CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims benefit of U.S. Provisional Application No. 61/695,205 filed August 30, 2012, and U.S. Provisional Application No. 61/783,367 filed March 14, 2013 both of which are herein incorporated by reference in their entirety for ail purposes.

ACCOMPANYING SEQUENCE LISTING

[0002] The contents of the text file submitted electronically herewith are incorporated, herein by reference in their entirety: A computer readable format copy of the Sequence Listing (file name: MONT-139_00US_SeqList.txt, date recorded Mar. 13, 2013, file size 3.19 kilobytes).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant number 2010-34397- 21391 awarded by United States Department of Agriculture (USD A). The government has certain rights in the invention

FIELD OF THE INVENTION

[0004] The present invention is generated in the field of vaccine development, and specifically provides new vaccines for brucellosis.

BACKGROUND OF THE INVENTION

[0005] Brucellosis is an infectious and contagious disease of animals and humans caused by infection by bacteria of the genus Brucella. Brucella infects a significant number of people and livestock in developing countries, and infects wild as well as domestic animals in the United States. These bacteria infect animals such as sheep, goats, cattle, pigs, deer, elk, dogs, and horses. Humans can acquire the disease by coming into contact with infected animals or contaminated animal products. Brucellosis in animals is generally typified by late -term abortions in females and inflammatory lesions in the male reproductive tract. For example in cattle, brucellosis causes abortions and decreased meat and milk production. Brucellosis causes heavy economic losses in animal production resulting from abortions, sterility, decreased milk production, veterinary attendance, and the cost of replacer animals. The disease can therefore be the cause of serious health problems and substantia] economic losses. In the U.S. the primary brucellosis concern is the transmission of B. abortus from infected bison to cattle, and transmission among wild-life (bison and elk) which can act as reservoirs for the strains that can then infect, livestock. The disease is not only an impediment to free animal movement and export but also presents a hazard to human health. For example, in humans, brucellosis causes intermittent or irregular fever also known as undulant, Malta, or MediteJTanean fever. Also, Brucella is a potential biowarfare agent; strains of Brucella have been constmcted with resistance to multiple antibiotics that are typically used to treat the disease in humans. These strains pose a significant threat to those exposed.

[0006] The genus Brucella currently contains six species: B. abortus, B. melitensis, B. canis, B. ovis, B. suis and B. neolomae which vary in their ability to infect host animals, B, abortus primarily infects cattle but is transmitted to buffaloes, camels, deer, dogs, horses, sheep and man. B. melitensis causes a highly contagious disease, mainly in sheep and goats, although cattle can be infected, and is the most common species for human infection. B. suis covers a wider host range than most other Brucella species and can infect swine, hares, reindeer, caribou, rodents, and humans. B. canis, which can also infect humans, causes inflammation of the epididymis and testicle in the male dog and abortion and metritis in the female dog. B. ovis is responsible for epididymitis in rams and occasionally abortion in ewes, but does not infect other animals or humans. Goats are susceptible to the disease by experimental infection. B. neotomae is only known to infect the desert wood rat under natural conditions.

[0007] Brucella infection can be particularly problematic since there is no effective way to detect infected animals by their appearance. The most obvious signs in pregnant animals are abortion, the birth of weak calves, and decreased milk production. Other signs of brucellosis include an apparent lowering of fertility with poor conception rates, retained afterbirths with resulting uterine infections, and (occasionally) enlarged, arthritic joints. Infected cows that have not been identified can continue to harbor and discharge infectious organisms may spread the infection,

[0008] Brucellosis is commonly transmitted to susceptible animals by direct contact with infected animals or with an environment that has been contaminated with discharges from infected animals. The disease may also be spread when wild animals or animals from an affected herd mingle with brucellosis-free herds. Humans are typically infected through contact with infected livestock, or by consumption of contaminated meat, or dairy products or by inhalation of infected aerosols.

[0009] The most preferred type of disease management is to avoid infection and to reduce the incidence and spread of the disease by vaccination. For livestock, at present vaccination consists of using an attenuated (weakened) vaccine strain such as the Brucella abortus strain 19, RB51 , the B. melitensis strain Rev ! and the killed H38 vaccine.

[0010] Vaccination by existing vaccine strains of Brucella, such as B. abortus strains 19 and RB51 , and B. melitensis strain Revl , can both protect against the Brucella species from which they were derived, and provide cross protection against infection by other species, such as B. abortus, B. melitensis, B. ovis, B. suis, B. canis and B. neotomae (Winter, A. J. et al., 1996, Am. j. Vet. Res., 57:677; P. Nicoletti in Animal Brucellosis, CRC Press (1990), pp. 284-296; J. M. Blasco in Animal Brucellosis, CRC Press (1990), pp. 368-370; and G. C. Alton in Animal Brucellosis, CRC Press (1990), pp. 395-400). However, none of the current brucellosis vaccines are completely effective. The best current vaccine against brucellosis in cattle is RB51 which is only approximately 50% effective, allowing herds to still become infected. The current cattle vaccines, strains RB51 and SI 9, are not effective in bison which may serve as a reservoir that can infect co-mingling livestock. Thus there is a current need to develop more effective Brucella vaccines that induce immunity in a wider range of hosts.

[0011] One of the most commonly used vaccines to prevent bovine brucellosis is B. abortus strain 19 which is an attenuated organism of smooth morphology that is unable to grow ^r in the presence of erythritol (Mingle, C. . 1941. J. Am. Vet. Med. Assoc. 99:203). Although it is one of the best vaccines for cattle, its effectiveness is limited since about 20% of vaccinated animals do not develop immunity to the bacterium. Subcutaneous vaccination of pregnant cattle can results in abortions in between 1% and 2.5% of vaccinated animals (Manthei, C.A. 1952. Proc. 56th Annu. Meet. Livestock Sanit. Assoc. 1 15). Another disadvantage of the vaccine is that serological tests used to detect brucellosis-infected cattle cannot differentiate between antibodies produced against the Strain- 19 Vaccine and antibodies produced against the wild-type brucellosis disease organism.

[0012] Another Brucella vaccine, H38, is composed of killed, smooth, virulent B. abortus cells in adjuvant. This vaccine is effective at, protecting against infection and can be administered to pregnant or lactating animals. However, because it stimulates a long lasting immune response which interferes with the serological diagnosis of brucellosis, and it induces a marked skin reaction on the injection site of the vaccine, it is not used very often (Alton, 1985 CEC seminar, Brussels, November 1984, 215-227; Plommet, 1991 Proceedings of a symposium held in Izmir, Turkey, on September 24-26, 77-85).

[0013] The Rev 1 vaccine is composed of living attenuated B. melitensis cells and is used in most countries that vaccinate small ruminants against B. melitensis. Rev 1 protects sheep and goats against B. melitensis infection, and rams against B. ovis infection. The use of Rev I in cattle has been investigated and results indicate that Rev 1 protects better than strain 19 (Van Drimmelsen et al. 1964. Bull. Off. Int. Epiz. 62:987; Horwell et al. 1971. S. Ak. Vet. Med. Assoc. 42:233; Garcia-Carrillo 1980. Zentralbl. Veterinaermed. 27: 131 ). While this vaccine is attenuated when compared to field strains, it retains some virulence and may cause abortion in pregnant sheep and goats, and it is excreted in the milk.

[0014] The Brucella abortus vaccine RB51 is a laboratory-derived Iipopolysaccharide (LPS) O- antigen-deficient mutant of the virulent B. abortus strain 2308 (S2308) [Schurig, G. G. et al. (1991) Vet. Microbiol. 28: 171-188] that is used as a vaccine against brucellosis and provides resistance to rifampin. Strain RB51 is as effective as Brucella abortus strain 19 vaccine but is much less abortigenic in cattle. It does not produce any clinical signs of disease after vaccination, nor does it produce a local vaccination reaction at the injection site. The organism is cleared from the blood stream within 3 days and is not present in nasal secretions, saliva, or urine. Immunosuppression does not cause recrudescence, and the organism is not spread from vaccinated to non-vaccinated cattle. The vaccine is safe in all cattle over 3 months of age.

[0015] Unlike Strain- 19 however, the RB51 vaccine does not stimulate antibodies that are detected by the standard brucellosis serological tests since it lacks the polysaccharide O-side chains on the surface of the bacteria which are responsible for the development of the diagnostic antibody responses of an animal to brucellosis infection and therefore does not show up on standard diagnostic tests (Cheville, N. F. 1993, supra; Jimenez de Bagues, M. P. et al. (1994) Infect. Irnmun. 62:4990-4996). This vaccine however does produce other types of antibodies that can be detected with a special assay to detect if an animal has been vaccinated.

[0016] Although RB51 is the best current vaccine against brucellosis in animals it, like all other brucellosis vaccines available today, is still not 100% effective. Thus, there is a need for a more effective vaccine which is able to provide long-term protection against brucellosis and infection by different Brucella strains. There is also a need for the development of effective, long-lasting vaccines that allow immunized animals to be distinguished from non-immunized animals using standard serological tests,

[0017] The present invention relates to the development of a brucellosis vaccine capable of preventing and possibly therapeutically controlling a Brucella infection. The present invention describes methods to modify wild-type or mutant Brucella strains by deleting at least one virulence gene or a combination thereof. These possible combinations of mutations permit the development of live attenuated Brucella strains that mimic virulent Brucella and infect the host by the same mechanism as a virulent strain, but causes mild, very little, or no disease. The vaccines of the invention may confer immunity against other homologous or heterologous Brucella infections. The present invention also provides vaccines comprising Brucella strains that express selectable marker polypeptides which allow the identification of immunized animals through standard serological tests. The present invention also provides for vaccines comprising Brucella strains comprising heterologous polypeptides and may provide protection against other pathogens.

SUMMARY OF THE INVENTION [0018] The present invention encompasses Brucella vaccines that protect against Brucella infections and brucellosis as well as the Brucella strains that can be used in vaccines to protect against Brucella infections and brucellosis. The present invention further encompasses methods of inducing immunity to a Brucella infection in a mammal with the Brucella vaccines or the Brucella strains of the invention. The present invention further encompasses a method of differentiating mammals immunized with the Brucella vaccines from unimmunized mammals by identifying antibodies produced to a heterologous polypeptide.

[0019] In one embodiment, the vaccines comprise Brucella strains comprising a loss of function mutation in at least one gene that significantly reduces the pathogenicity of the Brucella strain. In another embodiment, the vaccines comprise Brucella strains comprising a loss of function mutation in one or more virulence genes. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in either norD or btp . In another embodiment, the vaccine comprises any Brucella strain comprising a loss of function mutation in one or more virulence genes. In another embodiment, the vaccines comprise B. abortus, B. melitensis , or B, suis strains comprising a loss of function mutation in one or more virulence genes, including znuA, norD, and/or btpl loss of function mutations,

[0020] In another embodiment, the vaccines comprise Brucella strains comprising a selectable marker with or without a loss of function mutation in one or more virulence genes. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a selectable marker. In another embodiment, the vaccines comprise Brucella strains comprising a selectable marker, a zmtA loss of function mutation, and a second loss of function mutation in norD and/or btpl. In one embodiment, the vaccines comprise Brucella strains wherein the selectable marker may be inserted into the Brucella genome in an uncoded region or in a nonfunctional gene locus. In another embodiment, the selectable marker is lacZ, gfp, and/or rfpl. In another embodiment, the vaccine comprises any Brucella strain comprising a selectable marker and a loss of function mutation in one or more virulence genes. In another embodiment, the vaccines comprise B. abortus, B. melitensis, or B. suis strains comprising a selectable marker and a loss of function mutation in one or more virulence genes (e.g., znuA, norD and/or btpl).

