# Patent

## Title
Potomac horse fever isolates

## Abstract

The present invention discloses novel isolates of Neorickettsia risticii, compositions comprising such isolates, vaccines and methods for using such vaccines against Potomac Horse Fever.

## Background
RELATED APPLICATIONS
This application is a Continuation of U.S. patent application Ser. No. 14/101,659 filed on Dec. 10, 2013, now U.S. Pat. No. 9,339,532; which is a Divisional of U.S. patent application Ser. No. 13/221,356 filed on Aug. 30, 2011, now U.S. Pat. No. 8,628,947; which depends for priority on U.S. Provisional Application No. 61/378,261 filed Aug. 30, 2010 and U.S. Provisional Application No. 61/381,326 filed Sep. 9, 2010, all of which are hereby incorporated by reference in their entireties.


FIELD OF INVENTION
The present invention relates to novel isolates of Neorickettsia risticii and compositions comprising such isolates, and methods of using such compositions in vaccines against Potomac Horse Fever.
BACKGROUND
Potomac Horse Fever (PHF), an acute infectious disease of horses, was reportedly first recognized in 1979 in the region of the Potomac River in Maryland and Virginia. The disease is also identified as Equine Monocytic Ehrlichiosis and Equine Intestinal Ehrlichiosis. The causative agent is a gram-negative, obligate intracellular bacterium which was first identified in 1984 as Ehrlichia risticii (E. risticii), but has been renamed Neorickettsia risticii (N. risticii). The disease is characterized by a wide variety of intestinal symptoms, along with elevated temperature and inflamed mucus membranes. In many cases it results in severe pain, and sometimes death.
The bacteria infect the enterocytes of the small and large intestine, resulting in acute colitis syndrome, and producing symptoms of mild colic, fever, depression, anorexia and diarrhea in horses of all ages. The disease can also cause abortion in pregnant mares, laminitis, and death. N. risticii has been isolated from trematodes infecting fresh water snails, and from caddisflies, mayflies, damselflies, dragonflies and stoneflies. The route of infection appears to be inadvertent ingestion of the aquatic insects carrying N. risticii, and the incubation period is 10 to 18 days.
An antigen for use in an assay to detect the presence of N. risticii has been claimed in U.S. Pat. No. 4,759,927, which is herein incorporated by reference in its entirety. Its source was later identified as the 25-D strain. A second strain identified as 90-12, was disclosed in U.S. Pat. No. 6,375,954, which is herein incorporated by reference in its entirety. U.S. Pat. No. 6,375,954 claims a method for protecting against N. risticii by administering a particular 90-12 protein antigen. Equine vaccines are commercially available, but provide only partial or no protection against newer strains of N. risticii, and against strains we have isolated relating to the present invention.
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.
SUMMARY OF THE INVENTION
The present invention provides three novel strains of N. risticii that infect horses, and possibly other mammals. We have identified these three novel N. risticii isolates as N. risticii Oregon (OR), N. risticii New York (N.Y.) and N. risticii Michigan (MI), and deposited them with the ATCC, Manassas, Va., USA, as N. risticii Oregon (ATCC No. PTA-11232), N. risticii New York (ATCC No. PTA-11231) and N. risticii Michigan (ATCC No. PTA-11404). We have characterized these strains and have shown them to be biologically and structurally distinguishable from previously known strains.
The invention also provides novel protein antigens that characterize the new strains, as well as nucleic acids encoding these protein antigens, expression vectors that comprise such nucleic acids and express the protein antigens, vaccines comprising the new strains and/or the protein antigens, and/or the expression vectors, compounds comprising their protective antigens, methods for protecting animals, methods for producing the new strains and assays for detecting the novel strains. There is therefore a need for new vaccines against Potomac Horse fever.
These and other aspects of the present invention will be better appreciated by reference to the following figures and Detailed Description.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a Western Blot showing antibody reactivity and antigen banding with rabbit serum against the recombinant 85 kD strain specific antigen (SSA) of the N. ristcii 90-12 strain, FEF cells used to propagate N. risticii, and the 90-12, OR, NY, MI and 25-D strains.
FIG. 2 depicts a Western Blot showing antibody reactivity and antigen banding with horse serum against the recombinant 85 kD SSA of the N. ristcii 90-12 strain, FEF cells used to propagate N. risticii, and the 90-12, OR, NY, MI and 25-D strains.
FIG. 3 depicts a Western Blot showing reactivity, or lack thereof, of the same antigens against seronegative horse serum.
FIGS. 4A-1, 4A-2, 4A-3, 4B-1, 4B-2, 4B-3, 4C-1, 4C-2, 4C-3, and 4D depict a strain specific antigen (SSA) amino acid sequence alignment for the SSA 1 proteins of six N. risticii isolates. From the top to bottom:

    
    
        N. risticci ILL stain-CP001431, SEQ ID NO: 1
        N. risticci Oregon, SEQ ID NO: 2
        N. risticci 25-D, SEQ ID NO: 3
        N. risticci, 90-12 SEQ ID NO: 4
        N. risticci Michigan, SEQ ID NO: 5
        N. risticci New York, SEQ ID NO: 6
    
    


FIGS. 5A-1, 5A-2, 5A-3, 5B-1, 5B-2, 5B-3, and 5C depict a SSA amino acid sequence alignment for the SSA 2 protein of six N. risticii isolates. From the top to bottom:

    
    
        N. risticci ILL stain-CP001431, SEQ ID NO: 7
        N. risticci New York, SEQ ID NO: 8
        N. risticci 90-12, SEQ ID NO: 9
        N. risticci, 25-D, SEQ ID NO: 10
        N. risticci Oregon, SEQ ID NO: 11
        N. risticci, Michigan, SEQ ID NO: 12
    
    


FIGS. 6A-1, 6A-2, 6A-3, 6B-1, 6B-2, 6B-3, and 6C depict a SSA amino acid sequence alignment for SSA 3 protein of six N. risticii isolates. From the top to bottom:

    
    
        N. risticci ILL stain-CP001431, SEQ ID NO: 13
        N. risticci New York, SEQ ID NO: 14
        N. risticci 90-12, SEQ ID NO: 15
        N. risticci, 25-D, SEQ ID NO: 16
        N. risticci, Michigan, SEQ ID NO: 17
        N. risticci, Oregon, SEQ ID NO: 18
    
    


FIGS. 7A-1, 7A-2, 7A-3, 7B-1, 7B-2, 7B-3, 7C-1, 7C-2, 7C-3, 7D-1, 7D-2, 7D-3, 7E-1, 7E-2, 7E-3, 7F-1, 7F-2, 7F-3, 7G-1, 7G-2, and 7G-3 depict a SSA nucleic acid sequence alignment for the genes encoding SSA 3 of six N. risticii isolates. From the top to bottom:

    
    
        N. risticci ILL stain-CP001431, SEQ ID NO: 19
        N. risticci New York, SEQ ID NO: 20
        N. risticci 90-12, SEQ ID NO: 21
        N. risticci, 25-D, SEQ ID NO: 22
        N. risticci, Michigan, SEQ ID NO: 23
        N. risticci, Oregon, SEQ ID NO: 24
    
    


FIG. 8 depicts the nucleic acid sequence (SEQ ID NO: 25) for the coding sequence of the SSA#1 protein of N. risticii Oregon.
FIG. 9 depicts the nucleic acid sequence (SEQ ID NO: 26) for the coding sequence of the SSA#2 protein of N. risticii Oregon.
FIG. 10 depicts the nucleic acid sequence (SEQ ID NO: 27) for the coding sequence of the SSA#1 protein of N. risticii New York.
FIG. 11 depicts the nucleic acid sequence (SEQ ID NO: 28) for the coding sequence of the SSA#2 protein of N. risticii New York.
FIG. 12 depicts the nucleic acid sequence (SEQ ID NO: 29) for the coding sequence of the SSA#1 protein of N. risticii Michigan.
FIG. 13 depicts the nucleic acid sequence (SEQ ID NO: 30) for the coding sequence of the SSA#2 protein of N. risticii Michigan.
FIG. 14 depicts the nucleic acid sequence (SEQ ID NO: 31) for the coding sequence of the SSA#1 protein of N. risticii Illinois (CP001431).
FIG. 15 depicts the nucleic acid sequence (SEQ ID NO: 32) for the coding sequence of the SSA#2 protein of N. risticii Illinois (CP001431).



