Avirulent whole cell bacterial inactivated antigen vaccines against tick-borne disease

Abstract

The present disclosure provides immunogenic compositions and vaccines effective for reducing the incidence of or severity of clinical signs or symptoms of infection by Rickettsia rickettsii. In preferred forms, the immunogenic composition comprises an inactivated whole cell avirulent strain of Rickettsia rickettsii such as the Iowa strain, together with an adjuvant.

Description

AVIRULENT WHOLE CELL BACTERIALINACTIVATED ANTIGEN VACCINES AGAINSTTICK-BORNE DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 708,248 filed on October 16, 2024, the content of which (text, drawings, and claims) is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Al 152417 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Rocky Mountain Spotted Fever (RMSF) is caused by the rickettsial pathogen, Rickettsia rickettsii. R. rickettsii is endemic throughout North and Central America. The pathogen is transmitted primarily through the bites of infected ticks. In the USA, Dermacentor variabilis (the American dog tick) and D. andersoni (the Rocky Mountain wood tick) are the primary vectors for transmitting R. rickettsii. Seroprevalence for this pathogen in domestic and stray dogs in disease-endemic regions is estimated to be between 68-81%. Because of the likely high rates of exposure of dogs with ticks, they can serve as sentinels of risk for RMSF in humans. Moreover, clinical and hematological symptoms of RMSF in dogs are very similar to R. rickettsii infections in humans. Clinical signs or symptoms following the infection generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, rash, and can lead to death. The typical treatment for RMSF in dogs and humans is very similar; doxycycline at a dose rate of 5-10mg / kg / day for 10-21 days or 10-20mg / kg twice a day for a week. Relapse of RMSF in dogs after doxycycline therapy has also been reported. Sub-clinically infected dogs can also serve as a potential source of infection to ticks and human beings. A vaccine to prevent dog infection can reduce clinical diseases in dogs.The reduced disease in the canine host will also have positive implications in reducing the disease burden in people.

[0004] Recovery from aR. rickettsii infection confers long lasting protective immunity against subsequent reinfection, suggesting that the availability of a vaccine will be most useful in reducing the RMSF disease in both dogs and people. Currently, no effective vaccine is available to prevent the disease for either people or dogs. There have been attempts to develop a killed vaccine prepared from R. rickettsii cultured in ticks or embryonated yolk sac or from cell cultures using non-human primates and guinea pigs as animal models with varying efficacies. These studies, however, are 75- 90 years old. Recent studies in mice suggest that the outer membrane protein B (OmpB) and adhesion 2 (Adr2) protein of R. rickettsii alone or as in combination serve as protective antigen(s) in reducing the bacterial load. Considering the non-availability of the vaccines for both dogs and people, the goal of the current proposal is to demonstrate the use of an avirulent or non-pathogenic strain of R. rickettsii as an effective vaccine or immunogenic composition against RMSF.SUMMARY

[0005] The present disclosure provides an immunogenic composition or vaccine for R. rickettsii (which causes Rocky Mountain Spotted Fever (RMSF)). The immunogenic composition or vaccine for a tick-borne pathogen of the disclosure preferably comprises one or more inactivated tick-bome avirulent pathogens of the disclosure. An avirulent pathogen strain is a strain of a microorganism that has minimal or no virulence in the host of interest when compared to other strains of its species. In the present disclosure, avirulent and non-pathogenic are equivalent.

[0006] An immunogenic composition or vaccine for RMSF preferably comprises one or more inactivated R. rickettsii of an avirulent strain, such as the Iowa strain. In one aspect, the immunogenic composition or vaccine can further comprise one or more additional elements selected from the group consisting of pharmaceutical earners, stabilizers, adjuvants, compositions known to increase immunity, diluents, preservatives, and any combination thereof. In some forms, the one or more additionalelements is selected from the group consisting of stabilizers, adjuvants, compositions known to increase immunity, preservatives, and any combination thereof. In a preferred embodiment, the immunogenic composition or vaccine for RMSF comprises one or more R.rickettsii and an adjuvant. In preferred forms, the R. rickettsii is the Iowa strain.