[0021] In another embodiment, the invention provides a method of differentiating immunized animals from unimmimized animals by identifying antibodies that are produced to the selectable marker. In another embodiment, the antibodies produced to the selectable marker bind to lacZ, rfpl, and/or gfp. The antibodies produced to the selectable marker may be assayed using standard techniques known in the art.

[0022] In one embodiment, the vaccines comprise Brucella strains comprising a gene encoding a protective antigen. In another embodiment, the vaccines comprise Brucella strains comprising a gene encoding a protective antigen and a loss of function mutation in one or more vimlence genes, including, but not limited to, znuA, norD and/or btpl. In another embodiment, the vaccines comprise Brucella strains comprising a gene encoding a protective antigen and a znuA loss of function mutation. In another embodiment, the vaccines comprise Brucella strains comprising a gene encoding a protective antigen, a znuA loss of function mutation, a second loss of function mutation in norD and/or btpl. In another embodiment, the vaccines of the invention comprise Brucella strains wherein the gene encoding the protective antigen is inserted into the genome in an uncoded region or in a nonfunctional gene locus. In another embodiment, the protective antigen is overexpressed and confers resistance to a heterologous pathogen. In another embodiment, the gene encoding the protective antigen in the vaccines of the invention is cfaB, potD, potF, hotA, cafl, and/or IcrV. In another embodiment, the vaccine comprises any Brucella strain comprising a gene encoding a protective antigen and a loss of function mutation in one or more vimlence genes. In another embodiment, the vaccines comprise B. abortus, B. melitensis, or B. suis strains comprising a gene encoding a protective antigen (e.g. CfaB, PotD, PotF, BotA, Cafl, and/or lcrV and a loss of function mutation in one or more virulence genes (e.g., znuA, norD and/or btpl ).

[0023] The present invention also provides vaccines comprising Brucella strains comprising a mutation in one or more genes encoding LPS. In one embodiment, the vaccines comprise Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene (e.g. wbka, gmd, IpsA and/or manB). In another embodiment, the vaccines comprising Brucella strains comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced the araBAD (P _BAD ) promoter. In another embodiment, the vaccine comprises any Brucella strain comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene. In another embodiment, the vaccines comprise B. abortus, B. melitensis, or B. suis strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene.

[0024] In another embodiment, the vaccines comprise Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene and a loss of function mutation in one or more virulence genes including, but not limited to, znuA, norD and/or btpl. In another embodiment, the vaccine comprises any Brucella strain comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene and a loss of function mutation in one or more virulence genes. In another embodiment, the vaccines comprise B. abortus., B. melitensis, or B. suis strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, and further comprise a loss of function mutation in one or more virulence genes (e.g. znuA, norD and/or btpl).

[0025] The present invention also provides a method of inducing immunity to a Brucella infection in a mammal comprising administering to the mammal the vaccines or strain of the invention. In another embodiment, the administration of the vaccines or strain of the invention to a mammal confers immunity to a Brucella infection from the same strain as was used to immunize the mammal. In another embodiment, the administration of the vaccines or strain of the invention to a mammal confers immunity to a Brucella infection from a different strain as was used to immunize the mammal. In another embodiment, the mammal in which immunity has been induced is human, cattle, goat, sheep, and/or swine. [0026] The present invention also provides Brucella strains comprising a loss of function mutation in at least one gene that significantly reduces the pathogenicity of the Brucella strain. In another embodiment, the Brucella strain comprises a. loss of function mutation in one or more virulence genes. In another embodiment, the Brucella strain comprises a znuA loss of function mutation. In another embodiment, the Brucella strain comprises a znuA loss of function mutation and a second loss of function mutation in norD and/or btpl . In another embodiment, any Brucella strain may comprise loss of functions mutations in at least one or more virulence genes (e.g. znuA, norD and/or btpl). In another embodiment, the Brucella strain comprising a loss of function mutation in at least one virulence gene is B. abortus, B, melitensis, or B. suis.

[0027] In one embodiment, the Brucella strains of the invention comprise a selectable marker. In another embodiment, the Brucella strains comprise a selectable marker and a loss of function mutation in one or more virulence genes. In another embodiment, the Brucella strains comprise a selectable marker and a znuA loss of function mutation. In another embodiment, the Brucella strains comprise a selectable marker, a znuA loss of function mutation, and a second loss of function mutation in norD and/or btpl. In another embodiment, the Brucella strain comprises a selectable marker inserted into the Brucella genome in an uncoded region or in a nonfunctional gene locus. In another embodiment, the selectable marker is lacZ, gfp, and/or rfp. In another embodiment, any Brucella strain may comprise a selectable marker and a loss of function mutations in one or more virulence genes. In another embodiment, the Brucella strain comprising a selectable marker and a loss of function mutation in at least one virulence gene (e.g. znuA, norD and/or btpl) is B. abortus, B. melitensis, or B. suis.

[0028] In one embodiment, the Brucella strains comprise a gene encoding a protective antigen. In another embodiment, the Brucella strains comprise a gene encoding a protective antigen and a loss of function mutation in one or more virulence genes (e.g. znuA, norD and/or btpl). In another embodiment, the Brucella strains comprise a gene encoding a protective antigen and a znuA loss of function mutation. In another embodiment, the Brucella strains comprise a gene encoding a protective antigen, a znuA loss of function mutation, and a second loss of function mutation in either norD and/or btpl. In another embodiment, the Brucella strains comprise the gene encoding the protective antigen inserted into the genome in an uncoded region or in a nonfunctional gene locus. In another embodiment, the protective antigen is overexpressed and confers resistance to a heterologous pathogen. In another embodiment, the gene encoding the protective antigen is cfaB, potD, potF, botA, cafl , or lcrV. In another embodiment, any Brucella strain may comprise a gene encoding a protective antigen and a. loss of function mutation in one or more virulence genes. In another embodiment, the Brucella strain comprising a gene encoding a protective antigen and a loss of function mutation in at least one virulence gene (e.g. znuA, norD and/or btpl) is B. abortus, B, melilensis, or B. suis.

[0029] The present invention also provides Brucella strains comprising a mutation in one or more genes encoding LPS. In one embodiment, the Brucella strains comprise a mutation in a gene encoding LPS (e.g., wbka, gmd, IpsA and or rnanB) wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene. In another embodiment, the Brucella strains comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the araBAD (PBAD) promoter. In another embodiment, any Brucella strain may comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene. In another embodiment, B. abortus, B. meliiensis, or B. suis strains comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene,

[0030] In one embodiment, the Brucella strains comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, and a loss of function mutation in one or more virulence genes. In another embodiment, the Brucella strains comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, and a loss of function mutation in znuA, norD and/or btpl . In another embodiment, any Brucella strain may comprise a mutation in a gene encoding LPS (e.g., wbka, gmd, IpsA and or rnanB) wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, and a loss of function mutation in one or more virulence genes. In another embodiment, B. abortus, B. melitensis, or B, suis strains may comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, and a loss of function mutation in one or more virulence genes (e.g., znuA, norD and/or btpl) .

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1A-B demonstrate that the AznuA AnorD B. abortus-lacZ mutant constitutively expresses β-galactosidase in the presence (Fig. 1A) or absence (Fig. IB) of arabinose. Spots 1 and 2 depict, AznuA AnorD B. abortus mutants expressing β-galaetosidase and Spot 3 depicts wild-type B. abortus 2308 showing no β-galactosidase activity.

0032] Figure 2 shows AznuA AnorD B. abortus mutants are attenuated in RAW264.7 macrophages. Values are the means of quadruplicate wells + SEM. The differences in macrophage colonization are significant: * P< 0.0001 ; ** P 0.003; ⁴ Ρ<0.001.

[0033] Figure 3 demonstrates that the AznuA AnorD B, abortus-lacZ strain is attenuated in human peripheral blood macrophages. Values are the means of triplicate wells + SEM. The differences in growth between the AznuA AnorD B. abortus-lacZ mutant and RB51 are significant (*P<0.005). The differences in growth between the AznuA AnorD B. abortus-lacZ mutant and the wild-type 2308 strain are also significant P<_0.001).

[0034] Figure 4 demonstrates that the AznuA Abtpl B. abortus-LacZ vaccine and its parental vaccine (AznuA B, abortus) were greatly attenuated and unable to replicate in RAW26.7 macrophages, unlike the RB51 or wild-type B. abortus strain 2308. Values are the means of quadruplicate wells + SEM. Differences in macrophage colonization versus wild-type B. abortus 2308, *P < 0.001 , **P = 0.003; differences in macrophage colonization versus RG51 vaccine, *P < 0.001.

[0035] Figure 5 demonstrates that the AznuA AnorD B. abortus-lacZ mutant was readily cleared from, the host at a rate similar to that of the conventional RB51 vaccine. Values are the mean CFUs from individual mice + SEM. and differences in colonization were determined when compared to S19 vaccine, *P<0.001 , **P<0.009, ***P<0.029.

[0036] Figure 6A demonstrates that a single dose of the AznuA AnorD B. abortus-lacZ vaccine was sufficient to reduce colonization by wild-type .5. abortus relative to naive (PBS) control mice, or mice vaccinated with RB51 . Animals that received two doses of vaccine were completely protected against the wild -type B. abortus challenge. Values are the means of individual mice + SEM; for colonization, *P<0.001, **P=0.005 versus PBS-dosed mice, and ^tt P<G,008 versus RB51 -vaccinated mice.

[0037] Figure (SB demonstrates that vaccination with one or two doses of the AznuA AnorD B. abortus-lacZ vaccine resulted in less splenic inflammation than vaccination with the RB51 vaccine. Values are the means of individual mice + SEM; for colonization, *P<0.001 versus PBS-dosed mice, and ^t P= 0.003 versus RB51 -vaccinated mice.

[0038] Figure 7A demonstrates the AznuA Abtpl B. abortus-lacZ vaccine was greatly attenuated and unable to replicate in the RAW26.7 macrophages. RAW264.7 macrophages at a bacteria to macrophage ratio of 30:1 were infected with wild-type strain 2308, live RB51 vaccine, B. abortus AznuA mutant, and the AznuA. Abtpl B. abortus-lacZ, and sampled 0, 4, 24, or 48 hours after infection. Values are the means of quadruplicate well + SEM. Differences in macrophage colonization versus wild type B. abortus 2308, *P<0.0()1, **P~0.003; differences in macrophage colonization versus RB5I vaccine, ⁴ Ρ<0.001.

[0039] Figure 7B demonstrates that mice immunized with the AznuA AnorD B. abortus-lacZ or the AznuA Abtpl B. abortus-lacZ vaccines showed greatly reduced colony forming units (CFUs) levels relative to PBS-im unized mice. The dashed horizontal line depicts sensitivity of CPU detection. Values are the means of individual mice + SEM; for colonization, *P<0.001.

[0040] Figure 8 demonstrates the AznuA AnorD B, abortus-lacZ vaccinated mice show a positive IgG anti- gal antibody titer relative to RB51 -vaccinated mice. Significant differences versus RB51 -vaccinated mice are shown: *P=0.011.