DETAILED DESCRIPTION OF THE INVENTION
Commercially available vaccines presently comprise antigens from the Illinois strain of N. risticii. Commercially available vaccines include POTOMAVACTM from Merial, Athens, Ga. and POTOMACGUARDTM from Pfizer Inc, Fort Dodge, Iowa. The available vaccines do not presently provide full protection against all current Potomac Horse Fever (PHF) outbreaks. Therefore, we have sought to identify currently circulating strains that are immunogenically distinguishable from the known vaccine strains. For this purpose, we have solicited serum samples from animals showing clinical signs of PHF, from which we have isolated and characterized the three new N. risticii strains of the invention. These three new strains, N. risticii Oregon, N. risticii New York and N. risticii Michigan, can be distinguished both by their strain specific antigens (SSA's), i.e., expressed surface antigens that characterize the different strains of N. risticii, and by the failure of available vaccines to fully protect animals against infection upon challenge with these novel strains.
The novel Oregon strain was isolated from a blood sample from a horse in Oregon that had been immunized with a commercially available vaccine, but still exhibited clinical signs of PHF. We found that horses immunized with the N. risticii 90-12 strain were not protected when challenged with the newly isolated N. risticii Oregon strain. In addition, N. risticii Oregon did not to react with monoclonal antibodies raised against the 90-12 strain.
The novel New York (N.Y.) strain was isolated from the blood sample of a horse from New York State exhibiting signs of PHF. We found that horses immunized with the N. risticii 90-12 strain showed a significant reduction in clinical disease and bacteremia upon heterologous challenge with the N. risticii New York strain. N. risticii New York does react with a monoclonal antibody to the 90-12.
The novel Michigan (MI) strain was isolated from the blood sample of a horse from Michigan exhibiting signs of PHF. The MI strain is shown to be unique by its antigen banding pattern (FIGS. 1 and 2) and by its SSA #1 protein sequence when compared with the other isolates.
N. risticii bacteria are typically characterized by their strain specific antigens, and the Oregon strain has a SSA #3 of approximately 50 kDa, the New York strain has a SSA #3 of approximately 55 kDa, and the Michigan strain has a SSA#3 of approximately 60 kDa, whereas the 90-12 strain has a SSA #3 of approximately 85 kDa.
In addition to the novel strains of N. risticii and their antigens, the present invention also relates to immunogenic compositions and vaccines. The therapeutic agent (also referred to as the antigen, active agent, or the immunogenic composition) that can serve as the basis for a vaccine can be one or more of the following:
a) harvested cultures of host cells that are infected with N. risticii bacteria;
b) extracts or fractions of (a) that are enhanced with respect to the concentration of the N. risticii bacteria contained within the infected host cells;
c) N. risticii bacteria enhanced extracts of (a) that contain remnants of the host cells;
d) isolated and purified N. risticii bacterial extracts of (a) that do not contain remnants of the host cells;
e) attenuated or inactivated bacteria;
f) isolated bacterial immunogens;
g) recombinant N. risticii proteins;
h) recombinant expression vectors that comprise nucleotide sequences, under the control of one or more promoters, that encode one or more recombinant N. risticii proteins (e.g., N. risticii strain specific antigens) which can be expressed by the recombinant expression vector; and
i) N. risticii strain specific antigens.
In some embodiments an N. risticii isolate of the present invention, encodes an SSA #1 protein comprising an amino acid sequence that comprises 80% or greater, 90% or greater, 95% or greater, 98% or greater, and/or 99% or greater identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments an N. risticii isolate of the present invention, encodes an SSA #2 protein comprising an amino acid sequence that comprises 80% or greater, 90% or greater, 95% or greater, 98% or greater, and/or 99% or greater identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments an N. risticii isolate of the present invention, encodes an SSA #3 protein comprising an amino acid sequence that comprises 90% or greater, 95% or greater, 98% or greater, and/or 99% or greater identity with the amino acid sequence of SEQ ID NO: 18. In other embodiments an N. risticii isolate of the present invention, encodes an SSA #1 protein comprising an amino acid sequence that comprises 80% or greater, 90% or greater, 95% or greater, 98% or greater, and/or 99% or greater identity with the amino acid sequence of SEQ ID NO: 2, encodes an SSA #2 protein comprising an amino acid sequence that comprises 80% or greater, 90% or greater, 95% or greater, 98% or greater, and/or 99% or greater identity with the amino acid sequence of SEQ ID NO: 11, and encodes an SSA #3 protein comprising an amino acid sequence that comprises 90% or greater, 95% or greater, 98% or greater, and/or 99% or greater identity with the amino acid sequence of SEQ ID NO: 18.
As used herein the following terms shall have the definitions set out below:
“Isolated” when used herein means removed from its naturally occurring environment. Hence, isolated N. risticii bacterial cells broadly include those that have been removed from their naturally occurring environments, including without limitation arthropods, insects, infected animals and specimens from infected animals. Isolated N. risticii bacterial cells also include those that are contained within host cells as described herein, or separated therefrom, as well as those that are substantially free of other microorganisms, e.g., in a culture.
“Isolated bacterial immunogens” refers to bacterial immunogens that have been completely or partially separated from their respective source bacteria. Compositions of isolated bacterial immunogens can include some whole intact bacteria, portions or components of bacteria, whole intact host cell, portions or components of host cells comprising bacterial antigens, as well as antigens produced by physical, chemical, biological or molecular biological processes.
“N. risticii bacterial immunogens” as used herein include whole bacteria, as well as parts thereof, including proteins (lipoproteins, membranous proteins, cytosolic proteins), immunogenic fragments of such proteins, nucleic acids, lipids, saccharides, lipopolysaccharides or other biological molecules derived from the N. risticii bacteria. They may be present in live host cells and host cells that are killed or inactivated. The skilled artisan is generally familiar with techniques by which bacteria or host cells can be killed or inactivated. Such techniques include physical, chemical and biological means. Non-limiting examples of inactivation techniques include sonication, freeze-thaw techniques, pressure, treatment with heat, chemicals or enzymes. Non-limiting examples of chemical inactivation agents include treatment with binary ethyleneamine (BEA) and formalin (formaldehyde solution). Immunogens may also be the products of chemical, biological or molecular biological processes.
As used herein one amino acid sequence is 100% “identical” to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% “identical” to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.
As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program using the default parameters.
As used herein, the term “vaccine(s)” means and refers to a product, the administration of which is intended to elicit an immune response that can prevent and/or lessen the severity of one or more infectious diseases.
As used herein, an “immune response” refers to the subject animal's active immunity due to having received one or more vaccines. The immune response can include the production of antibodies to the antigen or immunogen present in the vaccine. “Immune response” in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses may be measured using standard immunoassays and neutralization assays, which are known in the art.
“Preventing infection” and like terms means to prevent or inhibit the replication of the bacteria that cause the identified disease, to inhibit transmission of the bacteria or virus, to prevent the bacteria from establishing itself in its host animal or to alleviate the symptoms of the disease caused by infection. The treatment is considered therapeutic if there is a reduction in bacterial load.
“Protection,” “Protecting” and the like, as used herein with respect to bacteria, mean that the vaccine prevents or reduces the symptoms of the disease caused by the organism from which the antigen(s) used in the vaccine is derived. The terms “protection,” “protecting” and the like also mean that the vaccine may be used to “treat” the disease or one of more symptoms of the disease that already exists in a subject.
“Treating” refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, condition or disease to which such term applies, or to preventing one or more symptoms of such disorder, condition or disease.
“Pharmaceutically acceptable” as used herein refers to substances (e.g., adjuvants, immunostimulants, carriers, diluents, emulsifying or stabilizing agents) that are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use. Pharmaceutically acceptable substances do not interfere with the effectiveness of the therapeutic agent.
A vaccine contains an antigen (or, “active agent,” “immunogen,” “therapeutic agent,” or “immunogenic composition”), including a host cell infected with N. risticii bacteria, whole intact bacteria, or bacterial fractions or parts or biomolecules that act to stimulate the immune system in an animal, particularly the SSA's of the novel strain. An antigen may be a live attenuated or killed preparation of bacteria-infected host cells, live attenuated or killed bacteria, living irradiated cells, crude fractions or purified bacterial immunogens. A vaccine can comprise enriched, isolated or purified antigen. The vaccines can be made from inactivated or killed cultures of infected host cells, or inactivated or killed bacteria or alternatively, comprise a recombinant expression vector that comprises one or more nucleotide sequence, under the control of one or more promoters, that encode one or more recombinant N. risticii proteins (e.g., N. risticii strain specific antigens) which can be expressed by the recombinant expression vector.