[0007] The present disclosure further provides for a method of preventing the incidence of or lessening the severity of RMSF in canine or human where the method comprises the step of administering an immunogenic composition or vaccine comprising one or more inactivated avirulent R.rickettsii and an adjuvant to an animal in need thereof, preferably a canine or human. In preferred forms, the R. rickettsii is the Iowa strain. In preferred forms, the prevention of the incidence of and / or lessening the severity of RMSF is in comparison to an animal that does not receive an administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent R.rickettsii and an adjuvant but has the same or a similar challenge.

[0008] The present disclosure additionally provides for a method of reducing clinical symptoms of RMSF or reducing the incidence and / or severity of clinical symptoms of RMSF, where the step of the method includes administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent R.rickettsii and an adjuvant to an animal in need thereof, preferably a canine or human. In preferred forms, the R. rickettsii is the Iowa strain. In preferred forms, the reduction in clinical symptoms and reducing the incidence and / or severity of clinical symptoms of RMSF is in comparison to an animal that does not receive an administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent R.rickettsii and an adjuvant but has the same or a similar challenge.

[0009] A method for preventing subclinical infection of R. rickettsii in a canine is also provided in the present disclosure. The method for preventing subclinical infection of R.rickettsii includes the step of administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent R.rickettsii and an adjuvant to an animal in need thereof, preferably a canine or human. In preferred forms, the R. rickettsii is the Iowa strain.BRIEF DESCRIPTION OF THE FIGURES

[0010] FIG. 1A provides a comparison of CD4 T cell proliferation levels in vaccinated and control groups just after vaccination.

[0011] FIG. IB provides a comparison of CD8 T cell proliferation levels in vaccinated and control groups just after vaccination.

[0012] FIG. 2A provides a comparison of CD4 T cell proliferation levels in vaccinated and control groups seven days after infection with either Sheila Smith, Morgan, or Iowa strains of R. rickettsii.

[0013] FIG. 2B provides a comparison of CD8 T cell proliferation levels in vaccinated and control groups seven days after infection with either Sheila Smith, Morgan, or Iowa strains of R. rickettsii.

[0014] FIG. 3A provides a comparison of measured body temperatures in control groups for the first seven days after infection with either Sheila Smith, Morgan, or Iowa strains of R. rickettsii.

[0015] FIG. 3B provides a comparison of body temperatures in two Iowa vaccinated groups and one control group for the first seven days after infection with either Sheila Smith, Morgan, or Iowa strains of R. rickettsii.

[0016] FIGS. 4A and 4B provide comparative photographs of dog ears from a control group three days post-infection and from a vaccinated group three days postinfection, showing visible clinical signs of disease in the control group.

[0017] FIG. 5 A provides IgG response levels measured across primary7vaccination, booster, and challenge periods for control groups and groups vaccinated with a whole cell antigen (WCA) Sheila Smith vaccine.

[0018] FIG. 5B provides IgG response levels measured across primary7vaccination, booster, and challenge periods for control groups and groups vaccinated with a WCA Morgan vaccine.

[0019] FIG. 5C provides IgG response levels measured across primary vaccination, booster, and challenge periods for control groups and groups vaccinated with a WCA Iowa vaccine.

[0020] FIG. 6A provides comparative microscopy images of the thymus taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine.

[0021] FIG. 6B provides comparative microscopy images of testes tissues taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine.

[0022] FIG. 6C provides comparative microscopy images of lung tissues taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine.

[0023] FIG 6D provides comparative microscopy images of liver tissues taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine.

[0024] FIG 6E provides comparative microscopy images of kidney tissues taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine.

[0025] FIG 6F provides comparative microscopy images of heart tissues taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine. FIGS.

