phosphatase Activity of OMVs. Fifty microliters of OMV sample of equal protein concentration, was added to 50μL of p-NPP reagent and incubated for 1 hr. The reaction was stopped with 0.5N sodium hydroxide and then read at 405nm. A-G are positive control standards (potato acid phosphatase), F is the negative control (PBS), G is F. novicida OMVs, and H is F. philomiragia OMVs (This experiment was done in triplicate) .

FIG. 8. Hemolytic Effect of OMVs isolated from Francisella spp. Equal protein concentrated Francisella OMVs were added to a 2% suspension of horse erythrocytes in PBS. After 1 hr, the mixture was read at 540nm and then % hemolysis was calculated. A is positive control (unlysed red blood cells), B is negative control (lysed red blood cells), C is F. novicida OMVs, and D is F. philomiragia OMV s (This experiment was done in triplicate).

FIG. 9. PLC Activity found in isolated OMVs from Francisella spp. Fifty microliters of equal protein concentrated OMV sample, was added to 50μL of p-NPPC reagent and incubated for 1 hr. The reaction was stopped with 0.5N sodium hydroxide and then read at 405nm. A-F is the positive control (PLC from C. perfringens) , G is the negative control, H is F. novicida OMVs, and I is F. philomiragia OMVs (This experiment has been done in triplicate).

FIG. 10. Macrophage - Francisella OMV Interaction. Cytopathic effects of equally protein concentrated Francisella OMVs on J774A.1 cells at 1 xlO⁵ cells/mL were determined by using the LDH Assay (Promega). A is Lysed-cells; B is non-lysed cells; C is cells treated with F. novicida OMVs; D is cells treated with F. philomiragia OMVs (LDH assay was carried out in triplicates) .

FIG. 11. Agarose Gel Electrophoresis. Samples were treated with 5X loading dye (Bioline), and then 15μL of sample was loaded to each well in a 1% agarose gel and run at 90 V for 1 hr. Lane 1 is the DNA Hyperladder I (Bioline), lane 2 is F. novicida OMV DNA, and lane 3 is F. philomiragia OMV DNA. The DNA fragments from the OMV samples were above 10 kbp.

FIG. 12. Coomassie Blue Stain of SDS-PAGE of F. novicida - and F. philomiragia- OMVs. CCB stain was used to detect the presence of protein in OMVs from Francisella spp. Lane #1 is the protein molecular weight standards, lane #3 is F. noυicida OMVs, lane #4 is F. novicida WCE, lane #5 is Ft-Ag, lane #6 is F. philomiragia OMVs, and lane #7 is F. philomiragia WCE. Arrows are pointing to the most visible protein bands.

FIG. 13. Silver Stain.

(a) F. noυicida OMVs. Lane 1: Protein Standard, lane 2: F. philomiragia OMV (15 μL) and lane 3: F. philomiragiaWCE (15 μL).

(b) F. philomiragia OMV s. Lane 1: Protein Standard, lane 2: F. philomiragia OMV (15 μL) and lane 3: F. philomiragiaWCE (15 μL).

Brackets represent the length of the carbohydrate smear, which is a characteristic of LPS.

FIG. 14. Western Blot for the Presence of LPS on F. novicida OMVs. Anti-F. novicida LPS Antibody was used as the primary antibody. Lane 1 : Molecular weight protein ladder, lane 3: F. novicida OMV (15μL), lane 4: F. novicida WCE (15 μL). This blot shows a smear between 75-25 kDa and various bands around ~ 15kDa, and ^ ~20kDa, which may represent the KDO core. Brackets represent the length of the carbohydrate smear which is a characteristic of LPS.

FIG. 15: Proteins found in Francisella tularensislNS OMVs

A) Coomassie Brilliant Blue Stain SDS PAGE gel of OMV samples.

Lane 1 & 8: Francisella Antigen 1 and 5 ul respectively (BD Biosciences) , Lane 2:

MW Markers,

Lane 3: 5 ul OMV prep,

Lane 4: 10 ul OMV prep,

Lane 5: 20 ul OMV prep,

Lane 6: "Whole Cell Extract" of F. tularensis LVS,

Lane 7: Blank.

B) Anti- Francisella Polyclonal Serum Western Blot of OMVs.

Lane 1 & 8: Francisella Antigen 1 and 5 ul respectively (BD Biosciences), Lane 2:

MW Markers,

Lane 3: 5 ul OMV prep,

Lane 4: 10 ul OMV prep, Lane 5: 100 ul OMV prep,

Lane 6: "Whole Cell Extract" of F. tularensis LVS,

Lane 7: Blank.

Without further elaboration, it is believed that one skilled in the art can, using the prceeding description, utilize the following invention to its fullest extent. The following specific preferred embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the forgoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.

EXAMPLES

The invention will be explained below with reference to the following non- limiting examples.

Example 1 (Isolation, purification, and analysis of OMV)

Bacterial Strains and Growth. The bacterial species used were F. noυicida (F. novicida) and F. philomiragia (Fph), and F. tularensis LVS (FtLVS) which were frozen in 5% trehalose solution and stored at -80⁰C. Bacteria were grown in 0.1% L- cysteine HCl (Fisher Scientific) brain heart infusion broth (C-BHI) (Becton- Dickinson) for 24-48 hr at 37°C with shaking at 200 rpm. Chamberlain's medium (CM) is a chemically defined medium that was used to grow Francisella spp., at 37°C for 1-2 days shaking at 200 rpm. Bacterial species were streaked onto Chocolate Agar II (Becton-Dickinson) plates and incubated at 37°C for 1-2 days depending on species. Isolated colonies were used to observe bacteria and presence of OMVs by AFM and TEM. Plates were wrapped with parafilm and then stored at 4⁰C until needed.

Isolation of OMVs. Thirty to fifty microliters of frozen stock of F. novicida, F. philomiragia, and FtLVS were transferred to 100 mL of C-BHI. Cultures were grown for 1-2 days to determine the time at which OMVs were produced. Broth cultures were centrifuged at 10,000 x g for 15 min, and supernatants were harvested. Supernatants were sequentially passed through a 0.45μm-, 0.22μm-, and 0. lOμm - filtration system. Filtered supernatants were subjected to ultracentrifugation for 2 hrs at 150,000 x g (Ti-70 rotor, Beckman Instruments, Inc.). The supernatant was removed and the pellet, which consists of OMVs was resuspended in IX phosphate buffered saline (PBS) and stored at -80⁰C. After determining the protein concentration of each sample, the volume was adjusted so that each sample had an equivalent concentration of protein.

Bradford Assay. Following the manufacturer's instructions, bovine serum albumin standard (BSA: 2 mg/mL) (Pierce, Inc) was diluted to make a standard curve. The range was from 25 μg/mL to 2000 μg/mL. Five microliters of standards or unknown samples were added to 500 μL of borate saline buffer (BS). Five hundred microliters of Coomassie Blue Better Bradford Reagent (Pierce) was added to each sample. The samples were then read at 595nm and a standard curve was generated. The concentration of protein was calculated using the equation from the standard curve. This method was used to determine protein concentration of F. novicida and F. philomirαgiα OMVs.

Time Point Analysis - Determination of OMV Production. Fifty microliters of F. noυicida was inoculated into 35OmL of C-BHI, and then processed for isolation of OMVs (see second method above) at different time points (0, 6, 12, 18, 24 hr,..etc). At certain time points, the optical density (O. D.) at 600 nm was read to generate a growth curve. For each time point, the protein concentration of the OMV samples was determined by the Bradford assay to determine at what time point OMVs are produced.

Atomic Force Microscopy (AFM). Mica slides were attached onto a glass slide, and to prepare the mica slide for AFM, a layer of mica is removed by scotch tape. Then, 1- 2 μL of bacterial and OMV samples were placed on mica glass slides (Polyscience, Inc), air-dried, rinsed with ddHaO and then air-dried. The samples were viewed by the Nano-R2 Atomic Force Microscope to determine size of vesicles and to visualize the vesicles shedding from the bacterial surface.

Transmission Electron Microscopy. Sample fixation was used to preserve the extracellular structures of OMVs as well as the bacterium. Five hundred microliters of 25% of glutaraldehyde (Sigma) was added to 2.5 mL of IX PBS, which made a 4.2% or 2X glutaraldehyde solution. This solution was then filtered through a 0.22μm filter to remove any particulate matter. Then 100 μL of the 4.2% solution was added to lOOμL of OMV or bacterial sample to make a 2.1% glutaraldehyde solution. Samples were incubated for 5-10 rain and stored at 4°C until shipped for transmission electron microscopy. Twenty microliters of purified OMVs and bacteria (fixed with glutaraldehyde or unfixed) were placed onto carbon- and Formvar-coated nickel grids, then stained with 2% aqueous uranyl acetate, rinsed, and examined with a JEOL IOOCXII transmission electron microscope (TEM) at the Advanced Microscopy Facility, UVA (Charlottesville, VA).

Results

A growth curve analysis showed that F. novicida begins to enter stationary phase after 24 hr (FIG. 2). When time versus OMV protein concentration over time as measure of OMV production is compared, F. novicida OMVs are formed by 48hrs and F. philomiragia produced OMVs by 36 hr and continued to produce OMVs at 48 hr (FIG. 3).

In one pilot experiment (FIG. 4), the size and shape of OMVs from Francisella sp were measured. Atomic Force Microscopy (AFM) image of Francisella tularensis novicida (formerly F. novicida) bacteria are shown in FIG. 4A. FIG. 4B (left to right) shows AFM images of OMVs generated by Francisella tularensis novicida (formerly F. novicida) and Francisella philomiragia, respectively. The results of these measurements are summarized in Table 1. Table 1: Volume of OMVs.

It should be noted that due to the drying of the sample before AFM is performed, the OMVs have assumed a flattened ovoid shape, much like a water balloon sitting on a flat surface. In order to correct for this distortion, the volume of the observed vesicles from their measured size in 3 dimensions was calculated and compared to the predicted volume of perfect spheres. Thus, using the formula, Volume V=4/3π(abc), where a and b are each half the measured diameter and c is half the measured height of each vesicle, the volume of each measured OMV was calculated, and then its "perfect sphere equivalent" diameter (i.e., the diameter of a sphere of equivalent volume) was determined. OMVs are reported to have diameters between 50 and 250 nm. Measuring 20 OMVs prepared from Francisella novicida, we calculated a median diameter of 121 nm for the perfect sphere equivalent. Measuring 20 different OMVs prepared from Francisella philomiragia, the median diameter was calculated to be 95 nm for the perfect sphere equivalent. It is not certain whether there is a statistically significant difference between the OMVs of F. novicida and F. philomiragia These studies would be facilitated by additional samples of OMVs from these two different species.

In a second experiment AFM showed F. novicida OMVs had an average diameter of 47 nm (FIG. 5a) (Table A - Appendix A). AFM of the colonies from Chocolate II agar revealed that OMVs are blebbing from the surface, and highly encapsulated (FIG. 5b). However, when compared with the 24 hr bacterial cell from liquid culture, this technique revealed that the bacterium has a coccobacillus shape and slightly encapsulated (FIG. 5c).

Moreover, the TEM - negative staining technique revealed that the OMVs were spherical bilayers. F, novicida OMVs had an average diameter of 77 nm (FIG. 6a) and F. philomiragia OMVs had an average of diameter of 70 nm (FIG. 6b and Table A - Appendix B). In addition to isolating OMVs, a fixed F. novicida bacterium from Chocolate II agar plates showed an OMV protruding off its surface (Fig. 6c).

Example 2 (Activity analysis)

Hemolysis Assay. The method used to detect hemolytic activity was adapted from Bergmann et al. (1989). A 2% horse erythrocyte suspension (Hemasource Inc.,) in IX PBS (Cellgro) was used for this assay. Fifty microliters of OMV sample were added to 50μL of erythrocyte suspension, and the sample incubated at 37°C for 60 min. Controls included the 2% erythrocyte solution alone with PBS, and 2% erythrocyte solution with 50μL of ddH^O for complete lysis. Unlysed erythrocytes were removed by centrifugation (2 min at 1000 x g). Then, absorbance of the supernatants was read at 540 nm. The formula to calculate % hemolysis was as follows: ((absorbance of sample-absorbance of no hemolysis) / (Absorbance of total hemolysis -absorbance of no hemolysis)) X 100.

Francisella OMVs showed hemolytic activity with an equal protein concentration (64 μg/μL). F. noυicida OMVs showed 34.5% hemolytic activity and F. philomiragia OMV s showed hemolytic activity of 39.3% (see FIG. 8).

