Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/098—Brucella
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

• the present invention is generated in the field of vaccine development, and specifically provides new vaccines for brucellosis.
• 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.
• 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.
• brucellosis causes intermittent or irregular fever also known as undulant, Malta, or MediteJTanean fever.
• 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.
• 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. abortus B. melitensis
• B. canis B. ovis
• B. suis B. neolomae which vary in their ability to infect host animals
• B, abortus primarily infect
• 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.
• 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,
• 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.
• the most preferred type of disease management is to avoid infection and to reduce the incidence and spread of the disease by vaccination.
• 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.
• Vaccination by existing vaccine strains of Brucella 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.
• 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.
• 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).
• 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.
• Rev I 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. Archivesbl. 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.
• 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.
• 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 does produce other types of antibodies that can be detected with a special assay to detect if an animal has been vaccinated.
• 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.
• 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.
• 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.
• the vaccines comprise Brucella strains comprising a loss of function mutation in one or more virulence genes.
• the vaccines comprise Brucella strains comprising a znuA loss of function mutation.
• the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a second loss of function mutation in either norD or btp .
• the vaccine comprises any Brucella strain comprising a loss of function mutation in one or more virulence genes.
• 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,
• the vaccines comprise Brucella strains comprising a selectable marker with or without a loss of function mutation in one or more virulence genes.
• the vaccines comprise Brucella strains comprising a znuA loss of function mutation and a selectable marker.
• 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.
• 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.
• the selectable marker is lacZ, gfp, and/or rfpl.
• the vaccine comprises any Brucella strain comprising a selectable marker and a loss of function mutation in one or more virulence genes.
• 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).
• the invention provides a method of differentiating immunized animals from unimmimized animals by identifying antibodies that are produced to the selectable marker.
• 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.
• the vaccines comprise Brucella strains comprising a gene encoding a protective antigen.
• 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.
• the vaccines comprise Brucella strains comprising a gene encoding a protective antigen and a znuA loss of function mutation.
• 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.
• 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.
• the protective antigen is overexpressed and confers resistance to a heterologous pathogen.
• the gene encoding the protective antigen in the vaccines of the invention is cfaB, potD, potF, hotA, cafl, and/or IcrV.
• 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.
• 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 ).
• the present invention also provides vaccines comprising Brucella strains comprising a mutation in one or more genes encoding LPS.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• the vaccines comprise B. abortus., B.
• 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).
• virulence genes e.g. znuA, norD and/or btpl
• 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.
• 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.
• 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.
• the mammal in which immunity has been induced is human, cattle, goat, sheep, and/or swine.
• 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.
• the Brucella strain comprises a. loss of function mutation in one or more virulence genes.
• the Brucella strain comprises a znuA loss of function mutation.
• the Brucella strain comprises a znuA loss of function mutation and a second loss of function mutation in norD and/or btpl .
• any Brucella strain may comprise loss of functions mutations in at least one or more virulence genes (e.g. znuA, norD and/or btpl).
• the Brucella strain comprising a loss of function mutation in at least one virulence gene is B. abortus, B, melitensis, or B. suis.
• the Brucella strains of the invention comprise a selectable marker.
• the Brucella strains comprise a selectable marker and a loss of function mutation in one or more virulence genes.
• the Brucella strains comprise a selectable marker and a znuA loss of function mutation.
• 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.
• the Brucella strain comprises a selectable marker inserted into the Brucella genome in an uncoded region or in a nonfunctional gene locus.
• the selectable marker is lacZ, gfp, and/or rfp.
• any Brucella strain may comprise a selectable marker and a loss of function mutations in one or more virulence genes.
• the Brucella strain comprising a selectable marker and a loss of function mutation in at least one virulence gene is B. abortus, B. melitensis, or B. suis.
• the Brucella strains comprise a gene encoding a protective antigen.
• 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).
• the Brucella strains comprise a gene encoding a protective antigen and a znuA loss of function mutation.
• 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.
• the Brucella strains comprise the gene encoding the protective antigen inserted into the genome in an uncoded region or in a nonfunctional gene locus.
• the protective antigen is overexpressed and confers resistance to a heterologous pathogen.
• the gene encoding the protective antigen is cfaB, potD, potF, botA, cafl , or lcrV.
• any Brucella strain may comprise a gene encoding a protective antigen and a. loss of function mutation in one or more virulence genes.
