Title:
Brucella melitensis mutants and methods
Kind Code:
A1


Abstract:
Certain attenuated mutants of Brucella, especially B. melitensis, B. abortus, B. suis and B. ovis, when administered to a human or animal trigger a protective immune response such that subsequent challenge with virulent Brucella of the same species does not result in disease or results in much less severe symptoms. Functional inactivation of galE, a virB gene or the operon (ORFs 1087-1090) comprising the gene encoding β-hexosaminidase (BMEI1087) and a lytic murein transglycosylase gene (BMEI1088). A specific example of the attenuated galE mutant which produces a protective immune response is B. melitensis GR024. The specific example of an inactivated ORF1087-1090 operon is B. melitensis GR026; it has an insertion mutation in the promoter region upstream of ORF 1090. Vaccination with live cells of either or both of these mutants results in a T cell response which protects the human or animal against challenge with virulent B. melitensis. Similar strategies for protective immunity using live attenuated mutants are useful for B. abortus, B. suis and B. ovis as well.



Inventors:
Rajashekara, Gireesh (Madison, WI, US)
Splitter, Gary (Brooklyn, WI, US)
Application Number:
11/633371
Publication Date:
05/08/2008
Filing Date:
12/01/2006
Primary Class:
Other Classes:
435/7.32, 435/252.3
International Classes:
A61K39/02; C12N1/21; G01N33/554; G01N33/569
View Patent Images:



Primary Examiner:
DUFFY, PATRICIA ANN
Attorney, Agent or Firm:
Leydig, Voit & Mayer, Ltd. (GS BOULDER) (Boulder, CO, US)
Claims:
We claim:

1. An attenuated strain of Brucella in which there is a mutation which functionally inactivates or prevents expression of at least one of the galE and having at least 85% nucleotide sequence identity to SEQ ID NO:28, the gene encoding lytic murein transglycosylase and having at least 85% nucleotide sequence identity to nucleotides 7908-10817 of SEQ ID NO:26, β-hexosaminidase and having at least 85% nucleotide sequence identity with nucleotides 6688-7740 of SEQ ID NO:26, or a gene encoding deoxyguanosinetriphosphate triphosphohydrolase and having at least 85% nucleotide sequence identity with nucleotides 2138-3346 of SEQ ID NO:27.

2. The attenuated strain of claim 1, wherein said mutation is in a Brucella melitensis 16M genetic background and wherein said a mutation which functionally inactivates the galE gene of BMEI1090, lytic murein transglycosylase gene of BME11088 and β-hexosaminidase of BMEI1087 or a gene encoding deoxyguanosinetriphosphate triphosphohydrolase and having at least 85% nucleotide sequence identity with nucleotides 2138-3346 of SEQ ID NO:27.

3. The attenuated strain of claim 1, wherein said strain is a Brucella melitensis in which there is a mutation which functionally inactivates ORF BME10971 or its counterpart in a species of Brucella other than B. melitensis, the ORF BME11090 or its counterpart in a species of Brucella other than B. melitensis, and the operon comprising ORFs BME11090, 1089, 1088 and 1087 or a counterpart operon in a species of Brucella other than B. melitensis.

4. The attenuated strain of Brucella of claim 1, wherein said mutation is a polar insertion mutation.

5. The attenuated strain of Brucella of claim 1, wherein said Brucella is Brucella melitensis.

6. The attenuated mutant strain of Brucella of claim 1, wherein said Brucella is Brucella abortus.

7. The attenuated strain of Brucella melitensis of claim 3, wherein said attenuated mutant is GRO24 or GRO26.

8. The attenuated strain of Brucella of claim 1 in which there is a deletion in the gene encoding deoxyguanosine triphosphate hydrolase.

9. The attenuated strain of claim 1, wherein said mutant strain expresses a listeriolysin O gene from Listeria monocytogenes.

10. An immunogenic composition comprising live cells of at least one attenuated mutant strain of the Brucella of claim 1 and a pharmaceutically acceptable carrier.

11. The immunogenic composition of claim 10, wherein said attenuated mutant strain of Brucella is a Brucella melitensis.

12. The immunogenic composition of claim 11, wherein said attenuated mutant is GR024 or GR026.

13. The immunogenic composition of claim 10 wherein said Brucella is Brucella abortus.

14. A method of protecting a human or animal against infection by administering an effective amount of the immunogenic composition of claim 10.

15. The method of claim 14, wherein said immunogenic composition comprises at least one attenuated mutant strain of Brucella melitensis.

16. The method of claim 13, wherein said attenuated mutant strain is GR024 or GR026.

17. The method of claim 14, wherein said immunogenic composition comprises at least one attenuated mutant strain of Brucella abortus.

18. A method of identifying epitopic peptides of B. melitensis comprising the steps of: (a) infecting macrophage cells in culture with B. melitensis; (b) culturing the macrophage cells infected with B. melitensis; (c) collecting MHC class I-peptide complexes from the cells cultured in step (b); (d) eluting the peptides from the collected complexes of step (c); and (e) characterizing the peptides eluted in step (d).

19. An epitopic peptide identified by the method of claim 18, wherein said peptide is derived from the HdeA protein of B. melitensis.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 60/741,282, filed Dec. 1, 2005, which application is incorporated by reference herein to the extent there is no inconsistency with the present disclosure.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant R01AI048490 awarded by the National Institutes of Health (NIH/NIAID), Grant AI057153 awarded by RCE for Biodefense and Emerging Infectious Diseases Research Program, and Grant 35204-14856 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING COMPACT DISK APPENDIX

The Sequence Listing filed herewith is incorporated by reference.

BACKGROUND OF THE INVENTION

The field of this invention is microbial genetics, especially as related to immunogenic compositions comprising attenuated bacterial pathogens or components thereof.

The Brucella species are important zoonotic pathogens affecting a wide variety of mammals. In agriculturally important domestic animals, these bacteria cause abortion and infertility, and they are of serious economic concern worldwide (5). In humans, Brucella species constitute potential bio-warfare agents. Brucella species that infect humans cause in undulating fever, which if untreated, can manifest as orchitis, osteoarthritis, spondylitis, endocarditis, and neurological disorders (11, 46). Currently there is no vaccine to protect against human brucellosis, especially that caused by B. melitensis. Treatment of brucellosis requires a prolonged combination of antibiotic therapy and is still problematic because of the potential for relapse.

Identifying Brucella virulence factors has been of great interest in understanding Brucella pathogenesis and immune evasion. After entry into macrophages virulent Brucella cells reside in an acidified vacuole, the Brucella containing vacuole (BCV). The BCV transiently interacts with early endosomes, followed by VirB-dependent sustained interaction with the endoplasmic reticulum (7). Thus, the BCV matures into a replicative niche in a VirB-dependent manner (7, 8). VirB proteins forming the type IV secretion system (T4SS) constitute important factors for Brucella virulence and intracellular replication (9, 14, 34). Lipopolysaccharide (LPS) is also an important virulence factor (27). Brucella LPS has minimal endotoxic effect, blocks complement activation, and protects against bactericidal cationic peptides (28). The O-chain is also important for the conventional entry of Brucella into macrophages through lipid rafts, a route which avoids fusion of the BCV with lysosomes (33, 37). Cyclic β-1, 2 glucan has been shown as an important virulence factor required for intracellular survival of Brucella (3). Although T4SS, cyclic β-1, 2 glucan, and LPS are clearly virulence factors of Brucella, the attenuated mutants lacking these virulence factors are either considered not safe or insufficient information is available to use them as vaccines for humans. This has necessitated identification of additional vaccine targets.

Several genetic loci that are required for Brucella replication in vitro have been identified (14, 24). In vitro conditions may not adequately reflect in vivo infection, and therefore, findings may have little or no in vivo relevance (45). In vivo screening methods have been used to identify Brucella genes required for survival and persistence (18, 26), however, these previous studies have relied on the conventional approach of determining tissue-specific cell counts (CFU) from multiple animals at different times, a process that is labor intensive and requires large numbers of animals. Because infection is a dynamic process and varies within individual mice, monitoring disease progression temporally within the same mouse provides a more comprehensive picture of pathogenic events. Further, such real-time analysis may reveal virulence determinants responsible for tissue specific replication of bacteria that would not be revealed using conventional CFU enumeration from liver and spleen.

Bioluminescent imaging of mice allows direct visualization of the infection process and is highly useful for bacterial pathogenesis studies (10), because the intensity of bioluminescence strongly correlates with the number of bacteria in the infected organs (16, 40). Bioluminescent imaging is useful in analyzing sub-acute and chronic infections that are often difficult to assess using conventional approaches because of uncertain bacterial locations (16, 40).

There is a long felt need in the art for safe and effective vaccines that protect humans and animals from infection by the pathogenic Brucella species, especially B. melitensis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides attenuated mutants of Brucella, including Brucella abortus and Brucella melitensis, which are useful in generating protective immunity to infection by virulent Brucella, including Brucella melitensis and Brucella abortus. In particular, mutants in which the galE gene (ORF BMEI0921 or the corresponding gene in other species of Brucella) is inactivated are useful in live vaccine formulations and mutants in which one or more peptidoglycan biosynthetic genes are functionally inactivated, i.e., the genes encoding the lytic murein transglycosylase and/or β-hexosaminidase are inactivated, for example polar mutations in the operon in which these genes are expressed, with the disruption eliminating all, four or three genes within the relevant operon (ORFs BMEI1087-1090 in B. melitensis or corresponding genes/operon in other species of Brucella) are not functionally expressed. The mutations resulting in the attenuated phenotype due to inactivation of galE can be insertion, substitution or deletion mutations. With respect to the peptidoglycan related genes, it is not entirely sufficient to eliminate functional expression of only the dGTP phosphohydrolase gene to produce a mutant which is attenuated enough to be a desirable vaccine strain. Where the galE-like mutant of B. melitensis is used, it is recommended that the genetic background into which the mutation is introduced is a 16M genetic background.

Also within the scope of the present invention, are attenuated mutants of other species of Brucella, including Brucella abortus, B. suis, B. ovis, etc, where the functionality of the corresponding gene(s) as described above are destroyed. The coding sequence identified by ORFs BMEI1087-1090 are presented in Tables 5 and 6; see also the corresponding regions of SEQ ID NO:26-27. In strains and species other than B. melitensis 16M, from which the sequence information of Tables 5 and 6 is derived, the corresponding genes will have at least 85% or higher nucleotide sequence identity, thus enabling the generation of equivalent mutants in these coding sequences. Such mutants, when administered as live vaccines, provide an immune response to the cognate species of Brucella.

Within the present invention, there is at least one attenuated strain of Brucella in which there is a mutation which functionally inactivates or prevents expression of at least one of the galE and having at least 85% nucleotide sequence identity to SEQ ID NO:28, the gene encoding lytic murein transglycosylase and having at least 85% nucleotide sequence identity to nucleotides 7908-10817 of SEQ ID NO:26, β-hexosaminidase and having at least 85% nucleotide sequence identity with nucleotides 6688-7740 of SEQ ID NO:26, or a gene encoding deoxyguanosinetriphosphate triphosphohydrolase and having at least 85% nucleotide sequence identity with nucleotides 2138-3346 of SEQ ID NO:27. Also encompassed are immunogenic compositions for administration to a human or animal comprising an attenuated strain of the present invention. The bacterial cells in the composition can be killed or live, advantageously alive.

Further embodied within the present invention are immunogenic compositions comprising live cells of attenuated Brucella cells, and a pharmaceutically acceptable carrier. These attenuated Brucella cells can be deficient in the functional expression of at least one gene selected from the group consisting of galE, lytic murein transglycosylase and β-hexosaminidase. Such compositions include vaccine compositions for use in humans, sheep, goats, cattle, bison and other susceptible animals. It is understood that the immunization with one particular species of Brucella results an immune response primarily to same species as administered. Thus, for protection against B. melitensis, it is desired to administer an immunogenic composition comprising at least one live attenuated B. melitensis mutant, as set forth herein. These compositions can further comprise an agent which stimulates the immune response, for example, an interleukin such as interleukin 12.

The present invention further provides methods for generating an immune response, especially a protective immune response in humans, sheep, goats, and other animals. Desirably, the immune response generated is a T cell response. A single injection with live cells of least one attenuated Brucella mutant strain (desirably from 103 to 108 viable cells of each strain) as set forth above can trigger a protective immune response in the human or animal to which it has been administered, due to the persistence of the bacterial within the body of that animal. The immunogenic composition can be administered by any route of administration, such as subcutaneous, intramuscular, intraperitoneal, intravenous, mucosal, intradermal and so on. Because of the ability of the attenuated mutants of the present invention to persist in the body, it is not necessary for there to be repeated administrations of the immunogenic composition, although booster immunizations may be given.