Acid Phosphatase (Acp) Activity. The method used to detect acid phosphatase activity was adapted from Bergmann et al. (1989). Fifty microliters of OMVs were added to 50 μL of 0.1 M sodium acetate buffer (pH 5.5) containing 10 niM p- nitrophenylphosphate (p-NPP). The suspension was incubated for 1 h at 37⁰C, and the reaction was stopped with 50 μL of 3 N sodium hydroxide (NaOH). The production of p-nitrophenol (pNP) was monitored at 405 nm. Potato acid phosphatase (Sigma) was used as a positive control and pNPP reagent was the negative control. The acid phosphatase activity was calculated by plotting a standard curve and using y=mx+b to determine the number of Units (U) of AcpA in the OMVs.

OMVs from Francisella ssp. exhibited acid phosphatase activity. This assay revealed that F. noυicida OMVs showed 3.68xlO"⁵ U of activity, and F. philomiragia OMVs showed 4.14xlO⁴ U of activity compared with the positive control (See FIG. 7). These results were based on OMV samples with equivalent protein concentrations of 86 μg/μL of OMV protein.

Phospholipase C (PLC) Activity. The method used to detect phospholipase C activity was adapted from Bergmann et al. 1989. Ten microliters of OMV sample was added to 90 μL of reagent. The reagent consisted of 0.25 M PBS buffer, pH 7.2, 1.0 μM ZnCl2, 10 m.M O- (4-nitrophenylphosphoryl) choline (p-NPPC) in a microtiter plate. Following incubation for 1 hr at 37⁰C, 50 μL of 3N NaOH was added to stop the reaction and the sample absorbances were read at 405 nm. The positive control for the experiment was phospholipase C from Clostridium perfringens (Sigma) and the negative control was PBS alone. The phospholipase C activity was calculated by plotting a standard curve and using y=mx+b to determine the number of U of PLC in the OMVs.

Francisella OMVs showed low PLC activity at an equal protein concentration (66 μg/μL). The PLC activity of Fn OMVs exhibited 2.66xlO"⁴ U and Fp OMVs exhibited about 1.37xlO-² U of PLC activity (Figure 9).

Cytopathic Effect of OMVs on a Monolayer of Cells. A lactate dehydrogenase (LDH) release assay (Promega CytoTox96® Non- Radioactive Cytotoxicity Assay), was carried out following the manufacturer's instructions to determine the cytopathic effects of OMVs on a monolayer of cells. A murine macrophage (J774A.1, ATCC TIB-67) cell line was grown to a monolayer in a 96-well round bottom microtiter plate with Dulbecco's Minimal Essential Media (DMEM) for 24 hrs. The next day, the DMEM was removed and PBS was added to all cells. Next, 50 μL of OMV sample were added to the monolayer of cells and the plate was incubated for 45 min at 37⁰C. After the 45 min incubation, the plate was centrifuged at 1200 x g. The supernantants were collected and substrate/ assay buffer was added to the supernatants and incubated for 30 min at room temperature (Note: Aluminum foil was used to cover the plate as the buffer is sensitive to light). The reaction was stopped with stop solution (1 M acetic acid) and the absorbance was measured at 490 nm. The formula for percent cytoxicity was: ((absorbance of OMVs plus cells - absorbance of non lysed cells - absorbance of OMVs alone) /(absorbance of lysed cells - non lysed cells)) X 100. The LDH assay was a correlate for Francisella- induced apoptosis in macrophages as shown by Lai et al. (2001).

Results of cytopathic effect of OMV are displayed in FIG. 10.

Example 3 (Biochemical characterization of OMV)

Presence of DNA in OMVs. To determine the presence of DNA in OMVs, OMVs were treated with phenol: chloroform to extract DNA (Wolf et al., 2006). Four hundred and sixty seven microliters of 1 X Tris-EDTA (TE) Buffer, pH 8.0 (Fluka) were added to the OMV pellet. Next, 30 μL of 10% (w/v) sodium dodecyl sulfate (SDS) solution and 3 μL of 20 mg/mL of proteinase K (Sigma) was added to the OMV sample and the sample incubated for 1 hr for 37⁰C. An equal volume of buffered-saturated phenol (Invitrogen) : chloroform (Sigma) (1: 1) was added to the OMV/DNA solution and then the tube was vortexed for 10 s. The OMV/DNA sample was centrifuged in a microfuge for 3 min at maximum speed. The aqueous layer was carefully removed and transferred to a new tube, being careful to avoid the interface between the two layers. To remove traces of phenol, an equal volume of chloroform was added to the aqueous layer and then spun for 3 minutes at maximum speed. The aqueous layer was added to a new tube and 0.3M sodium acetate (1/ 10 of the volume of the sample) and 100% cold ethanol (EtOH) (2-2.5 the volume of sample) were added and the sample was mixed. Next, the sample was placed on ice for 15- 20 min and then centrifuged at maximum speed in a microfuge for 10-15 min. The supernatant was carefully decanted and 1 niL of 70% EtOH was added, the sample mixed, and briefly centrifuged. The pellet was vacuum-dried and resuspended in 1000 μL of deionized water (Endotoxin-, DNase-, RNase- free) (Cellgro) and stored at -2O⁰C. The samples were placed into a spectrophotometer that calculated the concentration of DNA as well as the A260/A280 ratio to determine DNA purity. The positive control was 80 μg of lambda DNA and the negative control was deionized water. Samples were also run on a 1% agarose gel for visualization.

The A260/A280 ratio was calculated to determine the presence and purity of DNA in OMVs isolated from Francisella. The purity of F. novicida OMV-DNA was 1.7, and the F. philomiragia OMV - DNA was 1.68 (Table 2). The concentration of DNA for F. novicida OMVs was 83.8 ng/μL and for F. philomiragia OMVs, it was 36.55ng/μL (Table 2) in relation to lambda phage DNA (215 ng/μL). DNA ratios less than or equal to 1.6 indicates protein contamination, while a ration between 1.7- 1.8 indicates pure DNA, and greater than 1.8 indicates the presence of RNA.

Table 2. Determining the Purity of unknown DNA from Francisella ssp. OMVs.

Agarose gel electrophoresis was carried out to determine the size of DNA isolated from the OMVs. For both F. novicida OMVs and F. philomiragia OMVs, the DNA fragment was above 10 kbp (FIG. 11). Agarose Gel Electrophoresis. Agarose gel electrophoresis was adapted from Wolf et al. 2006. To prepare for agarose gel electrophoresis, first 1.0 g agarose was added to 10OmL of IX Tris - Acetate Buffer (IM Tris-Acetate and 0.025M EDTA) (TAE) in a flask. The flask was then microwaved until the agarose was dissolved and solution was clear. The solution was cooled to 56°C before pouring and 5μL of ethidium bromide (EtBr: 10mg/mL) (Invitrogen) was added to the gel solution. The comb was placed in the gel tray about 1 inch from one end of the tray and the comb was positioned vertically such that the teeth were about 1-2 mm above the surface of the tray. The gel was poured and solidified in about 20 min at room temperature. The comb was gently removed and the tray was placed in the electrophoresis chamber, and it was covered (just until wells were submerged) with IX TAE buffer. Next, 1 μL of 6X gel loading dye was added for every 5 μL of DNA solution and the samples were mixed. Ten microliters of DNA was added per well and then the samples were electrophoresed at 90-200 volts (V) until dye markers had migrated to an appropriate distance, a distance that is dependent on the size of DNA to be visualized. The gel was viewed in a UV box and then photographed to determine the size of the DNA compared to a DNA ladder (Hyperladder I - Bioline) . The fragments of DNA were cut out for purification.

Polyacrylamide Gel Electrophoresis. OMV samples were treated with 4X Sample Buffer (Boston BioProducts - Tris-HCl (250 mM, pH 6.8), SDS (8%), glycerol (40%), betamercaptoethanol (8%), Bromophenol blue (0.02%)). Whole cell extracts (WCE) of Francisella ssp. were prepared by adding 1-2 colonies into 4X sample buffer. Both OMV and WCE samples heated to 10O⁰C. Fifteen microliters of Precision Plus Protein™ Standards- Kaleidoscope™ (BioRad), 15 μL of OMV samples, 15 μL of Francisella spp. WCEs, and 15 μL of Francisella tularensis antigen (FtAg) (Becton- Dickinson (BD)) were loaded into a 10 well 12% Sodium Dodecyl Sulfate - polyacrylamide gel (SDS-PAGE) (Invitrogen). WCEs and FtAg were used as positive controls. The electrophoresis conditions were 200 V, 400 mA, for 35 min in 1 X Tris glycine-SDS (TGS) Buffer (BioRad). After electrophoresis, gels were transferred for Western blot, silver stained to detect LPS and stained by Coomassie Blue to detect the protein, as discussed below.

Coomassie Brilliant Blue Staining. OMVs were prepared for SDS-PAGE as described earlier, and the gel was stained with Coomassie Brilliant Blue stain (45% methanol, 45% dBbO, 10% acetic acid, 2.5 g/L of Coomassie Brilliant Blue (CBB)) (CBBS) for 30-60 minutes with constant agitation. Then, the gel was destained with a destaining solution (45% methanol, 10% acetic acid) for 60 min or overnight with constant agitation. Deionized water was used to rinse off any destaining solution on the gel. Then, the gel was photographed. This experimental protocol was used to determine whether the OMVs contain any proteins and to determine the size of proteins compared with a standard protein molecular weight ladder.

The Coomassie Blue staining technique revealed that proteins are present in OMVs isolated from Fn and Fph. Fn OMVs showed two major protein bands between 75-50 and 50-37 kDa and Fph OMVs showed protein three bands at 75- 50, 50-37, and 15-20 kDa (Figure 12).

Silver Stain OMV samples were run on SDS-PAGE and then stained with Gel Code^®

I Silver SNAP™ Stain (Pierce, Inc). Following the manufacturer's protocol, the gel was fixed in a solution containing 30% EtOH and 10% acetic acid for 30 min. The gel was washed twice with 10% EtOH and then washed three times with ultrapure water, 5-10 min per wash. Then, the gel was soaked in stain solution for 15-30 min with gentle shaking. Gels were washed in ultrapure water for 30 s to 1 min. Immediately, the gel was transferred to the developing solution and gently shaken until bands were visible (5-30 min). When the desired band intensity was reached, replacing the development solution with 5% acetic acid stopped the reaction.

The silver stain technique demonstrated that Fn OMVs (lane 3) and Fph OMVs (lane 5) have a carbohydrate smear between 75-25 kDa (Figure 13 a-b). There are some distinct bands around 20 kDa and 15 kDa in Fph OMVs.

Western Blot to Detect LPS. Western blot analysis was performed with anti-F. novicida LPS antibody (IPA009, IPA-F. novicidaovS.2 (raw ascites)). OMV samples were transferred from the SDS-PAGE gel to a PVDF membrane (at 100V, 30-40 mA, lhr) and incubated with anti-F. novicida LPS antibody (1:5,000) (primary antibody) in 3% BSA in IX phosphate buffered-saline with 0.1% Tween-20 (PBS-T), treated with an HRP labeled - goat anti -mouse antibody (1:2,000 - 1:5,000) (secondary antibody) (Chemicon), and an Opti-4CN kit from BioRad was used to view bands on the membrane. This technique was used to determine the presence of LPS.

Western blot analysis using an anti-FrαnciseZZα novicida LPS antibody revealed a smear between 25-75 kDa (Figure 14), which shows a repeating characteristic of LPS which can be seen in the experiment performed by Cowley et al. (2000). Also there are two distinct bands in immunoblot in lane 3, around 15 kDa, and the other above 20 kDa (Figure 14).

FIG. 15 (a) shows a commassie staining of proteins found in Francisella tularensis live vaccine strain (LVS) OMVs. FIG 15 (b) displays an immunoblot of OMVs using anti- Francisella polyclonal serum.

Example 5 (Determination of the components of OMV)

LC/ MS/ MS. LC/MS/MS is a technique that first measures the masses of the intact species eluting from the high performance liquid chromatography while the second scan fragments the peptides and measures the masses of the fragments, which are then analyzed to give the sequence of the peptide. This peptide sequence data is then compared to databases of protein sequences to make the appropriate protein identification. Protein bands were excised from the SDS-PAGE gel and digested with trypsin according to published procedures by Shevcheko et al. 1996. Tryptic peptides were analyzed by liquid chromatography nanospray tandem mass spectrometry using a Thermo Electron LTQ. Separations were performed using 100 μm i.d. x 10 cm long fused silica capillary columns packed in-house with 5 μm C 18 resin. The mass spectrometer was operated in a data-dependent MS/MS mode in which each full MS scan was followed by five MS/MS scans with dynamic exclusion. Tandem mass spectra were searched against the genome of F. tularensis Shu4 strain using tryptic cleavage constraints. For a peptide to be considered legitimately identified, it had to achieve cross correlation scores of 1.5 for [M⁺¹H]¹⁺, 2.0 for [M⁺²H]²⁺, 2.5 for [M⁺³H]³⁺, and a maximum probability of randomized identification of 0. This technique was used to determine the presence of proteins associated with OMVs.