[0041] Figures 9 A and 9B demonstrate that the AznuA AnorD B. melitensis-lacZ vaccine confers protection against wild-type Brucella strains and limit colonization of the spleens (Figure 9A) and lungs (Figure 9B). Values are the mean CFUs from individual mice + SEM relative to colonization by PBS-dosed mice, *P<0.001 , ^:;::;: P 0.003. ***P<0.05; NS = not significant.

DETAILED DESCRIPTION OF THE INVENTION

[0042] All publications and patent applications herein are incorporated by reference in their entirety for ail purposes. [0043] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described .

[0045] As used herein, "a," "an" or "the" can mean one or more than one. For example, a gene can mean a single gene or a multiplicity of genes.

[0046] Further, the term "about," as used herein when referring to a measurable value such as an amount of a compound or agent, dose, time, temperature, and the like, is meant to encompass variations of +/-.20%, +/-. !()%, +/-.5%, +-. !%, +/-.0.5%, or even +/-.0.1 % of the specified amount.

[0047] As used herein, the term "adjuvant" refers to a substance sometimes included in a vaccine formulation to enhance or modify the immune-stimulating properties of a vaccine.

[0048] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0049] As used herein, the terms "attenuation" and "attenuated" refer to diminution of the virulence in a strain of an organism, obtained through selection of variants that occur naturally or through experimental means.

[0050] As used herein, the term "attenuated Brucella vaccine" refers to an attenuated Brucella strain or serovar that has been sufficiently compromised to remain attenuated and to provide protection against homologous or heterologous Brucella species challenge. In addition, the live attenuated Brucella vaccine can also be genetically manipulated to carry a "foreign (native or non-native)" protein, carbohydrates, lipids, or protein encoded by nucleic acids for the expressed purpose of delivering a subunit vaccine. [0051] As used herein, the term "attenuated strain" refers to a strain of microorganism that has been weakened or treated in such a way as to decrease or eliminate the ability of a microorganism to cause infection or disease.

[0052] As used herein, the term "attenuated vaccine" refers to a vaccine in which live pathogens

(such as bacteria) are weakened through chemical, physical, or recombinant processes in order to produce an immune response without causing the severe effects of the disease.

[0053] As used herein, the term "antigen" refers to a foreign substance that when introduced triggers an immune system response, resulting in production of an antibody specific for the antigen.

[0054] As used herein, the term "Brucella" refers to any Brucella species, including, but not limited to B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, and B. suis. Brucella is an aerobic, gram-negative coccobacillus which causes the disease brucellosis in animals and humans.

[0055] As used herein, the term "brucellosis" refers to a bacterial disease caused by members of the Brucella genus that can infect humans but primarily infects livestock.

[0056] As used herein, the term Brucella vaccine" refers to a vaccine comprising a live, attenuated, or dead strain of Brucella to provide protection against homologous or heterologous Brucella species challenge. In addition, the Brucella vaccine can also be genetically manipulated to cany a "foreign (native or non-native)" protein, carbohydrates, lipids, or protein encoded by nucleic acids.

[0057] As used herein, the term "codon optimized" refers to the modification of a coding sequence to enhance its expression in a particular host. The codons that are used most often in a particular organism are "optimal codons". Codons can be substituted to reflect the preferred codon usage of the host. Optimized codon sequences can be prepared using standard methods in the art.

[0058] As used herein, the term "epitope" refers to the portion of a macromolecuie (antigen) which is specifically recognized by a component of the immune system, e.g., an antibody or a T- cell antigen receptor; epitopes are also called antigenic determinants. [0059] As used herein, the term "effective immune response" refers to an immune response that confers protective immunity. For instance, an immune response can be considered to be an "effective immune response" if it is sufficient to prevent a subject from developing a Brucella infection after administration of a challenge of dose of Brucella or administration of Brucella toxins. An effective immune response may comprise a humoral immune response and cell mediated immune response. The effective immune response refers to the ability of the vaccine of the invention to elicit the production of antibodies. An effective immune response may give rise to mucosal immunity. See, for instance, Holmgren and Czerkinsky, Nature Medicine 1 1 :S45~S53 (2005).

[0060] As used herein, the term "gene expression cassette" refers to a nucleic acid construct comprising a nucleic acid encoding one or more immunogenic peptides under the control of an inducible promoter. The nucleic acid encoding one or more immunogenic peptides may possess 99%, 95%, 90%, 85%, or 80% sequence identity to sequences known in the art. In one embodiment, the inducible promoter is an in vivo inducible promoter. The gene expression cassette may additionally comprise, for instance, one or more nucleic acids encoding a secretion tag and a nucleic acid encoding a peptide linker. A gene expression cassette may be contained on a plasmid or may be chromosomally integrated, for instance, at a gene deletion site. A microorganism may be constructed to contain more than one gene expression cassette.

[0061 As used herein, the term "heterologous Brucella infection" refers to infection by a Brucella strain different from the Brucella strain used to immunize the subject against brucellosis.

[0062] As used herein, the term "homologous Brucella infection" refers to infection by a Brucella strain the same as the Brucella strain used to immunize the subject against brucellosis.

[0063] As used herein, the term "expression" refers to the vaccine vector which is responsible for producing the vaccine.

[0064] As used herein, the term "immunity" refers to protection against infectious disease conferred either by the immune response generated by immunization or previous infection or by other non-immunologic factors. [0065] As used herein, the term "immunization" refers to a process by which a person or animal becomes protected against a disease; the process of inducing immunity by administering an antigen (vaccine and/or strain) to allow the immune system to prevent infection or illness when it subsequently encounters the infectious agent,

[0066] As used herein, "an immune response" is meant to encompass cellular and/or humoral immune responses that, are sufficient to inhibit or prevent infection, or prevent or inhibit onset of disease symptoms caused by a pathogenic microbial organism., particularly members of Brucella species,

[0067] As used herein, the term "loss of function mutation" is a mutation which results in either no polypeptide expression, or expression of a non-functional polypeptide from the mutated gene. The mutations may be made, for example, in the gene, the gene promoter, or in upstream regulatory elements required for gene expression. The mutations may be point mutations, deletions of one or more nucleic acids from the gene, or insertion of one or more different nucleic acids into the gene.

[0068] As used herein, the term "selectable marker" refers to genes used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. A selectable marker such as one providing for antibiotic resistance protects the organism from a selective agent that would normally kill it or prevent its growth. Examples of genes providing antibiotic resistance include, but are not limited to, ampR and neo. Selectable markers may also be used for screening to provide a way to visually identify transformed cells. For example, insertion of green fluorescent protein (gfp) makes cells glow green under UV light. Yellow iyfp) and red versions (rfp) are also commonly used in the art. Another commonly used marker gene is the bacterial lacZ gene which encodes beta-gaiactosidase and turns the transformed cell blue when grown on medium containing X-gal.

[0069] As used herein, the term "multivalent vaccine" refers to a Brucella vaccine that also confers immunity to a different organism by expressing an immunogenic polypeptide of the different organism.

[0070] As used herein, the term "mucosal" means any membrane surface covered by mucous. [0071] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081 ; Ohtsuka et al. (1985) J. Biol. Chem, 260:2605-2608; Cassol et al. (1992): Rossolmi et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g. , cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.

[0072] As used herein, the term "livestock" refers to any type of animals raised for home use or for profit. Examples include, but not limited to, alpaca, banteng, cattle, deer, reindeer, donkey, swine, gayal, goats, camels, buffalo, bison, guinea pigs, rabbits sheep, pigs, horses, llamas, mules, donkeys, dogs, cats, water buffalo and yaks. Livestock includes but is not limited to ruminants, including both large and small ruminants.

[0073] As used herein, the term "percentage sequence identity" of two nucleic acid sequences is the number of identical amino acids shared by these two sequences after a pairwise alignment divided by the total length of the shortest sequence of the pair.

[0074] As used herein the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0075] As used herein, the term "promoter" refers to a region of DNA involved in binding RNA polymerase to initiate transcription. [0076] As used herein, the term "protective antigen" refers to an antigen which when expressed by a Brucella strain enhances the protective immunity of the strain. The term "homologous protective antigen" refers to genes that are typically found in Brucella and which elicit an immune response in the host. Examples of these homologous protective antigens include, but are not limited to, potD, potF, bp26, omps, and trigger factor. The term "heterologous protective antigen" refers to genes that, are found in organisms other than Brucella. Examples of organisms from which heterologous protective antigens can be derived include other bacteria, viruses, and eukaryotic cells. Examples of these heterologous protective antigens include, but are not limited to the Clostridium botulinurn BotA, Yersinia pestis Cafl and IcrV, and enterotoxigenic Escherichia coli (ETEC) CfaB.

[0077] As used herein, the term "species" refers to organisms in the same genus that have similar characteristics.

[0078] As used herein, the term "strain" refers to a specific version of an organism.

[0079] As used herein, the term "sterile immunity" refers to the ability of an infectious agent, strain, vaccine, and/or antigen to confer immunity which exists even after the causative agent has been cleared from the host.

[0080] As used herein, the term "vaccine" means a preparation that contains an infectious agent or its components which is administered to stimulate an immune response that will protect animals, including human beings, from illness due to that agent. A therapeutic (treatment) vaccine is given after infection and is intended to reduce or arrest disease progression. A preventive (prophylactic) vaccine is intended to prevent initial infection. Agents used in vaccines may be whole -killed (inactive), live-attenuated (weakened) or artificially manufactured.

[0081] As used herein, the term "wildlife" refers to all non-domesticated mammals (e.g., wild buffalo, reindeer, deer, and wild mules), birds, reptiles, and amphibians living in a natural environment.

Attenuated Brucella Strains

[0082] The present invention describes Brucella vaccines for applications in livestock, humans, and wildlife to prevent brucellosis. The composition of these inventions are based on in-frame deletions of virulence genes from the Brucella genome which have been demonstrated to be effective in attenuating Brucella pathogenesis as described in part, in Yang et al. (Yang, et ai., 2006) and Clapp et al. (Clapp et al, 201 1) which are incorporated herein by reference in their entirety for ail purposes. The deletion of one or more virulence factors enhances the vaccine's safety, while not affecting its capacity for protection against wild-type Brucella challenge. The Brucella strains of the present invention can be any Brucella strain, including B. abortus, B. melitensis, B, suis, B. ovis, B. canis and B. neotomae. The Brucella strains of the invention are highly stable, have enhanced immunogenicity and retain Lipopolysaccharide (LPS) outside of the ceil. The Brucella strains described herein {See, for example, Examples 1-18) may be generated using standard recombinant techniques and can be adapted for human or veterinary medicine.

[0083] The present invention describes Brucella strains comprising mutations in one or more virulence genes. Examples of virulence genes that can be mutated include, but are not limited to, znuA, norD, bipl/tcpB, cfiG, cgs, ricA, hvrR, hvrS, the genes encoding the virB type IV secretion system {ΒΜΕΠ0025, B ME [10026, BMEIIQQ27, BMEl 10028, ΒΜΕΠ0029, BMEII0030, BMEII0031, ΒΜΕΠ0032, BME1I0033, BMEII0034, and BMEII0035), and the genes encoding lipopolysaccharide (gnid, man A, manC, per, pgm, pmn/manB, wbkA, whkB, whkC, wzm, and wzt). In one embodiment, the Brucella strain comprises at least a znuA loss of function mutation. In another embodiment, the Brucella strain comprises at least a znuA loss of function mutation in and a second loss of function mutation in the norD gene. In another embodiment, the Brucella strain comprises at least a znuA loss of function mutation and a second loss of function mutation in the htpl/tcpB gene. In another embodiment, the Brucella strains comprise at least a znuA loss of function mutation, a norD loss of function mutation, and a btpl/tcB loss of function mutation.