Also within the scope of the present invention are attenuated mutants of Brucella strains having the same or equivalent defects to those of GR024 and GR026, as described herein, in which the hly gene (listeriolysin O) of Listeria monocytogenes is expressed. This results in brucella-infected cells which are “leaky”, thus resulting in a more effective immune response.

The present invention further provides methods for identifying B. melitensis peptides that correspond to MHC class I-restricted T cell epitopes, especially those associated with MHC class I (H-2Kd).

Additionally, the present invention provides a number of peptides that are associated with intracellular survival strategies of Brucella. These include several derived from an extracellular serine protease (BMEEII0148), characterized by a carboxy terminal region (amino acids 2349-2554) with high sequence homology to the β-domains of autotransporters of the Type V Secretory Systems of bacterial pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a real-time analysis of the attenuated bioluminescent B. melitensis strains in IRF-1−/− mice. IRF-1−/− mice were infected with 1×107 CFU of B. melitensis strains GR019 (virB4), GR024 (galE) and GR026 (90-91IR) and imaged daily with a 10 min exposure. Numbers at the bottom indicate days PI. Unlike GR019, infection with GR024 or GR026 resulted in a localized bioluminescence suggesting a defect in systemic spread. Rainbow scale represents approximate photon counts.

FIGS. 2A-2B illustrate replication kinetics of bioluminescent B. melitensis strains. FIG. 2A: Stationary phase grown cultures (30 μl) were inoculated into 30 ml of brucella broth and grown at 37° C. with shaking and OD600 was determined. FIG. 2B: RAW264.7 macrophages were inoculated with a standardized bacterial suspension of different strains and growth monitored at specified times. The CFU counts were log transformed and values are average ±standard error for duplicate samples.

FIG. 3 provides a schematic representation of EZ::TN<lux> transposon insertion in the three attenuated bioluminescent B. melitensis strains (only relevant features are shown; picture not drawn to scale). EZ::TN<lux> transposon is indicated as a closed hexagon relative to the site of insertion. The relevant ORFs upstream and downstream of the insertion are shown in open boxes with arrows indicating direction of transcription with numbers corresponding to the B. melitensis 16M genome sequence. The orientation of the arrow below the transposon in each strain represents the direction of Lux expression based on our sequence data. The sites for ClaI restriction enzyme used in Southern hybridization experiment are shown by the letter C.

FIG. 4A illustrates complementation of GR019 with the virB operon fully restored growth in macrophages. FIGS. 3B and 3C show virulence in IRF-1−/− mice. RAW264.7 macrophages were inoculated with a standardized bacterial suspension of GR019 and GR019 complemented strains, and the growth was monitored at specified times. The CFU counts were log transformed and values are average ±standard error for duplicate samples. IRF-1−/− mice were inoculated i.p. with 1×107 CFU of GR019 and GR019 complemented strains, and mouse survival (FIG. 34) as well as CFU from livers, spleens and testes (FIG. 4C) were determined. The CFU counts from livers, spleen and testes were log transformed and the data are an average of 4 mice. Error bars represent the range of CFU.

FIG. 5 demonstrates that GR024 and GR026 protect IRF-1−/− mice from challenge with virulent B. melitensis. IRF-1−/− mice (n=9) immunized with different attenuated B. melitensis strains (1×107 i.p./mouse) were challenged with virulent B. melitensis GR023 (1×106) and monitored for survival.

FIG. 6A shows the results of bioluminescent monitoring of virulent B. melitensis infection in vaccinated IRF-1−/− mice. IRF-1−/− mice vaccinated with different attenuated strains were imaged for 10 min following GR023 challenge. Numbers at the bottom of each figure indicate days PI and images representing same PI day from different groups are shown. Rainbow scale represents approximate photon counts. FIG. 6B shows the results of bioluminescent imaging of surviving IRF-1−/− mice following challenge (upper panel) and the corresponding histological changes in livers and spleen (lower panel). Livers were scored by the number of focal granulomas observed per field of view (fov) at 4× magnification. Data represent the average number of granulomas from 8 fov. (+) 1-8; (++) 9-16; (+++) 17-24 granulomas. Spleens were scored on loss of white and red pulp architecture at 4× magnification; (−) normal spleen or no noticeable changes, (+) enlarged follicles, increased cellularity, and white pulp, (++) hyperplasia, with a significant increase in follicle size, and white pulp. (+++); increased red pulp and loss of white pulp architecture.

FIG. 7A illustrates real-time analysis of attenuated bioluminescent B. melitensis strains in C57BU6 mice. C57BU6 mice were infected with 5×107 cfu of B. melitensis strains GR019, GR024, and GR026 and imaged daily with a 10 min exposure. Numbers at the bottom indicate days PI and images representing same PI day from different groups are shown. Similar to IRF-1-1−/− mice, GR024 and GR026 resulted in only a localized bioluminescence suggesting a defect in systemic spread. Rainbow scale represents approximate photon counts. FIG. 7B shows the results of bioluminescent monitoring of the virulent B. melitensis infection in vaccinated C57BL/6 mice. C57BL/6 mice vaccinated with different attenuated strains were challenged with GR023 and imaged for 10 min. Rainbow scale represents approximate photon counts.

FIGS. 8A-8B shows the results of CFU counts from livers and spleens, respectively, of C57BU6 mice vaccinated with different attenuated strains followed by virulent GR023 challenge. The CFU counts were log transformed and the data are an average from 3-4 mice at each time point. Error bars represent the range of CFU of the samples from each time point.

FIG. 9A is a photomicrograph of large grossly visible focal calcified granulomas in C56BL/6 mice vaccinated with Rev-1. FIGS. 9B-9C are photomicrographs of large focal granulomas contained secondary changes including a central area of necrosis, neutrophil infiltration, and fibrosis with calcification.

FIG. 10 illustrates the generation of antigenic peptide MHC class I complexes. As shown in the control experiment, a recombinant E. coli expressing the Yersinia enterocolitica invasin (inv) and the Listeria monocytogenes listeriolysin O (Hly) as well as a green fluorescent protein (GFPuv) was allowed to infect macrophages in culture for 24 hrs.

FIG. 11 shows the strategy for the identification of relevant peptide epitopes of B. melitensis. Brucella infects macrophages in culture; MHC class I proteins complexed with epitopic peptides are purified by MHC I-specific immunoaffinity chromatography, the peptides are eluted and then those peptides are characterized by mass spectroscopy. The invasive listeriolysin-expressing E. coli are transformed with plasmids expressing the peptide epitopes identified; these cells are useful for immunizing humans or animals with Brucella-specific peptides.

FIG. 12 provides schematic illustrations of the Brucella extracellular serine protease (encoded by ORF BMEII0148), which contains β-domain (amino acids 2349-24554) and passenger domain (amino acids 1-2348). This structure is characteristic of autotransporters of Type V secretory proteins. The peptide of the Brucella extracellular serine protease was isolated from MHC class 1 molecules following a 24 hr infection of macrophage cells in culture; the peptide was identified by MALDI-TOF mass spectroscopy.

FIG. 13 summarizes the results of the demonstration project in which the Inv+ Hly+ E. coli expressing the GFPuv protein was cultured in macrophages. The MHC class I-peptide complexes were collected, the peptides were eluted and then characterized. Within the peptides isolated was NYNSHNVYIT (SEQ ID NO:25) from within the GFPuv protein.

FIGS. 14A-14B summarizes the experiment carried out with the invasive Inv+ Hly+ E. coli transformed with the B. melitensis hdeA (small chaperone protein functioning in Type II secretion) gene and the GFPuv (a green fluorescent protein) gene. These E. coli strains were injected into mice intraperitoneally. The invasive E. coli generates antigen-specific cytotoxic T lymphocytes in the mice. After 6 wks, cytotoxic lymphocyte (CTL) assays were performed with transduced target cells. FIG. 14A shows the results of the CTL assay carried out with serum from mice immunized with the Inv+ Hly+ E. coli expressing the GFPuv protein. FIG. 14B shows the results of the CTL assay carried out with serum from a mouse immunized with the Inv+ Hly+ E. coli expressing the B. melitensis HdeA protein.

FIG. 15 shows the bioluminescence transposon used to produce the GR019, GR024 and GR026 mutants described herein. GR023 is an insertion mutant which was not affected with respect to virulence in the mouse model. The strategy used for analyzing the bioluminescence transposon insertion mutants is also shown.

DETAILED DESCRIPTION OF THE INVENTION

Certain bioluminescent mutants of B. melitensis are avirulent in IRF-1−/− mice. IRF-1−/− mice are highly susceptible and succumb to virulent Brucella infection; however, their response varies with the virulence of the Brucella strains (21, 22). Therefore, attenuated strains can be readily identified using these mice. We tested the three EZ::TN/lux bioluminescent mutants, GR019, GR024 and GR026 in IRF-1−/− mice to determine the virulence and pathology associated with these strains. In addition, we also tested two other B. melitensis mutants, BM710, a rough strain and Rev-1, a vaccine strain, so that the bioluminescent mutants could be evaluated for their ability to confer protection against challenge with virulent B. melitensis. IRF-1−/− mice (n=4) infected with bioluminescent strains were monitored for bacterial dissemination and persistence. Bioluminescence spread systemically in GR019 infected mice by day 1 post infection (PI), however, in the GR024 or GR026 infected mice bioluminescence localized primarily at the injection site (FIG. 1). By day 2 stronger bioluminescence was observed in many areas including the submandibular region only in GR019 infected mice. However, by day 6 GR019 infected mice began to clear the infection indicated by reduced bioluminescence and by day 24 minimal bioluminescence was observed in the extremities (FIG. 1). In contrast, in both GR024 and GR026 infected mice bioluminescence was predominantly observed at the injection site. However, by day 12, bioluminescence began to appear in the tail as multifocal lesions and was more prominent in GR024 infected mice by day 24 (FIG. 1). Mice infected with all three bioluminescent strains appeared healthy and survived longer than 24 days suggesting attenuation of these strains. Similarly mice infected with rough B. melitensis strain BM710 survived greater than 24 days suggesting attenuation. However, all Rev-1 infected mice died by 7 days PI. Although Rev-1 is a commercial vaccine, it was fully virulent in these mice. To determine the relative pathology associated with different attenuated mutants, the livers and spleens were processed for CFU and histopathology. Livers and spleens from GR019 or BM710 infected mice had lower CFU counts compared to GR024 or GR026 infected mice (Table 2). However, except for the GR026 infected group, livers and spleens from other groups had no observable histological changes (Table 2, data not shown). GR026 infected mice displayed very few multi-focal granulomas in livers and minor changes in the white pulp of spleens.

GR019, but not GR024 or GR026, is attenuated in RAW macrophages. We examined the growth of bioluminescent mutants in RAW macrophage-like cells. All three insertion mutant strains exhibited growth rates similar to that of the virulent parent strain 16M with a duplication time of 2 hr in brucella broth suggesting no general growth defects in GR019, GR024 and GR026 (FIG. 2A). RAW macrophages were infected with each strain at a MOI of 1:50 and the growth was monitored for 72 hrs. Interestingly, only GR019 was defective in replication with a significant decrease in intracellular bacteria by 24 hr PI compared to 16M (FIG. 2B). On the other hand, both GR024 and GR026 displayed a growth distinct from GR019 or 16M. Both strains were phagocytosed more with no apparent intra-macrophage replication during 24 hr PI as bacterial levels remained constant. However, by 48 hr their growth was similar to 16M. The growth curves of GR024 and GR026 appeared as intermediate between a virulent (e.g., 16M) and rough (e.g., perA, GI-2 mutant) strains of Brucella (41, unpublished data). Rough strains of Brucella are phagocytosed at even higher number and are defective in intra-macrophagic replication though they persist in higher numbers for more than 3 days in culture (35, 20, and unpublished data). This led us to suspect that both GR024 and GR026 are defective in LPS. Therefore we tested the LPS phenotype of these two mutants by the acriflavin agglutination test (6). As suggested by the macrophage growth pattern, both strains resulted in fine amorphous agglutination particles that were less intense compared to known LPS rough strains of Brucella (data not shown). In addition, unlike GalE mutants from other bacteria, GR024 did not show any sensitivity to galactose (1, 36, data not shown).