LC/MS/MS revealed that F. novicida OMVs contained 449 different types of proteins (Appendix C), many of which are involved in protein folding, metabolism, and cell structure (Table 3). The more peptide hits a protein had, the more prevalent that protein was found in the sample (Table 3). The protein with the highest peptide hits in F. novicida OMVs was GroEL chaperone protein. Other proteins associated with F. novicida OMVs found by this technique include IgIB, IgIC, IgID, and MgIA (Table 3). Table 3. F. novicida OMV Proteins found by LC/MS/MS. This table shows the different proteins that were extracted from bands on a Coomassie stained SDS- polyacrylamide gel bands. A protein was extracted and was analyzed by LC/MS/MS.

Example 6 (Pharmaceutical compositions)

Outer Membrane Vesicles as a Vaccine against Tularemia.

Preliminary mouse immunization studies to test the efficacy of OMVs as a protective vaccine against Francisella infection will be performed. Intranasal vaccination followed by intranasal challenge will be employed as the first model. Organ bacterial loads will be evaluated. T and B cell responses will be measured. This small scale animal experiment will be performed two to three times with independent preparations of OMVs.

Mouse strain. Specific pathogen-free BALB /c mice will be purchased from Jackson Laboratories and will enter experiments when 8-12 weeks old. Inbred strains of mice are susceptible to infection by the various strains of Francisella, especially C57BL/6 and BALB/c (Rhinehart-Jones et al, 2004, Lopez et al, 2004). BALB/c mice will be selected for this study (Conlan, 2003). BALB/c mice demonstrate T- cell dependent immunity to intranasal vaccination by LVS (Wu et al, 2005). This intranasal immunization protocol used in this study will be followed as the positive control for the vaccine studies. The LD50 for intranasal infection by LVS of BALB/C mice was shown to be between 3000-5000 cfu (Lopez et al, 2004), which is similar to that reported elsewhere as between 1000-1500 cfu (Wu et al, 2005, Fortier et al, 1991)

Animal Vaccination. Intranasal (IN) vaccination with F. tularensis LVS will be used as the control, since IN LVS vaccination is protective against IN LVS challenge in mice (Chen et al, 2003). F. tularensis LVS will be grown in Mueller Hinton (MH) II broth supplemented with 1.2 mM CaCl₂, ImM MgCl₂, 335 nM Fe₄(P2θ₇)3, 0.1% glucose and 2% Isovitalex. Aliquots of bacteria will be prepared in 80% glycerol and frozen at -80°C prior to use. Viable bacterial counts are predicted to be approximately 5 x 10⁹ cfu/ml. Actual CFUs will be determined by serial dilution and plating as part of each experiment. Following Wu et al (Wu et al, 2005), BALB/c mice will be vaccinated IN with 2 x 10² CFU LVS, and by day 14 following vaccination, the mice will be clear of LVS bacteria and will have immunity against IN LVS challenge.

Vaccination with OMVs will also be performed IN. OMVs will be prepared for immunization from F. tularensis LVS bacteria grown overnight in TSB-C or supplemented MH broth. Purified OMVs will be pooled and split into aliquots such that the total protein concentration is approximately 250 μg protein per 50 μl. 50 μl aliquots will be prepared, using PBS to increase volume or dilute as necessary. Three different doses of OMVs will be tested in the first experiment (dose 1 = 250 μg/50 μl, dose 2= 125 μg /50 μl, dose 3 = 62.5 μg/50 μl) to establish the correct range for additional studies.

Challenge. Two weeks following vaccination, mice will be subjected to IN challenge with 10 LD50s of F. tularensis LVS from which the OMVs were prepared. Control mice will be included to verify the susceptibility of the mice to the Francisella LVS strain used. Sufficient numbers of mice will be included to generate statistically significant data in control and test groups (see calculations below). Following the challenge of the mice, the mice will be monitored for their survival for 14 days.

As an example of one experiment, the table below outlines an experiment immunizing mice with three different doses of OMVs (dose 1 = highest, dose 3 = lowest), and challenging with 10 LD50s of Franάsella LVS. The maximum probable adverse events (death) were estimated to be 1-2 mice (~20%) for unexplained reasons per group. Based on these predictions, a simple power computation was performed (see Sokahl and Rohlf, 1995, pg 768-9 for an overview on power computations) to determine how many subjects would be needed in each group for each comparison to detect a significant difference in expected survival rates. Computations were performed at a significance level of alpha=0.05, and power of (l-beta)=0.90.

Expected survival rates in each group were predicted based on a "best-case" scenario, and are outlined in the table below. In the "worst case" scenario, all the OMV vaccinate mice would die, and there would be a statistically significant difference between them and the control LVS vaccinated mice that could be easily determined by n=10 mice per group. The simple power computation was performed to determine how ' many subjects would be needed in each group for each comparison to detect a significant difference in expected survival rates compared to either 100 % survival or 0% survival. The higher number of animals was chosen.

* In groups where the statistical analysis predicted that less than 10 mice could be used, we will actually use 10 mice in case the actual outcome differs from the expected outcome.

Attached hereto as an Appendix are the results of AFM, TEM and LC/MS/MS analysis of outer membrane vesicles of Francisela spp. The Appendix is hereby incorporated by reference in its entirety. The entire disclosure of all applications, patents, and publications, cited herein and the U.S. Provisional Application Ser. No. 60/726,238, filed October 14, 2005 is incorporated by reference herein.

Pathology and hematology. Mice will be euthanized by CO2 asphyxiation. Necropsy will be performed at various times post exposure. Reticuloendothelial organs (liver, lung, spleen) will be removed and used for quantitation of bacterial load from 3 animals of each group. Organs will be removed aseptically and homogenized in PBS. 50 μl of the organ homogenate (and further dilutions as necessary) will be plated onto cysteine heart agar with 5% horse blood, grown for 48 hours, and counted to determine actual CFU. The organs from another 3 animals of each group will be immediately fixed (without perfusion) in 10% neutral buffered formalin, and then embedded in paraffin for HE (haematoxylin-eosin) staining by the contract facility. Cardiac blood will be collected and subjected to smears, staining with LeukoStat, and differential counts.

T and B cell assays. Both the quality and quantity of the antibody response will be measured. The total antibody production profile will be determined as well as the isotype class of antibodies will be determined 14 days post vaccination. Primarily, the amount of IgA as well as other classes of IgG that are produced will be determined. Antibodies will be tested for bactericidal activity. T-cell activation assays will be performed following standard procedures. Briefly, T cell activation will be measured by obtaining splenocytes, activating with OMV antigen and measuring proliferation. Cells secreting IFNγ in an Ag-specific manner may also be detected using an IFNγ enzyme-linked immunospot (ELISPOT) assay to measure T cell activation. In future studies, once it has been established whether OMVs induce protective immunity, a more detailed study of the immune response will be performed.

If positive results are obtained in these preliminary studies, this work will be extended in future proposals to the fully virulent strains of Francisella and their OMVs delivered by aerosol under BSL-3 conditions. The fully virulent F. tularensis Schu4 strain will be grown and OMVs prepared under BSL-3 conditions. Future experiments will include aerosol delivery and challenge in place of IN. Protection in mice will be tested via aerosol or IN OMVs against different routes of Francisella infection such as IP, ID, etc. Also, the potential to use OMVs in a prime-boost regimine if initial protection is low will be examined.

Example 7 (Isolation, purification, and analysis of OMV from Yersinia)

Bacteήal Strains and Growth. The bacterial species used are Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis which are frozen and stored at -80⁰C. Bacteria are grown in brain heart infusion broth (C-BHI) (Becton-Dickinson) or Luria-Bertani (LB) agar plates. Cultures are grown overnight with aeration in either LB broth or HI broth depending on the experimental conditions. Bacterial species are streaked onto plates and incubated for 1-2 days depending on species. Isolated colonies are used to observe bacteria and presence of OMVs by AFM and TEM, as previously described. Cultured bacterial plates are wrapped with parafilm and then stored at 4⁰C until needed.

Isolation of OMVs. Thirty to fifty microliters of frozen stock of Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis are transferred to 100 mL of C-BHI. Cultures are grown for 1-2 days to determine the time at which OMVs are produced, using techniques previously described. Broth cultures are centrifuged at 4,000-20,000 x g, and supernatants are harvested. Supernatants are sequentially passed through a filtration system comprising submicron filters. Filtered supernatants are subjected to ultracentrifugation for 1-3 hrs at 50,000-150,000 x g (Ti-70 rotor, Beckman Instruments, Inc.). The supernatant is removed and the pellet, which consists of OMVs is resuspended in IX phosphate buffered saline (PBS) and stored at -80⁰C. After determining the protein concentration of each sample, the volume is adjusted so that each sample had an equivalent concentration of protein.

Bradford Assay. Following the manufacturer's instructions, bovine serum albumin standard (BSA: 2 mg/mL) (Pierce, Inc) is diluted to make a standard curve. The range is from 25 μg/mL to 2000 μg/mL. Five microliters of standards or unknown samples are added to 500 μL of borate saline buffer (BS). Five hundred microliters of Coomassie Blue Better Bradford Reagent (Pierce) is added to each sample. The samples are then read at 595nm and a standard curve is generated. The concentration of protein is calculated using the equation from the standard curve. This method is used to determine protein concentration of Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis OMVs. Determination of OMV Production. Fifty microliters of Y. pestis is inoculated into 35OmL of C-BHI, and then processed for isolation of OMVs (see second method above) at different time points (0, 6, 12, 18, 24 hr, etc). At certain time points, the optical density (O. D.) at 600 nm is read to generate a growth curve. For each time point, the protein concentration of the OMV samples is determined by the Bradford assay to determine at what time point OMVs are produced.

Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) analysis of the outer membrane vesicles of Yersinia are then performed using the techniques previously described.

Using the previously-described methodology, OMVs isolated from Yersinia spp. will be biochemically characterized and its components (for example, proteins, nucleic acids, and lipids) analyzed using routine analytical techniques (for example, gel electrophoresis, LC/MS/MS, etc.).

Example 8 [Yersinia OMVs as a Vaccine against yersinosis and/or plague)

Using the techniques and strategies described previously, the vaccines prepared from outer membrane vesicles of Yersinia spp. (for example, Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis), will be evaluated against yersinosis. Studies will utilize routine in vitro and in vivo assays to determine the efficacy of OMV-derived vaccines, especially compared to other commercially available vaccines against Yersinia spp.

Preliminary mouse immunization studies to test the efficacy of OMVs as a protective vaccine against Yersinia infection will be performed. Intranasal vaccination followed by intranasal challenge will be employed as the first model. Organ bacterial loads will be evaluated. T and B cell responses will be measured. This small scale animal experiment will be performed two to three times with independent preparations of OMVs.

Animal Vaccination. Intranasal (IN) vaccination with Y. pestis will be used as the control. Vaccination with OMVs will also be performed IN. Preparations will be analogous to others described herein. Challenge. About two weeks following vaccination, hosts, e.g., mice will be subjected to IN challenge with Y. pestis from which the OMVs were prepared. Control mice will be included to verify the susceptibility of the mice to the strain used. Sufficient numbers of mice will be included to generate statistically significant data in control and test groups. Following the challenge of the mice, the mice will be monitored for their survival for 14 days.

As an example of one experiment, mice will be immunized with three different doses of OMVs (dose 1 = highest, dose 3 = lowest), and challenging with 10 LD50s of Yersinia, The maximum probable adverse events (death) will be estimated to be, e.g., 1-2 mice (~20%). Based on these predictions, a simple power computation will be performed (see Sokahl and Rohlf, 1995, pg 768-9 for an overview on power computations) to determine how many subjects would be needed in each group for each comparison to detect a significant difference in expected survival rates. Computations will be performed at a significance level of alρha=0.05, and power of (l-beta)=0.90.

Expected survival rates in each group will be predicted based on a "best- case" scenario, and are outlined in the table below. In the "worst case" scenario, all the OMV vaccinate mice would die, and there would be a statistically significant difference between them and the control vaccinated mice that could be easily determined by n=10 mice per group.

Pathology and hematology. Hosts will be euthanized by CO2 asphyxiation. Necropsy will be performed at various times post exposure. Reticuloendothelial organs (liver, lung, spleen) will be removed and used for quantitation of bacterial load from 3 animals of each group. Organs will be removed aseptically and homogenized in PBS. 50 μl of the organ homogenate (and further dilutions as necessary) will be plated onto cysteine heart agar with 5% horse blood, grown for 48 hours, and counted to determine actual CFU. The organs from another 3 animals of each group will be immediately fixed (without perfusion) in 10% neutral buffered formalin, and then embedded in paraffin for HE (haematoxylin-eosin) staining by the contract facility. Cardiac blood will be collected and subjected to smears, staining with LeukoStat, and differential counts.