[0084] The invention further describes Brucella strains that comprise selectable markers to permit identification of the strains and seram identification to determine the animal's vaccination status. These selectable markers can be inserted into the Brucella genome in an uncoded region, in a nonfunctional gene locus, or within a gene locus. These selectable markers allow the strains to be easily detected to distinguish the strains of the invention from wild-type Brucella, and to confirm whether a subject has been immunized with the strain of the invention by measuring the anti-marker antibody titers. Selectable markers that may be used include, but are not limited to, genes encoding β-galaetosidase (lacZ), red fluorescent protein (rfp), green fluorescent protein (gfp), yellow fluorescent protein, blue fluorescent protein, and cyan fluorescent protein. Selectable markers also include, but are not limited to, genes such as AmpR, PAC, hph, hsr and neo that confer resistance to antibiotics. The antibodies produced to the polypeptides produced by the selectable genes can be assayed by any standard assay commonly used in the art. In one embodiment, the Brucella strain comprises at, least one selectable marker and a loss of function mutation in at, least one virulence gene. In another embodiment, the Brucella strain comprises at least one selectable marker and at least a znuA loss of function mutation. In another embodiment, the Brucella strain comprises at least, one selectable marker and a znuA loss of function mutation and a second loss of function mutation in the norD and/or btpl genes. In another embodiment, the Brucella strain comprises lacZ, rfp and/or gfp and a znuA loss of function mutation. In another embodiment, the Brucella strain comprises lacZ, rfp and/or gfp, a znuA loss of function mutation, and a second loss of function mutation in norD and/or btpl .

[0085] The invention further describes Brucella strains comprising at least one homologous protective antigen to enhance the protective immunity of these mutant strains. These may be used to generate vaccines against other pathogenic organisms. The protective antigens encompassed by the invention include, but are not limited to, genes such as potD (e.g. SEQ ID NO: l), potF (e.g. SEQ ID NO:2), bp26, omps, and trigger factor. In one embodiment, the Brucella strain comprises at least one homologous protective antigen and a loss of function mutation in at least one virulence gene. In another embodiment, the Brucella strain comprises at least one homologous protective antigen and at least a znuA loss of function mutation. In another embodiment, the Brucella strain comprises at least one homologous protective antigen, a znuA loss of function mutation and a second loss of function mutation in the norD and/or btpl genes. In another embodiment, the Brucella strain comprises potD and/or potF, a znuA loss of function mutation, and a second loss of function mutation in the norD and/or btpl genes.

[0086] The present invention further describes Brucella strains that comprise heterologous protective antigens. In one embodiment, the Brucella strain overexpresses the heterologous protective antigens. The heterologous protective antigens may come from diverse sources including, but not limited to, bacteria, viruses, fungi, protozoa, and metazoan parasites. The structural genes may encode envelope proteins, capsid proteins, surface proteins, toxins, such as exotoxins or enterotoxins, enzymes, or oligosaccharide antigen. The protective antigens include, but are not limited to, the Clostridium botulinum BotA, Yersinia pestis Cafl , and lcrV, enterotoxigenic Escherichia coli (ETEC) CfaB, Human Immunodeficiency Virus vif, Plasmodium cireumsporozoite protein, and arboviral coat protein. The antibodies produced to the polypeptides produced by the heterologous protective antigens can be assayed by any standard assay used in the art. In one embodiment, the Brucella strain comprises at least one heterologous protective antigen and a loss of function mutation in at least one virulence gene. In another embodiment, the Brucella strain comprises at least one heterologous protective antigen and at least a znuA loss of function mutation. In another embodiment, the Brucella strain comprises at least one heterologous protective antigen, a znuA loss of function mutation, and a second loss of function mutation in the norD and/or btpl genes, in another embodiment, the Brucella strain comprises hot A, cafl, lcrV, and'Or cfaB, a znuA loss of function mutation, and a second loss of function mutation in the norD and/or btpl genes.

[0087] The present invention further describes Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene. In one embodiment, the Brucella strains comprise a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, araBAD (P _RAD )- After the promoters of one or more Brucella genes encoding LPS are replaced with P _RAD , the mutated strains will express LPS normally in medium in the presence of arabinose. However, when these strains infect the host, expression of the LPS genes or operons will be turned off due to the absence of arabinose in the host. Eventually, the mutated Brucella strain will stop replicating due to the laclv of LPS expression and will be cleared from the host, meanwhile, the immune response is elicited. There are at least three advantages associated with the regulated shutoff expression of LPS in constructing Brucella vaccines: (1) further attenuation of the bacterial organism because LPS is a virulence factor (Haag et al., 2010); (2) because LPS is a potent protective antigen (1.3 log protection units) (Bhattacharjee et al. 2006), the synthesis of LPS at initial infection stage will stimulate the animal to produce anti-LPS antibodies; (3) delayed shutoff expression of LPS may activate the innate immune system in a dynamic manner, and thus enhance the overall immunity, since LPS core serves as a shield to prevent the innate immune responses from occurrence (Conde-Alvarez et al., 2012). The promoters of the genes encoding LPS that may he replaced with P _BA r include, but are not limited to, wbkA whkB, wbkC, gmd, per, pgm, IpsA, man A, manB, manC, wzm, and wzt.

[0088] In another embodiment, the Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene further comprise a loss of function mutation in at least one virulence factor. In another embodiment, the Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene further comprise a loss of function mutation in znuA, norD and/or btpl .

Vaccines Comprising Attenuated Brucella Strains

[0089] The present invention describes Brucella vaccines that can be adapted for human, livestock, and wildlife use, which have advantages over the vaccines currently available. The

Brucella strains used in the vaccines can be any Brucella strain, including B, abortus, B. melitensis, B. suis, B. ovis, B. cants and B. neotomae. The vaccines described herein can confer cross-protection against heterologous Brucella infections. In a non-limiting example, immunization with an B. abortus strain of the present invention also protects the vaccinated subject from infection by B. melitensis. The vaccines of the invention are highly stable, have enhanced immunogenicity, and are subject to the insertion of selectable markers which will stimulate the production of antibodies in the immunized animals. In one embodiment, the

Brucella vaccines of the present invention allow immunized animals to be distinguished from unimmunized ones by identifying antibodies raised to the heterologous polypeptide using standard serological techniques. The vaccines can be adapted for human or veterinary medicine.

[0090] The present invention also encompasses a method of inducing immunity to a Brucella infection in a mammal comprising administering to the mamma; the vaccines of the invention. In

77 one embodiment, the vaccines of the present invention induce immunity in at least 50% of vaccinated subjects. In another embodiment, the vaccines of the present invention induce immunity in 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97,%, 98%, 99%, or 100% of vaccinated subjects. In one embodiment, the administration of the vaccines of the invention to a mammal confers immunity to a Brucella infection from the same strain as that used to immunize the mammal. In another embodiment, the administration of the vaccines of the invention to a mammal confers immunity to a Brucella infection from a different strain as that used to immunize the mammal. In another embodiment, the mammals in which immunity has been induced are humans, cattle, goats, sheep, or swine.

[0091] The present invention describes vaccines comprising Brucella strains comprising mutations in one or more virulence genes. Examples of virulence genes that can be mutated include, but are not limited to, znuA, norD, btpl/lcpB, cjiG, cgs, ricA, bvrR, bvrS, the genes encoding the virB type IV secretion system (BMEII0025, BMEII0026, BMEII0027, BMEII0028, ΒΜΕΠ0029, BMEII0030, BMEII0031, ΒΜΕΠ0032, BMEII0033, BMEJJ0034, and ΒΜΕΠ0035), and the genes encoding lipopolysaccharide (gmd, manA, manC, per, pgm, pmn/manB, wbkA, wbkB, wbkC, wzm, and wzt). In one embodiment, the vaccines comprise Brucella strains comprising at least a znuA loss of function mutation. In another embodiment, the vaccines comprise Brucella strains comprising at least a znuA loss of function mutation and a second loss of function mutation in norD. In another embodiment, the vaccines comprise Brucella strains comprising at least a znuA loss of function mutation and a second loss of function mutation in btpl/tcpB.

[0092] The invention further describes vaccines comprising Brucella strains comprising selectable markers to permit identification of the strains and seram identification to determine the animal's vaccination status. These selectable markers can be inserted into the Brucella genome in an uncoded region, in a nonfunctional gene locus, or within a gene locus. Because the Brucella genome does not naturally harbor these marker genes, the anti-marker antibody titer can be easily measured to distinguish the strains of the invention from wild-type Brucella, and to confirm whether a subject has been immunized with the strain of the invention by measuring the anti-marker antibody titers. Selectable markers that may be used include, but are not limited to, genes encoding β-gaiactosidase (lacZ), red fluorescent protein (rfp), green fluorescent protein (gfp), yellow fluorescent protein, blue fluorescent protein, and cyan fluorescent protein. Selectable markers also include, but are not limited to, genes such as AmpR, PAC, hph, bsr and neo that confer resistance to antibiotics. The antibodies produced to the polypeptides produced by the selectable genes can be assayed by any standard assay commonly used in the art. In one embodiment, the vaccines comprise Brucella strains comprising a loss of function mutation in at least one virulence gene and further comprise at least one selectable marker. In another embodiment, the vaccines comprise Brucella strains comprismg at least a znuA loss of function mutation and further comprise at least, one selectable marker. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in the norD or btpl genes and further comprise at, least one selectable marker. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and former comprise lacZ, rfp or gfp. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in norD or btpl and further comprise lacZ, rfp or gfp. The invention further describes a method of differentiating mammals immunized with the vaccines of the invention from unimmunized mammals by identifying the antibodies produced to the selectable marker.

[0093] The invention further describes vaccines comprising Brucella strains comprising at least one homologous protective antigen to enhance the protective immunity of these mutant strains. The protective antigens encompassed by the invention include, but are not limited to, genes such as pot D (e.g. SEQ ID NO: l), potF (e.g. SEQ ID NO:2), bp26, omps, and trigger factor. In one embodiment, the vaccines comprise Brucella strains comprising a loss of function mutation in at least one virulence gene and at least one homologous protective antigen. In another embodiment, the vaccines comprise Brucella strains comprising at least a znuA loss of function mutation and further comprise at least one homologous protective antigen. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in the norD or btpl genes and further comprise at least one homologous protective antigen. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in the norD or btpl genes and further comprise potD or potF.

[0094] Attenuated strains of pathogenic microorganisms can also be used as vector vaccines in which the attenuated organism further comprises a heterologous genome, gene, or DNA fragment which when expressed in the host will elicit an immune response against that heterologous polypeptide. This antibody response against the heterologous polypeptide can confer immunity to the pathogen from which the heterologous genome, gene, or DNA fragment was derived. The Brucella vaccine of the present invention can be utilized as a vaccine vector to carry and express other passenger polypeptides related to Brucella species or other bacterial, viral, fungal, or parasitic vaccine antigens. It can serve as either a mucosal or peripheral vaccine delivery vehicle. One or more of the desired antigens, or genes coding for these antigens, can be introduced into the live Brucella strains described herein for use as a vaccine, and can be used only to provide said antigen, i.e. as a delivery vehicle, or to provide protection as a vaccine and deliver the desired antigen. The desired gene or antigen can be introduced into the bacteria either as episomal DNA, or as part of the Brucella chromosome by recombination for example, advantageously inserted in the deletion site of the vaccine strain, or replacing the selectable marker used in selecting the vaccine strain.