To determine the EZ::TN/lux insertion site in GR019, GR024, GR026, we performed ‘rescue cloning’ of the R6KΔori present in EZ::TN transposon from genomic DNA of each strain. Nucleotide sequencing of the rescued R6K plasmid clones in both orientations identified the transposon insertion in VirB4 (BMEII0028) for GR019, GalE homolog (BMEI0921) for GR024, and in the intergenic region of BMEI1090-1091 for GR026 (FIG. 3). The type IV secretion system encoded by 11 ORFs virB1-11 are transcribed as a polycistronic message and the disruption of these genes has been shown to attenuate Brucella in macrophages as well as in mice (9, 14, 34). Similarly, in GR024, the insertion disrupted the GalE homolog (BMEI0921) that has been previously shown to attenuate Brucella (38). However, in GR026 the insertion was located in the intergenic region of two divergent ORFs BMEI1090 and BMEI1091. Annotation of the B. melitensis genome suggested that BMEI1090 is the first gene in a cluster of genes that are transcribed in minus orientation, whereas BMEI1091 is an independent transcriptional unit (FIG. 3). Based on the sequence data obtained by transposon mapping, we have concluded that the altered expression of BMEI1090 or its downstream genes is responsible for attenuation of GR026. Further, Southern blot analysis confirmed the sequencing results and also revealed the single copy insertion of the transposon in these strains.

To determine the gene(s) likely responsible for the observed phenotype of GR026, we created non-polar mutations in BMEI1090 and 1091 by allelic replacement. The respective ORFs were replaced with a kanr marker by homologous recombination and resulting strains, GR-1090Δ and GR-1091Δ, were tested for virulence in IRF-1−/− mice. IRF-1−/− mice infected with GR-1091Δ died within 10 days similar to virulent 16M; however, only two mice infected with GR-1090Δ died and the remaining mice survived for at least 21 days (Table 2). The livers and spleens from the surviving mice had an average CFU of 6.65E+04 and 1.14E+06, respectively. Therefore inactivation of 1090 resulted in partial attenuation suggesting the phenotype associated with GR026 is likely due to altered expression of I1090 and its downstream genes.

To confirm that the attenuation of bioluminescent mutants is due to disruption of transposon insertion targets and not due to secondary mutations, we complemented GR019, GR024 and GR026 with the corresponding ORFs. Because GR019 has a growth defect in RAW macrophages, the GR019 containing either pBBVirB4 or pBBVirB were tested for growth in these macrophages. Introduction of pBBVirB4 into GR019 resulted in partial restoration of the ability to grow in macrophages, as reflected by increase in intracellular bacteria at 24 hr Pi (FIG. 4A). However, addition of pBBVIrB, containing the entire virB operon (34), into GR019 resulted in complete restoration of growth (FIG. 4). In addition, GR019 complemented with pBBVirB but not pBBVirB4, was able to kill IRF-1−/− mice and restore complete virulence (FIG. 4B). Consistent with the in vitro results, mice infected with pBBVirB4-complemented GR019 did not die and contained more bacteria in livers and spleens compared to GR019 infected group (FIG. 4C).

Because both GR024 and GR026 agglutinated in the presence of acriflavin, we tested the GR024 and GR026 complemented strains for agglutination. GR024 complemented with pBBGalE resulted in no agglutination in the presence of acriflavin. Our earlier results suggested that the observed phenotype for GR026 is due to the altered expression of 11090 and its downstream genes, so we complemented GR026 with a plasmid containing 4 ORFs likely to form an operon (12). Surprisingly, addition of pBB1087-90 to GR026 resulted in much more pronounced agglutination, as seen with rough strains of Brucella. The functions encoded by BMEY1087-1090 are α-hexosamionidase, soluble lytic murein transglycosylase, arginyl tRNA synthetase and deoxyguanosinetriphosphate triphosphohydrolase. Consistent with the acriflavin agglutination results, both GR024 and GR026 were partially resistant to smooth-type specific Tbilisi (Tb) phage, and the addition of pBBGalE restored the susceptibility of GR024 to Tb phage. However, GR026 complemented with pBBI1087-90 was completely resistant to Tb phage suggesting a rough phenotype of the complemented strain.

GR024 and GR026 protect IRF-1−/− mice from virulent challenge. IRF-1−/− mice, though immuno-compromised, have been shown to generate a protective immune response following vaccination with attenuated strains (22). Therefore, we tested the abilities of the attenuated bioluminescent mutants to protect IRF-1−/− mice from virulent challenge. IRF-1−/− mice (n=9) were vaccinated by intraperitoneal injection with 1×107 CFU of each Brucella strain, and 60 days after vaccination, the mice were challenged with 1×106 CFU of virulent bioluminescent B. melitensis strain GR023 (40). IRF-1−/− mice vaccinated with attenuated bioluminescent mutants were challenged by intraperitoneal injection when no bioluminescent bacteria were detectable. The GR023 strain of B. melitensis was used for challenge studies to evaluate vaccine candidates for the ability to alter the dissemination and localization of virulent Brucella to different tissues as visualized temporally in individual mice by imaging. All mice vaccinated with either GR024 or GR026 survived for at least 44 days, where as only 2 mice vaccinated with GR019 and 3 mice vaccinated with BM710 survived for 44 days following challenge (FIG. 5). Fifty percent of GR019 vaccinated mice died by day 12, whereas 50 percent of the BM710 vaccinated mice died by day 9 following challenge. As expected, all the unvaccinated mice died within 2 weeks following challenge with fifty percent of mice being dead by 7 days (FIG. 5).

The livers and spleens from surviving mice vaccinated with different strains had very similar CFUs (CFU ranges; liver: 2.2E+02-1.2E+03, spleen: 1.5E+04 to 3.4E+04). Bacteria recovered from livers and spleens of mice vaccinated with bioluminescent strains were confirmed as the GR023 challenge strain by verifying the insertion site of EZ::TN<lux> using PCR. Bioluminescent imaging of vaccinated mice following i.p. challenge revealed strikingly different dynamics of persistence and spread of virulent bacteria. Unlike the unvaccinated mice, in all vaccinated groups, bacterial spread was less extensive (See FIGS. 6A and 6B), but correlated with ability of the vaccine strain to protect from challenge. In both BM710 and GR019 vaccinated groups, bioluminescence was pronounced with systemic spread; however, in both GR024 and GR026 groups, bioluminescence was observed at the site of injection and in the tail region (see FIGS. 6A-6B). By day 44 both GR024 and GR026 vaccinated mice had no detectable bioluminescent bacteria while both BM710 and GR019 vaccinated survivors still exhibited detectable bioluminescence (FIG. 6B). Consistent with IRF-1−/− mice survival data, the GR024 and GR026 vaccinated mice had the least histological changes in livers and spleens. The GR024 and GR026 vaccinated mice had only few focal granulomas (less than 3/field of view) in the liver sections, while the spleens of GR024 vaccinated mice appeared normal with only minimal disorganization of the splenic white pulp in GR026 vaccinated mice. However, both GR019 and BM710 vaccinated survivors had more histological changes in both livers and spleens compared to GR024 or GR026 groups (see FIG. 6B).

IRF-1−/− mice are defective in multiple aspects of the immune system (44). Therefore, to better correlate the immune protection provided by the different attenuated strains, we tested these bacterial strains in wild type C57BU6 mice, the parental strain of IRF-1−/− mice. C57BU6 mice are susceptible to virulent Brucella infection naturally and serve as a relevant model in which to study Brucella pathogenesis and immune protection. To assess the protection by different attenuated strains, we monitored bacterial clearance and histological changes in livers and spleens. In addition, the dynamics of infection by attenuated bioluminescent strains and their effects on virulent challenge were monitored by imaging. Similar to IRF-1−/− mice, GR019 vaccinated C57BU6 mice had bioluminescence in systemic organs by day 1 PI; however, in GR024 or GR026 vaccinated mice bioluminescence was detected primarily at the injection site (FIG. 7A). Bioluminescence began to diminish by day 5 in all groups and by 2 weeks PI minimal or no bioluminescence was observed (FIG. 7A). However, after challenge the dynamics of virulent Brucella spread was similar in all vaccinated groups being limited primarily to the injection site, although bioluminescence was stronger in GR019 and BM710 vaccinated groups (FIG. 7B). Consistent with image data, all vaccinated groups had at least 2 logs less CFUs from livers and spleens at 1 week post challenge with Rev-1 and GR024 vaccinated groups containing even lower numbers of CFUs (FIG. 8). Similarly, at 2 weeks post challenge, livers from Rev-1 and GR024 vaccinated groups had significantly lower CFU compared to other groups. However, spleens from GR024 and Rev-1 vaccinated mice had lower CFU at all times compared to other groups though Rev-1 vaccinated mice had significantly fewer CFU compared to other groups (FIG. 8). To correlate the bacterial clearance with the tissue damage, histological changes were assessed in livers and spleens from immunized mice following challenge. Consistent with the bacterial clearance, GR024 and Rev-1 vaccinated mice exhibited fewer granulomas in liver at all times; however, livers from GR019 and BM710 vaccinated mice contained more granulomas (Table 3). Surprisingly, the livers from all Rev-1 vaccinated mice had large grossly visible focal calcified granulomas (FIG. 9). On the other hand, histological changes in spleens were similar in all vaccinated groups but contained fewer changes compared to unvaccinated controls (Table 3).

Mice are used extensively to study Brucella pathogenesis; however, the interpretation of data is often limited to CFU or histological changes observed in specific tissues. These approaches have limited our understanding of the dynamics of Brucella dissemination and localization into tissues beyond those organs that are conventionally used for evaluation. In this report, we describe the infection dynamics of three attenuated bioluminescent mutants in mice by visualizing how infection disseminates, bacterial preference to organs, contribution of certain Brucella genes to pathogenesis, and effect of vaccination on the dynamics of virulent bacterial infection. GR019, GR024, GR026, and BM710 were all attenuated in IRF-1−/− mice; however, Rev-1 remained virulent in these mice. Imaging of mice infected with bioluminescent strains revealed striking differences in bacterial dissemination and persistence. GR019 (VirB4), unlike GR024 or GR026, spread systemically and bioluminescence was observed in liver, spleen, testes, submandibular region and extremities early in infection, suggesting that the VirB system is not important for establishing early infection. However, the VirB system is required for Brucella persistence because C57BU6 mice cleared GR019 infection faster than virulent Brucella. GR024 (GalE) and GR026 (90-911R), on the other hand, failed to disseminate systemically (FIG. 1). Interestingly, in both GR024 and GR026 infected mice, signals reappeared 12 days PI and localized in the joint-rich tail region during the later stages of infection (FIG. 1), suggesting that virulence is regulated differently in GR024 and GR026. Bioluminescent imaging is critical in identifying the contribution of Brucella genes to preferential tissue localization of Brucella. In addition, temporal bioluminescence analysis of infection revealed patterns of growth and clearance, as well as reemergence of bacteria, that is extremely difficult to observe with conventional methods. Thus, our study clearly demonstrates that conventional CFU enumeration is useful but not ideal to assess Brucella clearance. Importantly, only GR019 was attenuated in vitro in RAW macrophages (FIG. 2B). Therefore, in vivo imaging may provide a more comprehensive approach to identify Brucella virulence genes that are relevant to in vivo pathogenesis. Although GR024 and GR026 localized in the tail region in later stages of infection, no (GR024) or very minimal (GR026) histological changes in livers and spleens were observed, similar to GR019 or BM710 infected groups. Thus, these strains, individually or mixed together in combinations of two or three mutants, or two or three mutations in a single strain, are useful in the formulation of immunogenic compositions, including vaccines. candidates and bioluminescent imaging may be highly useful for vaccine selection.