T and B cell assays. Both the quality and quantity of the antibody response will be measured. The total antibody production profile will be determined as well as the isotype class of antibodies will be determined 14 days post vaccination. Primarily, the amount of IgA as well as other classes of IgG that are produced will be determined. Antibodies will be tested for bactericidal activity. T-cell activation assays will be performed following standard procedures. Briefly, T cell activation will be measured by obtaining splenocytes, activating with OMV antigen and measuring proliferation. Cells secreting IFNγ in an Ag-specific manner may also be detected using an IFNγ enzyme-linked immunospot (ELISPOT) assay to measure T cell activation. In future studies, once it has been established whether OMVs induce protective immunity, a more detailed study of the immune response will be performed.

If positive results are obtained in these preliminary studies, this work will be extended in future proposals to the fully virulent strains and their OMVs delivered by aerosol under BSL-3 conditions. Future experiments will include aerosol delivery and challenge in place of IN. Protection in hosts such as mice will be tested via aerosol or IN OMVs against different routes of Yersinia infection such as IP, ID, etc. Also, the potential to use OMVs in a prime-boost regimine if initial protection is low will be examined.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/ or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

APPENDIX

Appendix A AFM Measurements

Table 4: AFM Measurements.

Appendix B TEM Measurements

Table 5. TEM Measurements of OMVs from Francisella ssp.

Appendix C LC/MS/MS Analysis on F. novicida OMVs

Table 6. LC/MS/MS Analysis to determine Presence of Proteins associated with F. novicida OMVs.

Protein Probability MW Accession Peptide Hits hypothetical protein FTT0045 [Francisella tularensis subsp.

1 tularensis 8.49E-04 7455.3 56707225 1 5OS ribosomal protein L30 [Francisella tularensis subsp. tularensis SC 1.44E-11 6866.7 56707496 3 hypothetical protein FTTIlOO

[Francisella tularensis subsp. tularensis 7.62E-06 12549.5 56708182 1 hypothetical protein FTTl 153c [Francisella tularensis subsp. tularensi7.94E-04 8385.3 56708224 1 intracellular growth locus, subunit B [Francisella tularensis subsp. t 2.35E-06 58831.3 56708414 1 5OS ribosomal protein L32 [Francisella tularensis subsp. tularensis SC 4.09E-06 6856.6 56708424 1 Peptidyl-prolyl cis-trans isomerase. [Francisella tularensis subsp. tu 7.77E-15 10234.1 56708512 2 50S ribosomal protein L33 [Francisella tularensis subsp. tularensis SC 2.89E-04 6137.3 56708624 1 Glu-tRNAGln amidotransferase C subunit [Francisella tularensis subsp. 1.34E-08 10842.5 56707199 1 Glutaredoxin-related protein

[Francisella tularensis subsp.

10 tularensis 3.88E-06 12303.3 56707246 1 3OS ribosomal protein S 16 [Francisella

11 tularensis subsp. tularensis SC 6.56E-04 9076.1 56707320 1 3OS ribosomal protein SlO [Francisella

12 tularensis subsp. tularensis SC 5.85E-07 11916.6 56707477 4 3OS ribosomal protein S8 [Francisella

13 tularensis subsp. tularensis SCH 2.06E-04 14387.5 56707492 1 cold shock protein [Francisella

14 tularensis subsp. tularensis SCHU S4] 7.66E-12 7380.7 56707540 1 chorismate mutase [Francisella

15 tularensis subsp. tularensis SCHU S4] 5.57E-05 13521.2 56707943 1 dihydroneopterin aldolase [Francisella

16 tularensis subsp. tularensis SC 4.70E-04 13739.9 56708040 1 Thioredoxin 1 [Francisella tularensis

17 subsp. tularensis SCHU S4] 2.31E-07 12164.4 56708068 2 hypothetical protein FTTl 184c

18 [Francisella tularensis subsp. tularensi 1.50E-04 11788.4 56708250 1 Sigma-54 modulation protein

[Francisella tularensis subsp.

19 tularensis 2.93E-12 11174.7 56708341 3 histidine triad (HIT) family protein 0 [Francisella tularensis subsp. tu 1.13E-10 12491.6 56708358 1 outer membrane lipoprotein

[Francisella tularensis subsp. 1 tularensis S 1.81E-04 15836.1 56707373 1 deoxyuridine 5' -triphosphate nucleotidohydrolase [Francisella 2 tularens 5.76E-08 15932.3 56707472 2 3-dehydroquinate dehydratase [Francisella tularensis subsp. tularensis 1.33E-14 16319.5 56707613 4 hesB family protein [Francisella tularensis subsp. tularensis SCHU S4] 2.30E-08 13162.4 56707707 3 hypothetical protein FTT0970

[Francisella tularensis subsp. tularensis 7.22E-07 14936.9 56708063 1 hypothetical protein FTT 1041

[Francisella tularensis subsp. tularensis 2.03E-04 16067.2 56708128 1 hypothetical protein FTT 1045c [Francisella tularensis subsp. tularensi2.23E-04 16124.4 56708132 1 preprotein translocase family protein [Francisella tularensis subsp. t 8.69E-04 12857.1 56708194 1 hypothetical protein FTTl 140

[Francisella tularensis subsp. tularensis 8.84E-04 7158.6 56708214 1 N utilisation substance protein B [Francisella tularensis subsp. tular 6.83E-08 16410.2 56708465 2 Thioredoxin [Francisella tularensis subsp. tularensis SCHU S4] 2.37E-06 12031.0 56708487 2 outer membrane lipoprotein

[Francisella tularensis subsp. tularensis S 9.27E-04 13388.9 56708690 1 ferric uptake regulation protein [Francisella tularensis subsp. tulare 2.62E-08 16136.2 56707210 2 ATP synthase epsilon chain

[Francisella tularensis subsp. tularensis S 8.11E-05 15757.2 56707244 2 Zinc-binding domain protein

[Francisella tularensis subsp. tularensis 2.11E-04 17146.8 56707349 1 outer membrane lipoprotein

[Francisella tularensis subsp. tularensis S 1.14E-06 17996.2 56707363 1 3OS ribosomal protein SI l [Francisella tularensis subsp. tularensis SC 6.63E-12 13743.4 56707501 3 heat shock protein 15 (HSP 15) [Francisella tularensis subsp. tularensi 5.13E-05 14257.8 56707854 1 transcription regulator [Francisella tularensis subsp. tularensis SCHU 5.80E-04 48308.7 56708159 1 hypothetical protein FTT1639c [Francisella tularensis subsp. tularensi4.90E-04 20494.6 56708656 1 preprotein translocase, subunit B, chaperone protein [Francisella tula 9.22E-11 16889.4 56708747 2 NADH dehydrogenase I, E subunit [Francisella tularensis subsp. tularen 8.89E-06 18159.2 56707215 2 NADH dehydrogenase subunit I

[Francisella tularensis subsp. tularensis 8.63E- 10 18849.5 56707219 4 N-acetylmuramoyl-L-alanine amidase [Francisella tularensis subsp. tula 2.66E-06 19783.8 56707332 2 hypothetical protein FTT0667

[Francisella tularensis subsp. tularensis 1.59E-04 19068.7 56707788 2 hypothetical protein FTT0704

[Francisella tularensis subsp. tularensis 6.43E-08 20961.6 56707824 1 glycine cleavage system protein H [Francisella tularensis subsp. tular 9.46E-04 14500.2 56707923 1 transcriptional elongation factor [Francisella tularensis subsp. tular 2.74E-09 17692.2 56708370 2 hypothetical protein FTT 1347

[Francisella tularensis subsp. tularensis 7.17E- 13 17491.2 56708403 3 Acetyltransferase [Francisella tularensis subsp. tularensis SCHU S4] 9.38E-08 18935.7 56708433 1 Integrase/recombinase XerC

[Francisella tularensis subsp. tularensis S 8.14E-04 33472.8 56708541 1 hypothetical protein FTT 1540c [Francisella tularensis subsp. tularensi 2.60E- 10 22455.4 56708572 2 hypothetical protein FTT1550

[Francisella tularensis subsp. tularensis 1.25E-09 20329.3 56708578 1 peptide deformylase [Francisella tularensis subsp. tularensis SCHU S4] 8.32E-04 19811.3 56708685 1 transcription antitermination protein nusG [Francisella tularensis sub 3.33E-15 20027.7 56707309 3 Hypoxanthine-guanine phosphoribosyltransferase [Francisella tularensis 5.00E- 10 20297.6 56707369 2 glycine cleavage system H protein [Francisella tularensis subsp. tular 1.57E-10 13902.0 56707557 1 translation initiation factor IF- 3 [Francisella tularensis subsp. tula 3.63E-12 17557.3 56707929 2 deoxycytidine triphosphate deaminase [Francisella tularensis subsp. tu 8.87E-09 21110.5 56708085 1 3-deoxy-D-manno-octulosonate 8- phosphate phosphatase [Francisella tula 4.92E-08 20075.6 56708116 1 Type IV pili lipoprotein. [Francisella tularensis subsp. tularensis SC 2.65E-04 22992.3 56708228 1 5-formyltetrahydroformate cycloligase family protein [Francisella tula 6.50E-05 21275.3 56708282 1 NH (3) -dependent NAD(+) synthetase [Francisella tularensis subsp. tular 9.27E-04 28068.1 56708321 1 macrophage growth locus, subunit A [Francisella tularensis subsp. tula 1.07E-05 23606.8 56708335 1 hypothetical protein FTTl 506

[Francisella tularensis subsp. tularensis 9.66E-06 21969.3 56708544 1 hypothetical membrane protein [Francisella tularensis subsp. tularensi 7.86E- 11 22482.0 56708593 1 lipoprotein releasing system, subunit A, outer membrane lipoproteins c 8.66E-04 23481.1 56708653 1 Ferredoxin [Francisella tularensis subsp. tularensis SCHU S4] 5.62E-07 12046.4 56708759 1 hypothetical protein FTTOO 14c [Francisella tularensis subsp. tularensi 3.32E-07 14592.2 56707194 1 trp repressor binding protein [Francisella tularensis subsp. tularensi 6.4 IE- 13 21240.7 56707374 3 peptide deformylase [Francisella tularensis subsp. tularensis SCHU S4] 4.49E-07 24260.1 56707552 2 orotate phosphoribosyltransferase [Francisella tularensis subsp. tular 2.13E-07 23329.2 56707581 3 Glutaredoxin 2 [Francisella tularensis 9.90E-04 25166.8 56707772 1 subsp. tularensis SCHU S4] peptidyl-tRNA hydrolase [Francisella

74 tularensis subsp. tularensis SCHU 5.00E-06 21124.0 56707800 1 Recombination protein recR

[Francisella tularensis subsp.

75 tularensis S 9.66E-04 22005.6 56707920 1 hypothetical protein FTTlO 15

[Francisella tularensis subsp.

76 tularensis 3.89E- 14 22442.9 56708104 7 isochorismatase hydrolase family

77 protein [Francisella tularensis subsp 9.18E-06 21318.8 56708195 2 tRNA-(ms(2)io(6)a)-hydroxylase

78 [Francisella tularensis subsp. tularens 1.61 E-05 23699.3 56708382 1 hypothetical protein FTT 1370

[Francisella tularensis subsp.

79 tularensis 9.52E-08 19557.0 56708423 3 hypothetical lipoprotein [Francisella

80 tularensis subsp. tularensis SCH 7.73E-06 12163.9 56708622 1 hypothetical protein FTT 1651

[Francisella tularensis subsp.

81 tularensis 6.66E-15 23084.0 56708663 2 phosphoheptose isomerase [Francisella

82 tularensis subsp. tularensis SCH 8.34E-07 21043.5 56708691 4 NADH dehydrogenase [Francisella

83 tularensis subsp. tularensis SCHU S4] 1.07E-09 24974.8 56708699 2 adenine phosphoribosyltransferase

84 [Francisella tularensis subsp. tular 5.68E-04 18783.1 56707257 2 Deoxyribose-phosphate aldolase

85 [Francisella tularensis subsp. tularens 1.20E-05 27597.3 56707288 1 elongation factor P [Francisella

86 tularensis subsp. tularensis SCHU S4] 2.55E-04 20889.6 56707386 1 Oxygen-insensitive NAD(P)H nitroreductase [Francisella tularensis

87 subs 3.17E-06 24780.9 56707457 2 uridylate kinase [Francisella tularensis

88 subsp. tularensis SCHU S4] 8.92E- 10 26642.8 56707468 2 undecaprenyl pyrophosphate synthetase [Francisella tularensis

89 subsp. t 7.04E-11 29687.3 56707470 2 3OS ribosomal protein S3 [Francisella

90 tularensis subsp. tularensis SCH 6.16E-11 24838.5 56707484 2 phenol hydroxylase [Francisella

91 tularensis subsp. tularensis SCHU S4] 1.23E-04 27695.2 56707516 1 hypothetical protein FTT0484

[Francisella tularensis subsp.

92 tularensis 6.22E-11 25666.6 56707626 3 hypothetical protein FTT0485

[Francisella tularensis subsp.