• the Brucella strain comprising a gene encoding a protective antigen and a loss of function mutation in at least one virulence gene is B. abortus, B, melilensis, or B. suis.
• the present invention also provides Brucella strains comprising a mutation in one or more genes encoding LPS.
• 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.
• 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.
• PBAD araBAD
• 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.
• 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,
• 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.
• 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 .
• 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.
• LPS e.g., wbka, gmd, IpsA and or rnanB
• 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) .
• virulence genes e.g., znuA, norD and/or btpl
• Figures 1A-B demonstrate that the AznuA AnorD B. abortus-lacZ mutant constitutively expresses ⁇ -galactosidase in the presence (Fig. 1A) or absence (Fig. IB) of arabinose. Spots 1 and 2 depict, AznuA AnorD B. abortus mutants expressing ⁇ -galaetosidase and Spot 3 depicts wild-type B. abortus 2308 showing no ⁇ -galactosidase activity.
• 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 ⁇ 0.001.
• 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).
• 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.
• 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 ⁇ 0.001.
• FIG. 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.
• a can mean one or more than one.
• a gene can mean a single gene or a multiplicity of genes.
• 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.
• adjuvant refers to a substance sometimes included in a vaccine formulation to enhance or modify the immune-stimulating properties of a vaccine.
• 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.
• 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.
• 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.
• 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.
• the term "attenuated vaccine” refers to a vaccine in which live pathogens
• 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.
• 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.
• the term "brucellosis” refers to a bacterial disease caused by members of the Brucella genus that can infect humans but primarily infects livestock.
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• heterologous Brucella infection refers to infection by a Brucella strain different from the Brucella strain used to immunize the subject against brucellosis.
• homologous Brucella infection refers to infection by a Brucella strain the same as the Brucella strain used to immunize the subject against brucellosis.
• the term "expression” refers to the vaccine vector which is responsible for producing the vaccine.
• 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.
• 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,
• 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,
• 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.
• 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.
• multivalent vaccine refers to a Brucella vaccine that also confers immunity to a different organism by expressing an immunogenic polypeptide of the different organism.
• nucleic acid As used herein, the term “mucosal” means any membrane surface covered by mucous.
• nucleic acid 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.
• 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.
• 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.
• nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
• nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
• 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.
• 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.
• 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.
• 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.
• promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
• protecting antigen refers to an antigen which when expressed by a Brucella strain enhances the protective immunity of the strain.
• 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.
• heterologous protective antigen refers to genes that, are found in organisms other than Brucella.
• heterologous protective antigens examples include other bacteria, viruses, and eukaryotic cells.
• heterologous protective antigens include, but are not limited to the Clostridium botulinurn BotA, Yersinia pestis Cafl and IcrV, and enterotoxigenic Escherichia coli (ETEC) CfaB.
• the term "species” refers to organisms in the same genus that have similar characteristics.
• strain refers to a specific version of an organism.
• 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.
• 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.
• 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.
• 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.
• LPS Lipopolysaccharide
• the present invention describes Brucella strains comprising mutations in one or more virulence genes.
• 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 ⁇ 0025, B ME [10026, BMEIIQQ27, BMEl 10028, ⁇ 0029, BMEII0030, BMEII0031, ⁇ 0032, BME1I0033, BMEII0034, and BMEII0035), and the genes encoding lipopolysaccharide (gnid, man A, manC, per, pgm, pmn/manB, wbkA, whkB, whkC, wzm, and wzt).
• 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.
• 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 ⁇ -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.
• the Brucella strain comprises at, least one selectable marker and a loss of function mutation in at, least one virulence gene.
• 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 .
• 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.
• the Brucella strain comprises at least one homologous protective antigen and a loss of function mutation in at least one virulence gene.
• the Brucella strain comprises at least one homologous protective antigen and at least a znuA loss of function mutation.
• 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.
• 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.
• the present invention further describes Brucella strains that comprise heterologous protective antigens.
• 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.
• the Brucella strain comprises at least one heterologous protective antigen and a loss of function mutation in at least one virulence gene.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 .
• the present invention describes Brucella vaccines that can be adapted for human, livestock, and wildlife use, which have advantages over the vaccines currently available.
• 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.
• 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.