Both GR024 and GR026 exhibited growth patterns in macrophages intermediate between those of smooth and rough strains of Brucella (41), and both strains produced very fine agglutination particles in the presence of acriflavin and were partially resistant to smooth-type specific Tb phage, suggesting that they have an altered surface structure (30). In GR024, the transposon insertion is in ORF BMEI0921, a NAD dependent epimerase/dehydratase family member that is closely related to enterobacterial galE. The galE gene is an important virulence factor in many Gram negative bacteria and is involved several cellular processes including cell membrane biogenesis (15, 17, 29, 32, 39, 42). The galE mutants in other bacteria possess defective LPS, reflecting a contribution of galE to LPS biogenesis. Likewise, acriflavin agglutination and phage susceptibility tests suggest a defect in the GR024LPS; however, GR024 was not sensitive to galactose. The galE mutants of other bacteria display a variable response to galactose, with some being sensitive while others are not sensitive to galactose (15, 19, 39). The B. melitensis genome contains another member of the NAD dependent epimerase/dehydratase family, BMEII0730. BMEII0730 is more closely related to UDP-glucose 4-epimerases from members of the α-proteobacteria and shares no homology with BMEI0921. A few bacterial species have two functional galE genes. In Yersinia enterocolitica one galE gene is linked to galactose utilization genes and the other linked to the LPS synthesis genes (39). However, neither of the Brucella galE genes is linked to galactose metabolic genes or to LPS biosynthetic genes. Although our results indicate that BMEI0921 plays a role in cell membrane biogenesis, whether it is involved in galactose utilization is not clear because the growth of GR024 was not inhibited in galactose-containing medium. Brucella genome annotation suggest that Brucella BMEI10730 is linked to sugar metabolism genes and may be involved in galactose utilization.

GR026 has an insertion in the intergenic region between BMEI1090 and 1091. Further, selective allelic replacement of BMEI1090 or BMEI10191 supported the conclusion that loss of function of BMEI1090 and its downstream genes is responsible for the attenuation of GR026 (Table 1). BMEI1090 or BMEI10191 encode HesB protein and a theoretical protein, respectively. Without wishing to be bound by any particular theory, we have concluded that 1090 and its downstream genes (1087-1090) form an operon. BMEI1087 encodes α-hexosaminidase A, while BMEI1088 encodes soluble lytic murein transglycosylase, and these are involved in amino sugar metabolism and N-glycan biosynthesis (kegg database). Therefore, this operon may contribute to cell membrane and/or wall biogenesis. Consistent with this observation the acriflavin agglutination and Tb phage susceptibility tests suggested that GR026 has a surface structure defect. Complementation of GR026 with a plasmid containing BMEI1087-1090 ORFs resulted in more pronounced agglutination and complete resistance to Tb phage suggesting that the expression of these genes are under strict regulation.

Both GR024 and GR026 protected IRF-1−/− mice from virulent B. melitensis challenge, whereas highly attenuated GR019 and BM710 failed to protect these mice. In addition, GR024 and GR026 vaccinated mice displayed minimal changes in livers and spleens and no bioluminescence was observed at 44 days post-challenge. IRF-1−/− mice are defective in multiple immune components with reduced numbers of CD8+ T cells, functionally impaired natural killer cells, and dis-regulation of IL-12 p40 and inducible nitric oxide synthase (44). Though these mice are severely immuno-compromised, they mount an adaptive immune response sufficient to protect against virulent challenge and protection is vaccine strain dependent. Unlike, GR019, both GR024 and GR026 produced a localized but persistent infection in these mice (FIG. 1) and induced a protective immune response against virulent Brucella that may require some persistence of the vaccine strain. Similar results have been observed with two field vaccines stains, S19 and RB51 (23, 43). S19 persist longer and is more protective than RB51 in mice and other models (23, 43). However, S19 still possess residual virulence in domestic animals and in IRF-1−/− mice (22, 31), whereas RB51 is highly attenuated (22). GR024 and GR026 are highly attenuated in IRF-1−/− mice similar to RB51; however, they cause no or very minimal pathological changes in livers and spleen and are protective. Consistent with the IRF-1−/− mice data, both GR024 and GR026 provided greater protection to C57BLU6 mice than GR019 or BM 710 suggesting that IRF-1−/− mice may serve as an important model to rapidly assess vaccine efficacy of Brucella strains. Interestingly Rev-1 vaccinated mice had fewer CFU in both livers and spleens compared GR024 or GR026 vaccinated mice; however, Rev-1 vaccinated mice displayed severe liver damage with grossly visible lesions (FIG. 9) that was not seen in other groups. These lesions are likely vaccine induced as they were apparent even at 1 week post challenge. Rev-1 vaccine is used in domestic animals where B. melitensis is endemic with varying degrees of success (4). Although Rev-1 protected wild type mice, Rev-1 was highly virulent to IRF-1−/− mice (Table 2) and caused severe liver damage in wild type mice. In summary, our study revealed contribution of Brucella genes to in vivo pathogenesis and identified a new set of virulence genes (BMEI1090 and its downstream genes). Further, the galE deficient GR024 has altered LPS structure, results in no or very minimal tissue damage, and protects against virulent B. melitensis challenge making it an interesting vaccine candidate for brucellosis.

While the immunization strategy has been described using particular mutants of B. melitensis it is understood that corresponding mutants can be made in other species of Brucella, for use in immunogenic compositions and vaccination strategies for protection of the cognate species of Brucella. It is understood that there may be some immunological cross reactivity between species of Brucella, the most effective protection is afforded by immunization with an attenuated mutant of the same species as that for which protection is sought.

Further to the particular insertion and deletion mutants or those having equivalent loss of function as GR024 and GR026 described herein, immunogenic compositions and vaccines can be prepared using such mutants in which the listeriolysin (hly) derived from Listeda monocytogenes is expressed. Expression of this protein results in phagosomes which are “leaky”. The intracellular bacteria from the phagosomes are released into the cytoplasm of the cells in which they are reproducing, and there is a better immune response triggered. See, for example, Grode et al. (2005) J. Clin. Invest. 115: 2472-2479. For further discussions of listeriolysin, see also Giammerini et al. 2003. Protein Expr. Purif. 28:78-85; Dancz et al. 2002. J. Bacteriol. 184:5935-5945, Mengaud et al. 1988. Infect. Immun. 56:766-772 among others.

We have identified and analyzed B. melitensis-specific MHC class I-restricted T cell epitopes. There is additional data of MALDI-TOF Mass spectral analysis of such peptides naturally processed and associated with MHC class 1 molecules from macrophages infected with Brucella for 24 hrs. We have identified over 2500 peptides identified as Brucella associated with MHC class I (2 Kd). These include peptides derived from the ORFs, as identified in Table 4.

Analysis of the peptides associated with MHC class I (2 Kd) has revealed that a number of the peptides are likely associated with proteins previously unknown to be a part of Brucella's intracellular survival strategies. For example, one of the identified peptides is from an extracellular serine protease (BME10148). This protein has a conserved β-domain at the carboxy-terminal region that has high sequence homology to the β-domains of autotransporters of the Type V secretory system of bacterial pathogens (see FIG. 10). Without wishing to be bound by theory, we believe that Brucella uses this Type V secretory system protein as an intracellular survival or virulence strategy in macrophages. Peptide epitopes identified by this strategy can be expressed by nonreplicating, nonpathogenic E. coli cells which have been genetically modified to express the Yersinia enterocolitica inv gene and the hly gene from Listeria monocytogenes. The invasin confers the ability to invade nonprofessional phagocytic cells. Binding of invasion to β1 integrin expressed on mammalian cells is necessary and sufficient to induce phagocytosis of the bacteria. After internalization, E. coli is taken into the phagosome/lysosome where lysis of the bacterium occurs. Among the various bacterial proteins released into the lysosomal vesicle, listeriolysin present in the cytoplasm of the invasive E. coli gains access to the phagosomal membrane, perforating it a low pH. The cytoplasmic contents of the bacteria can then escape into the cytosolic compartment of the mammalian cell through the pores generated by listeriolysin. Using this mechanism, it was demonstrated that invasive E. coli can be used as a delivery vector for therapeutic proteins. Furthermore, invasive E. coli can elicit a specific CTL response and thus, expression of Brucella proteins or peptides which can elicit an effective and protective T cell response within a mammalian cell and release from the phagosome via listeriolysin provides for a useful vaccine against brucella infections. See FIG. 13A and FIG. 13B for the results of CD8 CTL assays carried out with serum from mice immunized by intraperitoneal injection with live Inv+Hly+E. coli expressing the GFPuv protein or the HdeA protein of B. melitensis. Such vaccine(s) are valuable for protection of humans and animals against the cognate species of Brucella; human welfare and improved animal health, with benefits to agriculture, animals in captivity and the like are achieved.

As used herein, attenuated means that a bacterial strain is reduced in virulence as compared to a “wild-type” clinical strain that causes disease in a human or particular animal; the attenuated strain does not cause disease in the human or particular animal.

With reference to a mutation, functional inactivation of a gene means that there is little or no activity of the gene product. For example, where the gene encodes an enzyme, the encoded product has less than 10%, desirably less than 5% or less than 1% of the enzymatic activity of the product from the wild type gene or there is less than 10%, less than 5% or less than 1% of the expression product. That is to say that the coding sequence can be interrupted with an inserted nucleotide or sequence, partly or wholly deleted or there can be a substitution mutation that changes the amino acid sequence of the encoded protein such that activity is significantly reduced. Alternatively, there can be an insertion, deletion or change in transcription and/or translation regulatory sequences such that expression is reduced or prevented at the level of gene transcription and/or translation of mRNA.

When a compound is claimed, it should be understood that compounds known in the art including the compounds disclosed in the references disclosed herein are not intended to be included. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.

Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of compounds and/or genes or mutants are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. One of ordinary skill in the art will appreciate that methods, starting materials, mutagenic methods, compositions, vaccine regiments and immunogenic composition ingredients other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, starting materials, genetic methods, and formulations and vaccination regiments are intended to be included in this invention. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.

As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

An immunogenic composition is one which triggers either a humoral immune response or a cellular (T cell) response, or both, in a human or animal to which the compositions has been administered. A vaccine (or vaccine composition) is an immunogenic composition, which after administered to a human or animal, which results in either no infection or infection without less severe or no symptoms upon challenge with a virulent strain of the same microorganism as the vaccine composition contained. In the context of the present invention, cellular immune responses are especially important in protecting a human or animal against infection by virulent B. melitensis.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art, unless otherwise defined herein.

In the present context, functionally inactivated means that a gene does not produce a biologically active gene product (there is less than 10% of the normal enzymatic activity or ligand binding activity). In the present context biological activity does not encompass triggering an immune response in a mammalian host in which the functionally inactivated gene product is expressed. However, it is intended that functionally inactivated includes those cases in which the gene is not expressed, for example, due to a large (or polar) insertion in a promoter region or other untranslated sequence.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g. Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. 1). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions, or to other negative effects. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and dose frequency, also varies according to the age, body weight, and response of the individual patient or animal. A program comparable to that discussed above also may be used in veterinary medicine.

Use of pharmaceutically acceptable carriers to formulate the immunogenic compositions herein disclosed for the practice of the invention into dosages suitable for administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection. Appropriate compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions, including those formulated for delayed release or only to be released when the pharmaceutical reaches the small or large intestine.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods and accessory methods described herein are representative of preferred embodiments and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

Monoclonal or polyclonal antibodies, preferably monoclonal, specifically reacting with a protein or other cellular component of interest may be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986 and subsequent editions) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York.

Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.

All references cited herein are hereby incorporated by reference to the extent there is no inconsistency with the present disclosure. These references indicate the level of skill in the relevant arts.

The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention.

EXAMPLES

Example 1

Bacterial Strains, Plasmids, and Growth Conditions

Bacterial strains and plasmids used in this study are listed in Table 1. Strains GR019, GR024, and GR026 are the EZ::TN-lux transposon insertional mutants of B. melitensis 16M containing the promotorless lux operon. A schematic illustration of the bioluminescence transposon and the mutagenesis and analysis strategy is shown in FIG. 15. B. melitensis Rev-1 is an attenuated strain of virulent B. melitensis 6056 (2, 13) and is used as a vaccine for brucellosis in small ruminants (4). B. melitensis 710 is a spontaneous rough mutant of Rev-1 isolated from vaccinated sheep and is phenotypically identical to Rev-1 except for the rough LPS. GR023 is a virulent bioluminescent strain of B. melitensis 16M (40) used for challenge studies. All Brucella strains were grown in brucella broth (Difco, Detroit, Mich.). Ampicillin 100 μg/ml, chloramphenicol 20 μg/ml, kanamycin 50 μg/ml, and zeocin; 50 μg/ml for E. coli and 250 μg/ml for Brucella were added to the medium as necessary. Brucella strains were grown at 37° C. with shaking unless otherwise stated. E. coli strain DH5α (Invitrogen) and EC100D™pir+ (Epicentre, Madison, Wis.) were grown in LB broth (Difco).