93 tularensis 1.23E-07 25037.5 56707627 1 CutC family protein [Francisella

94 tularensis subsp. tularensis SCHU S4] 1.14E-04 26390.8 56707635 1 cytidylate kinase [Francisella

95 tularensis subsp. tularensis SCHU S4] 5.69E-09 24856.2 56707689 1 hypothetical protein FTT0571

[Francisella tularensis subsp.

96 tularensis 7.43E-11 27330.0 56707700 2 hypothetical protein FTT0596c

97 [Francisella tularensis subsp. tularensi 5.79E-09 24054.1 56707723 5

98 DJ-1/PfpI family protein [Francisella 1.30E-08 23677.0 56707776 2 tularensis subsp. tularensis SCH holliday junction DNA helicase,

99 subunit A [Francisella tularensis subs 7.84E -04 23693.5 56707779 1 2-dehydro-3-deoxyphosphooctonate

100 aldolase [Francisella tularensis subs 6.99E-04 30962.656707821 1 2-C-methyl-D-erythritol 4-phosphate

101 cytidylyltransferase [Francisella 7.33E-05 25906.356707830 1 MutT protein [Francisella tularensis

102 subsp. tularensis SCHU S4] 8.86E-04 25368.956707934 1 dethiobiotin synthetase [Francisella

103 tularensis subsp. tularensis SCHU 9.97E-06 25173.156708031 1 ThiJ/PfpI family protein [Francisella

104 tularensis subsp. tularensis SCH 3.21 E- 11 24227.056708056 4 adenylate kinase [Francisella

105 tularensis subsp. tularensis SCHU S4] 4.74E-09 24346.956708232 1 Oxidoreductase, short-chain dehydrogenase family protein

106 [Francisella 9.90E-08 25972.5 56708264 5

ABC transporter, ATP-binding protein

107 [Francisella tularensis subsp. tu 8.14E-11 28899.1 56708310 2 Chaperone protein grpE (heat shock

108 protein family 70 cofactor) [Franci 2.70E-04 22038.3 56708330 1 3-methyl-2-oxobutanoate hydroxymethyltransferase [Francisella

109 tularens 2.21E-04 28717.556708438 1 conservered hypothetical protein

110 [Francisella tularensis subsp. tulare 3.41E-04 25285.756708471 1 hypothetical protein FTT 1686c

111 [Francisella tularensis subsp. tularensi5.32E-04 25090.1 56708695 1 tryptophan synthase alpha chain

112 [Francisella tularensis subsp. tularen 7.92E-04 29050.2 56708766 2 peptide methionine sulfoxide reductase

113 msrA [Francisella tularensis su 2.13E-05 26830.2 56708785 1 ATP synthase gamma chain [Francisella tularensis subsp.

114 tularensis SCH 1.00E-08 33222.3 56707242 10 hypothetical protein FTT0086 [Francisella tularensis subsp.

115 tularensis 5.18E-11 33294.7 56707264 2

5OS ribosomal protein Ll [Francisella

116 tularensis subsp. tularensis SCH 1.18E-08 24481.1 56707311 4 UDP-3-O-[3-hydroxymyristoyl] N- acetylglucosamine deacetylase

117 [Francise 9.62E-06 31970.3 56707354 2 universal stress protein [Francisella

118 tularensis subsp. tularensis SCH 1.37E-05 30168.9 56707402 2 3OS ribosomal protein S2 [Francisella

119 tularensis subsp. tularensis SCH 7.53E-10 26452.9 56707466 3 methionine aminopeptidase [Francisella tularensis subsp.

120 tularensis SC 6.85E-06 28341.5 56707542 1 beta-lactamase [Francisella tularensis

121 subsp. tularensis SCHU S4] 2.53E-12 31962.7 56707736 4 hypothetical protein FTT0663 [Francisella tularensis subsp.

122 tularensis 8.51E-06 32064.2 56707784 3

Enoyl- [acyl-carrier-protein] reductase

123 (NADH) [Francisella tularensis 1.32E-07 27787.156707893 6

124 Cyanophycinase [Francisella tularensis 1.88E- 13 29284.256707913 2 subsp. tularensis SCHU S4] FKBP-type peptidyl-prolyl cis-trans

125 isomerase family protein [Francise 1.01E-09 29308.9 56708130 7 ribosomal large subunit pseudouridine

126 synthase B [Francisella tularens 4.89E-05 31140.6 56708141 2 bifunctional methionine sulfoxide

127 reductase A/B protein [Francisella t 2.70E-10 32589.9 56708184 3 glutamine amidotransferase class-II

128 family protein [Francisella tulare 6.97E-04 30279.5 56708236 1 Fructokinase [Francisella tularensis

129 subsp. tularensis SCHU S4] 2.48E-10 32759.5 56708387 3 hypothetical protein FTT 1343c

130 [Francisella tularensis subsp. tularensi 5.71E-04 30107.0 56708399 1 3-deoxy-manno-octulosonate cytidylyltransferase [Francisella

131 tularensi 6.85E-09 28167.9 56708518 1 hypothetical protein FTT1536c

132 [Francisella tularensis subsp. tularensi 3.96E- 10 34521.5 56708568 5 Pyrroline-5-carboxylate reductase

133 [Francisella tularensis subsp. tular 9.59E-05 30051.6 56708586 1 septum site-determining protein MinD

134 [Francisella tularensis subsp. tu 5.70E-13 30101.8 56708626 3 uroporphyrinogen decarboxylase

135 [Francisella tularensis subsp. tularens 1.56E-06 38850.0 56707226 1 Type IV pili nucleotide-binding protein

136 [Francisella tularensis subsp. 3.86E-05 38008.1 56707266 1 D-alanine— D-alanine ligase B

137 [Francisella tularensis subsp. tularensi 1.45E-05 32751.4 56707350 1 UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase >

138 [Francisell 3.12E-08 37360.5 56707440 2 hypothetical protein FTT0311c

139 [Francisella tularensis subsp. tularensi 5.90E-08 34926.9 56707464 2 elongation factor Ts [Francisella

140 tularensis subsp. tularensis SCHU S4 1.67E-10 30968.5 56707467 7 preprotein translocase SecY

[Francisella tularensis subsp.

141 tularensis 1.45E-04 48432.8 56707498 1 hypothetical protein FTT0483c

142 [Francisella tularensis subsp. tularensi 1.08E-09 33220.2 56707625 1 SIS domain protein [Francisella

143 tularensis subsp. tularensis SCHU S4] 6.27E-07 37222.7 56707726 1 hypothetical protein FTT0655

[Francisella tularensis subsp.

144 tularensis 1.13E-07 26823.3 56707777 1 Glycerol-3-phosphate dehydrogenase

145 [NAD(P)+] [Francisella tularensis s 4.77E-06 36620.1 56707975 1 chorismate synthase [Francisella

146 tularensis subsp. tularensis SCHU S4] 8.67E-08 37996.6 56707980 4 coproporphyrinogen III oxidase

147 [Francisella tularensis subsp. tularens 3.95E-09 35831.7 56708148 1 transaldolase [Francisella tularensis

148 subsp. tularensis SCHU S4] 4.56E-11 35665.9 56708176 11 conserved hypothetical lipoprotein

149 [Francisella tularensis subsp. tula 4.83E-05 36139.3 56708258 1 glycosyl transferase family 8 protein

150 [Francisella tularensis subsp. t 4.07E-07 35233.1 56708299 2 cystathionine beta-synthase (cystein

151 synthase) [Francisella tularensis 8.86E-07 34483.5 56708346 1 glucose kinase [Francisella tularensis

152 subsp. tularensis SCHU S4] 1.07E-08 37568.3 56708354 3 malonyl coA-acyl carrier protein

153 transacylase [Francisella tularensis 1.40E-11 33480.2 56708427 4 NAD dependent epimerase [Francisella

154 tularensis subsp. tularensis SCHU 7.77E-15 36355.0 56708500 4 Adenine-specific methylase, HemK

155 family [Francisella tularensis subsp. 2.86E-05 35751.0 56708636 3 aspartate carbamoyltransferase

156 [Francisella tularensis subsp. tularens 4.46E-09 35051.2 56708676 7 DNA polymerase III, beta chain

157 [Francisella tularensis subsp. tularens 6.78E- 10 41648.4 56707189 8 NADH dehydrogenase beta subunit

158 [Francisella tularensis subsp. tularen 8.34E-04 17246.7 56707212 1 aminomethyltransferase [Francisella

159 tularensis subsp. tularensis SCHU 1.59E-06 39559.3 56707556 4 aspartate-semialdehyde dehydrogenase [Francisella tularensis

160 subsp. tu 2.39E-10 40520.1 56707572 3 phosphoserine aminotransferase

161 [Francisella tularensis subsp. tularens 6.45E- 12 39245.0 56707690 4 selenocysteine lyase [Francisella

162 tularensis subsp. tularensis SCHU S4 1.43E-06 45024.4 56707706 1 3-phosphoshikimate 1- carboxyvinyltransferase [Francisella

163 tularensis s 1.39E-07 46843.8 56707715 1 4-hydroxy-3-methylbut~2-en- 1 -yl

164 diphosphate synthase [Francisella tula 6.57E-04 43823.8 56707732 1 hypothetical protein FTT0632c

165 [Francisella tularensis subsp. tularensi 2.02E-06 44994.3 56707757 3 OmpA family protein [Francisella

166 tularensis subsp. tularensis SCHU S4] 6.29E-05 46725.2 56707940 1 ToIB protein precursor [Francisella

167 tularensis subsp. tularensis SCHU 1.69E-07 48834.9 56707949 5 Glutamate- 1 -semialdehyde-2 , 1 - aminomutase [Francisella tularensis

168 subsp 1.80E-07 47155.7 56708027 1 Aldo/keto reductase [Francisella

169 tularensis subsp. tularensis SCHU S4] 7.73E-06 40760.7 56708044 2 hypothetical protein FTT0980

[Francisella tularensis subsp.

170 tularensis 2.84E-06 47037.8 56708072 3 phenylalanyl-tRNA synthetase alpha

171 subunit [Francisella tularensis sub 8.28E-08 38452.6 56708093 2 aspartate aminotransferase

[Francisella tularensis subsp.

172 tularensis S 5.58E-13 44097.5 56708235 7 hypothetical membrane protein

173 [Francisella tularensis subsp. tularensi 5.27E-04 51013.9 56708300 1 intracellular growth locus, subunit D

174 [Francisella tularensis subsp. t 4.31E-08 46805.7 56708412 1 phosphogylcerate kinase [Francisella

175 tularensis subsp. tularensis SCHU 9.38E-06 41910.5 56708420 2 Aminotransferase [Francisella

176 tularensis subsp. tularensis SCHU S4] 1.02E-06 42191.1 56708460 3 DNA-directed RNA polymerase alpha

177 subunit [Francisella tularensis subs 1.02E-05 35119.4 56708484 3 Galactose- 1-phosphate

178 uridylyltransferase [Francisella 9.66E-05 39565.9 56708515 1 tularensis subs tryptophanyl-tRNA synthetase [Francisella tularensis subsp.

179 tularensis 3.97E-10 37819.7 56708527 5 carbamoyl-phosphate synthase small

180 subunit [Francisella tularensis sub 6.50E-04 43237.1 56708674 1 hypothetical membrane protein

181 [Francisella tularensis subsp. tularensi2.92E-06 37446.2 56708686 2 tryptophan synthase subunit beta

182 [Francisella tularensis subsp. tulare 1.35E-05 43051.3 56708767 1 diaminopimelate decarboxylase

183 [Francisella tularensis subsp. tularensi 6.19E- 11 47368.0 56707207 1 adenylosuccinate synthetase

[Francisella tularensis subsp.

184 tularensis 1.3 IE- 12 46869.5 56707368 5 ATP-dependent protease ATP-binding

185 subunit [Francisella tularensis sub 6.33E-04 46294.8 56707750 2 ATP-dependent protease ATP-binding

186 subunit [Francisella tularensis sub 6.00E-06 51205.4 56707807 1 aspartate aminotransferase

[Francisella tularensis subsp.

187 tularensis S 2.95E-13 44281.8 56707985 5 conserved hypothetical lipoprotein

188 [Francisella tularensis subsp. tula 3.28E-05 38696.3 56708183 2 formate dehydrogenase [Francisella

189 tularensis subsp. tularensis SCHU S 8.15E-04 26583.8 56708707 1 Glutamyl- tRNA(G In) amidotransferase

190 subunit A [Francisella tularensis 6.34E-09 52270.5 56707200 1 aspartyl/ glutamyl- tRNA amidotransferase subunit B

191 [Francisella tularen 7.85E-05 53059.7 56707201 1 ATP synthase subunit A [Francisella

192 tularensis subsp. tularensis SCHU 1.59E-09 55358.0 56707241 9 anaerobic glycerol-3-phosphate dehydrogenase [Francisella tularensis

193 s 2.77E-06 57780.5 56707303 1 UDP-N-acetylglucosamine pyrophosphorylase/ glucosamine- 1 -

194 phosphate N-ac 3.-33E- 14 49625.3 56707536 5 hypothetical membrane protein

195 [Francisella tularensis subsp. tularensi 4.78E-04 44405.2 56708454 1 Cysteinyl-tRNA synthetase [Francisella

196 tularensis subsp. tularensis SC 2.56E-09 53100.6 56708635 3 dihydroorotase [Francisella tularensis

197 subsp. tularensis SCHU S4] 5.43E-13 50333.5 56708672 2 amidophosphoribosyltransferase

198 [Francisella tularensis subsp. tularens 1.96E-08 55314.6 56708727 3 glutamate decarboxylase [Francisella

199 tularensis subsp. tularensis SCHU 4.35E-09 50782.7 56708728 3 outer membrane protein tolC precursor [Francisella tularensis

200 subsp. t 7.11E-04 57220.2 56708730 1 hypothetical protein FTTO 199

[Francisella tularensis subsp.