• the vaccines described herein can confer cross-protection against heterologous Brucella infections.
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• the mammals in which immunity has been induced are humans, cattle, goats, sheep, or swine.
• the present invention describes vaccines comprising Brucella strains comprising mutations in one or more virulence genes.
• 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, ⁇ 0029, BMEII0030, BMEII0031, ⁇ 0032, BMEII0033, BMEJJ0034, and ⁇ 0035), and the genes encoding lipopolysaccharide (gmd, manA, manC, per, pgm, pmn/manB, wbkA, wbkB, wbkC, wzm, and wzt).
• 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.
• 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.
• 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 ⁇ -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.
• 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.
• 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.
• 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.
• 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.
• the vaccines comprise Brucella strains comprising a loss of function mutation in at least one virulence gene and at least one homologous protective antigen.
• the vaccines comprise Brucella strains comprising at least a znuA loss of function mutation and further comprise at least one homologous protective antigen.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the vaccines comprise Brucella strains comprising heterologous protective antigens that are overexpressed.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• livestock or wildlife species 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.
• 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.
• the vaccine further comprises one or more immunogenic peptides from a second pathogenic organism or which is capable of expressing one
• the bacterial vaccine vector of the invention can be engineered to additionally express an immunogenic peptide from a second, third or fourth enteric pathogen.
• 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.
• the composition may comprise the microorganism as described, and a pharmaceutically acceptable carrier, for instance, a pharmaceutically acceptable vehicle, excipient and/or diluent.
• 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.
• 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.
• 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.
• the compositions further comprise at least one adjuvant or other substance useful for enhancing an immune response.
• 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.
• 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.
• 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 dir
• cholera toxin or enterotoxin surface-charged poly(lactide-co-glycolide) microparticles, nanoparticles, glycolipids (e.g. alpha-galactosylceramide (alpha-GalCer)), and polysaccharides (e.g. chitosan).
• glycolipids e.g. alpha-galactosylceramide (alpha-GalCer)
• polysaccharides e.g. chitosan
• 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.
• the microorganism may be administered as a suspension in a solution containing sodium bicarbonate and ascorbic acid (plus aspartame as sweetener).
• 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.
• compositions of the present invention can be administered to any livestock or wildlife animal.
• the compositions are administered to animals younger than one year.
• the compositions are administered to female animals before their first pregnancy.
• the compositions are administered to female animals after their first pregnancy.
• the compositions are administered to laetating animals.
• the compositions are administered to human subjects before they come into contact with potentially infected animals.
• the compositions are administered to human subjects after they come into contact with potentially infected animals.
• 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.
• 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.
• the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for deliver ⁇ ' into the cell.
• 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.
• 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.
• 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,
• the present invention includes methods of preventing and treating a. brucellosis infection comprising administering a composition comprising an attenuated microorganism of the invention.
• the method of the invention induces an effective immune response in the subject, which may include a mucosal immune response against Brucella.
• the method of the invention may reduce the incidence of (or probability of) recurrent brucellosis infection.
• 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.
• compositions of the invention are advantageously useful in veterinary medicine.
• 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.
• 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.
• 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.
• the compositions are administered orally through the addition of the compositions to the animal's feed, food and/or water.
• 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.
• 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.
• composition comprising agents of the invention may be assayed in vitro and/or in vivo, for the desired therapeutic or prophylactic activity.
• 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.
• 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.
• 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.
• 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).
• 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.
• compositions of the invention for transmucosai administration are solutions in sterile isotonic aqueous alcohol buffer.
• the compositions may also include a flavoring agent.
• 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.
• the delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
• 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.
• 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.
• 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.
• 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.
• the vaccine dosage is about 1.0 x 10 s to about, 1.0 x lQ ij
• 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 !
• 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.
• 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.
• 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.
• a second dose is administered about one month after the first administration.
• 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.
• 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.
• the delivery agent can increase antigen retention time at the site of delivery (e.g., delay expulsion of the antigen).
• a delivery agent may be a bioadhesive agent.
• 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, beauin, dextrin, dextran) , cross-linked derivatives of poly(aerylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (including mucin and other mucopolysaccharides) cellulose derivatives (e.g.
• glycosaminoglycans e.g., chondroitin sulfate, dermatan sulf
• the mucoadhesive agent is a polysaccharide, such as chitosan , a chitosan salt, or chitosan base (e.g. chitosan glutamate).