[0095] Thus, the nucleic acids encoding for a subunit vaccine ferried by the attenuated Brucella species of the present invention can also be adapted for expression of passenger antigens (vaccines) to protect against homologous or heterologous disease (infectious agent). Production of these vaccines adheres to conventional practices for propagating bacteria. Thus, the present invention also provides Brucella strains and/or vaccines that can be modified to transfer or immunize a host against any number of infectious agents or autoimmune diseases as long as the relevant protective epitopes can be stably ferried by the attenuated Brucella. Therefore, the present invention provides brucellosis vaccines that can also serve as a vaccine vectors to elicit mucosal and systemic immunity.

[0096] The present invention further describes vaccines comprising Brucella strains that comprise heterologous protective antigens. The heterologous protective antigens may come from diverse sources including, but not limited to, bacteria, viruses, fungi, protozoa, and metazoan parasites. The structural genes may encode envelope proteins, capsid proteins, surface proteins, toxins, such as exotoxins or enterotoxins, enzymes, or oligosaccharide antigen. The protective antigens include, but are not limited to, the Clostridium botuiinum BotA, Yersinia pestis Cafl, and IcrV, enterotoxigenic Escherichia coli (ETEC) CfaB, Human Immunodeficiency Virus vif, Plasmodium circumsporozoite protein, and arbo viral coat protein. The antibodies produced to the polypeptides produced by the heterologous protective antigens can be assayed by any standard assay commonly used in the art. In one embodiment, the vaccines comprise Brucella strains comprising heterologous protective antigens that are overexpressed. In one embodiment, the vaccines comprise Brucella strains comprising a loss of function mutation in at least one virulence gene and further comprise at least one heterologous protective antigen. In another embodiment, the vaccines comprise Brucella strains comprising at least a znuA loss of function mutation and further comprise at least one heterologous protective antigen. In another embodiment, the vaccmes comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in the norD and/or btpl genes and further comprise at least one heterologous protective antigen. In another embodiment, the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in the norD and/or btpl genes and further comprise botA, cafl, IcrV, and/or cfaB.

[0097] The present invention further describes vaccines comprising Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arahinose metabolic pathway gene. In one embodiment, the vaccines comprise Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene, araBAD (P _BAD )- After the promoters of one or more Brucella genes encoding LPS are replaced with P _BAD , the mutated strains will express LPS normally in medium in the presence of arabinose. However, when these strains infect the host, expression of the LPS genes or operons will be turned off due to the absence of arabinose in the host. Eventually, the mutated Brucella strain will stop replicating due to the lack of LPS expression and will be cleared from the host. The promoters of the genes encoding LPS that may be replaced with P _BAD include, but are not limited to, wbkA wbkB, wbkC, gmd, per, pgm, IpsA, man, A, manB, manC, wzm, and wzt.

[0098] In another embodiment, the vaccines comprise Brucella strains comprising a mutation in a. gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene and further comprise a loss of function mutation in at least one virulence factor. In another embodiment, the vaccines comprise Brucella strains comprising a mutation in a gene encoding LPS wherein the promoter of the gene encoding LPS has been replaced with the promoter of an arabinose metabolic pathway gene and further comprise a loss of function mutation in znuA, norD and/or btpl.

[0099] The compositions of the invention may be used to either treat or prevent the disease brucellosis. The effect may be prophylactic in terms of completely or partially preventing the disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for brucellosis and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of brucellosis in an animal, e.g., cattle, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. The methods and compositions of the present invention induce a strong immune response and may be used to elicit a humoral and/or a cell- mediated response against the antigen(s) of the vaccine in a subject.

[00100] The subjects to which the present invention is applicable may be human and any livestock or wildlife species, which include, but are not limited to, cattle, sheep, goats, pigs, cats, dogs, horses, mules, donkeys, elk, deer, bison, both wild-life and domestic, bison/cattle hybrids (beefalo and/or cattaio), antelope, water buffalo, camels, yaks, and bears.

Vaccine Formulation and Administration

[00101] The microorganisms of the invention may be formulated as a composition for delivery to a subject, such as for oral or nasal delivery to a subject.

[00102] In one embodiment of the invention, the vaccine further comprises one or more immunogenic peptides from a second pathogenic organism or which is capable of expressing one

n or more immunogenic peptides from a second pathogenic organism. For instance, the bacterial vaccine vector of the invention can be engineered to additionally express an immunogenic peptide from a second, third or fourth enteric pathogen. In one embodiment, the second enteric pathogen is enterotoxoxigenic E. coli (ETEC) and the peptide is the ETEC heat labile toxin or heat stable toxin or variant or fragment thereof.

[00103] The composition may comprise the microorganism as described, and a pharmaceutically acceptable carrier, for instance, a pharmaceutically acceptable vehicle, excipient and/or diluent. The pharmaceutically acceptable carrier can be any solvent, solid or encapsulating material in which the vaccine can be suspended or dissolved. The pharmaceutically acceptable carrier is non-toxic to the inoculated individual and compatible with the live, attenuated microorganism.

[00104] Suitable pharmaceutical carriers known in the art, include, but are not limited to, liquid carriers such as saline and other non-toxic salts at or near physiological concentrations. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical vehicles, excipients and diluents are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, which is hereby incorporated by reference in its entirety.

[00105] In one embodiment of the invention, the composition comprises one or more of the following carriers: di sodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, sterile saline and sterile water.

[00106] In certain embodiments, the compositions further comprise at least one adjuvant or other substance useful for enhancing an immune response. For instance, the invention includes a. composition comprising a live, attenuated Brucella bacterium of the invention with a CpG oligodeoxynucleotide adjuvant. Adjuvants with a. CpG motif are described, for instance, in U.S. Patent Publication 20060019239, which is herein incorporated by reference in its entirety. [00107] Other adjuvants that can be used in a vaccine composition with the attenuated microorganism of the invention, include, but are not limited to, aluminum salts such as aluminum hydroxide, aluminum oxide and aluminum phosphate, oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant , mycolate-based adjuvants (e.g., trehalose dirnyco!ate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g. , mureins, mucopeptides, or glycoproteins such as N-Opaea, muramyl dipeptide [MDP], or MDP analogs), proteoglycans ( e.g. , extracted from Klebsiella pneumoniae ), streptococcal preparations (e.g. , GK432), mxiramyldipeptides, Immune Stimulating Complexes (the "Iscoms" as disclosed in EP 109 942, EP 180 564 and EP 231 039), plant derivatives (such as saponins), DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), oil emulsions (such as MF59), MPL (rnonophosphoryl lipid A), small molecule immunopotentiators, virosom.es, liposomes, polyols, the Ribi adjuvant, system (see, for instance, GB-A-2 189 141), vitamin E, cholera toxin adjuvant, maltose binding protein (MBP), carbopol or interleukins, particularly those that stimulate cell mediated immunity, archaeosome vaccine adjuvants, mucosal adjuvants, antigens (e.g. cholera toxin or enterotoxin), surface-charged poly(lactide-co-glycolide) microparticles, nanoparticles, glycolipids (e.g. alpha-galactosylceramide (alpha-GalCer)), and polysaccharides (e.g. chitosan).

|00108] In certain embodiments, the compositions may comprise a carrier useful for protecting the microorganism from the stomach acid or other chemicals, such as chlorine from tap water that may be present at the time of administration. For example, the microorganism may be administered as a suspension in a solution containing sodium bicarbonate and ascorbic acid (plus aspartame as sweetener).

[00109] Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof. Gelatin capsules can serve as carriers for lyophilized vaccine.

Dosage & Routes of Delivery

Therapeutic/Prophylactic Administration and Compositions [00110] The compositions of the present invention can be administered to any livestock or wildlife animal. In one embodiment, the compositions are administered to animals younger than one year. In a further embodiment, the compositions are administered to female animals before their first pregnancy. In another embodiment, the compositions are administered to female animals after their first pregnancy. In another embodiment, the compositions are administered to laetating animals. In yet a further embodiment, the compositions are administered to human subjects before they come into contact with potentially infected animals. In a further embodiment, the compositions are administered to human subjects after they come into contact with potentially infected animals.

[00111] The compositions of the present invention can be administered via mucosal, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal and buccal routes. Alternatively, or concurrently, administration may be noninvasive by either the oral, inhalation, nasal, or pulmonary route. In one embodiment, the compositions are administered nasally. In a further embodiment, the compositions are administered orally.

[00112] Suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyi cellulose, sorbitol and dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for deliver} _' into the cell.

[00113] In certain embodiments, the compositions of this invention may be coadministered along with other compounds typically prescribed for the prevention or treatment of a brucellosis infection or related condition according to generally accepted medical practice.

[00114] In a second aspect, the invention provides a method for vaccinating a subject against brucellosis by administering an attenuated microorganism of the invention, or composition comprising the same, to a subject. For example, the microorganism may be orally administered to a subject, such as a subject at risk of acquiring a brucellosis infection, or a subject having a brucellosis infection, including a. subject having a recurrent infection, Accordingly, the present invention includes methods of preventing and treating a. brucellosis infection comprising administering a composition comprising an attenuated microorganism of the invention.

[00115] The method of the invention induces an effective immune response in the subject, which may include a mucosal immune response against Brucella. In certain embodiments, the method of the invention may reduce the incidence of (or probability of) recurrent brucellosis infection. In other embodiments, the vaccine or composition of the invention is administered to a subject post-infection, thereby ameliorating the symptoms and/or course of the illness, as well as preventing recurrence.

Dosage & Routes of Delivery

Therapeistic/Propiiylactk Administration And Compositions

[00116] Due to the increased bioavailability and potential lower dose or other advantage resulting from transmucosal administration of compositions of the invention, the compositions are advantageously useful in veterinary medicine. As described herein, the methods of transmucosal administration of compositions of the invention are useful for the treatment or prevention of conditions, illnesses, or disorders capable of being treated and/or prevented with a vaccme.

[00117] One embodiment of the invention encompasses methods of treatment or prophylaxis by transmucosal administration to the oral mucosa of an animal, in need thereof, of a therapeutically or proph lactically effective amount of a composition comprising a vaccine of the invention.

[00118] The compositions, which comprise a vaccine of the invention, are administered transmucosally, preferably to the oral mucosa, and more preferably to the buccal mucosa. The compositions of the invention can be administered by any convenient route, for example, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa or buccal mucosa) and may be administered together with an additional therapeutic agent. Various delivery systems are known that can be used to administer an active agent of the invention. Methods of administration include, but are not limited to, mucosal administration, particularly to the oral mucosa, preferably to the buccal mucosa, for example, using a pump spray or aerosol spray. In one embodiment, the compositions are administered orally through the addition of the compositions to the animal's feed, food and/or water. As used herein, animal feed refers to any food given to animals, including but not limited to wild, semi-domesticated and domesticated animals. Animal feed includes but is not limited to fodder and forage. As used herein, fodder refers to food given to animals rather than what they forage themselves. Fodder includes but, is not limited to food that comprises hay, straw, silage compressed and pelleted feeds, oils, mixed rations, household or commercial (e.g., from, restaurants or food processors) food scraps, and sprouted grains and legumes. Forage includes but is not limited to any plant material consumed by an animal, including but not including the plant leaves and stems. Forage includes plants eaten directly by animals (e.g., pasture, crop residue, immature cereal crops) and plants or plant, material cut for fodder and carried to the animals, especially as hay or silage. The preferred mode of administration can be left to the discretion of the practitioner, and may depend in part upon the specific type of the medical conditions of interest. In most instances, administration will result in the release of the vaccine of the invention into the bloodstream.

|00119] The methods of mucosa! administration of composition comprising agents of the invention may be assayed in vitro and/or in vivo, for the desired therapeutic or prophylactic activity. For example, in vi ro assays can be used to determine whether administration of a specific agent of the invention or a combination of agents of the invention is preferred for vaccine. The active agents of the invention may also be demonstrated to be effective and safe using laboratory animal model systems.