201 tularensis 3.38E-05 29891.9 56707364 1 bifunctional purine biosynthesis

202 protein [Francisella tularensis subsp 2.36E-09 56184.2 56707367 6 acid phosphatase (precursor)

203 [Francisella tularensis subsp. 1.18E-07 57713.1 56707380 1 tularensis

Glutamate— cysteine ligase [Francisella

204 tularensis subsp. tularensis S 4 50E-06 56871.3 56707518 2 hypothetical membrane protein

205 [Francisella tularensis subsp. tularensi4 18E-04 69761.1 56707644 1 ribonucleotide-diphosphate reductase

206 alpha subunit [Francisella tulare 3 3.11E-04 66559.856707667 1 hypothetical protein FTT0918 [Francisella tularensis subsp.

207 tularensis 4.22E-08 58709.756708019 1 AMP-binding family protein [Francisella tularensis subsp.

208 tularensis S 8.19E-04 52527.6 56708041 1

NADH oxidase [Francisella tularensis

209 subsp. tularensis SCHU S4] 3.49E-07 62359.7 56708472 2 hypothetical protein FTT 1577 [Francisella tularensis subsp.

210 tularensis 5.32E-09 41239.0 56708603 3 fumerate hydratase [Francisella

211 tularensis subsp. tularensis SCHU S4] 1.78E-14 54939.2 56708620 6 soluble lytic murein transglycosylase

212 [Francisella tularensis subsp. t 1.68E-10 76856.9 56707549 5 Arginyl-tRNA synthetase [Francisella

213 tularensis subsp. tularensis SCHU 8.91E-05 65953.7 56707608 1 penicillin binding protein (peptidoglycan synthetase) [Francisella

214 tul 9.80E-04 62659.8 56707817 1 chitin binding protein [Francisella

215 tularensis subsp. tularensis SCHU 3.19E-06 33387.3 56707927 1 hypothetical protein FTT1022c

216 [Francisella tularensis subsp. tularensi δ 8.29E-10 69400.1 56708111 4 GTP binding translational elongation

217 factor Tu and G family protein [F 3.00E-06 67418.2 56708247 2 glucose-inhibited division protein A

218 [Francisella tularensis subsp. tu 1.09E-04 69768.3 56708268 2 GTP pyrophosphokinase [Francisella

219 tularensis subsp. tularensis SCHU S 4.48E-04 74033.6 56708546 1 ABC transporter, ATP-binding protein

220 [Francisella tularensis subsp. tu 9.87E-04 62855.7 56708774 1 NADH dehydrogenase gamma subunit

221 [Francisella tularensis subsp. tulare 1.94E-10 87317.1 56707217 4 DNA topoisomerase IV subunit A

222 [Francisella tularensis subsp. tularens 2.00E-09 84017.3 56707545 1 lysine decarboxylase, inducable

223 [Francisella tularensis subsp. tularen 2.13E-07 81841.0 56707555 2 maltodextrin phosphorylase [Francisella tularensis subsp.

224 tularensis S 2.07E-13 86425.9 56707565 6

DNA-binding, ATP-dependent protease

225 La [Francisella tularensis subsp. 2.74E-05 86161.2 56707751 1 leucyl-tRNA synthetase [Francisella

226 tularensis subsp. tularensis SCHU 3.01E-10 93371.3 56708082 3 Phenylalanyl-tRNA synthetase, beta

227 subunit [Francisella tularensis sub 1.28E-12 88021.8 56708092 2 Chitinase [Francisella tularensis

228 subsp. tularensis SCHU S4] 9.98E-06 65718.3 56708763 1 translation initiation factor IF-2

229 [Francisella tularensis subsp. tula 2.99E-08 92349.0 56707229 4

230 hypothetical protein FTT0066 8.08E-10 105253. 56707245 7 [Francisella tularensis subsp. 7 tularensis

DNA polymerase I [Francisella 100986.

231 tularensis subsp. tularensis SCHU S4] 4 33E-12 5 56707285 9 translocase [Francisella tularensis 103731.

232 subsp. tularensis SCHU S4] 346E-04 8 56707883 1 Fusion protein PurC/PurD [Francisella

233 tularensis subsp. tularensis SCH 9 47E-05 86298.5 56707995 1 Adenosylmethionine-8-amino-7- oxononanoate aminotransferase

234 [Francisell 5. 47E-05 51121.1 56708035 1 DNA excision repair enzyme, subunit A 104964.

235 (UvrABC system protein A), ABC t 146E-05 7 56708369 1 Ribonuclease E [Franciselia tularensis 100642.

236 subsp. tularensis SCHU S4] 186E-05 9 56708289 3 ABC transporter, ATP-binding and

237 membrane protein [Francisella tularen 7 01E-04 62016.4 56708392 1 tRNA/rRNA methyltransferase [Francisella tularensis subsp.

238 tularensis 5 26E-04 23672.4 56707583 1 glycosyl transferase family protein

239 [Francisella tularensis subsp. tul 1 05E-04 37540.6 56707908 1 hypothetical protein FTT0369c

240 [Francisella tularensis subsp. tularensi 1 00E-30 39865.4 56707520 1,6 ,1 Chaperone protein dnaK (heat shock 1,3,2, 23,17,1

241 protein family 70 protein) [Francis 555E-16 69211.856708329 0,3,1,1 cell division protein FtsZ [Francisella

242 tularensis subsp. tularensis S 1 18E-09 39720.2 56707353 1,2 pyruvate dehydrogenase, El component [Francisella tularensis 100165. 1 ? o, ? 137,

243 subsp. tu 1 00E-30 2 56708524 42, 26 ,11,7 Acetyl-CoA carboxylase, biotin

244 carboxylase subunit [Francisella tulare 7 21E-11 49934.9 56707615 1,2 succinate dehydrogenase, catalytic

245 and NAD/flavoprotein subunit [Franc 1 32E-07 65829.0 56707253 1,1 ,1 5OS ribosomal protein L29 [Francisella

246 tularensis subsp. tularensis SC 1 65E-04 7768.3 56707486 1,2 3OS ribosomal protein S 17 [Francisella

247 tularensis subsp. tularensis SC 6 70E-10 9857.2 56707487 1,1 5OS ribosomal protein L31 [Francisella

248 tularensis subsp. tularensis SC 1 99E-10 8099.0 56707517 2,3 glycogen synthase [Francisella

249 tularensis subsp. tularensis SCHU S4] 5 18E-04 53502.0 56707564 1,1 Histone-like protein HU form B

250 [Francisella tularensis subsp. tularens 2 04E-10 9468.1 56707752 2,2 3OS ribosomal protein S21 [Francisella

251 tularensis subsp. tularensis SC 8 78E-07 7835.4 56708125 1,3 3OS ribosomal protein S 18 [Francisella

252 tularensis subsp. tularensis SC 3 69E-04 8346.5 56708146 1,1 5OS ribosomal protein L28 [Francisella

253 tularensis subsp. tularensis SC 2 56E-04 8933.9 56708623 1,1 hypothetical protein FTT 1637c

254 [Francisella tularensis subsp. tularensi 3 96E-05 7987.2 56708654 1,1 5OS ribosomal protein L7/L12 [Francisella tularensis subsp.

255 tularensis 3 84E-08 12839.756707313 1,1,2 5OS ribosomal protein L23 [Francisella

256 tularensis subsp. tularensis SC 2.86E-13 11029.956707480 1,6,2

257 3OS ribosomal protein S 19 [Francisella 4.28E-09 10492.656707482 1,5,1 tularensis subsp. tularensis SC

3OS ribosomal protein S 14 [Francisella

258 tularensis subsp. tularensis SC 5.59E-05 11710.4 56707491 2,2,2 3OS ribosomal protein S 15 [Francisella

259 tularensis subsp. tularensis SC 1.78E-13 10352.7 56707818 2,5, 1 Chaperonin protein, groES [Francisella

260 tularensis subsp. tularensis SC 1.68E-08 10238.4 56708704 1,2,2 hypothetical protein FTT0022

[Francisella tularensis subsp.

261 tularensis 2.78E-14 16131.9 56707202 1,2,6, 1 3OS ribosomal protein S 13 [Francisella

262 tularensis subsp. tularensis SC 1.93E-10 13369.3 56707500 2,2,5,3 5OS ribosomal protein L21 [Francisella

263 tularensis subsp. tularensis SC 1.14E-07 11612.3 56707884 1,1,4, 1 hypothetical protein FTT0902

[Francisella tularensis subsp.

264 tularensis 7.63E-10 17681.3 56708003 2,2,9,8 hypothetical membrane protein

265 [Francisella tularensis subsp. tularensi2.38E-09 13732.1 56708771 1,6,2,3,1 5OS ribosomal protein LI l [Francisella

266 tularensis subsp. tularensis SC 2.10E-13 15246.1 56707310 1,2,1,6, 1 AhpC/TSA family protein [Francisella

267 tularensis subsp. tularensis SCHU 1.51E-11 19685.8 56707687 1, 1,2,1,7,3 conserved hypothetical lipoprotein

268 [Francisella tularensis subsp. tula 1.89E-14 15793.1 56708002 1,1,3,3,7,5, 1 hypothetical protein FTT 1441

[Francisella tularensis subsp . 3,11,10,33,1

269 tularensis 1.00E-30 18495.6 56708483 3,1,2 hypothetical lipoprotein [Francisella 3, 14,3,1,3, 1,

270 tularensis subsp. tularensis SCH 4.90E-10 13018.5 56708211 1 hypothetical membrane protein

271 [Francisella tularensis subsp. tularensi 8.37E-08 14526.5 56708770 1,2,1 3OS ribosomal protein S7 [Francisella

272 tularensis subsp. tularensis SCH 4.22E-11 17796.5 56707475 1,6,7,7,1, 1 elongation factor Tu (EF-Tu)

[Francisella tularensis subsp. 2,1,1,2, 14,1,

273 tularensis 1.49E-12 43377.1 56707307 1,1

3,1,2,2,4,5,2 1,28,14,6,14, Chaperone protein, groEL [Francisella 81,31,23, 10,

274 tularensis subsp. tularensis SCH 1.00E-30 57392.7 56708705 9,8,3 5OS ribosomal protein L20 [Francisella

275 tularensis subsp. tularensis SC 8.03E-06 13310.5 56707931 1,1 Peptidoglycan-associated lipoprotein

276 [Francisella tularensis subsp. tu 4.55E-14 23246.2 56707951 1,1,2,8,8,3 5OS ribosomal protein L16 [Francisella

277 tularensis subsp. tularensis SC 1.46E-08 15674.7 56707485 1,2 phoH-like protein [Francisella

278 tularensis subsp. tularensis SCHU S4] 1.29E-07 36608.0 56707742 1,3 glutamine synthetase [Francisella

279 tularensis subsp. tularensis SCHU S4 3.94E- 10 38260.8 56707361 1,2, 1 deoxyguanosinetriphosphate triphosphohydrolase [Francisella

280 tularensis 2.24E-09 50368.3 56707838 1,2,7 thioredoxin reductase [Francisella

281 tularensis subsp. tularensis SCHU S 6.92E-12 33976.2 56707631 3,1 L-threonine 3-dehydrogenase

[Francisella tularensis subsp.

282 tularensis 3.60E-05 38356.5 56707832 2,1 hypothetical protein FTT0960 [Francisella tularensis subsp.

283 tularensis 1.00E-30 37391.6 56708054 18,1 3-deoxy-7-phosphoheptulonate synthase [Francisella tularensis subsp.

284 t 4.06E-10 40825.2 56708057 6,1 choloylglycine hydrolase family protein

285 [Francisella tularensis subsp. 6.50E-07 42506.9 56708296 1, 1 acetyl-CoA acetyltransferase

[Francisella tularensis subsp.