• 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.
• 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).
• 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.
• 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.
• 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.
• 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).
• ABSC periplasmic binding protein-dependent and ATP-binding cassette
• 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).
• 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.
• 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.
• lacZ can express constitutively, which allows a blue color to appear in the medium.
• lacZ 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.
• 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.
• the genotype of the znlacZ strain is znuA ' , norD ' , kicZT B. abortus.
• Figures 1A and IB show that the AznuA AnorD B. abortus -lacZ mutant constitutively expresses ⁇ -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 ⁇ -galaetosidase activity
• Spot 3 depicts wild-type B. abortus 2308 showing no ⁇ -galactosidase activity.
• 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.
• the !acZ expression cassette was inserted in the locus of BAB1_0048 as described in Example 1.
• lacZ can express constitutively, which allows a blue color to appear in the medium.
• the genotype of the zblacZ strain is znuA " btpl ' , lacZ 1' B. abortus.
• 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.
• the genotype of the znCfaB3 strain is znuA ' , norD ' , cfaB ' B. abortus.
• 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).
• 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).
• the genotype of the zbZ strain is znuA " , btpl " , lacZ/ B. melitensis.
• 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 ).
• 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).
• the genotype of the znZ strain is znuA " , norD " , lacZ B. melitensis.
• 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.
• the genotype of the znT strain is znuA " , norD ' , rfp " B. melitensis. E am le n 7. Construction of Brucella melitensis zb-botA strain
• 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 ⁇ 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.
• the genotype of the zb-botA strain is znuA " , btpl, botA ' B, melitensis.
• 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.
• the genotype of the zb-F+V strain is znuA " , btpl " , cafl IcrV B. melitensis.
• 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.
• the genotype of the zb-potD strain is znuA ' , btpl ' , potD T B. melitensis.
• 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.
• the genotype of the zb-potF strain is znuA ' , btpF, potF 1 B. melitensis.
• 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.
• LPS lipopolysaccharide
• PBAD arabinose metabolic pathway genes araBAD
• 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.
• VwbkA 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.
• the genotype of the pA___wbkA strain is Vwbkcf P / MO ' B. melitensis
• 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.
• the genotype of the pZA wbkA strain is znuA ' , VwkbA ' V B/I D ⁇ B. melitensis. Ex3 ⁇ 4m]3
• 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.
• the genotype of the pA_gmd strain is Pgmd ' T?BAD + B. melitensis.
• Example 14 ConslrnetioK of Brucella melitensis pZA_gmd strain.
• 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.
• the genotype of the pZA_gmd strain is znuA " , Pgmd " PBAD 1 B. melitensis.
• 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.
• the genotype of the pA IpsA strain is Pips A ' PBAD B. melitensis.
• 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.
• the genotype of the pZA JpsA strain is znuA ' , Pips A ' ⁇ & ⁇ B. melitensis. Ex m ⁇ Ie ⁇ !?. Construction of Brucella melitensis pA_manB strain
• 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 .
• the genotype of the pAjmanB strain is VmanB ' VBAD ' B. melitensis.
• 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 .
• 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
• FIG. 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.
• FIG. 3 shows that the AznuA AnorD B. abortus-lacZ strain is also attenuated in human peripheral blood macrophages.
• Macrophages lX10 well
• Macrophages 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.
• 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.
• Example 20 The zb!acZ strain ⁇ AznuA Ahipl B. abortus-lacZ) is attenuated in macrophages in vitro
• Example 21 The ziiiaeZ strain (AznuA AnorD ⁇ > court abortus) is attenuated in vivo
• 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
• 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.
• PBS na
• 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.
• Collectively these data demonstrate that the ⁇ 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
• 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).
• Example 24 The zalacZ strain (AznuA AnorD B. abortus-lacZ) vaccinated mice show positive antibody titer to ⁇ -galactosidase ( ⁇ -gal) relative to RBSl-vaccinated mice
• 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 ⁇ -galactosidase three times. The results indicate mice vaccinated with the AznuA.
• Example 25 The doable Brucella mutants confer protection against wild-type Brucella strains
• 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.
• Zinc uptake system (znuA locus) of Brucella abortus is essential for intracellular survival and virulence in mice. J. Vet. Med. Sci. 66: 1059-1063.

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