Suicide vectors pGR026-90K and pGR026-91K for generating deletions in BMEI1090 and BMEI1091, respectively, were created using pZErO-1. To construct pGR026-90K, approximately 1 kb DNA sequences upstream and downstream of the deletion target was amplified by PCR (upstream: forward 5′atcaacggtaccCGTTCAGCGCGTCGAGATCG (SEQ ID NO:1) and reverse 5′gctctaggatccGACTGATMTTATGCCGTGCG (SEQ ID NO:2), downstream: forward 5′acagtcgaatccATMCCGMGCCTATTCCTTC (SEQ ID NO:3) and reverse 5′ggtaacctqcagCGMCGTGCCCGCAT CAT (SEQ ID NO:4)) and cloned into pZErO-1 to generate plasmid pGR026-90. Appropriate restriction sites were included in the PCR primers to facilitate the insertion of the kanamycin resistance (kanr) gene from pUC4K between the 2 fragments to generate pGR026-90K. Bases added to the 5′ end of each primer to provide restriction sites are underlined. To construct pGR026-91K, the desired deletion target was amplified with approximately 1 kb upstream and downstream sequences using specific primers (forward 5′agatacggtaccTCTTCCATCGTTCCGGGCCT (SEQ ID NO:5) and reverse 5′catgcatctaga GACGCCGTTGATGTTCCATGTA (SEQ ID NO:6)) and cloned into pZErO-1 to generate pGR026-91. Then, inverse PCR was performed on pGR026-91 using primers (5′tcttgagaattcCCCMTGCGACCGCTT (SEQ ID NO:7) and 5′gattcagaattcTTlGGCGATCCGCCTGGCA (SEQ ID NO:8)) designed to amplify all but the deletion target. The inverse PCR product was digested with restriction enzyme and ligated to the kanr gene fragment to generate the final suicide vector pGR026-91 K.

To construct plasmids pBBVirB4, pBBGalE, and pBBI1087-90, DNA sequences encoding the respective ORFs were amplified using primers (VirB4: forward-5′agagagGGTA CCCATGTTCATATTGCCGCTGATCG (SEQ ID NO:9) and reverse-5′agagagGGATCCTGCTGGTTACA GTCAGGGCGAAT (SEQ ID NO:10); GalE: forward 5′agagagGGTACCAAAGCCCGGTAAAACGATTGATG (SEQ ID NO:11) and reverse 5′ agagagGGATCCGTTCCGGCATTTTCTGGCAAA (SEQ ID NO:12); 1087-90: forward-5′agagag ACTAGTTGTGCCGTCGTTTCCACCTG (SEQ ID NO: 13) and reverse-5′ agagagCTCGAGAGGGACGGGGA TCGGGTTAT (SEQ ID NO:14). PCR products were digested with restriction enzymes and ligated to pBBR-MCS4 to generate the complementation plasmids.

Example 2

Mapping of the EZ::TN/lux Transposon Insertion Site

The site of transposon insertion in GR019, GR024, and GR026 mutants was identified by rescue cloning. Two micrograms of genomic DNA from each strain was digested to completion with NcoI to generate a fragment with intact transposon and flanking sequences. Digested DNA was religated using a FastLink DNA ligation kit (Epicentre). Ligations were dialyzed and transformed into electrocompetent EC100 Dpir+ cells (Epicentre) and plated on LB agar containing kanamycin. Two independent kanr colonies were selected, the plasmid was extracted and the site of insertion was identified by sequencing the plasmid DNA bi-directionally using outward primers (40). Sequencing was performed using dye terminators at the DNA sequencing core facility, University of Wisconsin Biotechnology Center. Sequences were compared to the 16M genome sequence to determine the site of insertion.

For Southern hybridization, 10 μg of genomic DNA was digested with ClaI and separated in a 0.7% agarose gel by electrophoresis. The single copy insertion of the transposon at the expected location was detected using the kanr gene as a probe. A 700 bp internal fragment of the kanr gene was amplified from pUC4K using primers, KanF2; 5′GCTCGAGGCCGC GATTAAAT (SEQ ID NO:15) and KanR2; 5′TCACCGAGGCAGTTCCATAGGA (SEQ ID NO:16), labeled with North2South Direct HRP detection and labeling kit (Pierce) and used as a probe.

Example 3

Inactivation of BMEI1090 and BMEI1091 in 16M

To generate specific deletions, suicide vectors pGR026-90K and pGR026-91 K were electroporated into B. melitensis 16M. Cells were plated on brucella agar containing kanamycin. To select for double recombinants, the kanr colonies were checked for sensitivity to zeocin (zeos). The resulting kanr and zeos clones were streak purified, and one such purified clone was used for further study.

Example 4

Macrophage Infection

The macrophage-like RAW 264.7 cells were cultured in RPMI supplemented with 10% heat-inactivated fetal calf serum. For macrophage growth assays, 24-well microtiter plates were seeded with 5×105 macrophages/well and infected with different B. melitensis strains at 1:50 multiplicity of infection. Cells were incubated for 1 hr at 37° C. in 5% CO2, extracellular bacteria were removed with 3 washes of PBS followed by treatment with gentamicin 25 μg/ml for 30 min. Following gentamicin treatment, the cells were maintained with medium containing 5 μg of gentamicin/ml. At specified times, cells were washed with PBS three times, lysed with 0.1% Triton-X, and plated on brucella agar to determine intracellular bacterial counts. All experiments were performed in duplicate.

Example 5

IRF-1−/− Mice Virulence Assay

Groups of 6-9 week old IRF-1−/− (n=4) were infected intra-peritoneally (i.p.) with 1×107 CFU of GR019, GR024, GR026, Rev-1 and BM710 strains. Infected mice were housed in a biosafety level 3 facility and monitored for survival (virulent Brucella kills these mice within 14 days; 21). For imaging, mice were anesthetized with isoflurane, and bioluminescence was recorded after a 10 min exposure using a CCD camera (Xenogen, Alameda, Calif.). From the surviving mice, livers and spleens were collected aseptically, homogenized in PBS and plated on brucella agar. Plates were incubated at 37° C. for 4 days, and CFU were determined. For histology, a portion of livers and spleens were collected and fixed in 10% formalin, 5 μm sections were prepared, stained with hemotoxylin and eosin and microscopically examined.

Example 6

Vaccination and Challenge Studies

IRF-1−/− mice 6-9 weeks old (n=9/group) were vaccinated with 1×107 CFU i.p. with B. melitensis strains GR019, GR024, GR026, or BM710 in 200 μl PBS. As a control, a group of 10 mice were injected with 200 μl PBS. Similarly, C57BU6 mice (n=20/group) were vaccinated i.p. with 5×107 CFU with each of the above strains and the Rev-1. Mice were imaged daily using a CCD camera until challenge. After 60 days, both IRF-1−/− and C57BL/6 mice were challenged with 1×106 CFU of virulent bioluminescent B. melitensis GR023i.p. Following challenge, mice were imaged with 10 min exposure using a CCD camera and dissemination of virulent bioluminescent GR023 in different groups was monitored.

For IRF-1−/− mice, the survival was recorded in different groups following virulent challenge. At 44 days post challenge, livers and spleens from surviving mice were processed for CFU enumeration. For C57BU6 mice, to determine CFU in livers and spleens, 4 mice from each group were killed at weekly intervals. Portions of the livers and spleens were weighed and then homogenized in PBS. Homogenates were serially diluted, plated on brucella agar with or without antibiotic and colonies were counted after 72 hr of incubation at 37° C. To determine the histological changes at each time, a portion of livers and spleens were collected, fixed in 10% formalin, and 5 μm sections were prepared and stained with hematoxylin and eosin.

Example 7

Identification of Brucella-Specific MHC Class I-Restricted T Cell Epitopes

Raw264.7 mouse macrophage cells (haplotype H2d) are infected (MOI 1000) with B. melitensis cells for 48 hrs. Infected cells, along with uninfected control cells (2×109 each) are harvested by scraping, and membrane proteins are extracted using Mem-PER (Pierce Chemical Co., Rockford, Ill.). The extract is dialyzed overnight in 0/5% CHAPS buffer to prepare for immunoprecipitation. H2-Dd/peptide co-immunoprecipitation is performed using Seize Primary Immunoprecipitation Kit (Pierce) couple with anti-mouse H-2Dd monoclonal antibody that recognizes a conformationally sensitive epitope of H-2Dd (5589125, BD Biosciences Pharmingen, San Diego, Calif.). After elution of the MHC I/peptide complex in acidic conditions, the peptides are separated from MHC I components by passing through a 5 kDa MWCO filter (Millipore, Billerica, Mass.). Micro BCA protein assays are performed on the peptide mix, and the peptides are separated, sequenced and analyzed by liquid chromatograph/mass spectrometry.

Alternatively, the RAW264.7 mouse macrophage cells are infected with invasive E. coli expressing GFPuv for identification of infected cells (MOI 100 24 hr infection). The results demonstrated that MHC I and associated peptide can be identified, and the invasive E. coli vaccine vector can be used to deliver antigen to cells for processing and presentation by MHC class I. In this example, of eight H2-Dd nonamers from Infected Raw264.7 cells, one (NYNSHNVYI, SEQ ID NO:17) was specific to the GFPuv protein. Other sequenced peptides included HYLSTQSAL (SEQ ID NO:18), LFTGWPIL (SEQ ID NO:19), KFICTTGKL (SEQ ID NO:20), DFKEDGNIL (SEQ ID NO:21), LPVPWPTLV (SEQ ID NO:22), EYNYNSHNV (SEQ ID NO:23) and TPIGDGPVL (SEQ ID NO:24).

TABLE 1
Bacterial strains and plasmids used in this study.
Strains or PlasmidsDescriptionsReference or source
Strains
16MWild type strain of B. melitensisATCC
DH5αE. coli strain used for cloningInvitrogen
EC100Dpir+E. coli strain used for rescue cloningEpicenter
GR019Bioluminescent B. melitensis with EZ::TNThis study
transposon inserted in the virB4 gene
GR023Bioluminescent B. melitensis strain used for40
mice challenge studies
GR024Bioluminescent B. melitensis with EZ::TNThis study
transposon inserted in the galE homolog
GR026Bioluminescent B. melitensis with EZ::TNThis study
transposon inserted in the intergenic region of
BMEI1090-1091
BM710Spontaneous rough mutant of B. melitensisThis study
Rev-1 strain
Rev-1B. melitensis 16M vaccine strain 2
GR-1090ΔB. melitensis 16M with BMEI1090 replacedThis study
with Kanr
GR-1091ΔB. melitensis 16M with BMEI1091 replacedThis study
with Kanr
Plasmids
pBBR-MCS4Broad host range plasmid. Ap25
pZErO-1Cloning vector. ZeoInvitrogen
pUC4KSource of kanamycin resistance marker. KmAmersham
pGR026-90pZero-1 containing BMEI1090 withThis study
approximately 1kb upstream and downstream
sequences. Zeo
pGR026-90KpGR026-90 containing kanr marker replacingThis study
ORF I1090. Zeo, Km
pGR026-91pZero-1 containing BMEI1091 withThis study
approximately 1kb upstream and downstream
sequences. Zeo
pGR026-91KpGR026-91 containing kanr marker replacingThis study
ORF I1091. Zeo, Km
pBBVirBpBBR-MCS1 containing the virB region used34
for complementation. Cm
pBBVirB4pBBR-MCS4 containing B. melitensis virB4This study
ORF used for complementation. Ap
pBBGalEpBBR-MCS4 containing B. melitensis galEThis study
ORF used for complementation. Ap
pBBI1087-90pBBR-MCS4 containing B. melitensisThis study
BMEI1087-90 ORFs used for
complementation. Ap

Ap; Ampicillin,

Zeo; Zeocin,

Km, Kanamycin,

Cm; Chloramphenicol,

TABLE 2
IRF-1−/− mouse virulence assay.
# of mice# of miceTissue damagea,bCFU countsa
StrainsinfectedsurvivedLiverSpleenLiverSpleen
16M40SevereSevere1.9E+101.6E+10
GR02340SevereSevere1.5E+101.4E+10
GR01944NoneNone1.3E+031.0E+04
GR02444NoneNone9.5E+031.2E+04
GR02644MinimalMinimal1.3E+043.2E+05
BM71044NoneNone2.0E+024.5E+03
Rev-140NDdNDNDND
GR-1090Δ42c (1090)NDND6.6E+041.1E+06
GR-1091Δ40 (1091)NDNDNDND

aCFU counts and tissue damage were assessed two weeks post infection except for the 16M and GR023 infected groups for which tissues were collected when mice were moribund.

bLiver damage was assessed based on the number of focal granulomas and spleen damage was assessed based on the loss of architecture of white and red pulp. Tissue sections were visualized at 4X magnification and damage was assessed by observing more than 5 fields of view.

cSurvived for at least 3 weeks.

dNot determined

TABLE 3
Liver and spleen damage in C57BL/6 mice vaccinated with different
Brucella strains following a virulent challenge.
Weeks PC
TreatmentLiveraSpleenb
groups1234123 4
GR019++++++++++++++++++++
GR024++++++++++++++
GR026+++++++++++++
Rev 1++++++++++++++
710++++++++++++++++++
Control+++++++++++++++++++++++++++

aLivers were scored by the number of focal granulomas observed per field of view at 4X magnification. At each time, 8 fields were counted to determine the number of granulomas: (+) 1-8; (++) 9-16; (+++) 17-24; (++++) 25-32; (+++++) 33-40 granulomas.

bSpleens were scored on loss of white and red pulp architecture. At each time, 8 fields (4X) were scored using the following criteria: (−) normal spleen or no noticeable changes; (+) enlarged follicles, increased cellularity, and white pulp; (++) hyperplasia, with a significant increase in follicle size, and white pulp; (+++) increased red pulp, early loss of architecture, and the diminution of white pulp;
# (++++) severe loss of architecture, and a dramatic reduction in the number of follicles.