286 tularensis 1.80E-08 41697.6 56708565 7,3 2-amino-3-ketobutyrate coenzyme A

287 ligase [Francisella tularensis subsp 2.22E-15 43908.3 56707833 10,1,1 transketolase [Francisella tularensis

288 subsp. tularensis SCHU S4] 7.55E-14 73274.8 56708422 1,22,9,2 ATP synthase subunit B [Francisella

289 tularensis subsp. tularensis SCHU 7.77E-15 49769.8 56707243 10,1 Glutamyl-tRNA synthetase [Francisella

290 tularensis subsp. tularensis SCH 7.26E-07 52919.8 56707460 1, 1 glycine dehydrogenase subunit 1

291 [Francisella tularensis subsp. tularen 1.02E-13 49667.2 56707558 2,5 glutathione reductase [Francisella

292 tularensis subsp. tularensis SCHU S 1.08E-10 49486.3 56708049 2,3 3-oxoacyl-[acyl-carrier-protein]

293 synthase II [Francisella tularensis s 2.57E- 12 44052.2 56708430 10,6 fusion product of 3-hydroxacyl-CoA 100465.

294 dehydrogenase and acyl-CoA-binding 3.33E-15 8 56708564 1,5,41, 14,2, 1 GMP synthase (glutamine-hydrolyzing)

295 [Francisella tularensis subsp. tu 1.83E-06 57591.9 56708108 4, 1 phosphoglyceromutase [Francisella

296 tularensis subsp. tularensis SCHU S4 3.05E-08 57562.9 56708385 1,4 Glucose-6-phosphate isomerase

297 [Francisella tularensis subsp. tularensi 3.93E- 10 61114.1 56708372 7,3, 1 aldehyde dehydrogenase [Francisella

298 tularensis subsp. tularensis SCHU 1.11E-15 54435.9 56707682 11,2, 1 L-glutaminase [Francisella tularensis

299 subsp. tularensis SCHU S4] 4.07E-11 57174.7 56707360 2,2 3OS ribosomal protein Sl [Francisella

300 tularensis subsp. tularensis SCH 5.70E-12 61551.3 56707348 2,5 phosphoglucomutase [Francisella

301 tularensis subsp. tularensis SCHU S4] 3.27E-08 59598.5 56707563 1,1 dihydroxy-acid dehydratase

[Francisella tularensis subsp.

302 tularensis S 8.12E-10 58964.8 56707764 8,1 Carbamoyl-phosphate synthase large 120626.

303 chain [Francisella tularensis subsp 1.49E-13 9 56708675 1,3,32,9, 1 heat shock protein 90 [Francisella

304 tularensis subsp. tularensis SCHU S 2.67E-12 72303.1 56707508 20,2 hypothetical protein FTT0989

[Francisella tularensis subsp.

305 tularensis 1.33E-14 73156.6 56708081 13,4 RNA polymerase sigma-70 factor

306 [Francisella tularensis subsp. tularens 1.37E-07 67591.2 56708122 2,2 phosphate acetyltransferase

[Francisella tularensis subsp.

307 tularensis 6.23E-08 77050.9 56708752 1,2 CIpB protein [Francisella tularensis

308 subsp. tularensis SCHU S4] 4.45E-12 95869.3 56708764 1,8,5

309 polyribonucleotide 4.05E-11 75348.3 56707819 6,7,2,1, 1,1 nucleotidyltransferase [Francisella tularensis subs

Glucosamine— fructose-6-phosphate

310 aminotransferase [Francisella tulare 4.87E-07 67402.0 56707537 3,1 Oligopeptidase A [Francisella

311 tularensis subsp. tularensis SCHU S4] 1.33E-04 77137.1 56708000 3,1 Alanyl-tRNA synthetase [Francisella

312 tularensis subsp. tularensis SCHU 1.12E-09 96000.8 56708179 6,5 isocitrate dehydrogenase [Francisella

313 tularensis subsp. tularensis SCH 4.11E-12 83401.5 56708560 10,3 Aminopeptidase N [Francisella

314 tularensis subsp. tularensis SCHU S4] 2.58E-08 98786.0 56708781 4,2 cyanophycin synthetase [Francisella 103874.

315 tularensis subsp. tularensis SCHU 7.17E-11 3 56708207 3,16,2 aconitate hydratase [Francisella 102550.

316 tularensis subsp. tularensis SCHU S4] 1.00E-30 6 56707265 1,26,5,3 Valyl-tRNA synthetase [Francisella 104723.

317 tularensis subsp. tularensis SCHU S 2.22E-15 9 56707452 1,15,2, 1 hypothetical protein FTT 1344

[Francisella tularensis subsp.

318 tularensis 4.87E-13 95294.1 56708400 5,11,2,3 DNA gyrase, subunit A [Francisella

319 tularensis subsp. tularensis SCHU S 1.65E-13 97123.5 56708601 12,8,1,3 elongation factor G (EF-G) [Francisella

320 tularensis subsp. tularensis S 9.99E-15 77648.3 56707476 17,10,2,1,1 DNA topoisomerase I [Francisella

321 tularensis subsp. tularensis SCHU S4] 7.62E- 12 87196.9 56708007 6, 1,1 2-oxoglutarate dehydrogenase El component [Francisella tularensis 105962.

322 subs 2.87E-12 3 56707255 12,3 Isoleucyl-tRNA synthetase [Francisella 106899.

323 tularensis subsp. tularensis SC 1.28E-12 4 56708016 20,4 Exodeoxyribonuclease V beta chain 140905.

324 [Francisella tularensis subsp. tular 2.52E-12 3 56708443 1,14 Exodeoxyribonuclease V gamma chain 125820.

325 [Francisella tularensis subsp. tula 2.27E-07 1 56708446 5,1 DNA-directed RNA polymerase beta 151219.

326 chain [Francisella tularensis subsp. 2.22E-16 7 56707314 1,28,20,7 Multifunctional protein, transcriptional repressor of proline 150171.

327 utilizat 6.28E-12 8 56708221 9,2 ATPase, AAA family [Francisella

328 tularensis subsp. tularensis SCHU S4] 8.03E-04 33999.6 56708485 1,1 DNA-directed RNA polymerase, beta 157318.

329 subunit [Francisella tularensis subs 2.03E-11 3 56707315 30,13,6 hypothetical lipoprotein [Francisella

330 tularensis subsp. tularensis SCH 1.11E-16 72729.5 56707857 3,8,3 phosphoribosylformylglycinamidine 141399.

331 synthase [Francisella tularensis sub 1.96E-12 2 56708726 9,2,2 hypothetical protein FTT0613c

332 [Francisella tularensis subsp. tularensi l.09E-06 15690.1 56707738 3,1 5OS ribosomal protein L24 [Francisella

333 tularensis subsp. tularensis SC 1.17E-10 11527.3 56707489 2,3 50S ribosomal protein L25 [Francisella

334 tularensis subsp. tularensis SC 2.43E-06 10838.9 56707795 3,1 translation initiation inhibitor

335 [Francisella tularensis subsp. tulare 3.41E-11 13744.1 56708394 2,4 hypothetical protein FTT 1346

336 [Francisella tularensis subsp. 1.75E-10 14493.5 56708402 1,3 tularensis conservered hypothetical protein

337 [Francisella tularensis subsp. tulare 7.45E-13 15377.9 56708517 2, 1 50S ribosomal protein L19 [Francisella

338 tularensis subsp. tularensis SC 2.09E-11 13314.2 56707323 1,5,2 host factor I for bacteriophage Q beta

339 replication [Francisella tulare 1.06E-08 12517.2 56707755 2,5,3 3OS ribosomal protein S6 [Francisella

340 tularensis subsp. tularensis SCH 2.54E-10 13045.7 56708147 1,1,2 3OS ribosomal protein S9 [Francisella

341 tularensis subsp. tularensis SCH 8.61E-08 15085.0 56708334 1,2,2 5OS ribosomal protein L17 [Francisella

342 tularensis subsp. tularensis SC 7.73E-10 16743.9 56707504 1,2,1,2 3OS ribosomal protein S5 [Francisella

343 tularensis subsp. tularensis SCH 8.88E-15 17548.3 56707495 1,1, 1,8,1 outer membrane protein OmpH

[Francisella tularensis subsp.

344 tularensis 5.02E-10 18753.7 56708598 1,1,1,6,2 ATP-dependent protease peptidase

345 subunit [Francisella tularensis subsp 3.33E-16 19789.3 56707808 3,1,3,9,7,5,2 SNO glutamine amidotransferase

346 family protein [Francisella tularensis 2.66E-08 20305.6 56707651 2,1, 12,2 hypothetical lipoprotein [Francisella

347 tularensis subsp. tularensis SCH 4.38E-05 15645.2 56707443 1,1 Asparaginase [Francisella tularensis

348 subsp. tularensis SCHU S4] 5.21E-13 30939.5 56707914 1,6 phosphoribosylaminoimidazole carboxylase,catalyic subunit

349 [Francisella 1.00E-30 17184.8 56707997 1,6 nucleoside diphosphate kinase

350 [Francisella tularensis subsp. tularensi 1.00E-30 15517.0 56707524 2,1,5 Ferritin-like protein [Francisella

351 tularensis subsp. tularensis SCHU S 1.82E-13 19013.6 56707774 2,3,5 chorismate mutase [Francisella

352 tularensis subsp. tularensis SCHU S4] 3.62E-13 20762.1 56708662 1,1,11,5 Single-strand binding protein

353 [Francisella tularensis subsp. tularensi 5.79E- 12 17512.5 56708750 1,3, 1,3,1 hypothetical lipoprotein [Francisella

354 tularensis subsp. tularensis SCH 1.00E-12 21034.7 56708083 1,3 glycine dehydrogenase subunit 2

355 [Francisella tularensis subsp. tularen 1.00E-30 52705.1 56707559 1,1,5,17,3, 1 5OS ribosomal protein Ll 8 [Francisella

356 tularensis subsp. tularensis SC 1.50E-07 13028.0 56707494 1, 1 hypothetical protein FTT0364c

357 [Francisella tularensis subsp. tularensi 4.12E-08 16793.4 56707515 1,2 Hypothetical lipoprotein [Francisella

358 tularensis subsp. tularensis SCH 3.86E-04 14873.5 56708463 2,1 preprotein translocase, subunit B,

359 chaperone protein [Francisella tula 3.27E- 10 16688.3 56708539 4, 1 riboflavin synthase beta subunit (6,7-

360 dimethl-8-ribityllumazine syntha 1.28E-12 16279.5 56708684 1,6 5OS ribosomal protein Ll 5 [Francisella

361 tularensis subsp. tularensis SC 1.11E-15 14991.2 56707497 1,4,2 5OS ribosomal protein LlO [Francisella

362 tularensis subsp. tularensis SC 1.12E-05 18707.0 56707312 1,3,5,2 outer membrane protein 26

[Francisella tularensis subsp.

363 tularensis SC 7.31E-12 19773.2 56708574 1,1,4, 13,1, 1

364 3-oxoacyl-(acyl-carrier-protein) 8.98E-11 26340.4 56708428 1,1,3,1,3,8, 1 reductase [Francisella tularensis sub Acetyl-CoA carboxylase, biotin

365 carboxyl carrier protein subunit [Franc 3.73E- 13 16483.7 56707614 2,1,7, 1,2, 1 intracellular growth locus, subunit C

366 [Francisella tularensis subsp. t 2.55E-11 22418.6 56708413 1, 1, 1,6,1 Pyrrolidone-carboxylate peptidase

367 [Francisella tularensis subsp. tular 1.47E-11 24617.9 56707449 2, 1,2, 12,2,1 superoxide dismutase [Fe] [Francisella

368 tularensis subsp. tularensis SC 2.22E-14 21925.9 56707247 2,3,6 short chain dehydrogenase

[Francisella tularensis subsp.

369 tularensis SC 3.18E-09 21691.4 56707688 1,3,3

1, 15,21,30, 1 glutamate dehydrogenase [Francisella 5,11,50,23,2

370 tularensis subsp. tularensis SCHU 1.00E-30 49076.9 56707529 4, 10,4,1,2 methionyl-tRNA synthetase

[Francisella tularensis subsp.

371 tularensis SC 1.21E-06 76552.0 56708349 1,3 methionine sulfoxide reductase B

372 [Francisella tularensis subsp. tulare 1.25E-11 19619.9 56707982 2, 1 50S ribosomal protein L9 [Francisella

373 tularensis subsp. tularensis SCH 8.99E-14 16065.6 56708145 1,6 5OS ribosomal protein L13 [Francisella

374 tularensis subsp. tularensis SC 2.47E-10 16929.1 56708333 1,6 acyl carrier protein [Francisella

375 tularensis subsp. tularensis SCHU S4 3.58E-07 10653.2 56708429 1, 1,2 Gamma-glutamyltranspeptidase

[Francisella tularensis subsp.