A vaccine of the present invention may also comprise a combination of antigens from more than one N. risticii bacterial species and/or a combination of N. risticii antigens. For example, a vaccine of the present invention can comprise a combination of two or more of the following strains: N. risticci Oregon, N. risticci 90-12, N. risticci Illinois, N. risticci New York, N. risticci 90-12, N. risticci, 25-D, and/or N. risticci, Michigan. In a particular embodiment of this type, the vaccine can comprise N. risticci Oregon, and N. risticci 90-12. In another such embodiment, the combination vaccine can comprise N. risticci Oregon, and N. risticci New York. In yet another embodiment, the combination vaccine can comprise N. risticci New York and N. risticci, Michigan. In still another embodiment, the vaccine can comprise N. risticci Oregon, N. risticci 90-12, and N. risticci New York. All other such combinations are further envisioned by the present invention. In addition, any N. risticii vaccine of the present invention can further include antigens from other pathogens (e.g. viral, bacterial parasitical or fungal), as described further below.
Vaccines made from material cultured according to the present invention comprise a therapeutically effective amount of the antigen. In the context of this disclosure, a “therapeutically effective amount” refers to an amount of an antigen or vaccine that would induce an immune response in an animal receiving the antigen or vaccine that is adequate to prevent or ameliorate signs or symptoms of disease caused by infection with a N. risticii bacterium. Humoral immunity or cell-mediated immunity, or both humoral and cell-mediated immunity, may be induced. The immunogenic response of an animal to a vaccine may be evaluated, e.g., indirectly through measurement of antibody titers, via microscopic analysis, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine may be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, body temperature and overall physical condition and overall health and performance of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular antigen used, or the condition of the subject, and may be determined by one skilled in the art through well known means.
The novel N. risticii strains according to the present invention can also be used to diagnose diseases or illnesses caused by N. risticii bacteria. Non-limiting examples of such diagnostic applications include use of bacterial fractions, proteins or other biomolecules in antibody binding assays. The bacterial fractions, proteins or other biomolecules may also be used to generate polyclonal or monoclonal antibodies for such assays.
Host Cell Growth
Host cells for culturing bacterial organisms according to the present invention are first prepared prior to infecting with the desired bacterial organism. Examples of appropriate cell lines include: feline embryonic fibroblast cells, mouse macrophage P388D1 cells (ATCC No.: TIB 63), or a human histiocyte (HH) cell line (ATCC No. U937).
In one embodiment a sample of an isolated feline embryonic fibroblast cell line is seeded into media for either suspended or adherent growth. As used herein, adherent growth conditions exist when a layer of cells coats surfaces contained within the vessel in which the cells are cultured. The surfaces can include the interior surface of the vessel itself, or surfaces of glass or polymeric beads contained within the vessel to increase surface area. Microcarriers can also be used to increase surface area and host cell growth. In contrast to adherent growth, host cells may also be grown in suspension, in which the host cells need not bind to surfaces within the culturing vessel.
The skilled artisan is generally familiar with the varieties of culturing media that may be used to grow the host cells. The host cell growth media may be derived from animals. Alternatively, the host cell growth media may be vegetable or yeast based, and may be animal protein-free. The growth media may be derived from soy bean extracts or from other protein-rich plants or protein-rich plant food products including, for example, legumes. Non-limiting examples of specific media useful for growing host cells include Dulbecco's Modified Eagle's Medium (D-MEM), Eagle's Minimal Essential Media (MEM), Glasgow-Minimal Essential Media, RPMI1640, OptiMEM, and AIM V. The growth media may contain or be supplemented with fetal bovine serum (FBS), tryptose solution, lactos-albumin hydrosolate solution, L-glutamine, sodium bicarbonate; lactalbumin hydrolysate, Polymyxin B, sodium pyruvate, glucose, and/or magnesium sulfate.
Fresh growth media may be fed or replenished to the host cells prior to or after infection or exposure of the host cells to the bacteria. Cells may be grown at 36-38° C. for 2-9 days at 5% CO2.
Infecting the Host Cells
The host cells may be exposed to or infected with bacterial organisms by bringing the host cells into contact with other eukaryotic cells known to be infected with the bacterial organisms. The skilled artisan is familiar with determining whether such other eukaryotic cells from a mammal, for example, are infected with such bacterial organisms. The infected mammalian cells may be derived from any tissue, including the spleen, liver, pancreas, lungs, heart or other muscle tissue, brain, gall bladder, blood, kidneys, lymph nodes or stomach. The infected mammalian cells may be prepared from a tissue extract via blender homogenization in an appropriate isotonic solution. The homogenate can then be used to innoculate (i.e., infect) a culture of host cells, applied as a layer over the host cells or simply brought into contact with them.
Alternatively, the host cells may be exposed to or infected with isolated bacterial organisms. The skilled artisan is familiar with techniques of isolating such bacterial organisms, or can obtain stocks of isolated bacterial organisms from a biological depository.
The growth medium used to prepare host cells prior to contact with bacteria may be the same as the medium used to propagate the host cells after such contact. The bacteria-exposed (or infected) host cells may be cultured for up to 95 days, up to 35 days, or for about 5 to 14 days, to achieve a titer of ≧1×104 TCID50 (Tissue Culture Infectious Dose), and then the culture may be harvested and processed.
Harvesting
The bacteria infected host cells may be harvested by collecting the tissue cell culture fluids and/or cells. The host cells may be harvested from the media (and the culture vessels) with the bacterial cells contained with the walls of the host cells. Alternatively, during harvesting the concentration of the bacteria may be enriched by techniques that improve the liberation of the infective bacterial cells from the growth substrate, e.g., sonication, freeze thawing, heating, pressure or chemical or selective enzymatic lysis of the eukaryotic host cells. An enriched harvest of bacteria can include material that is free of host cells or host cell material. Alternatively, an enriched harvest of bacteria can include material that contains host cells or host cell material.
Inactivating
The skilled artisan is generally familiar with the techniques by which bacteria or host cells can be killed or inactivated. Such techniques include, physical, chemical and biological means. Non-limiting examples of inactivation techniques include sonication, freeze-thaw techniques, pressure, treatment with heat, chemicals or enzymes. Non-limiting examples of chemical inactivation agents include treatment with binary ethyleneimine (BEI), formalin (formaldehyde solution), beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate, or the like, or a mixture thereof. The host cells can also be inactivated by heat or psoralen in the presence of ultraviolet light. These chemical inactivation agents or physical inactivation means can also be used to inactivate the bacterial cells after their having been extracted or separated from the host cells.
Formulating
The inactivated, infected host cells or enriched bacterial cells can serve as the antigen and may be formulated as a liquid suspension or may be lyophilized for its use in the preparation of a vaccine against diseases caused by the organisms. Material cultured according to the present invention can be formulated with any pharmaceutically acceptable adjuvants, immunostimulants, carriers, diluents, emulsifying or stabilizing agents, non-limiting examples of which are discussed below. The skilled artisan, however, would recognize that other adjuvants, immunostimulants, carriers, diluents, emulsifying agents or stabilizing agents may be used in formulating vaccines based upon material cultured according to the present invention.
Adjuvants & Immunostimulants
An adjuvant in general is a substance that boosts the immune response of the target in a non-specific manner. Many different adjuvants are known in the art. Non-limiting examples of adjuvants that may be used in the formulation of a vaccine made with material according to the present invention include aluminum salts (e.g., alum, aluminum hydroxide, aluminum phosphate, aluminum oxide), cholesterol, monophosphoryl lipid A adjuvants, amphigen, tocophenols, monophosphenyl lipid A, muramyl dipeptide, oil emulsions, glucans, carbomers, block copolymers, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, muramyl dipeptide, Freund's Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers and polyamines such as dextransulphate, carbopol, pyran, saponins and saponin derivatives, block co-polymers, and adjuvants such as those identified in U.S. Pat. Nos. 4,578,269, 4,744,983, 5,254,339, which are all herein fully incorporated by reference. Non-limiting examples of peptides that can serve as adjuvants include muramyldipeptides, dimethylglycine, or tuftsin. Non-limiting examples of oils that can serve as adjuvants include mineral oils, vegetable oils, animal oils and emulsions thereof.
Vaccines made from material according to the present invention may be formulated as oil-in water emulsions, as water-in-oil emulsions or as water-in-oil-in-water emulsions. Non-limiting examples of oil-in-water emulsions include paraffin oil-in-water emulsions, or emulsions made from one or more of squalene, block copolymers of ethylene oxide and propylene oxide, polysorbate surfactants, and/or threonyl analogs of muramyl dipeptide.
Oils used as adjuvants may be metabolizable by the subject receiving the vaccine such as vegetable or animal oils. Such oils typically consist largely of mixtures of triacylglycerols, also known as triglycerides or neutral fats. These nonpolar, water insoluble substances are fatty acid triesters of glycerol. Triacylglycerols differ according to the identity and placement of their three fatty acid residues.