[0026] FIG 6G provides comparative microscopy images of brain tissues taken from dogs in control groups and groups vaccinated with the WCA Iowa vaccine.DETAILED DESCRIPTION

[0027] The present disclosure provides an immunogenic composition or vaccine for Rickettsia rickettsii, where the immunogenic composition or vaccine comprises one or more inactivated avirulent strains of R.rickettsii. In preferred forms, the immunogenic composition or vaccine further includes or comprises an adjuvant. In some preferred forms, the R.rickettsii strain is the Iowa strain.

[0028] The present disclosure further provides for a method of preventing or lessening the severity of . rickettsia, in canine or human where the method comprises the step of administering an immunogenic composition or vaccine comprising one ormore inactivated avirulent strains of R.rickettsii and an adjuvant to a canine or human. In some preferred forms, the R. rickettsii strain is the Iowa strain.

[0029] The present disclosure additionally provides for a method of reducing clinical symptoms of infection from R. rickettsia, in a dog or reducing the incidence or severity of clinical symptoms of infection from R. rickettsia in canine or human, wherein the method includes administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent strains of R. rickettsia and an adjuvant to a canine or human. In some preferred forms, the Rrickettsii strain is the Iowa strain.

[0030] The clinical symptoms of RMSF caused by R. rickettsia reduced or reduced in incidence or severity include, but are not limited to, loss of appetite, fever, pain in muscles and joints, depression, swollen lymph nodes, edema, coughing, difficulty breathing, vomiting, blood in the stool, dizziness, ataxia, seizures, nose bleeds, retina bleeding, blood in the urine, pinpoint bruises that appear on the lining of the eyelids and mouth, irregular heartbeat, difficulty with clotting leading to shock or death, and any combination thereof. Preferably the clinical symptoms associated with RMSF are reduced in frequency and / or severity by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or reduced by 100% in comparison to an animal or group of animals that has been subjected to the same challenge conditions but did not receive an administration of the immunogenic composition or vaccine.

[0031] The present disclosure also provides a method for preventing subclinical infection of at least one of Rrickettsii in a canine or human. The method for preventing subclinical infection of at least one of Rrickettsii includes the step of administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent strains of R.rickettsii and an adjuvant to a canine or human. In some preferred forms, the Rrickettsii strain is the Iowa strain.

[0032] In other aspect, the present disclosure provides a method for reducing the bacterial load of at least one tick -borne pathogen of the disclosure in a canine orhuman, where the method comprises the step of administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent strains of tickhome pathogen Rickettsia rickettsi and an adjuvant to a canine or human. Preferably, the bacterial load is reduced at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and 100% in comparison to an animal or group of animals that has been subjected to the same challenge conditions but did not receive an administration of the immunogenic composition or vaccine.[0033j In another aspect of the disclosure, a method of lowering the incidence of RMSF infection in the human population is provided. The method for lowering the incidence of RMSF infection in the human population includes the step of administration of an immunogenic composition or vaccine comprising one or more inactivated avirulent strains of R.rickettsii and an adjuvant to a canine. In some preferred forms, the R.rickettsii strain is the Iowa strain. The incidence of RMSF infection in humans is reduced by the reduced incidence of RMSF infection in canines as the overall number of host animals having or carrying RMSF is reduced. The administration of the immunogenic composition or vaccine to canines reduces the transmission of RMSF from RMSF-carrying ticks to canines and stops the retransmission from canines that would have been infected by the RMSF-carrying ticks to subsequent ticks that feed on the canines. Without the administration, more canines would be infected, which would lead to more ticks being infected, which then poses an increased risk to the human population.

[0034] The one or more inactivated avirulent tick-bome pathogen strains of the present disclosure may be inactivated using any method known to prevent the bacteria from replicating in a host. Inactivation may be accomplished by a method selected from, but not limited to, heat inactivation, formalin inactivation, or any method used to provide a killed bacteria.

[0035] The one or more inactivated tick-bome pathogens of the present disclosure may be any whole cell avirulent strain of Rickettsia rickettsii. Preferred strains of R. rickettsia, include, but are not limited to the Iowa strain.

[0036] In a preferred aspect, the one or more R.rickettsii have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity or homology with the Iowa strain of R.rickettsii. Further, in an embodiment where more than one avirulent strain of R.rickettsii is present, the immunogenic composition or vaccine of the present disclosure may include more than one strain within a single composition.