[00120] The compositions will contain a therapeutically or prophylactically effective amount of a vaccine of the invention, optionally more than one vaccine of the invention, preferably in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle so as to provide the form for transmucosal administration to the animal.

[00121] The compositions to be used in the methods of the invention can take the form of solutions, suspensions, emulsions, aerosols, dry powders or particulates, sprays, mists, capsules, or any other form suitable for use in transmucosally administering a drug to the oral mucosa, preferably the buccal mucosa of an animal. In one embodiment, the pharmaceutically acceptable vehicle is a buccal spray (see, e.g. ., U.S. Pat. No. 6,676,931 , which is incorporated herein by reference in its entirety).

[00122] In an illustrative embodiment, the vaccine of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for transmucosai administration to the oral mucosa of an animal. Typically, compositions of the invention for transmucosai administration are solutions in sterile isotonic aqueous alcohol buffer. Optionally, the compositions may also include a flavoring agent. Generally, the ingredients are supplied either separately or mixed together in unit, dosage form, for example, as an aerosol spray or pump spray indicating the quantity of active agent. The optional flavoring agents include, for example, animal flavoring or flavor enhancement agents, agents that improve the palatability or odor of the compositions to an animal, and preserving agents, to provide a pharmaceutically palatable preparation.

[00123] The delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.

[00124] The amount of a vaccine of the invention that will be effective in the treatment of a particular disorder, disease, or condition disclosed herein can often depend on the nature of the disorder, disease, or condition, and can be determined by standard clinical techniques, in addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions may also depend on the route of administration and the seriousness of the disease, disorder, or condition and on the animal being treated, and should be decided according to the judgment of the practitioner and each animal's particular circumstances. However, suitable dosage ranges for transmucosai administration to the oral mucosa are generally from about 0.001 milligram to about 200 milligrams of a vaccine of the invention per kilogram body weight. In specific preferred embodiments of the invention, the oral dose of vaccine is from about 0.005 milligram to about 150 milligrams per kilogram body weight, more preferably from about 0.01 milligram to about 100 milligrams per kilogram body weight, more preferably from about 0.05 milligram to about 50 milligrams per kilogram body weight, for example from about 0.1 milligram to about 25 milligrams per kilogram body weight. In a most preferred embodiment, the oral dose is from about 0.2 milligrams to about 10 milligrams of a vaccine of the invention per kilogram body weight. The dosage amounts described herein refer to total amounts administered; that is, if more than one agent of the invention is administered, the preferred dosages correspond to the total amount of each agent administered. Oral compositions typically contain about 10% to about 95% active ingredient by weight.

[00125] In certain embodiments, the vaccine dosage is about 1.0 x 10 ^s to about, 1.0 x lQ ^ij

CFU/ml or cells/ml. For instance, the invention includes a vaccine with about 1.0 x 10 ³ , about 1.5 x 10 ⁵ , about 1.0 x l O ⁶ , about 1.5 x 10 ⁶ , about 1.0 x ! 0 ⁷ , about 1.5 x 10 ⁷ , about 1 .0 x 10 ^s , about 1.5 x 10 ⁸ , about 1.0 x 10 ⁹ , about 1.5 x l O ⁹ , about 1.0 x 10 ^3G , about 1.5 x 10 ³⁰ , about 1.0 x 10 ¹¹ , about 1 .5 x 10 ¹¹ , about 1 .0 x 10 ¹² , about 1 .5 x 10 ¹² , about 1 .0 x l O ¹³ , about 1.5 x 10 ³³ , about 1.0 x 10 ¹⁴ , about 1.5 x 10 ¹⁴ or about 1.0 x 10 ^lj> CFU/ml or cells/ml. In one embodiment, the vaccine dosage is about 1.0 x l O ⁶ to about 1.0 x 1Q ⁹ CFU/ml . In another embodiment, the vaccine dosage is about 1.0 x 10 ^s CFU/ml. In certain embodiments, the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

|00126] The vaccine may be administered to the subject, or may be administered a plurality of times, such as one, two, three, four or five or more times. The compositions of the invention may also be administered on a dosage schedule, for example, an initial administration of the vaccine composition followed by subsequent booster administrations. In one embodiment, a second dose of the composition is administered anywhere from one week to one year, preferably from about 1 , about 2, about 3, about 4, about 5 to about 6 months, after the initial administration. In one embodiment, a second dose is administered about one month after the first administration. Additionally, one or more subsequent boosters may be administered after the second dose and from about three months to about two years, or even longer after the initial administration.

Delivery Ageat

[00127] The vaccines of the present invention may further comprise a delivery agent to enhance antigen uptake based upon, but not restricted to, increased fluid viscosity due to the single or combined effect of partial dehydration of host mucopolysaccharides, the physical properties of the delivery agent, or through ionic interactions between the delivery agent and host tissues at the site of exposure, which provides a depot effect. Alternatively, the delivery agent can increase antigen retention time at the site of delivery (e.g., delay expulsion of the antigen). Such a delivery agent may be a bioadhesive agent. In particular, the bioadhesive may be a mucoadhesive agent selected from the group consisting of glycosaminoglycans (e.g., chondroitin sulfate, dermatan sulfate chondroitin, keratan sulfate, heparin, heparan sulfate, hyaluronan), carbohydrate polymers (e.g., pectin, alginate, glycogen, amylase, amylopectin, cellulose, chitin, staehyose, unulin, dextrin, dextran) , cross-linked derivatives of poly(aerylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (including mucin and other mucopolysaccharides) cellulose derivatives ( e.g. , hydroxypropyl methylcelMose, carboxymethylcelMose), proteins ( e.g. lectins, fimbria! proteins), and deoxyribonucleic acid. Preferably, the mucoadhesive agent is a polysaccharide, such as chitosan , a chitosan salt, or chitosan base (e.g. chitosan glutamate).

[00128] Chitosan, a positively charged linear polysaccharide derived from chitin in the shells of crustaceans, is a bioadhesive for epithelial cells and their overlaying mucus layer. Formulation of antigens with chitosan increases their contact time with the nasal membrane, thus increasing uptake by virtue of a depot effect (Ilium et at. 2001 ; 2003; Davis et al. 1999; Bacon et al. 2000; van der Lubben et al. 2001; 2001 ; Lim et ai. 2001). Chitosan has been tested as a nasal delivery system for several vaccine, including influenza, pertussis and diphtheria, in both animal models and humans (Ilium et al. 2001 ; 2003; Bacon et ai. 2000; Jabbal-Gill et al. 1998; Mills et al. 2003; McNeela et al. 2004). In these trials, chitosan was shown to enhance systemic immune responses to levels equivalent to parenteral vaccination. In addition, significant antigen-specific IgA levels were also measured in mucosal secretions. Thus, chitosan can greatly enhance a nasal vaccine's effectiveness. Moreover, due to its physical characteristics, chitosan is particularly well suited to intranasal vaccines formulated as powders (van der Lubben et al. 2001 ; Mikszta et al. 2005; Huang et al. 2004).

[00129] Accordingly, in one embodiment, the present invention provides an antigenic or vaccine composition adapted for intranasal administration, wherein the composition includes antigen and optionally an effective amount of adjuvant. In preferred embodiments, the invention provides an antigenic or vaccine composition comprising an attenuated Brucella strain of the invention, in combination with at least one delivery agent, such as chitosan, and at least one adjuvant, such as MPL®, CPGs, imiquimod, gardiquimod, or synthetic lipid A or lipid A mimetics or analogs.

[00130] The molecular weight of the chitosan may be between 10 kDa and 800 kDa, preferably between 100 kDa and 700 kDa and more preferably between 200 kDa and 600 kDa. The concentration of chitosan in the composition will typically be up to about 80% (w/wj, for example, 5%, 10%, 30%, 50%, 70% or 80%. The chitosan is one which is preferably at least 75% deacetylated, for example 80-90%, more preferably 82-88% deacetylated, particular examples being 83%, 84%, 85%, 86% and 87% deacetylation.

Examples

|00131] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[00132] The invention is described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be constraed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1. Construction of Brucella abortus znlacZ strain

[00133] The B. abortus znlacZ strain as constructed by disrupting the znuA and norD genes and inserting lacZ in the wild-type B. abortus 2308 strain. Deletion mutations in Brucella abortus 2308 were accomplished using conventional molecular biologic approaches in which homologous regions upstream and downstream of the gene of interest were recombined with an exogenous homologous DNA plasmid. The recombination or exchange of genetic material (requiring a double crossover event) facilitated the loss of the target gene resulting in the B. abortus deletion mutant for that particular ge e. The AznuA B. abortus strain was constructed as described in Yang et al., 2006. The znuA gene codes for a high-affinity periplasmic binding protein-dependent and ATP-binding cassette (ABC) transport system for zinc (Lewis et al., 1999, Beard et al, 2000, Kim et al., 2004). To further cripple the attenuated AznuA B. abortus strain, a second mutation was introduced which deleted the norD gene, a member of the norEFCBQD operon encoding a nitric oxide reductase (Loisel-Meyer et al., 2006).

[00134] Based on AznuA B. abortus, the gene norD was in-frame deleted from the genome. The protocol, mutant selection procedure, and mutant, verification used for knocking out norD were identical to the process described to create the AznuA B. abortus mutation described in Yang et al. (2006). A total of 1,176 base pair inner DNA fragment sequence of norD was deleted from AznuA B. abortus. The new double mutant was named AznuA AnorD B. abortus. The resulting strain comprises two mutations that contribute to attenuating the wild-type pathogen. To prevent reversion to wild-type phenotypes, in-frame gene deletions were created. No known transposon sites were found near the deleted genes. To differentiate detection between the AznuA AnorD B. abortus strains of the invention and wild-type B. abortus strains, the E. co!i lacZ gene was incorporated within chromosome I. Subsequently, the marker gene lacZ was inserted into the genome of this double mutant. The lacZ gene was excised from pcDNA3.1 His/lacZ, installed with a promoter F _BAD that regulates arahinose metabolic pathway, and the expression cassette was inserted in the locus of BAB1 0048 via allele exchange. BABl 0048 is the last gene of a 10 gene-operon which encodes a hypothetical 71 amino acid polypeptide, and its disruption does not cause any observable impact on the bacterium. In the presence of X-gal in rich medium, such as Pi A or Brucella broth, lacZ can express constitutively, which allows a blue color to appear in the medium. Thus the final strain, AznuA AnorD B. abortus-lacZ, has two virulence genes attenuated to enable minimal infection and a marker gene to enable selection of vaccine from wild-type B. abortus.

[00135] The genotype of the znlacZ strain is znuA ^' , norD ^' , kicZT B. abortus.