Adjuvants may also consist of components that cannot be metabolized by the body of the animal subject to which the emulsion is administered. Non-metabolizable oils suitable for use in the emulsions of the present invention include alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof. The individual compounds of the oil may be light hydrocarbon compounds, e.g., compounds having 6 to 30 carbon atoms. The oil may be synthetically prepared or purified from petroleum products. Non-limiting examples of non-metabolizable oils for use in the preparation of vaccines based upon material cultured according to the present invention include mineral oil, paraffin oil, and cycloparaffins, for example. The term “mineral oil” refers to a non-metabolizable adjuvant oil that is a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with “liquefied paraffin,” “liquid petrolatum” and “white mineral oil.” The term is also intended to include “light mineral oil,” i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil.
Other compounds capable of enhancing a humoral immunity response that may be used in the formulation of vaccines based upon material cultured according to the present invention include, without limitation, ethylene maleic anhydrate (EMA) copolymer, latex emulsions of a copolymer of styrene with a mixture of acrylic acid and methacrylic acid.
In addition to the adjuvant, a vaccine based upon material cultured according to the present invention can include immunomodulatory agents such as, e.g., interleukins, interferons, or other cytokines (e.g., Th1-related cytokines, such as interleukin-12 (IL-12), interleukin-18 (IL-18), or gamma interferon).
The amount of adjuvant or immunostimulant added in a vaccine formulation based upon material cultured according to the present invention depends on the nature of the adjuvant or immunostimulant itself. The skilled artisan is capable of selecting an amount that is sufficient to enhance an immune response to the bacterial immunizing agent.
Carriers
Pharmaceutically acceptable carriers suitable for use in vaccines comprising material according to the present invention may be any conventional liquid carrier suitable for veterinary pharmaceutical compositions, including balanced salt solutions suitable for use in tissue culture media. Pharmaceutically acceptable carriers are understood to be compounds that do not adversely effect the health of the animal to be vaccinated, at least not to the extent that the adverse effect is worse than the effects seen when the animal is not vaccinated. Suitable carriers also include sterile water, saline, aqueous buffers such as PBS, solvents, diluents, isotonic agents, buffering agents, dextrose, ethanol, mannitol, sorbitol, lactose and glycerol, and the like.
Vehicle
Vaccines formulated from material according to the present invention may also comprise a vehicle. A vehicle is a compound to which the host cells, bacterial cells, or proteins, protein fragments, nucleic acids or parts thereof adhere, without being covalently bound to it. Non-limiting examples of such vehicles include bio-microcapsules, micro-alginates, liposomes and macrosols. Some materials that serve as adjuvants can also serve as vehicles such as aluminum-hydroxide, aluminum phosphate, aluminum sulphate or aluminum oxide, silica, kaolin, and bentonite, all known in the art.
Stabilizers
Often, a vaccine is mixed with stabilizers, e.g., to protect degradation-prone components from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency. Non-limiting examples of stabilizers that may be added to vaccine formulations based upon material cultured according to the present invention include SPGA, skimmed milk, gelatins, bovine serum albumin, carbohydrates (e.g., sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (e.g., albumin, casein or degradation products thereof), non-animal origin stabilizers, and buffers (e.g., alkali metal phosphates).
Multivalent Vaccines
An immunogen according to the present invention may be formulated in a vaccine comprising one or more additional antigens. Additional immunoactive component(s) may be whole parasites, bacteria or viruses (inactivated or modified live), or a fractionated portion or extract thereof (e.g., proteins, lipids, lipopolysacharides, carbohydrates or nucleic acids).
Where the immunogen according to the present invention is used in an equine vaccine, antigens from other pathogens may be added into the formulation. Non-limiting examples of other pathogens for which additional antigens may be added include one or more (including all) of the following: Tetanus, Rabies, Eastern Encephalomyelitis, Western Encephalomyelitis, influenza virus, herpesvirus, West Nile virus (including a yellow fever virus/west nile virus chimeric flavivirus, live or killed, see e.g., US 2009/0246233, hereby incorporated by reference in its entirety) and Venezuelan Encephalomyelitis. Alternatively, a vaccine based upon material according to the present invention may be administered simultaneously with other live or inactivated vaccines.
Freeze-Drying/Reconstitution
For reasons of stability or economy, vaccines based upon material cultured according to the present invention may be freeze-dried. In general this will enable prolonged storage at temperatures above 0° C., e.g., at 4° C. Procedures for freeze-drying are known to persons skilled in the art. Equipment for freeze-drying at different scales is available commercially. To reconstitute the freeze-dried vaccine, it may be suspended in a physiologically acceptable diluent. Such diluents may be as simple as sterile water, a physiological salt solution or other carrier as discussed above.
Dosaging
Vaccines based upon material according to the present invention may be formulated in a dosage unit form to facilitate administration and ensure uniformity of dosage. A dosage unit as it pertains to the vaccine composition refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of bacterial immunogen calculated to produce the desired immunogenic effect in association with the required adjuvant system and carrier or vehicle.
The effective immunizing amount of bacterial immunogen can vary depending upon the chosen strain or strains and may be any amount sufficient to evoke a protective immune response. For example, amounts wherein the dosage unit comprises at least about 1×104 TCID50 inactivated bacterin are suitable.
Administering
Administration of the vaccine to a subject results in stimulating an immune response in the subject mammal. The route of administration for vaccines according to the present invention may be administered to the mammalian target according to methods known in the art. Such methods include, but are not limited to, intradermal, intramuscular, intraocular, intraperitoneal, intravenous, mucosal, oral, oronasal, and subcutaneous, as well as inhalation, suppository, or transdermal. The vaccine may be administered by any means that includes, but is not limited to, syringes, nebulizers, misters, needleless injection devices, or microprojectile bombardment gene guns.
Alternative routes of application that are feasible are by topical application as a drop, spray, gel or ointment to the mucosal epithelium of the eye, nose, mouth, anus, or vagina, or onto the epidermis of the outer skin at any part of the body; by spray as aerosol or powder. Alternatively, application may be via the alimentary route, by combining with the food, feed or drinking water, e.g., as a powder, a liquid, or tablet, or by administration directly into the mouth as a liquid, a gel, a tablet, or a capsule, or to the anus as a suppository. The preferred application route is by intramuscular or by subcutaneous injection.
The vaccine according to the invention may be in several forms, e.g., a liquid, a gel, an ointment, a powder, a tablet, or a capsule, depending on the desired method of application to the target. The scheme of the application of the vaccine according to the invention to the target mammal may be in single or multiple doses, which may be given at the same time or sequentially, in a manner compatible with the dosage and formulation, and in such an amount as will be immunologically effective.
Challenge Model
In order to effectively study and evaluate the pathogenic mechanisms of the bacteria and the defense mechanisms of the host mammals, and thereby to advance the vaccine art and improve vaccine products, an effective challenge model should be employed.
A challenge model, for example, may be based upon the percentage of test animals that demonstrate persistent and severe clinical symptoms commonly associated with the disease.
Several other cellular diagnostic methods exist to determine the presence of infection. For example, the presence of infection may be determined by direct or indirect immunofluorescence. Other methods to detect infection include staining, e.g., Giemsa, Wright/Giemsa. Acridine Orange can also be utilized to stain the organisms.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are hereby wholly incorporated by reference.
For a clearer understanding of the invention, examples are set forth below. These examples are merely illustrative and are understood to not limit the scope or underlying principles of the invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the examples set forth hereinbelow and the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
EXAMPLES
Example 1
Identification and Characterization of Neorickettsia risticii Isolates from Oregon, New York and Michigan
A blood sample was obtained from a horse in Oregon showing clinical signs of Potomac Horse Fever. We found the Oregon (OR) sample to be positive for N. risticii by PCR and by immunofluorescent antibody testing (IFA). We discovered this unique N. risticii Oregon strain to be distinct from the 90-12 strain. When tested by IFA, it did not react with a monoclonal antibody to the 90-12 strain.
Horses vaccinated with the 90-12 strain of N. risticii (study E-05-09-PHF) were challenged with the N. risticii Oregon. The vaccinated horses were not protected from disease. Serologically, the 90-12 vaccinated horses had no detectable antibody response to N. risticii Oregon after vaccination, and most of the unvaccinated control horses challenged with N. risticii Oregon did not have a detectable antibody response to the 90-12 strain 23 days after challenge.
A blood sample was obtained from a horse in New York showing clinical signs of Potomac Horse Fever. Unlike the corresponding Oregon isolate disclosed above, the New York (N.Y.) sample was shown to react with the monoclonal antibody to the 90-12 strain by IFA. When horses vaccinated with the 90-12 strain of N. risticii (study E-01-09-PHF) were challenged with N. risticii New York, the vaccinated horses showed a significant reduction in clinical disease and bacterial isolation.
A blood sample was obtained from a horse in Michigan showing clinical signs of Potomac Horse Fever. We found the Michigan (MI) sample to be positive for N. risticii by PCR and by immunofluorescent antibody testing. We discovered this unique N. risticii strain to be distinct from the 90-12 strain. When tested by Western Blot, it did not react with a monoclonal antibody to the 90-12 strain.
Example 2
N. risticii Host Animal Protection Studies