TABLE 4
Brucella peptides identified from MHC I molecules*
BMEII0160Flagellar Hook-associated protein
BMEII0148Extracellular serine protease
BMEII0793Multidrug resistance efflux pump
BMEI1895Outer membrane protein
BMEII0976ABC transporter ATP-binding protein
BMEI1715Maltose transport permease
BMEII0862Dihydrodipicolinate synthase
BMEII0126Amino acid permease
BMEII03484-aminobutyrate aminotransferase
BMEI1744Glucose-resistance amylase regulator
BMEI1427UDP-4-dehydro-6-deoxy-2-actamido-D-glucose
BMEI0943Vitamin B12-dependent ribonucleotide reductase
BMEI0864NifR3 Nitrogen regulation
BMEII0749Hypothetical protein
BMEI0903Hypothetical protein
BMEI1718Hypothetical protein

*Examples from over 2,500 different peptides isolated from MHC I H-2Kd

TABLE 5
Nucleotide sequence encompassing two open reading frames of
the Brucella melitensis 16M genome (SEQ ID NO:26).
LOCUSAE009548 10975 bp DNA linear BCT 20-MAR-2003
DEFINITIONBrucella melitensis 16M chromosome I,
section 105 of 195 of the complete sequence.
ACCESSIONAE009548 AE008917
VERSIONAE009548.l GI:17983048
ORGANISMBrucella melitensis 16M
AUTHORSDelVecchio et al. 2002. The genome sequence
of the facultative intracellular pathogen
Brucella melitensis. Proc. Natl.
Acad. Sci. USA 99:443-448)
FEATURESLocation/Qualifiers
genecomplement(6688. . .7740)
/gene = “BMEI1O87”
CDScomplement(6688. . .7740)
/gene = “BMEI1087”
/EC_number = “3.2.1.52”
/Product = “BETA-HEXOSAMINIDASE A”
/protein_id = “AAL52268. 1”
/db_xref = “GI:17983056”
/translation = “MIQQDNSRKSRMKECKAWIAGISGTKLTPDEIAFFRDETPWGFILFARNVESLEQV
SELTAHLRDLTGLDQTPVFIDQEGGRVQRLRPPLVPNYPSASEIGAIYARDKEKGLRAAWLHARLHAFDLL
KVGVNADCLPVLDVPVEGAHDVIGMRAYSKNPHAVAEMGRAAAEGLLAGGVLPVVKHMPGHGRAFSDTHKE
LARVSVALNELVAHDFVPFKALNDLPMAMTAHVVFDCIDPERPSTLSPTVINTIIRDVIGFDGLVISDDIS
MKALSGDLGDITDGIVTAGCDIVLYCSGVMEELVAARVPVLDGKAKRRAELAEVYAGDPDLSDEDEVRAEF
NAMFEPIA”
genecomplement(7908. . .10817)
/gene = “BMEI1O88”
CDScomplement(7908. . .10817)
/EC_number = “3.2.l.”
/product = SOLUBLE LYTIC MUREIN TRANSGLYCOSYLASE”
/protein id = “AAL52269.1”
/db_xref = “GI:17983057”
ORIGIN
1aggctgccat tgctcaaaat caatgcaact gaagccgttc cgacaaaagc gcgaagcggt
61tttttggaat catcctcaaa caaaatcttg gagcgggatg atggttggac ttaaattcaa
121cccgttttag agcgcgtttc gatctgattg aatcagatcg gcgctctaat cctttgtttt
181gacgcgcatc ttttccgaaa accgtttcac acttttcggg atgcgctcta aagaacggaa
241gacgtgcctt cgatgaacgg ctgatatcga accggcatga ggtcttcctg ttcaaaacgg
301cttccaacct ttgaaatccg cgtcatgatc tggcgtccat cgccagggcc gatcggggct
361atcaggacgc catgggtggc gagcagttca acgaaatggc gcggcacctc atcgcatgcg
421agccagatga caatgcggtc aaacggcccg cccggcatac cgtggcgccc gtctgtatgt
481ttcaccatga tattctcgcg cttcagcgaa acgaactgct ggagagcgtg gtcgcagagt
541tttcgatacc gttccaccgt cgttacacgg ccggacagca aggacataac ggcggcggta
601aagccggagc cggtgccgat ttccagaacc cgatggccgg gctcaagctt cagggcggaa
661atgacgcgcg cctgatcgtc tatgccttcc atatattcac cgcaatcaag cggcgcggtt
721cgcgggctat aggcaagatg cgaccatgcc gccgccagaa agctctggcg cggcgttgct
781tcaattgccg caaaaagttg cggatcatca atgctgtgcc cacgcatccg cagaacaaag
841gatgcaaatc cctcccggtc cgaaagccgc gggcgttcag acgttgcctg cctcatgctt
901ccactccaag cgccgcgccc agttctgcac gaaccttatg agcggtcaga tcaaggtgga
961gtggggtcac tgaaatgcaa cccgaacgga tggcagcaat atcgctgtcg tcggcaaccg
1021gagccttgcc gcgaccgaaa tgcagccaga aataagggaa accacgtcca tcgcggcgct
1081cgtcaaggcg cgcatcatgg ctaagcttgc cttgtgccgt gacgcgcacg cccttcactt
1141cttccggagc gcaattcggg aaattgaggt tcaacagcac gccttccggc cagcccgcct
1201ccatcagcct cccgataagc tcaggcgcat gagcttccgc cgtttcccac ggcacgatcc
1261ggcgatcgcc cgcatattca tattcctgcg acaaagcgat ggctcgcaca ccaagcaatg
1321tcccctccat cgcaccggca accgtgcccg aataggtcac atcgtcggcc atgttcgccc
1381cggaattgac gccggagagg acgagatcgg gcgcgcccgg caatacatgg cgcaccccca
1441tgatgacgca atcggtcgga gtgccgcgca gggcaaaatg acgggcatcg atctggcgaa
1501ggcgaagcgg ctccgacagt gtcagtgagt gggcaagccc gctctggtcc gtttcagggg
1561ccaccaccca cacatcgtcg gagagcttgc gtgcaattcg ctccagaaca gcgaggcctt
1621cagcgtggat accgtcatcg ttcgtcagca gaatacgcaa tttgtcactc cttcgccgaa
1681atggataaga cacttaagac actacagcgg ttccagttga aatgggatcg ttgaaactgc
1741tctctctttg ttctttcgca tgtccccaaa accggttccc acttttgggg gcatgctata
1801attccagatc aagcggcttt ttcgatccgc gtgaggccgc ccatatatgg ctgtaatgct
1861tcaggaatat gaatgctgcc gtcttcctgc tggtaatttt ccataaccgc aatcagcgcg
1921cgcccgacag cagcgcccga cccgttgagg gtgtgcacga agcgcgtgga tttttcgcct
1981tccgggcgat agcgggcatt catgcggcgg ccctggaaat caccgcaggt cgaacagctt
2041gaaatttcgc gataggtgtt ctgccccggc aaccagacct cgatatcata ggtccgctgt
2101gcgccaaagc ccatgtcgcc cgtgcaaagc acaacggtac ggaacggcag gcccagccgc
2161ttcagcactt cttccgcgca agccgtcatg cgctcatgct cggcaacgga gctttccgca
2221tcggtgatcg ataccatctc cactttcagg aactgatgct ggcgcaacat gccgcgcgta
2281tcgcgcccgg ccgaccccgc ttccgagcga aaacatgggg tcagcgccgt gaagcgcagc
2341ggcagcccct tcatatcgac aatttcttcg gcaaccagat tggtgagcgg cacctccgcc
2401gtcgggatca gccagcggcc atccgtcgtg cggaaaagat cttctgaaaa cttcggcaat
2461tgccccgtgc catagaccgc ttcgtcgcgc accatcagcg gcggcatgac ttcggtataa
2521ccgtgttctg tcgtgtgaag atcgagcatg aactggccaa gcgcgcgctc aagacgggcg
2581agcgggcctt tcagcaccgt aaagcgcgca ccggcaagct tggccgcgcg ctcgaaatcc
2641atgtatccaa gcgcctcgcc aagctcaaaa tgctctttcg gctggaagga gaaattgtgc
2701gggttgccaa tgcggcgcag ctcaacattg tcgctttcat ccttgccgag cggcacatca
2761tcaagcggaa tattgggaat ggtggacaat gcgtcgctca gttccttgct gaggcggcgc
2821tcgtcttctt ccgcatgggc gagaaaatct ttcagttcgc ccacttcggc cttcagcttt
2881tcagccgtgc ccatgtcctt tgcggccatg gccttgccga tttccttcga ggcggcattg
2941cggcgctcct gcgctgcctg caccttgccg acatgctcgc ggcgcttttc atccagcgca
3001atcagttcgg acgaaagcgg agcagcccca cgctttgcga gcgccttgtc gagggtttcc
3061gggttttcgc gaatccattt gatgtcgagc atggaaaaaa gccatttcgt gaaattgaac
3121agaagcgagg ctaaacgatc ttcagcccca aagatgcctg acgtcagatc aggtggagga
3181agcgttgtta tcagcgtcgg cagatgcctg cgcctcatcc cgcttcttct cgatcatgcg
3241cgccagaaag atcgaaatct cgtaaagaag gatcgtcggc aaggcaagac cgatctggct
3301cgccgggtcc ggcggggtca gcaccgcagc cgcgacgaag gcaatgacga tcgcatattt
3361gcgcttgtcc ttcagccccg ccgaagtcac cagccccaca cgcgccatga ggctcgtcac
3421caccggcaac tggaagacca ggccaaaagc aaagatgagc gtcatgatga ggctcagata
3481ttccgacact ttcggcagaa gcgaaatctg gacctcgccg ctgccgccgg tctgctgcat
3541ggcgaggaag aaccacatca ccatgggcgt gaaaaagaaa tagacgagcg cgccgccgat
3601caggaacaga atgggcgacg cgatcaggaa cggcagaaat gcagtgcgtt cgtgcttgta
3661gagaccggga gccacgaatt tataaatctg tgcggcgatg accgggaggg ccagcacaat
3721gccgccgaac atggccacct tcacctgcgt gaagaagaat tcctgaggtg cggtatagat
3781caattccgcc ttggagcggt ccatgccggc ccagtcgatg gcccattgat acggcaccac
3841aagcaggttg aagagctgtt ttgcgaaagc aaagcagaaa atgaatgcca cgaaaaaagc
3901caggatagcc caaataaggc ggcggcgcag ttcgatcagg tgttcaagca gaggcgctgc
3961gctctgttcg atttcatcct cgtcccggtt cacgctttgg ttcctgtctt ctttgtggtc
4021tttttaaccg gcgttgcggt cttgtctgcc gtcggcttgg gggtagctcc ggtttttttg
4081gcagtctttg tcgtcgtcgg tttcggcccg gcttttgcag ccggacgcgg tgatgttttc
4141ctaggcttgg cgggttcttc gggcgcggtg atcattggta cgggaactgg cggcgcggga
4201actggcgttc cgcccggctc aaccggcgtc gtaacctcac ccaccttgtt ctcggtgact
4261ggcgacattg atgttgcgga ctggagacca gaccgcaaat cctcgccagc actgcgaatc
4321gggtcaaaaa cctgtgtcag ccttgtgcgc ggatcaaggc ttctggcttc atcgatgatg
4381gtcttgacgt cttcaagttc cgcctctttc aaggcctcgt tgaattgatg gcgaaactcg
4441ttggcggtgg tgcgcatgcg tgcagtcgcc ttgccgaacg cgcgaagcat tttcggcaaa
4501tccttgggac cgaccaccac aatcatgaca attgcgataa tcagcagttc agaccaagcg
4561atatcgaaca taatttgata ccttgcgctc tgcgcgcaca tcctgtctct tggcgaaaag