[00136] Figures 1A and IB show that the AznuA AnorD B. abortus -lacZ mutant constitutively expresses β-galactosidase in the presence (Fig. 1A) or absence (Fig. IB) of arabinose. Cells were grown on potato infusion agar (PIA) co taining 1 ,0 mm IPTG and X-gai. Spots 1 and 2 depict AznuA AnorD B. abortus mutants showing β-galaetosidase activity, and Spot 3 depicts wild-type B. abortus 2308 showing no β-galactosidase activity.

Example 2. Construction of Brucella abortus zblacZ strain

[00137] The B. abortus zblacZ strain was constructed by disrupting the znuA and btpl genes and inserting lacZ in the wild-type B. abortus 2308 strain. Based on the AznuA B. abortus mutant, the btpl gene was in-frame deleted from the genome. The protocol, mutant selection procedure, and mutant verification used for knocking out btpl are identical to the procedure described in Yang et al. (2006). A total of 669 base pair inner DNA fragment sequence of btpl was deleted from AznuA B. abortus, and the new double mutation strain was named AznuA A btpl B. abortus. Subsequently, the !acZ expression cassette was inserted in the locus of BAB1_0048 as described in Example 1. In the presence of X-gal in a rich medium such as PIA. or Brucella broth, lacZ can express constitutively, which allows a blue color to appear in the medium.

[00138] The genotype of the zblacZ strain is znuA ^" btpl ^' , lacZ ^1' B. abortus.

Example 3. Construction of Brucella abortus znCfaB3 strain

[00139] The B. abortus znCfaB3 strain was constructed by disrupting the znuA and norD genes and inserting the ETEC major subunit encoding gene cfaB in the wild-type B. abortus 2308 strain. The AznuA AnorD B. abortus strain was constructed as in Example 1. Subsequently, the cfaB expression cassette was inserted in the locus of BAB1 0048 as described in Example 1.

[00140] The genotype of the znCfaB3 strain is znuA ^' , norD ^' , cfaB ^' B. abortus.

Example 4. Construction of Brucella melitensis zbZ strain

[00141] The B. melitensis zbZ strain was constructed by disrupting the znuA and btpl genes and inserting lacZ in the wild-type B. melitensis 16M strain. A 834 base pair inner DNA fragment sequence of znuA in B. melitensis 16M genome was constructed as described in Clapp et al. (201 1). Based on AznuA B. melitensis, the btpl gene was in-frame deleted from the genome. The protocol, mutant selection procedure, and mutant verification used for knocking out btpl are identical to that previously described for deleting znuA (Yang et al., 2006). As a result, a total of 306 base pair inner DNA fragment sequence of btpl was deleted from AznuA B. melitensis, and the new double mutation strain was named AznuA A btpl B. melitensis. Subsequently, the lacZ expression cassette was inserted in the uncoded region between BMEI1800 and BMEI1801. In the presence of X-gal in rich medium, such as PIA or Brucella broth, LacZ can express constitutively, which allows a blue color to appear in the medium.

[00142] The genotype of the zbZ strain is znuA ^" , btpl ^" , lacZ/ B. melitensis.

Example 5, Construction of Brucella melitensis znZ strain

[00143] The B. melitensis znZ strain was constructed by disrupting the znuA and norD genes and inserting lacZ in the wild-type B. melitensis 16M strain. A 834 base pair inner DNA fragment, sequence f znuA in B. melitensis 16M genome was constructed as described in Clapp et al. (201 1 ). Based on AznuA B. melitensis, the norD gene was in-frame deleted from the genome. The protocol, mutant selection procedure, and mutant verification used for knocking out btpl are identical to that previously described for deleting znuA (Yang et al., 2006). As a result, a total of 1 ,1 6 base pair inner DNA fragment sequence of norD was deleted from AznuA B. melitensis, and the new double mutation strain was named AznuA AnorD B. melitensis. Subsequently, the lacZ expression cassette was inserted in the uncoded region between ΒΜΕΠ 800 and BMEI1801. In the presence of X-gai in rich medium, such as PIA or Brucella broth, lacZ can express constitutively, which allows a blue color to appear in the medium.

[00144] The genotype of the znZ strain is znuA ^" , norD ^" , lacZ B. melitensis.

Example 6. Construction oi Brucella melitensis znT strain

[00145] The B. melitensis znT strain was constructed by disrupting the znuA and norD genes and inserting turbo rfp in the wild-type B. melitensis 16M strain. Strain AznuA AnorD B. melitensis was constructed as described in Example 5. Subsequently, the turbo rfp expression cassette was inserted in the uncoded region between BMEII 800 and BMEI1801. On rich medium, such as PIA or Brucella broth, znT exhibits a color between red and orange.

[00146] The genotype of the znT strain is znuA ^" , norD ^' , rfp ^" B. melitensis. E am le _n 7. Construction of Brucella melitensis zb-botA strain

[00147] The B. melitensis zb-botA strain was constructed by disrupting the znuA and btpl genes and inserting the protective antigen encoding gene botA heavy chain from Clostridium botulinum in the wild-type B. melitensis 16M strain. Strain ΔζηνΛ btpl B, melitensis was constructed as described in Example 4, Subsequently, the botA heavy chain expression cassette was inserted in the uncoded region between BMEIl 800 and BMEIl 801.

[00148] The genotype of the zb-botA strain is znuA ^" , btpl, botA ^' B, melitensis.

Example 8. Construction of Brucella melitensis zb-F+V strain

[00149] The B, melitensis zb-F+V strain was constructed by disrupting the znuA and btpl genes and inserting the protective antigen encoding genes cafl and IcrV from. Yersinia Pestis in the wild-type B, melitensis 16M strain. Strain AznuA Abtpl B. melitensis was constructed as described in Example 4. Subsequently, the expression cassette of the fused genes of cafl -IcrV was inserted in the uncoded region between BMEI1800 and BMEI1801.

[00150] The genotype of the zb-F+V strain is znuA ^" , btpl ^" , cafl IcrV B. melitensis.

Example 9. Construction of Brucella melitensis zb-potD strain

[00151] The B. melitensis zb-potD strain was constructed by disrupting the znitA and btpl genes and inserting the protective antigen potD from Escherichia coli in the wild-type B. melitensis 16M strain. Strain AznuA Abtpl B. melitensis was constructed as described in Example 4. A potD expression cassette comprising a potD sequence that was codon-optimized towards Brucella melitensis (SEQ ID NO: 1 ) was inserted in the uncoded region between BMEIl 800 and BMEIl 801.

[00152] The genotype of the zb-potD strain is znuA ^' , btpl ^' , potD ^T B. melitensis.

Example 10. Construction at ^" Brucella melitensis zb-potF strain

[00153] The B. melitensis zb-potF strain was constructed by disrupting the znuA and btpl genes and inserting the protective antigen potF from Escherichia coli in the wild-type B. melitensis 16M strain. Strain AznuA Abtpl B, melitensis was constructed as described in Example 4. A potF expression cassette comprising a potF sequence that was codon-optimized towards Brucella melitensis (SEQ ID NO:2) was inserted in the uncoded region between BMEI1800 and BMEI1801.

[00154] The genotype of the zb-potF strain is znuA ^' , btpF, potF ¹ B. melitensis.

Example 11. Construction of Brucella melitensis pA_wbkA strain

[00155] The mutant strain was generated by replacing the lipopolysaccharide (LPS) gene promoter with the promoter of arabinose metabolic pathway genes araBAD (PBAD) i the wild- type B. melitensis 16M strain. Deletion mutations in B. melitensis 16M were accomplished using conventional molecular biologic approaches in which homologous regions upstream and downstream of the gene of interest, were recombined with an exogenous homologous DNA plasmid. The recombination or exchange of genetic material (requiring a double crossover event), facilitated the loss of the target gene resulting in the B. melitensis 56M deletion mutant for that particular gene. The principle of replacing VwbkA is the same as deleting the AznuA gene from the B. abortus genome as described in Yang et al,, 2006. Specifically, the flanking sequences f VwbkA in B. melitensis were cloned, in which the PBAD was inserted between these two sequences to replace the native VwbkA . This sandwich DNA fragment was carried by a suicide vector and then transferred to wild-type B, melitensis 16M. After a series of selections, the colony with its VwbkA replaced by PB _A D was selected and this mutant strains was named pA W'bkA.

[00156] The genotype of the pA___wbkA strain is Vwbkcf P _/ MO ^' B. melitensis

Example 12. Construction of Brucella melitensis pZA__wbkA strain

[00157] The B. melitensis pZA wbkA strain was constructed by replacing the promoter of the wbkA gene with PBAD in the mutant AznuA B. melitensis strain. The procedure for generating pZA-wbkA was identical to the construction of pA_wbkA described in Example 1 1 , except the strain used was AznuA B. melitensis 16M instead of wild- type B. melitensis 16M.

[00158] The genotype of the pZA wbkA strain is znuA ^' , VwkbA ^' V _B/I D ^÷ B. melitensis. Ex¾m]3|e J3. Construction of Brucella melitensis pA_gmd strain

[00159] The B. melitensis pA gmd strain was constructed by replacing the promoter of the gmd gene with PBAD in the wild-type B. melitensis 16M strain. The procedure for generating pA_gmd was identical to the construction of pA_wbkA described in Example 1 1 , except the promoter replaced was Pgmd instead of PwbkA.

[00160] The genotype of the pA_gmd strain is Pgmd ^' T?BAD ⁺ B. melitensis.

Example 14, ConslrnetioK of Brucella melitensis pZA_gmd strain.

[00161] The B. melitensis pZA_gmd strain was constructed by replacing the promoter of the gmd gene with PBAD in the mutant AznuA B. melitensis strain. The procedure for generating pZAjpnd was identical to the construction of pA_wbkA described in Example 12, except the promoter replaced was Pgmd instead of PwbkA.

[00162] The genotype of the pZA_gmd strain is znuA ^" , Pgmd ^" PBAD ¹ B. melitensis.

Example 15. Construc ion of Brucella melitensis pA_LpsA strsm

[00163] The B. melitensis pA_lpsA strain was constructed by replacing the promoter of the IpsA gene with PBAD i the wild-type B. melitensis 1 6M strain. The procedure for generating pA IpsA was identical the construction of pA wbkA described in Example 1 1 , except the promoter replaced was Pips A. instead of PwbkA.

[00164] The genotype of the pA IpsA strain is Pips A ^' PBAD B. melitensis.

Example 16. Construction at ^" Brucella melitensis pZA IpsA strain

[00165] The B. melitensis pZA IpsA strain was constructed by replacing the promoter of the IpsA gene with PBAD in the mutant AznuA B. melitensis strain. The procedure for generating pZA IpsA was identical to the constmction of pA wbkA described in Example 12, except the promoter replaced was Pips A instead otPwbkA.

[00166] The genotype of the pZA JpsA strain is znuA ^' , Pips A ^' Ρ _&ίί B. melitensis. Ex m^Ie _^ !?. Construction of Brucella melitensis pA_manB strain

[00167] The B. melitensis pA manB strain was constructed by replacing the promoter of the manB gene with PB _A D in the wild-type B. melitensis 16M strain. The procedure for generating pA manB was identical to the coiistmction of pA wbkA described in Example 1 1 , except the promoter replaced was VmanB instead of VwbkA .

[00168] The genotype of the pAjmanB strain is VmanB ^' VBAD ^' B. melitensis.