TABLE 1







PHF Vaccine Formulations











Component
Lot Number
Volume











E-13-06-PHF Vaccine












N. risticii (90-12) antigen
120306
300
mL



Havlogen Adjuvant
024
200
mL



PBS #2
095
1276
mL



Glycerin
005
200
mL



EDTA (240 mM)
006
20
mL



Thimerosal (10% stock solution)
025
2
mL



Phenol Red (1% stock solution)
005
2
mL



Total Volume:

2000
mL







E-01-09-PHF Vaccine












N. risticii (90-12) antigen
101608
52
mL



Carbopol 971P Adjuvant
002
300
mL



PBS #2
436
2642
mL



Thimerosal (10% stock solution)
043
2.95
mL



Phenol Red (1% stock solution)
008
3
mL



Total Volume:

3000
mL







E-05-09-PHF Vaccine












N. risticii (90-12) antigen
013009
48
mL



Carbopol 971P Adjuvant
003
200
mL



PBS #2
504
1748
mL



Thimerosal (10% stock solution)
043
1.95
mL



Phenol Red (1% stock solution)
008
1.95
mL



Total Volume:

2000
mL







E-03-10-PHF Vaccine












N. risticii (Oregon) antigen
120809
5.2
mL



Carbopol 971P Adjuvant
003
10
mL



PBS #2
601
84.6
mL



Thimerosal (10% stock solution)
062
0.095
mL



Phenol Red (1% stock solution)
008
0.095
mL



Total Volume:

100
mL










Example 2a
90-12 Vaccination/90-12 Challenge (Study E-13-06-PHF)
The purpose of this study was to evaluate the efficacy of an inactivated vaccine containing the 90-12 strain of N. risticii, against challenge with the homologous 90-12 N. risticii isolate, in host animals.
Eight month-old horses, antibody negative to N. risticii, were randomized into two treatment groups, with fifteen horses per group. The horses were vaccinated twice intramuscularly three weeks apart with 1.0 mL of vaccine containing the inactivated 90-12 strain of N. risticii (Lot 120306), or with 1.0 mL of a placebo vaccine. The horses were challenged with the 90-12 strain of N. risticii at twenty-one days following the second vaccination. Horses were observed for 24 days post-challenge for clinical signs of PHF, and whole blood samples were collected daily from 6 to 24 days post-challenge for bacterial isolation and white blood cell (WBC) counts. The clinical signs of PHF disease include rectal body temperature of >1.5° F. over baseline temperature (established as the average of the rectal temperatures for 3 days prior to challenge), anorexia, depression, or diarrhea, and laminitis.
Clinical Disease
Horses that exhibited at least one clinical sign of PHF post-challenge were classified as affected. Fifteen of the fifteen (100%) placebo-vaccinated control horses were affected. None of the fifteen (0%) horses vaccinated with N. risticii 90-12 vaccine were affected.
The prevented fraction was 1.00 with a 95% confidence interval of [0.77, 1.00], therefore the data supports the claim of “aid in the prevention of disease caused by N. risticii”. 
N. risticii Bacteremia
Horses with a positive isolation from a buffy coat sample were classified as affected. Fifteen of the fifteen (100%) placebo-vaccinated control horses were bacteremic for 5-8 days following challenge. Six of the fifteen (40%) horses vaccinated with N. risticii 90-12 vaccine were bacteremic for only one day following challenge.
The prevented fraction was 0.60 with a 95% confidence interval of [0.32, 0.80], therefore the data supports the claim of “aid in the prevention of bacteremia caused by N. risticii”. 
Conclusion
The data demonstrated that inactivated N. risticii 90-12 strain vaccine is protective against challenge with homologous N. risticii strain 90-12.
Example 2b
90-12 Vaccination/NY Challenge (Study E-01-09-PHF)
The purpose of this study was to evaluate the efficacy of an inactivated vaccine containing the 90-12 strain of N. risticii, against challenge with the heterologous NY N. risticii strain, in host animals.
Eleven month-old horses, antibody negative to N. risticii, were randomized into two treatment groups, with fifteen horses per group. The horses were vaccinated twice intramuscularly three weeks apart with 1.0 mL of vaccine containing the inactivated 90-12 strain of N. risticii (Lot 101608), or with 1.0 mL of a placebo vaccine. The horses were challenged with the NY strain of N. risticii fourteen days following the second vaccination. Horses were observed for 21 days following challenge for clinical signs of disease associated with PHF, and whole blood samples were collected daily from 6 to 18 days post-challenge for bacterial isolation. The clinical signs of PHF disease include rectal body temperature of >1.5° F. over baseline temperature (established as the average of the rectal temperatures for 3 days prior to challenge), anorexia, depression, diarrhea, colic or laminitis, when associated with the other clinical signs of PHF.
Clinical Disease
Horses that exhibited at least one clinical sign of PHF post-challenge on at least one day post-challenge were classified as affected.
The severity of clinical disease was considered to be the number of post-challenge days with at least one clinical sign, and was analyzed using the Wilcoxon Rank Sum test. The mitigated fraction was 0.54 with a lower confidence interval of 0.17, therefore the data supports the claim of “aid in the reduction of severity of clinical disease caused by N. risticii”. 
N. risticii Bacteremia
Horses with a positive isolation from a buffy coat sample were classified as affected. The placebo-vaccinated control horses had an average of 6.4 days of positive isolations following challenge. The horses vaccinated with inactivated N. risticii 90-12 strain vaccine had an average of 3.5 days of positive isolations following challenge.
The duration of bacteremia was analyzed using the Wilcoxon Rank Sum test. The mitigated fraction was 0.59 with a lower confidence interval of 0.24, therefore the data supports the claim of “aid in the reduction of bacteremia caused by N. risticii”. 
Conclusion
The data demonstrated that inactivated N. risticii 90-12 strain vaccine reduces the severity of clinical disease and bacteremia, following challenge with heterologous N. risticii strain NY.
Example 2c
90-12 Vaccination/OR Challenge (Study E-05-09-PHF)
The purpose of this study was to evaluate the efficacy of an inactivated vaccine containing only the OR strain of N. risticii, against challenge with the OR N. risticii isolate, in host animals.
Five to six month-old horses, antibody negative to N. risticii, were randomized into two treatment groups, with fifteen horses per group. The horses were vaccinated twice intramuscularly three weeks apart with 1.0 mL of the inactivated vaccine containing the inactivated 90-12 strain of N. risticii (Lot 013009), or with 1.0 mL of a placebo vaccine. The horses were challenged with the heterologous OR strain of N. risticii fourteen days following the second vaccination. Horses were observed for 21 days post-challenge for clinical signs of PHF, and whole blood samples were collected daily from 6 to 18 days post-challenge for bacterial isolation. The clinical signs of PHF disease include rectal body temperature of >1.5° F. over baseline temperature (established as the average of the rectal temperatures for 3 days prior to challenge), anorexia, depression, or diarrhea, and additionally colic or laminitis, when associated with the other clinical signs of PHF.
Clinical Disease
Horses that exhibited at least one clinical sign of PHF post-challenge were classified as affected. Twelve of the fifteen (80%) placebo-vaccinated control horses were affected, and twelve of the fifteen (80%) horses vaccinated with inactivated N. risticii 90-12 strain vaccine were affected. The inactivated N. risticii 90-12 strain vaccine provided no protection from clinical disease caused by heterologous OR strain N. risticii. 
N. risticii Bacteremia
Horses with a positive isolation from a buffy coat sample were classified as affected. Two of the fifteen (13%) placebo-vaccinated control horses had a positive isolation on one day following challenge. Eight of the fifteen (53%) horses vaccinated with inactivated N. risticii 90-12 strain vaccine had a positive isolation on at least one day following challenge. The inactivated N. risticii 90-12 strain vaccine provided no protection from bacteremia caused by heterologous OR strain N. risticii. 
Conclusion
The data demonstrates that the inactivated N. risticii 90-12 strain vaccine did not protect against, or reduce the severity of, clinical disease or bacteremia following challenge with heterologous N. risticii strain OR.
Example 2d
OR Vaccination/OR Challenge (Study E-03-10-PHF)
The purpose of this study was to evaluate the efficacy of an inactivated vaccine containing only the OR strain of N. risticii, against challenge with the homologous OR N. risticii isolate, in host animals.
Ten to twelve month-old horses, antibody negative to N. risticii, were enrolled in the study and were randomized into treatment groups, with eight horses per group. The horses were vaccinated twice intramuscularly three weeks apart with 1.0 mL of the inactivated vaccine containing the OR strain of N. risticii (Lot 120809), or with 1.0 mL of a placebo vaccine. The horses were challenged with the OR strain of N. risticii at twenty days following the second vaccination. Horses were observed for 24 days post-challenge for clinical signs of PHF, until resolution of the clinical signs, and whole blood samples were collected daily from 7 to 18 days post-challenge for bacterial isolation. Horses were weighed prior to challenge and following the post-challenge period, as an additional non-subjective indication of illness, anorexia, and diarrhea caused by PHF, and percent weight gains or losses during the post-challenge period were determined. The clinical signs of PHF disease include rectal body temperature of >1.5° F. over baseline temperature (established as the average of the rectal temperatures for 4 days prior to challenge), anorexia, depression, or diarrhea, and additionally colic or laminitis, when associated with the other clinical signs of PHF.
Clinical Disease
Horses that exhibited at least one clinical sign of PHF post-challenge were classified as affected. Seven of the eight (88%) placebo-vaccinated control horses were affected. Three of the eight (38%) horses vaccinated with experimental N. risticii OR strain vaccine were affected.
The Fisher test p-value was 0.0594. The proportion of affected horses vaccinated with N. risticii OR strain vaccine was marginally less than the proportion of horses vaccinated with placebo.
Severity of Disease
To evaluate disease severity, the number of post-challenge days with at least one clinical sign of PHF was analyzed using the Wilcoxon Rank Sum test. The p-value was 0.0485, therefore the number of post-challenge days with at least one clinical sign of PHF for horses vaccinated with N. risticii OR strain vaccine was marginally less than for the placebo-vaccinated horses.
The Wilcoxon Rank Sum test was also used to evaluate the number of clinical signs of PHF present during the post-challenge period, as an additional measure of disease severity. The p-value was 0.0485, indicating that the total number of clinical signs of PHF present during the post-challenge period was marginally less for the horses vaccinated with experimental N. risticii OR strain vaccine than for the placebo-vaccinated control horses.
N. risticii Bacteremia
Horses with a positive isolation from a buffy coat sample were classified as affected. Two of the eight (25%) placebo-vaccinated control horses had a positive isolation on one day following challenge. One of the eight (13%) horses vaccinated with experimental N. risticii OR strain vaccine had a positive isolation on one day following challenge.
The Fisher test p-value was 0.3354, therefore there was no statistical difference in bacteremia between the horses vaccinated with experimental N. risticii OR strain vaccine and the placebo-vaccinated horses.
Weight
Horses were weighed prior to and following challenge. Four of the eight placebo-vaccinated control horses lost weight during the post-challenge period, and the average percent of body weight gain for all eight horses was only 0.3%. The horses vaccinated with experimental N. risticii OR strain vaccine had an average percent weight gain of 5.2%, and none of the eight vaccinated horses lost weight.
Conclusion
This data demonstrates that vaccination of horses with N. risticii OR strain vaccine reduces the incidence and severity of clinical PHF disease, compared to placebo-vaccinated control horses, following challenge with homologous OR strain N. risticii. Table 2 provides a comparison of the clinical results of sixteen horses challenged with N. risticii Oregon, eight of which received a vaccine containing the N. risticii Oregon isolate and eight of which received a placebo.