4621ccgcactgcc cacaaacctg ccatgcgcgt tttcagccca tggcagttca tcccggaagg
4681atcaggactt ggtggtcttc ttgacgtcct tgacgggttc ttccgctttg gcgtcgatcg
4741tacgcggatc ttccttggcg tcttcgtcag ccatgccctg cttaaaattc ttgataccct
4801tggcgacatc gcccatcagc tcggggatct tgccgcggcc gaacagaaga agcacaaccg
4861ccagaacgat cagccagtgc cagatggaaa agctacccat attattcctc tcagtgccgc
4921ccaaggcgcg gcatatgcct gctatctccg atacgattta agcgctttca acaaatcttt
4981caaacagaag tgtgatgatg aacggcttca aaccggatta attcgtcgca ggcagaaatt
5041ttgttctatt ctcccctggg tgcaagcaaa cccagtccct ccagatcaat atcctccagc
5101gggtcctccc cttcggtcag ctcgtccggg tcgatattgg ggatcggtac ggcaaaactg
5161gaaggaatgc gcgccgagag aagccctgcg ccgcgcaatt cctcaagacc gggcagatcg
5221cggatttccg gcaggccaaa atggtcgagg aaagcgtcgg tggtgccata tgttaccggg
5281cgccctggcg tgcgcctgcg cccgcgcagc ttgatccagc cggtttccat caagacatca
5341agcgtcccct tggatgtttc cacgccgcga atatcctcaa gttcggcgcg tgtcaccggc
5401tggtgatagg caatgatggc aagcacctcc atggtcgcgc gcgaaagctt gcgctgctga
5461acagtctcgc ggttcatgat gaaggcgaga tctggcgcgg tgcgaaacgc ccagccactg
5521cccaccttca caaaatgcac gcccctgccc tcgtaaacct tctggagatg gttcaaaacc
5581ggagcaatat ccacattggc gggaagccgc tcggcaagtg cgcgctcgca aacaggctgc
5641gaagacgcaa aaacaatcgc ctccacaatg cgggcaagct cggcaagcgt caccggcgag
5701gcaggccccg cctgctcttc ttccccaacg ccttccatat ccatcaaatc gcggcgctct
5761gcttcaggca ttttcgtcct catcgaattc atcgagttcg cgggtcgcgc gcatatagat
5821cggctcgaac ggagcgttct ggcgtacttc aagcttgcct tcgcgcacca gctcgaggca
5881tgcggcgaaa gaactggcaa gcgccgacgc cctctcctgc ggagaaagtg cataatcgat
5941caaaaaacgg tccagcgaaa cccagtcgcc caccgcgccc atcaggcgca caagcgccgt
6001gcgtgcctcc ttgagggacc agacgctgcg tttttctatc tgtacctggg aaaccgcctg
6061gcgctggcgc tgcgacgcat aagcgctaag cagatcgtaa agcgttgcgg aaaaacggct
6121ggcgcggtcc accaccacca tttccggcat gccgcgcggg aaaacatcgc ggccgagccg
6181atgacgattg acgagtgccg ccgccgcatc gcgcatggct tcaagccgtt tcaaccggaa
6241ttgcagggag gcaacgagtt cctcgcccgt ggcgccatcg tcgccctgct gcttcgggat
6301cagcagcttg gatttcagat aggcaagcca tgccgccata acgagataat cggcggcaag
6361ctccagacgc agcgcgcgcg cctgctccac gaaaccgaga tattgttcgg caagcgccag
6421cacggaaatg cgcgcaagat cgacgcgctg gttacgcgca agatgcagaa gaaggtcgag
6481cggaccttca aagccctgca catcgatcag cagtgacggc tcgcctgcgc ctcgcccggc
6541ctcattttgc cacagggtat ccatcggcac gcgtgtgccg tcgtttccac ctgtatgtgc
6601atccgatgct gccaagcctg tcgtcctgct gttgcccctg ccggcacaga ttctgccagc
6661aggaacaagt ttatcaagtt ttgccgatta cgctatcggt tcaaacatgg cattgaattc
6721ggcgcgcacc tcgtcctcgt cggaaagatc ggggtcgccc gcatagactt ccgccagttc
6781ggcccggcgc tttgctttgc catccagaac cggcacacgg gcggcaacct ttaccagttc
6841ctccataacg cctgagcaat agagcacgat atcgcagccc gccgtaacga tgccgtcggt
6901tatatcgcca agatcgccgg acaatgcctt catggaaatg tcgtcactga tgacaaggcc
6961gtcgaacccg atcacatcgc gaatgatcgt attaataacc gtcggcgaaa gcgtggacgg
7021tctttccggg tcgatgcaat cgaacaccac atgggcggtc atggccatcg gcagatcatt
7081gagcgccttg aacggcacga aatcatgcgc aaccagttcg ttgagcgcaa cgctgacccg
7141cgccagttcc ttatgcgtat cggaaaaggc gcggccatgg cccggcatat gcttcacgac
7201gggaagaacg ccgccagcca gaagaccttc ggcggcagcg cgtcccattt ccgcaaccgc
7261atgggggttt ttggaatagg cccgcattcc gatcacatca tgcgcgccct ccaccggcac
7321atccagaacc ggcaggcaat ccgcattgac gccgaccttc aacagatcga aagcatggag
7381ccgggcatgg agccaggcgg cacgcaaccc cttttccttg tcgcgtgcat agatcgcgcc
7441aatttcggac gcggacggat agttcggtac cagcggcggg cgaaggcgct gcacgcgccc
7501gccctcctga tcgatgaaaa ccggcgtctg gtccagcccc gtcaggtcac gcagatgggc
7561ggtgagctcg ctcacctgtt cgaggctttc cacattgcga gcaaaaagaa tgaagcccca
7621cggggtttca tcccggaaga aggcaatctc gtccggggtg agcttcgtgc cggatatacc
7681ggcaatccat gccttgcact ctttcatgcg gcttttcctc gaattgtctt gctgaatcaa
7741actcgcctcc gccgggttta gccgaaaaaa cggccgcttg gtaggctgta ggctgtggtg
7801acgaattaac ttatggttcg gtatgaacga aaatgctcaa tagcccggca gatgcgaaaa
7861gggcggctga cgccgccctc aaatggctgg aaccttgctg gttgttttta ctgcgtcacg
7921aaacagcttc cgccagccga cttgagacgg ctgcaaagcg ccaatgcatc ttccttcgag
7981ccagcctgta cgcgaacacg gtaataggtg cccttgccct gaatgtcggc gcgcttgata
8041tcgacgctat gaccgccaat cacactggca tatttctggg ctatgttggc ataggacttc
8101tgcgccagct cagcagaagg ctgcgaggca atctggatga aataaccacc cgccccggct
8161gccgatgcga cctgcggtgc agccgatgcc tgcgcgcgct gcggcacgtt accgacgata
8221ttgacgggct gttcagcggg gcgcgacggc acgatcggag cgcgggtcgg aaggcgtggg
8281gtctccggcg tcgcggattg ctgtgcctgt ggcgctggcg gttcatttcc tgccgcgagt
8341gcgccgattt catcccgtgc tgccggtgcg gcaggcggcg ccatattgtc ggctaccgac
8401ggctgtgctg catgtccgaa cgaaggctga atgatcgtgc catccgggcg aacgatcatc
8461gtttcgacct cacgcggctg gatcagcggt tcatgcgtgc cggaatgagc ctgttgcgca
8521tcgttctgcg gtacattgcc acccggttct tctgtggcat tatattcgct gtcatcggta
8581ccggaaatat cgaccggttc ttcaccggat gtaatcaggg ctttctgttc cgggttgttc
8641ggaagcgttc cggccacacg gtcatagacc gccttatcct ggttcggaac cgtggttccg
8701cccggatttt ccggctgcat cttgatgggc tggttatcgg cgcgaatcac aaccggctca
8761ccggagcctc cgccgccgag gaaatgatag ccgattccgc cgagcagaac cgccacaccg
8821gcaacacttg ccaaaatcaa gccacggcga ccacgaaccg ggcgattgcg gtaagcttcc
8881gccgcgccgc ccagatcgtc ttccgtcggc attgcggcgc gctcgccata atcgccgcct
8941tccatggtct gcgcaccctg cgcggcccag tgattgtaga aatcatcctg gctggcagtc
9001gttgcagcgg caggtgcagc ttcagacccg gcgcgtcggt aggaagcagc agccgctgcc
9061gccgcagcac ccaagccggc cgcggccatg ccgctattcg gcatataggt cgatgcgctt
9121tcacggaaga tgtcctcaaa agccctgtcg gcttcgctct ggccttccgt gatctgcaca
9181ttctcatcaa caccaatcgt gctgaaaact tccgcgaact ccgcttccag ctcactgaga
9241ttggtgcccg cttcctcttc gccgtaattc acttccggca gatcgagcga atgcgtctgt
9301tcgaccttgt tttccgtgac cgtgagggtc tcaacctccg gtgcaggctc cggtgcgggc
9361atacgcgccg gagcctgcat gtcggtataa gcggcaaagg agacagcagg gcgataatcc
9421tcgccggaat ggattgcgtg cgcataggct ggcgcgggag aagcttcttg ctcctcctca
9481tggagatcaa gttcaacatc ggtgaagaaa tcttcatcgt tgaaaaaatc ttcatccgca
9541gtatcggcga cgtctgcgtc gaatgtgtct ccagacggct cgaaaccgaa atcatcttcg
9601gtcaggctga tttcgtcgag ccccgacagg tcatcatccg tcgattcggc ggcggcagcc
9661gtttccggct ctgcttcaaa ggtaaaatcg tcctcaagcg aaaactcatc ctcgattggc
9721cggaacggat cctgggcaac aaaatcctgc acaataggcg cctggggcgt tttcagttgc
9781ggccccgagg agagcacacc gggggcggct acacccggcg caaaattgct gcgcggataa
9841tagggatagg ccggcgcctc gccagtgcgt gacgcatagc cctgcccggc atgggacgga
9901gcatccagat gcggttcgct gaccggcgct tccggttcgc cctgatgcgg ttgttccggc
9961gcgtaggaaa caggctcgac gtgatccgaa tagctgttcc gggatgccac aggctgcggc
10021tcatcgccaa acagaaggtt ttccagctcg tcttcaagcg aaagcggttg ctgtgcaggc
10081tcagccgcga aatgcgtagc gggagcatct acaggctgct gcgcattcca attattctgt
10141gctggccagt cattctgttc cagcctgtca ttctgtgcag gccactcatc ctgtgcctgc
10201caattgtcct gcacgccggt atcttcacgc caagcttgct cacccggctc aatggcaggc
10261tgatagacgg tcggatcata ttcgcccgca tctgcctcta tcggctgctg gcgatcacca
10321taagccgcag gtgatgccat atgggcgtgt tcgctcgaat cacgagcctg cgaataatcg
10381ttatagtcga attgcggaac gggctcggag cgatccacgt cctcgaactc gaaaatttcc
10441ggtgacggtg ctgcttctgc cgagccaagg tcgagatcga attcttcttc cagcgcggca
10501gcgaacgcat cctcctccaa cggagactgc tcgccatagg cccgctcccc gtgcacgcca
10561aaggttgcgg tcgaaacggt ttcgctatga gctgtaggct gcgtataatc gtcgaaatgc
10621cccataagct cgcgctcaag atcgagaacg ggatcaaaag aggggtcatc ctgcgccgaa
10681tcaaagcgcg gctctgctcg gccctgatcc tcgaattgac tgtcatggcg gcgctcattc
10741cgagcgacgt tatcatcagc aggcgtgtcg aagtccataa tccgcgaaag ttccatcagc
10801ggatcatctt cgtgcaccgg acgctcgccg taattacggg gatttgcact gctgtccgtc
10861atggcgtgtt cctaactcaa accctggacg ccgcaagacg tctccataca ttgcatatta
10921gcgaggcaat gtgggcaaaa gttgacggaa gtttcctgca caggaaggaa gatcc