376 tularensis 1.33E-14 65018.9 56708248 1,3,2,1,12 cytosol aminopeptidase family protein

377 [Francisella tularensis subsp. t 1.11E-14 52791.8 56708177 1,4,1,1, 13,2 heat shock protein [Francisella

378 tularensis subsp. tularensis SCHU S4] 1.36E- 11 16701.5 56708782 3,2 hypothetical protein FTT1798c

379 [Francisella tularensis subsp. tularensi6.81E-06 14237.1 56708786 1, 1 (3R)-hydroxymyristoyl-(acyl-carrier

380 protein) dehydratase [Francisella 5.38E-12 18138.5 56708596 6,1,7,3,1,2,1 5OS ribosomal protein L6 [Francisella

381 tularensis subsp. tularensis SCH 1.32E-12 19077.4 56707493 3,3,3 hypothetical protein FTT0825c

382 [Francisella tularensis subsp. tularensi l.24E- 10 12219.2 56707936 2,4,2,1 pyridoxine biosynthesis protein 4,6,4,3,29, 13

383 [Francisella tularensis subsp. tularen 1.89E-14 30798.9 56707650 ,5,1 Glyceraldehyde-3-phosphate dehydrogenase [Francisella tularensis 1,4,8,7,4,26,

384 subsp 1.00E-30 37261.3 56708421 9, 1 hypothetical protein FTT0975

[Francisella tularensis subsp. 2,3,3,11,1,3,

385 tularensis 1.05E-11 25714.1 56708067 1 outer membrane associated protein

386 [Francisella tularensis subsp. tular 2.85E-10 41391.3 56707711 1,3, 1,6,3, 1 5OS ribosomal protein L3 [Francisella

387 tularensis subsp. tularensis SCH 5.55E-15 22479.6 56707478 1, 1, 10 Two-component response regulator

388 [Francisella tularensis subsp. tulare 4.98E-12 25489.9 56708585 1,2,11,4 purine nucleoside phosphorylase

389 [Francisella tularensis subsp. tularen 7.96E-12 26874.5 56707880 2,2,14,2 hypothetical protein FTT1614c

390 [Francisella tularensis subsp. tularensi 7.41 E-04 25274.0 56708633 1,1 hypothetical protein FTTl 534c

391 [Francisella tularensis subsp. tularensi6.77E-07 53176.9 56708567 1,1 LemA-like protein [Francisella

392 tularensis subsp. tularensis SCHU S4] 1.56E-09 22002.7 56707969 2,1 5OS ribosomal protein L5 [Francisella

393 tularensis subsp. tularensis SCH 3.47E-08 20013.7 56707490 3,1 ATP-dependent CIp protease subunit P

394 [Francisella tularensis subsp. tu 1.33E-14 22136.4 56707749 8,4 hypothetical protein FTT0903

[Francisella tularensis subsp.

395 tularensis 5.12E-09 19339.3 56708004 3,1 inorganic pyrophosphatase

[Francisella tularensis subsp.

396 tularensis SC 2.16E-09 19588.8 56708117 5,3 hypothetical protein FTT 1240c

397 [Francisella tularensis subsp. tularensi 1.12E-05 21483.6 56708302 2,2 outer membrane protein [Francisella

398 tularensis subsp. tularensis SCHU 8.84E-13 20931.6 56708745 11,3 carbonic anhydrase [Francisella

399 tularensis subsp. tularensis SCHU S4] 5.08E-11 25407.9 56707719 1,4, 10, 1 choloylglycine hydrolase family protein

400 [Francisella tularensis subsp. 3.33E-14 37150.8 56708187 1,3,2, 12, 1 Succinyl-CoA synthetase, alpha

401 subunit [Francisella tularensis subsp. 2.71E-12 30075.7 56707642 3,4,1,2,18,3 lactate dehydrogenase [Francisella 4,2,1,1, 14,3,

402 tularensis subsp. tularensis SCHU S 7.44E-14 34069.0 56707668 2 glycerophosphoryl diester phosphodiesterase family protein

403 [Francisell 1.55E-14 39018.0 56707843 1,2, 1,5,16 hypothetical protein FTT0781c

404 [Francisella tularensis subsp. tularensi 1.59E- 11 26450.6 56707892 1,7 Periplasmic L-asparaginase II precursor [Francisella tularensis

405 subsp. 2.31E-08 38471.6 56707606 1,2,2 delta-aminolevulinic acid dehydratase

406 [Francisella tularensis subsp. t 1.25E-10 35788.1 56707604 1,9, 1 4'-phosphopantothenoylcysteine decarboxylase,phosphopantothenoylcy

407 stei 6.09E-08 43156.7 56708218 1,6 NADH dehydrogenase I [Francisella

408 tularensis subsp. tularensis SCHU S4 2.60E-11 25170.9 56707213 1, 1 GTP cyclohydrolase I [Francisella

409 tularensis subsp. tularensis SCHU S4 9.98E-08 23665.9 56708045 6, 1 transcriptional regulator [Francisella

410 tularensis subsp. tularensis SC 1.73E-11 27904.9 56708441 2,4 conserved hypothetcial protein

411 [Francisella tularensis subsp. tularens 4.19E- 10 25546.6 56708630 3,3 fructose-bisphosphate aldolase

412 [Francisella tularensis subsp. tularens 5.55E-15 38121.1 56708418 1, 1,7,1 D-alanyl-D-alanine carboxypeptidase

413 (Penicillin binding protein) famil 1.00E-30 48069.5 56708118 2, 1,2, 10, 1 3OS ribosomal protein S4 [Francisella

414 tularensis subsp. tularensis SCH 5.03E-10 23222.2 56707502 1,8, 1 succinyl-CoA synthetase subunit beta 5,3,2, 19,3, 1,

415 [Francisella tularensis subsp. tu 2.22E-15 41515.9 56707643 1 DNA-directed RNA polymerase alpha

416 subunit [Francisella tularensis subs 1.00E-30 35335.8 56707503 1,1, 10 Peroxidase/ catalase [Francisella 1,2,1, 13,20,4

417 tularensis subsp. tularensis SCHU S4] 1.33E-14 82448.8 56707839 ,3,2 Inositol- 1 -monophosphatase [Francisella tularensis subsp.

418 tularensis S 1.20E-05 28850.2 56708431 1,1 adenylosuccinate lyase [Francisella

419 tularensis subsp. tularensis SCHU 3.04E-10 49345.4 56707195 1,3,10 Enolase (2-phosphoglycerate dehydratase) [Francisella tularensis

420 subsp 2.19E-13 49506.3 56707828 1,1,3 triosephosphate isomerase [Francisella

421 tularensis subsp. tularensis SC 1.11E-14 27638.4 56707259 6, 1 transcriptional regulator [Francisella

422 tularensis subsp. tularensis SC 4.44E-15 27967.4 56707286 5, 1 Aldolase/ adducin class II family

423 protein [Francisella tularensis subsp 1.00E-30 22707.7 56707786 7,2 Rhodanese-like family protein

424 [Francisella tularensis subsp. tularensi2.94E-08 28219.2 56708204 1,1 uridine phosphorylase [Francisella

425 tularensis subsp. tularensis SCHU S 1.01E-09 29628.9 56708383 4,3 Carbon-nitrogen hydrolase [Francisella

426 tularensis subsp. tularensis SC 6.14E-11 30102.0 56708464 4,3 UDP-N-acetylglucosamine ' acyltransferase [Francisella tularensis

427 subsp. 3.33E-15 28106.6 56708595 5,4 hypothetical protein FTT1693c

428 [Francisella tularensis subsp. tularensi4.47E- 10 29367.4 56708702 1,4 conserved membrane hypothetical

429 protein [Francisella tularensis subsp. 2.11E-14 38374.3 56707276 1, 11,3 dihydrolipoamide dehydrogenase 2,2,3,3, 1,37,

430 [Francisella tularensis subsp. tularens 1.1 IE- 16 50453.4 56708522 11,5,3, 1 citrate synthase [Francisella tularensis

431 subsp. tularensis SCHU S4] 4.44E-15 47367.8 56707250 1,1, 16,1,1 serine hydroxymethyltransferase

432 [Francisella tularensis subsp. tularen 1.11E-16 45287.3 56708303 1,1,4,10,2 pyruvate dehydrogenase, E2 component [Francisella tularensis 1, 1,2,5,3,6,5,

433 subsp. tu 1.11E-15 67211.1 56708523 32,21,33,21 Acetyl-CoA carboxylase beta subunit

434 [Francisella tularensis subsp. tul 8.73E-07 33625.2 56707523 2, 1 Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha

435 [Fran 9.00E-10 35441.4 56708537 4,2 pyruvate kinase [Francisella tularensis

436 subsp. tularensis SCHU S4] 6.62E-13 51703.1 56708419 2,1, 1,28,4 dihydrolipoamide succiny transferase 2,6,3, 1,7,21,

437 component of 2-oxoglutarate dehyd 1.11E-15 52715.5 56707256 7,4,3, 1 Aspartyl-tRNA synthetase [Francisella 3, 1, 1,12,13,2

438 tularensis subsp. tularensis SCH 3.85E-12 66931.4 56707192 , 1 Carbohydrate/ purine kinase pfkB

439 family protein [Francisella tularensis 3.33E-15 40350.7 56707912 1,8 NAD -dependent malic enzyme

[Francisella tularensis subsp.

440 tularensis S 8.79E-13 67387.5 56708018 1, 1,4, 14, 1 UDP-glucose/GDP-mannose dehydrogenase [Francisella tularensis

441 subsp. t 2.49E-13 48835.4 56708501 1,3,4 cytosol aminopeptidase [Francisella

442 tularensis subsp. tularensis SCHU 5.55E-15 51955.4 56708375 2,19, 12, 1,2 hypothetical protein FTTl 539c

443 [Francisella tularensis subsp. tularensi 1.00E-30 52024.5 56708571 1, 1, 15,1,3, 1 seryl-tRNA synthetase [Francisella

444 tularensis subsp. tularensis SCHU S 1.67E-14 48554.9 56708386 1,13,1 soluble pyridine nucleotide transhydrogenase [Francisella

445 tularensis s 3.35E-10 52833.3 56707804 1,6 hypothetical protein FTT 1402c

446 [Francisella tularensis subsp. tularensi l.38E- 13 59851.1 56708450 1,8 trigger factor [Francisella tularensis

447 subsp. tularensis SCHU S4] 1.51E-08 49554.9 56707748 1,4,2 Inosine-5-monophosphate dehydrogenase [Francisella tularensis

448 subsp. t 4.55E-14 52045.4 56708374 1, 16, 1,1 Peptidase, M24 family protein

449 [Francisella tularensis subsp. tularensi9.99E-15 68638.8 56707734 1,6,5

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Claims

What we claim:What we claim is:

Claim 1. An immunogenic composition for the immunization of an individual comprising outer membrane vesicles of Gram-negative bacteria, Francisella or Yersinia spp.

Claim 2. An immunogenic composition according to claim 1 wherein, said Francisella spp is F. tularensis or F. philomiragia

Claim 3. An immunogenic composition according to claim 2 wherein, said F. tularensis is F. tularensis subspecies tularensis (Group A), F. tularensis subspecies holarctica (Group B), or F. tularensis subspecies novicida.

Claim 4. A method for preparing native outer membrane vesicles from Francisella, comprising,

(i) culturing said cells;

(ii) harvesting the supernatant by centrifuging the culture of (i);

(iii) filtering the supernatant of (ii) using a .45μm-, 0.22μm-, and O. lOμm - filtration system; and (iv) ultracentrifuging the filtrate of (iii) .

Claim 5. A vaccine for protection against infection with Gram negative bacteria, Francisella or Yersinia spp, comprising outer membrane vesicles of said bacteria in an amount effective to elicit protective antibodies in an animal to said Gram negative bacteria, and a pharmaceutically acceptable carrier.

Claim 6. A vaccine for protection against infection with Gram negative bacteria according to claim 5 wherein said Francisella spp is F. tularensis or F. philomiragia

Claim 7. A vaccine for protection against infection with Gram negative bacteria according to claim 6 wherein said F. tularensis is F. tularensis subspecies tularensis (Group A), F. tularensis subspecies holarctica (Group B), or F. tularensis subspecies novicida.

Claim 8. A vaccine for protection against infection with Gram negative bacteria according to claim 5 wherein said Yersinia spp is Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis.

Claim 9. A vaccine for protection against Gram negative bacteria according to claim 5, wherein said carrier is adapted for intranasal administration.

Claim 10. Isolated outer membrane vesicles from a strain of Francisella or Yersinia.

Claim 11. Vessicles of claim 10 from a Francisella strain which expresses iron uptake proteins.

Claim 12. A method of preventing infection by Francisella or Yersinia Gram- negative bacteria in an animal comprising administering a vaccine of claim 5.

Claim 13. A method of claim 12 wherein said animal is a human.

Claim 14. A method for reducing Francisella infection symptoms, comprising administering to a patient in need of such treatment an effective amount of antibodies against native outer membrane vesicles of Francisella in a pharmaceutically acceptable excipient.

Claim 15. A method of eliciting an immune response against Francisella comprising administering outer membrane vesicles from Francisella.