TABLE 2






Control
Vaccinated



Horses
Horses


Treatment Group
(Placebo)
(Oregon strain)







No. of Horses with ≧1 Clinical Sign of PHF
7/8
3/8


No. of Horses with Fever
5/8
1/8


No. of Horses with Anorexia
4/8
2/8


No. of Horses with Depression
1/8
0/8


No. of Horses with Diarrhea
3/8
1/8


No. of Horses with Laminitis
1/8
0/8


No. of Horses with Bacteremia
2/8
1/8


Average % Weight Gain
0.3
5.2









These data show that vaccination of horses with N. risticii Oregon strain vaccine reduces the incidence and severity of clinical PHF, compared to placebo-vaccinated control horses.

Example 3
Serological Cross-Reactivity of N. risticii Strains
Immunofluorescent antibody (IFA) testing was performed on sera collected from horses following vaccination and/or challenge with various strains of N. risticii, to measure the antibody reactivity against the five different strains of N. risticii. For the IFA assay, fixed feline embryonic fibroblast (FEF) cells, infected with one of the N. risticii strains (OR, 90-12, NY, MI, or 25-D), were reacted with serial dilutions of the equine serum samples. The plates were read using a fluorescence microscope for positive fluorescence, as indicated by bright green, specific cytoplasmic staining. The antibody titer was the reciprocal of the highest serum dilution that showed positive fluorescence.
The IFA results show that for most strains of N. risticii there is some serological cross-reactivity to other strains of N. risticii, but there appears to be very little cross-reactivity in sera from horses challenged with the OR strain of N. risticii, to other strains of N. risticii. The OR strain of N. risticii appears to be highly unique antigenically.







TABLE 3







Oregon Challenged Horses (21, 22 or 24 days post-challenge)

















New York
90-12
Michigan






Oregon IFA
IFA
IFA
IFA
25D IFA
OSU* IFA


Study No.
Horse ID
Results
Results
Results
Results
Results
Results

















E-05-09
121
1280
80
40
40
80
40


E-05-09
123
2560
320
160
320
320
2560


E-05-09
126
640
80
<20
80
80
320


E-05-09
127
320
40
<20
40
40
40


E-05-09
128
1280
40
<20
<20
20
<20


E-05-09
130
640
<20
<20
<20
40
<20


E-05-09
132
640
80
<20
20
40
<20


E-05-09
133
320
<20
<20
<20
<20
<20


E-05-09
136
1280
<20
<20
<20
<20
<20


E-05-09
137
1280
40
<20
40
<20
<20


E-05-09
138
640
20
<20
40
40
<20


E-05-09
140
320
<20
<20
<20
<20
<20


E-05-09
144
640
20
<20
<20
<20
<20


E-05-09
147
2560
80
<20
80
160
<20


E-05-09
148
640
40
<20
160
40
<20


E-07-08
2
1280
<20
<20
<20
<20
NT


E-07-08
5
1280
<20
<20
<20
<20
NT


E-07-08
10
640
<20
<20
<20
<20
NT


E-07-08
22
640
<20
<20
<20
<20
NT


E-07-08
11
1280
80
<20
40
<20
NT


E-07-08
12
2560
80
<20
<20
160
NT


E-07-08
17
640
<20
<20
<20
<20
NT


E-07-08
21
640
<20
<20
<20
<20
NT


E-10-10
328
80
<20
<20
<20
<20
NT


E-10-10
331
640
<20
<20
<20
<20
NT


E-10-10
333
160
40
<20
<20
40
NT


E-10-10
337
320
80
<20
<20
<20
NT


E-10-10
336
1280
160
<20
40
<20
NT


E-10-10
338
80
<20
<20
<20
<20
NT


E-10-10
339
640
80
<20
40
80
NT


E-03-10
162
640
80
80
40
80
NT


E-03-10
165
640
<20
20
<20
80
NT


E-03-10
167
1280
80
40
40
20
NT


E-03-10
173
640
80
80
80
<20
NT


E-03-10
177
320
40
40
40
<20
NT


E-03-10
179
640
40
40
40
40
NT


E-03-10
214
640
80
80
80
80
NT


E-03-10 
216
640
40
40
40
40
NT


Average:

859
53
27
42
46
208
















TABLE 4







Oregon Vaccinated and Challenged Horses (24 days post-challenge)















Oregon
New York
90-12
Michigan
25D


Study
Horse
IFA
IFA
IFA
IFA
IFA


No.
ID
Results
Results
Results
Results
Results
















E-03-10
171
1280
640
320
640
640


E-03-10
175
2560
640
640
1280
1280


E-03-10
204
1280
320
160
320
640


E-03-10
208
1280
320
320
320
640


E-03-10
213
1280
320
320
320
320


E-03-10
217
1280
320
320
320
640


E-03-10
218
1280
320
80
80
640


E-03-10
219
2560
2560
2560
2560
2560


Average:

1600
680
590
730
920
















TABLE 5







New York Challenged Horses (22 days post-challenge)

















New York
90-12
Michigan






Oregon IFA
IFA
IFA
IFA
25D IFA
OSU IFA


Study No.
Horse ID
Results
Results
Results
Results
Results
Results

















E-07-08
4
160
640
320
320
640
NT


E-07-08 
6
40
160
80
<20
<20
NT


E-07-08 
14
160
1280
320
640
640
NT


E-07-08 
19
40
320
160
160
320
NT


E-07-08 
9
80
320
160
160
320
NT


E-07-08 
15
80
640
160
320
640
NT


E-07-08 
23
160
1280
320
320
320
NT


E-07-08 
24
80
1280
640
320
320
NT


E-01-09 
74
<20
1280
1280
320
640
640


E-01-09 
76
160
1280
1280
640
640
640


E-01-09 
77
80
640
640
320
320
160


E-01-09 
78
80
640
1280
320
640
320


E-01-09 
79
<20
640
1280
640
640
320


E-01-09 
81
160
320
640
160
320
80


E-01-09 
87
160
640
1280
640
640
320


E-01-09 
92
<20
1280
1280
1280
640
1280


E-01-09 
93
<20
2560
640
640
1280
640


E-01-09 
96
640
1280
640
1280
1280
1280


E-01-09 
97
<20
1280
640
320
640
320


E-01-09 
98
160
2560
2560
1280
2560
1280


E-01-09 
100
<20
2560
640
320
640
320


E-01-09 
102
80
1280
1280
640
320
320


E-01-09 
106
<20
2560
640
<20
<20
320


Average:

105
1162
790
481
627
549
















TABLE 6







90-12 Challenged Horses (21 days post-challenge)















Oregon 
New York
90-12
Michigan
25D


Study 
Horse
IFA
IFA
IFA
IFA
IFA


No.
ID
Results
Results
Results
Results
Results
















E-08-08 
27
80
1280
640
320
320


E-08-08 
30
640
1280
2560
1280
320


E-08-08 
32
80
1280
1280
640
640


E-08-08 
40
640
2560
5120
2560
5120


E-08-08 
45
320
2560
2560
2560
2560


E-08-08 
46
640
5120
2560
2560
2560


E-08-08 
48
320
2560
2560
1280
2560


E-08-08 
49
160
1280
640
640
1280


E-08-08 
52
160
1280
1280
640
1280


E-08-08 
53
160
1280
640
640
1280


E-08-08 
55
80
1280
640
640
320


E-08-08 
57
80
1280
640
640
640


E-08-08 
59
160
2560
5120
2560
2560


E-08-08 
66
640
2560
2560
5120
5120


E-08-08 
71
40
640
640
640
640


Average:

280
1920
1963
1515
1813
















TABLE 7







90-12 Vaccinated and Challenged Horses (21 days post-challenge)















Oregon
New York
90-12
Michigan
25D


Study
Horse
IFA
IFA
IFA
IFA
IFA


No.
ID
Results
Results
Results
Results
Results
















E-08-08
26
1280
10240
10240
5120
2560


E-08-08
29
1280
10240
10240
2560
5120


E-08-08
34
1280
5120
5120
2560
5120


E-08-08
35
640
10240
10240
2560
5120


E-08-08
36
1280
10240
10240
5120
10240


E-08-08
37
1280
10240
10240
10240
10240


E-08-08
38
1280
10240
10240
5120
5120


E-08-08
39
2560
10240
10240
10240
10240


E-08-08
41
1280
5120
2560
5120
10240


E-08-08
44
2560
10240
10240
10240
10240


E-08-08
50
1280
10240
10240
5120
10240


E-08-08
51
640
5120
5120
5120
10240


E-08-08
60
1280
2560
5120
5120
2560


E-08-08
64
1280
5120
10240
10240
5120


E-08-08
69
2560
5120
10240
10240
10240


Average:

1451
8021
8704
6315
7509
















TABLE 8







Michigan Challenged Horses (21 days post-challenge)















Oregon 
New York 
90-12
Michigan
25D


Study
Horse
IFA
IFA
IFA
IFA
IFA


No.
ID
Results
Results
Results 
Results
Results
















E-10-10
326
320
640
320
640
1280


E-10-10 
327
<20
<20
<20
80
80


E-10-10 
335
320
2560
640
640
1280


Average:

218
1072
325
453
880
















TABLE 9







25D Challenged Horses (21 days post-challenge)















Oregon
New York
90-12
Michigan
25D


Study
Horse
IFA
IFA
IFA
IFA
IFA


No.
ID
Results
Results
Results
Results
Results
















E-10-10
329
40
160
40
40
320


E-10-10 
332
<20
160
80
40
320


E-10-10 
334
<20
320
160
160
640


E-10-10 
340
<20
160
40
80
640


Average:

21
200
80
80
480





NT = Not Tested, NA = Not Applicable


<20 assigned a value of 15 for average titer calculations


Antigen Lots: Oregon Lot 012010, 90-12 Lot 101910, Michigan Lot 100510, New York Lot 101310, 25-D Lot 101210


*OSU = Oregon State University Veterinary Diagnostic Laboratory






Example 4
Western Blot Analysis of N. risticii Strains
Western Blots were performed to evaluate the antibody reactivity and antigen banding patterns of rabbit and horse serum against the recombinant 85 kDa protein of the N. risticii 90-12 strain with FEF cells used to propagate N. risticii and against the 90-12, OR, NY, MI and 25-D strains of N. risticii. Rabbit serum lot 02×1808a was a pool of sera collected from four rabbits following hyperimmunization with purified recombinant 85 kDa protein from the 90-12 strain of N. risticii. Horse serum lot 112310 was a pool of sera collected from four horses following challenge with the 90-12, OR, NY, or MI strains of N. risticii. Horse serum lot 012809 was collected from a horse that had not previously been exposed to, vaccinated or challenged with N. risticii, and served as a negative control to identify non-specific reactivity to FEF cells and N. risticii. 
The pooled serum from rabbits immunized with the recombinant 85 kDa protein of the N. risticii 90-12 strain reacted with the 85 kDa recombinant protein on the Western Blot at the expected molecular weight, and at the same approximate molecular weight for the 90-12 strain of N. risticii. (FIG. 1). The serum reacted at differing, lower molecular weight bands of approximately 50, 55, 60 and 55 kDa for the OR, NY, MI and 25-D strains of N. risticii, respectively. No reactions were observed for the FEF cells.
The pooled serum from horses challenged with one of four strains of N. risticii, reacted with the recombinant 85 kDa protein at a lower than expected molecular weight of approximately 33 kDa on the Western Blot. (FIG. 2). The predominant unique bands for strain 90-12 of N. risticii had molecular weights of approximately 177, 89, and 22 kDa. The serum reacted at differing molecular weight bands of approximately 47 for the OR strain, 180 and 39 kDa for the NY strain, 180 and 31 kDa for the MI strain, and 79 kDa for the 25-D strain of N. risticii. No reactions were observed for the FEF cells. These banding patterns indicate that the challenge strains are distinctive and unique.
The bands that appear at the molecular weights of approximately 61-63 kDa and 54-56 kDa of the N. risticii strains are non-specific bands that also react with seronegative horse serum.
Example 5
DNA Sequencing Analysis of N. risticii Strains
GeneWiz generated consensus sequences for MAH for the strain specific antigen (SSA) locus of the 90-12, OR, NY, MI and 25-D strains of N. risticii. The sequences were analyzed and compared with each other and with the currently published sequences for the N. risticii Illinois strain. Both nucleotide sequences and predicted protein sequences were analyzed using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST). In addition, DNASTARTM LASERGENE MEGALIGN analysis software (version 8.0.2) was used to align and compare sequences with one another.