TABLE 6
Nucleotide sequence encompassing four open reading frames
from the Brucella melitensis 16M genome (SEQ ID NO:27)
LOCUSAE009549 10209 bp DNA linear BCT 20-MAR-2003
DEFINITIONBrucella melitensis 16M chromosome I,
portion of section 106 of 195 of the
complete sequence.
ACCESSIONAE009549 AE008917
VERSIONAE009549.1 GI:17983058
SOURCEBrucella melitensis 16M
REFERENCE1 (bases 1 to 10209)
AUTHORSDelVecchio et al. 2002. The genome sequence
of the facultative intracellular pathogen
Brucella melitensis. Proc. Natl.
Acad. Sci. USA 99:443-448 (2002)
FEATURESLocation/Qualifiers
source1. . .4500 (excerpted herein)
/organism = “Brucella melitensis 16M”
/mol_type = “genomic DNA”
/strain = “16M”
CDScomplement(159. . .1916)
/gene = “BMEI1089”
/EC_number = “6.1.1.19”
/product = “ARGINYL-TRNA SYNTHETASE”
CDScomplement(2138. . .3346)
/gene = “BMEI1090”
/EC_number = “3.1.5.1”
/codon_start = 1
/product = “DEOXYGUANOSINETRIPHOSPHATE
TRIPHOSPHOHYDROLASE”
/protein_id = “AAL52271.1”
/db_xref = “GI:17983060”
CDS3513. . .3839
/gene = “BMEI1091”
/product = “HESB PROTEIN”
CDScomplement(3920. . .4411)
/gene = “BMEI1092”
/product = “hypothetical protein”
/protein_id = “AAL52273.1”
ORIGIN
1tattagcgag gcaatgtggg caaaagttga cggaagtttc ctgcacagga aggaagatcc
61atgactttca atcatactga tcttccttca cattagtctt actgtcactc atccaaaggc
121gtctttcgac tttcagtact tcggacgacc gtgtaatcct agcgcatctc cgtaggagca
181tccgcgccga taatcgtcaa tcctgacgtc agcacatcgg aaacaacctg caccagccct
241agcctggcca gcgacaagtc tggatcgtta accttaataa aacgtaagtc cggattttcc
301gcgcctctgt tccattgcga atggaacgaa ctggcgaggt cgtagaggta gaaagccagg
361cgatgcggct cctgatgaat ggctgccgat tcgatcaggc gcgggtattc cgcaagcttg
421cgaacgagcg caatttcgct ctcgtcggtc agcttttcaa aatgcgaccc catggccacg
481cggtcaagat cgacaagccc aagctggtcc gcagcctgac ggaaaaccga atggcagcgc
541gcggaagcat attgcacata gaaaaccgga ttgtccttgg actgctccgt caccttggcg
601aagtcgaagt ccaatggcgc atcgttcttc cggtaaagca tcatgaagcg gaccggatcg
661cgaccgacct cgtccaccac atcgcgcagc gtaatgaact cgcctgcccg cttggacatg
721cgcaccggct cgccattgcg gaacagcttc acgagctggc acaggagcac ggtcaatttg
781gccttgccat cggaaacggc acgcgcaacg gcttccagac gcttgacata accgccatga
841tccgcgccga gcacatagat catctcattg aagccgtggt cgtacttgtc cttgaaatag
901gccacgtcac ccgcaaaata ggtgaacgag ccatcggact tcatcagcgg acggtcaata
961tcatcgccca cttccgtaga acggaacagc gtctgctcac ggtcttccca atcttccggc
1021aactgcccct tcggaggcgg cagcttgccc ttataaacat ggcccttgag cgtcagatca
1081ttgatcgcgt tacggatcgc gcgcgcatgg tcgacatgta gcttgcgctc ggaatagaag
1141acatcatgat gcacgttcag cgcgtcgaga tcagcgcgga tcattgccat catggcgtcg
1201atcgtgcggt ccttcacgat ggccagtgct tcggcttcag gcatttccag aagttttgtg
1261ccaaactcac cggcaagctc ctgcccgacc cgcacgagat aatcaccggg gtaaagcccc
1321gccggaatct cgccgatgct ttcgcccagt gcctcacgat agcgcagcat cacagaacgc
1381gcgagaacat cgatctgcgc gcccgcatcg ttgatgtaat attccttgac gacgtcatag
1441cccgcgaatt tcagcaggtt cgccagcaca tcacccacaa ccgcgccccg gcaatggccg
1501acatgcatcg ggcccgtagg gttggccgat acatattcga cattgacctt cttgcccgcg
1561ccaagcctgg agcggccaaa atccgttccc tcgttcagca tcaccaaaag ctcgcgctgc
1621caatagctgg ccttgaggcg cagattgatg aagcccggac cggcgacatc gacggattcg
1681acatcctcat cggccttcag cgcctcggca atgcgggcag caagctcgcg cgggttctgg
1741ccgaccgcct tggaaagcac cattgcggca ttggtcgcga tatcgccatg cgaagcatcg
1801cgcgggggct cgacacctat gcgtgaaagg tcaagttcac caccatcttt tggtttcaga
1861tcaatatctt gcaacgtttt tttaatacgt gcatcgaaat ctgcaaagat attcatggtc
1921tgtcctgtca ggctagcgcg gttcctgttt taacagaatc gccggaacca ctctaactat
1981tgttttgtcg cattttccaa cgcaaaaccg tttcacactt ttggctcgaa aatactctaa
2041cgcctggatt tttttccagt tttcccggcg cgggttcatc caaaaaacgc ggatattcga
2101tgcatttgcg tatcgaagcc gcttcgtccg gccccgatta agctaaatcg ggggttcggt
2161caaacaatcg tcggtgttcg cgcacggcat aattatcagt catcccggcc agataatcgg
2221ctacgcggcg tgcgagtgct gccttatcca gtgcctcaca gcccaaacgc cattcatcag
2281gcatcaatga gggatcggtg aaacaggcat cgaagagatc ctgcacgatc ctgtcggctg
2341cgtgcctgcg caccaccacg ctttcgtgaa aatagagatt cttgaacaaa aagcgcttca
2401gcaccttttc ctcggcccgc atggcgtcgg aaaagccaac cagcgcgcgc ggctggttgt
2461gcacgtcttc catcgttccg ggcctggcgg atgcaaggcg gcgctgcgcc tcctcgatca
2521cgtcttccac catgatcgtg atctggcggc gcaccagttc gtgtccagtg cggacggggt
2581cgagattggg ataacgtgtc cgcacaatat caagcagccg tttggcgagc ggtacttcgt
2641ccagcgattc gagggtcaag agccctgccc gcaagccatc atcaatgtca tgcgcattgt
2701aggcaatgtc gtcggcaatg gccgcgcatt gcgcctcaag gctcgcaaag cgtgaaagct
2761ccagatcata gcgcgcgtta aaatccagaa tgggttgcgg aaccgggata tcgggatggg
2821ctgcatatgg ccccagcaac gggccattat gcttcaccag accttccagc gtttcccacg
2881aaaggttgag gccatcgaaa tcagcgtagc gatgctcaag cttcgtgacg atcctgagcg
2941actgggcatt atggtcgaaa ccgccgaaat tcttcatgcg ctcgttgagt gcgtcctcgc
3001cggtatggcc gaagggcgtg tggccgaaat catgaacgag agcgacagct tcagcgaggt
3061cttcatccag gcgcagcgcg cgcgccagcg cccgcgcaat ctgcgccacc tcgatggtgt
3121gcgtcagcct cgtgcggtaa tgatcgccct catgcgcgat gaaaacctgc gtcttgtgct
3181ttaaacgccg gaaagccgtg gagtggataa tacggtcacg gtcccgctgg aacggcgtgc
3241gggtcgggct ttccggttcc ggcaccagcc ggccacggct gaaagcagga ttgctggcat
3301aaggcgcacg ttcgcgataa ccgaagccta ttccttccag cgacattgcg atgttttcct
3361cactgtaata tgattacgtc aaattggtgc gtcattgact tccgcaacct gcgttcatag
3421ctatcagcta aacatgaagg caagtacgcg gctatcggaa aatctcaaga acgcataacc
3481cgatccccgt ccctgcaaac ggaacaaggc aaatgacagg cattaccgtt tcagattccg
3541ctgccaggcg gatcgccaaa attctcgatt cggagccggg aaagaccgcg cttcgcgttt
3601ctgtcgaagg tggcggctgc tccggctttt cctataaata tgacctcgtc gacgcacaga
3661ccgaggatga catcgtcatc gaaaaactcg gcgccagagt gctgattgat tccatctccg
3721tgccttatat ggacggctct gaaattgatt tcgtcgatga tctgatgggg caatcattcc
3781agatccgcaa ccccaatgcg accgcttcct gcggctgcgg caccagcttc gcgatctgag
3841cggcgcaaca aaacccgtga tgcaaaaccg gcggccagat ggccgccgtt tttttaacca
3901tggcaacaag cggacagttt cagactttca ctgaagcaac ggtcgcttcg atgtggtcca
3961ccagcgcatc ttgcaggcca agccgcccgg caagcatatc cagatagccg cgttcggcgc
4021ggttatctgg atcgatagcc agccgcgatg ccgtataaag ttcaactttc tgctcttccg
4081tctgcgctgc ggcaaccagc acatcgagat cgacgggttc ggccagttcc cttgcaagga
4141aggcctcagc ctcgtcgtcc agaccggaaa tcttcacctt ttccatgatg cgggcacgtt
4201cggcatcatc aatataacca tcagccctgg cggcggcgat catggcctga accagcgtca
4261gcgcgaaact attgctcatc gcgggagaat gcggatggaa gggtgaatcg gccggtggcg
4321ccggaagaag ctccggctct tttgccaccg gctgttccgc ctcctgcggg gcctgaccgg
4381acttataatt cttgtaggca agatagccca atccggctat ggcggcgatg ccgccgacag
4441ttgctacatt gccagcaagt ttgcggcccg ttttcgtgcc aaaaatggct gcggctatgg

TABLE 7
cading sequence af gale-like cading sequence
af B. melitensis 16M (SEQ ID NO:28).
atgacaattcttgtaacaggtggtgctggctatatcggctcccacacgtg
tgtgcagttgatcgaggcaggccatgaagtggttgtggtcgataatttcg
acaacagccatcctgaggcactgcatcggattgaaaagatcacgggccgc
gcgccgcgccgcgaaccgggcgatattcgcgatcgcgcccttatggaaca
gatgatcaaacgccataaatgcactgcggttatccattttgccgggctga
aggccgtgggtgaatcgagcgaaaagccgctgctctattatgattgcaat
gtgctgggcacacttcggcttctgcaggccatggaagcgacaggcgtgaa
gaagctcgttttcagctcttcggccaccgtctatggcgacccggataaac
tgccgatcaccgaagatcagcccctttcggccaccaatccctatggccgg
accaagcttgtcatcgaagacatgctgcgcgacctttataacagtgacaa
tagctgggcgattgcgattctgcgctatttcaatcctgtcggcgctcatg
aaagcgggcttatcggtgaagacccgaagggtattcccaacaatctgatg
cccattattgctcaggtcgcaactggacgacgcgaaaagctgaacatctg
gggcaacgactatccgacaccggatggcaccggcgtacgcgactatatcc
atgtcaacgatctggctgccgggcacctcaaggccctgaaaaagctggat
aagcccaagtgcttcgccgtcaatcttggaacggggcagggctatagtgt
tcttgatgtgatcaaggcgtttgaacatgtctccaatcgcgagatcaaat
atgagattgcgccgcgccgtcccggcgatgttgccgaatgctatgccgat
cccggctttgcaaagaaatttctgggctggtcggctgagaaaaacctgcg
tgaaatgtgtcaggacatgtggaactggcaatcgaaaaatccgaacggct
acgaataa

Although the description herein contains many specific examples and descriptions, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of the invention. For example, thus the scope of the invention should be determined by the appended claims and their equivalents, rather than by the examples given.

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