Example 18. Construction of Brucella melitensis pZA_manB strain

[00169] The B, melitensis pZAjmanB strain was constructed by replacing the promoter of the manB gene with PBAD in the mutant AznuA B. melitensis strain. The procedure for generating pZAjmanB was identical to the construction of pA_wbkA described in Example 12, except the promoter replaced was VmanB instead oi VwbkA .

[00170] The genotype of the pZA_manB strain is znuA ^" , VmanB ^" PB _A D ' B. melitensis.

Example 19, The znlacZ strain (AznuA AnorD B. abortus) is attenuated in macrophages in vitro

[00171] Figure 2 shows AznuA AnorD B, abortus mutants were attenuated in RAW264.7 macrophages. RAW264.7 macrophages were infected with either wild-type B. abortus 2308, live RB51 vaccine, the AznuA B. abortus mutant, or the AznuA AnorD B. abortus-lacZ mutant at a bacteria to macrophage ratio of 30: 1. After 1 hour of incubation at 37 °C followed by treatment with gentamicin for 30 minutes, infected RAW264.7 cells were incubated in fresh medium for 0, 4, 24, or 48 hours. Infected macrophages were water lysed, and supernatants were diluted for CFU enumeration. The level of initial infection was the same for all B. abortus strains (t ^:=: Oh). As shown in Figure 2, after infection both RB51 and wild-type B. abortus grew exponentially, while both the AznuA AnorD B, abortus-lacZ and its parent the AznuA B. abortus mutant strain failed to replicate. Values are the means of quadruplicate wells ₊ SEM. The differences in macrophage colonization are significant: * P< 0.0001 ; ** P= 0.003: * Ρ<0.001.

[00172] Figure 3 shows that the AznuA AnorD B. abortus-lacZ strain is also attenuated in human peripheral blood macrophages. Macrophages (lX10 well) were differentiated from human peripheral blood mononuclear cells and infected 30: 1 with either wild-type B. abortus 2308, live RB51 vaccine, or the AznuA AnorD B. abortus-lacZ mutant. After 1 hour incubation at 37 °C followed by 30 minutes of gentamiciii treatment, human macrophages were incubated in fresh medium for 0, 24, or 48 hours. Infected macrophages were water lysed, and supernatants were diluted for CPU enumeration. The level of initial infection was the same for all B. abortus strains (t = Oh). As shown in Figure 3, after infection both RB51 and wild-type B. abortus grew exponentially while the AznuA AnorD B. abortus-lacZ mutant is attenuated and failed to replicate. Values are the means of triplicate wells + SEM. The differences in growth between the AznuA AnorD B, abortus-lacZ mutant and RB51 are significant (*P<0.005). The differences in growth between the AznuA AnorD B, abortus-lacZ mutant, and the wild-type 2308 strain are also significant P<_0.001 ).

Example 20, The zb!acZ strain {AznuA Ahipl B. abortus-lacZ) is attenuated in macrophages in vitro

[00173] To assess the level of virulence of the zblacZ strain (AznuA Abtpl B. abortus- lacZ), RAW26.7 macrophages were infected as described in Example 59. As shown in Figure 4, the AznuA Abtpl B. ahortus-iacZ vaccine and its parental vaccine (AznuA B. abortus) were greatly attenuated and unable to replicate in these cells, unlike the RB51 or wild-type B. abortus strain 2308. Values are the means of quadruplicate wells + SEM. Differences in macrophage colonization versus wild-type B. abortus 2308, *P < 0.001, **P = 0.003; differences in macrophage colonization versus RG51 vaccine, P < 0.001.

Example 21. The ziiiaeZ strain (AznuA AnorD ϋ>„ abortus) is attenuated in vivo

J00174) To assess its virulence in vivo, a kinetic analysis was performed to ascertain the rate the AznuA AnorD B, abortus-lacZ strain was eliminated from the host. Groups of BALB/c mice (4-6 mice/group/time point) were infected i.p. with 1X10 ^s CFUs of the AznuA AnorD B, abortus-lacZ mutant, RB51 or 1X10 ⁵ B. abortus S19 (smooth, live) vaccine. Colonization of the spleens was evaluated at 1 , 2, 4, 6, and 6 weeks post-infection. Figure 5 shows that both RB51 and the AznuA AnorD B. abortus-lacZ strain showed similar clearance rates, and by 4 weeks post-infection < 200 CFUs were detected. In contrast, mice infected with S19 continued to show elevated colonization, even by 8 weeks post-infection. Values are the mean CFUs from individual mice +JSEM, and differences in colonization were determined when compared to SI 9 vaccine, *P<0.001, **P<0.009, ***P<0.029. These data show that the AzrtuA AnorD B. abortus - lacZ mutant, was readily cleared from t e host at a rate similar to that of the conventional RB51 vaccine.

Example 22. The zs!acZ strain (AznuA AnorD B. abortus) protects against brucellosis in vivo

[00175] To assess its protective efficacy, groups of BALB/c mice were immunized i.p. with 1X10 ⁸ CFUs of the AznuA AnorD B. aborlus-lacZ strain (n=10), RB51 (n=5), or PBS (n=T0) on day 0. On day 28, one group of the AznuA AnorD B. abortus-lacZ mice (n=5) was given a second i.p, dose of I X 10 ^s CFUs of AznuA AnorD B. abortus-lacZ vaccine. On day 56, RB51 -immunized mice, half of the PBS-dosed mice, and the single dose-immunized AznuA AnorD B. abortus-lacZ mice were challenged i.p. with 5X10 ⁴ CFUs of wild-type B. abortus strain 2308. The remaining PBS-dosed mice and the twice-immunized AznuA AnorD B. abortus- lacZ mice were challenged 8 weeks after the second immunization. All mice were evaluated four weeks after challenge for the extent of splenic colonization by wild-type B. abortus strain 2308. As shown in Figure 6A, a single dose of vaccine was sufficient to reduce colonization by wild- type B. abortus relative to naive (PBS) control mice or mice vaccinated with RB51. Additionally, Figure 6A shows that animals that received two doses of vaccine were completely protected against the wild-type B. abortus challenge and showed a reduced colonization by more than 4 logs. The double immunization with the AznuA AnorD B, abortus-lacZ strain was significantly more effective than immunization with RB51.

[00176] Figure 6B shows the splenic weights of the vaccinated and unvaccinated mice after challenge by wild-type B. abortus strain 2308. The AznuA AnorD B. abortus-lacZ strain induced less splenic inflammation, which typically represents another correlate of protective immunity. Vaccination with one or two doses of this vaccine resulted in less splenic inflammation than RB51 vaccine. [00177] Collectively these data demonstrate that the ΔζηιιΑ AnorD B. ahortus-lacZ strain is highly protective and more effective than co ventional livestock RB51 vaccine and based upon these parameters, more attenuated than the RB51 vaccine. The AznuA AnorD B. abortus- lacZ strain is aviruient and is excluded from the Select Agent list by the CDC, NIH, and USDA.

Example 23. The zblacZ strain {AznuA Abtpl B. ahortus-lacZ) protects against brucellosis in vivo

[00178] To assess the level of virulence of the zblacZ strain in vitro, RAW26.7 macrophages were infected as described in Example 19, As shown in Figure 7 A, the AznuA Ahtpl B. abortus-!acZ vaccine was greatly attenuated and unable to replicate in these cells, behaving as its parental AznuA B. abortus vaccine.

[00179] To assess its protective efficacy in vivo, groups of BALB/c mice were immunized with the zblacZ strain (AznuA Abtpl B. abortus -iacZ) . Mice were immunized on days 0 and 28 with 1 X10 ⁸ CFUs of AznuA Abtpl B. abortus-lacZ (10/group), AznuA AnorD B. ahortus-lacZ (10/group), or PBS (10/group). Eight weeks after its last immunization, all mice were challenged i.p. with 5X10 ⁴ CFUs of wild-type B. abortus 2308 and four weeks later, spleens were evaluated for the extent of B. abortus colonization. As shown in Figure 7B, both vaccines showed greatly reduced CFU levels relative to PBS-imniunized mice, and in some cases sterile immunity: 40% in AznuA AnorD B. abortus-lacZ immunized mice, and 80% in AznuA Abtpl B. ahortus-lacZ immunized mice. None of the colonies detected in either AznuA. Abtpl B. abortus- lacZ or AznuA AnorD B. abortus-lacZ immunized mice showed β-galactosidase activity. While the AznuA Abtpl B, abortus-lacZ strain conferred slightly better protection, these data show that two potent vaccines for brucellosis have been generated.

Example 24. The zalacZ strain (AznuA AnorD B. abortus-lacZ) vaccinated mice show positive antibody titer to β-galactosidase (β-gal) relative to RBSl-vaccinated mice

[00180] Since the AznuA AnorD B. abortus-lacZ strain still bears its LPS, thus rendering the strain positive in Bang's test (Moore and Maekie (1945) Can J Comp Med Vet Sci. July 9(7): 192-6), the serum IgG anti-p-gaiactosidase antibody response in mice twice immunized with this strain was evaluated. As shown in Figure 8, the AznuA AnorD B. abortus-lacZ vaccinated mice show a positive IgG anti-Pgal antibody titer relative to RB51 -vaccinated mice. Briefly, mice were vaccinated as described in Example 22 with two doses of the AznuA AnorD B. abortus-lacZ strain on days 0 and 28 or a single dose of RB51 on day 0. Three weeks after the second immunization, mice were bled, and serum from, the individual mice was evaluated for IgG anti- -galactosidase endpoint titers (Log ₂ ) by standard ELISA methods. A positive control was included from mice given intramuscular DNA vaccine encoding β-galactosidase three times. The results indicate mice vaccinated with the AznuA. AnorD B. abortus-lacZ strain show an IgG antibody response similar to that of the DNA-vaccinated mice. Significant differences versus RB51 -vaccinated mice are shown; *P=0.01 1.

Example 25. The doable Brucella mutants confer protection against wild-type Brucella strains

[00181] To test whether the species of Brucella may influence its potency to protect against wild-type challenge, the zbZ strain (AznuA, AnorD, lacZ ^~ B. melitensis) was developed. Groups of BALB/c mice were initially vaccinated nasally with IxlO ⁹ CFUs of AznuA, AnorD, lacZ ^' B. melitensis (n-8) or AznuA, B. melitensis (n~5), 1x10' CFUs of B. melitensis Rev-1 vaccine (n ^::: 5), or given sterile PBS (n=5). Mice were boosted orally four weeks later with the same doses of vaccines. After resting for another four weeks, mice were challenged nasally with 2X10* CF Us wild- type B. melitensis 16M and evaluated four weeks later for the extent of tissue colonization of spleens (Figure 9A) and lungs (Figure 9B). No growth of vaccine strains was found as determined by β-galactosidase expression or by PGR. Values are the mean CFUs from individual mice + SEM relative to colonization by PBS-dosed mice, *P<0.001, **P=0.003, ***P<0.05; NS = not significant. Collectively, these data show AznuA, AnorD, lacZ ^÷ B. melitensis can confer sterile immunity, as do AznuA, AnorD, lacZ ^r B, abortus or AznuA, Ahtpl, lacZ ⁺ B. abortus vaccines. Further, its potency of protection is similar to those of the B. abortus double mutant strains. Moreover, the vaccines are effective when given mucosally, e.g., nasally or orally, and can protect against respiratory challenge with wild-type Brucella. [00182] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[00183] The foregoing detailed description has been given for clarity of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[00184] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[00185] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

References:

[00186] Ail references cited anywhere in this specification including those cited anywhere above, are incorporated herein by reference in their entirety and for all purposes.

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