TABLE 10







DNA Size comparison:











N. risticii Strain
SSA Locus
SSA-1
SSA-2
SSA-3





Illinois (acc#
~6600 bp 
1560 bp
1539 bp
1302 bp


CP001431)


90-12
6356 bp
1887 bp
1539 bp
1614 bp


OR
6871 bp
1422 bp
1464 bp
1305 bp (inverted)


NY
6916 bp
1638 bp
1539 bp
1614 bp


MI
6917 bp
1869 bp
1410 bp
1458 bp


25-D
6620 bp
1620 bp
1539 bp
1302 bp









The BLAST results differ slightly from those obtained using the DNASTARTM LASERGENE MEGALIGN program, which indicates that the NY strain shares the highest identity with the published Illinois strain. MEGALIGN software uses the CLUSTALW, SLOW_ACCURATE algorithm for aligning multiple sequences (FIGS. 4-7), while the NCBI BLAST search uses the BLASTN and BLASTP algorithms (TABLE 11). In FIG. 7 the nucleic acid sequence for OR SSA#3 is the reversed complimentary sequence from the wild type sequence in order to match it with the genes of the compared N. risticii strains.







TABLE 11







BLAST Analysis Results Summary:












Top Blast Hit
Genebank


Strain
Portion analyzed
(% identity)
Accession No.





90-12
whole fragment
92.5% to Illinois
CP00143



(DNA)
(94% to 90-12: 85 kd)*



SSA1 (amino acid
82.5% to 90-12: 85 kd
AAC31428



sequence)



SSA2 (amino acid
99.0% to Illinois
YP_0030



sequence)
(37% to 90-12: 85 kd)



SSA3 (amino acid
94.2% to Illinois
YP_0030



sequence)


OR
whole fragment
87.3% to Illinois
CP00143A



(DNA)



SSA1 (amino acid
52.6% to Illinois
YP_0030



sequence)



SSA2 (amino acid
63.5% to Illinois
YP_0030



sequence)



SSA3 (amino acid
88.1% to Illinois
YP_0030



sequence)


NY
whole fragment
94.0% to Illinois
CP00143



(DNA)



SSA1 (amino acid
86.3% to Illinois
YP_0030



sequence)



SSA2 (amino acid
98.8% to Illinois
YP_0030



sequence)



SSA3 (amino acid
94.2% to Illinois
YP_0030



sequence)


MI
whole fragment
97.0% to Illinois
CP00143



(DNA)



SSA1 (amino acid
69.0% to 25-D: 50 kd
AAC31427



sequence)
(67% to Illinois)



SSA2 (amino acid
90.9% to Illinois
YP_0030



sequence)



SSA3 (amino acid
85.6% to Illinois
YP_0030



sequence)


25-D
whole fragment
96.3% to Illinois
CP00143A



(DNA)



SSA1 (amino acid
98.0% to 25-D: 50 kd
AAC31427



sequence)



SSA2 (amino acid
99.8% to Illinois
YP_0030



sequence)



ssa3 (amino acid
99.8% to Illinois
YP_0030



seq.)





*Accession AF059673 (strain 90-12) 85 kd SSA gene nucleotide sequence is smaller than whole fragment sequenced.






BIOLOGICAL DEPOSIT
Cultures of the following biological materials have been deposited with the following international depository: American Type Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., under conditions that satisfy the requirements of the Budapest Treaty.















Organism
Accession No.
Date of Deposit









Neorickettsia risticii
PTA-11232
Jul. 27, 2010



Oregon



Neorickettsia risticii
PTA-11231
Jul. 27, 2010



New York



Neorickettsia risticii
PTA-11404
Oct. 13, 2010



Michigan




















SEQUENCE LISTING TABLE










SEQ ID NO:
STRAIN
SS#
Type













1
Illinois
1
AA


2
Oregon
1
AA


3
25-D
1
AA


4
90-12
1
AA


5
Michigan
1
AA


6
New York
1
AA


7
Illinois
2
AA


8
New York
2
AA


9
90-12
2
AA


10
25-D
2
AA


11
Oregon
2
AA


12
Michigan
2
AA


13
Illinois
3
AA


14
New York
3
AA


15
90-12
3
AA


16
25-D
3
AA


17
Michigan
3
AA


18
Oregon
3
AA


19
Illinois
3
NA


20
New York
3
NA


21
90-12
3
NA


22
25-D
3
NA


23
Michigan
3
NA


24
Oregon
3
NA


25
Oregon
1
NA


26
Oregon
2
NA


27
New York
1
NA


28
New York
2
NA


29
Michigan
1
NA


30
Michigan
2
NA


31
Illinois
1
NA


32
Illinois
2
NA


33
25-D
1
NA


34
25-D
2
NA


35
90-12
1
NA


36
90-12
2
NA





AA is an amino acid sequence;


NA is a nucleic acid sequence.






It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, provided to describe nucleic acids and polypeptides according to the invention are approximate within conventional measurement variations.

## Claims

1. A killed or inactivated Neorickettsia risticii (N. risticii) bacterium of the N. risticii Oregon strain that encodes:
a strain specific antigen #1 (SSA#1) comprising an amino acid sequence that has 99% or greater sequence identity with the amino acid sequence of SEQ ID NO: 2;
a strain specific antigen #2 (SSA#2) comprising an amino acid sequence that has 99% or greater sequence identity with the amino acid sequence of SEQ ID NO: 11; and
a strain specific antigen #3 (SSA#3) comprising an amino acid sequence that has 99% or greater sequence identity with the amino acid sequence of SEQ ID NO: 18.

2. An immunogenic composition comprising the killed or inactivated N. risticii bacterium of claim 1 and a carrier.

3. The immunogenic composition of claim 2, wherein the N. risticii bacterium was killed or inactivated by a method selected from the group consisting of sonication, freeze-thaw techniques, pressure, treatment with heat, treatment with ultraviolet light, a chemical method, and with an enzyme.

4. The immunogenic composition of claim 3, wherein the chemical method is selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate, and any mixture thereof.

5. The killed or inactivated N. risticii bacterium of claim 1, wherein the N. risticii bacterium is the N. risticii Oregon isolate having the ATCC deposit number PTA-11232.

6. A vaccine comprising an immunogenically effective amount of the killed or inactivated N. risticii bacterium of claim 5 and a carrier.

7. The vaccine of claim 6, wherein the N. risticii bacterium was killed or inactivated by a method selected from the group consisting of sonication, freeze-thaw techniques, pressure, treatment with heat, treatment with ultraviolet light, a chemical method, and with an enzyme.

8. The vaccine of claim 7, wherein the chemical method is selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate, and any mixture thereof.

9. The killed or inactivated N. risticii bacterium of claim 5, wherein the N. risticii bacterium was killed or inactivated by a method selected from the group consisting of sonication, freeze-thaw techniques, pressure, treatment with heat, treatment with ultraviolet light, a chemical method, and with an enzyme.

10. The killed or inactivated N. risticii bacterium of claim 9, wherein the chemical method is selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate, and any mixture thereof.

11. The killed or inactivated N. risticii bacterium of claim 1, wherein the N. risticii bacterium was killed or inactivated by a method selected from the group consisting of sonication, freeze-thaw techniques, pressure, treatment with heat, treatment with ultraviolet light, a chemical method, and with an enzyme.

12. The killed or inactivated N. risticii bacterium of claim 11, wherein the chemical method is selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, merthiolate, gluteraldehyde, sodium dodecyl sulfate, and any mixture thereof.

13. The killed or inactivated N. risticii of claim 1 comprising a nucleic acid comprising a nucleotide sequence encoding a SSA, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and any combination thereof.