[0037] The immunogenic composition or vaccine of the present disclosure preferably includes an adjuvant. The adjuvant for purposes of the present disclosure may be selected from any adjuvant suitable for use in canines. One preferred adjuvant is Quil A.

[0038] The immunogenic composition or vaccine of the present invention may be administered in any dose necessary to provide an immune response in or to a canine or human. Their administration modes, dosages and optimum pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to an animal such as, for example, the age or the weight, the seriousness of its general condition, the tolerance to the treatment and the secondary’ effects noted. Preferably, the vaccine of the present disclosure is administered in an amount that is protective against RMSF or infection by Rickettsia rickettsii.

[0039] According to a further embodiment, the immunogenic composition or vaccine is administered to canines or humans in one or two doses at an interval of about 2 to 4 weeks. For example, the first administration is performed when the animal or human is about 2 to 3 weeks to about 8 weeks of age. The second administration is performed about 1 to about 4 weeks after the first administration of the first vaccination. According to a further embodiment, revaccination is performed in an interval of 3 to 12 months after administration of the second dose. Administration of subsequent vaccinedoses is preferably done on a 6 month to an annual basis. In another preferred embodiment, animals vaccinated before the age of about 2 to 3 weeks should be revaccinated. Administration of subsequent vaccine doses is preferably done on an annual basis. In an alternate embodiment, the immunogenic composition or vaccine of the present disclosure is effective after a single dose administration, therefore, in such an embodiment, the immunogenic composition or vaccine would only need to be administered a single time.

[0040] In a further embodiment, the immunogenic composition of the present disclosure is administered in an amount of from about 102to about 109TCID50 per dose, preferably about IO3to about 108TCID50 per dose, more preferably, about 104to about 108TCID50 per dose, where values such aslO2TCID50 per dose, 103TCID50 per dose, 104TCID50 per dose, IO5TCID50 per dose, 106TCID50 per dose, 107TCID50 per dose, 108TCID50 per dose, and IO9TCID50 per dose are envisioned.

[0041] In another embodiment, the immunogenic composition of the present disclosure includes at least 103bacteria per administration or per dose, more preferably, 103- 1012bacteria per dose, still more preferably IO4- 1010bacteria per dose, even more preferably 105- 108bacteria per dose, and still more preferably 106- 107bacteria per dose.

[0042] These compounds can be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal or subcutaneous route, or by the oral route. In a more preferred manner, the immunogenic composition or vaccine according to the disclosure will be administered by the intravenous route.

[0043] Their administration modes, dosages and optimum pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to an animal such as, for example, the age or the weight, the seriousness of its general condition, the tolerance to the treatment and the secondary effects noted. Preferably, the vaccine of the present disclosure is administered in an amount that is protective against RMSF or against signs or symptoms of infection from Rickettsia rickettsii.

[0044] An “immunogenic composition” or “vaccine” refers to a composition of matter that comprises at least one antigen which elicits an immunological response in the host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or yd T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity' of the disease reduced. Such protection will be demonstrated by either a reduction in the severity or prevalence of. up to and including a lack of symptoms normally displayed by an infected host, a quicker recovery time and / or a lowered viral titer in the infected host in comparison to an animal receiving the same challenge but not having any administration of the immunogenic composition or vaccine.

[0045] “Incidence” in clinical signs or symptoms can refer to either the overall number of clinical signs or symptoms present in an individual animal or can refer to the relative number of animals in a group of animals that exhibit clinical signs or symptoms.

[0046] “Adjuvants” as used herein, can include montanide, Freund’s incomplete adjuvant, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane or squalene oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyd group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri- (caprylate / caprate) or propylene glycol dioleate; esters of branched fatty7acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0047] For example, it is possible to use the SPT emulsion described on page 147 of '‘Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

[0048] A further instance of an adjuvant is a compound chosen from the polymers of acry lic or methacry lic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U. S. Patent No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol ; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 97 IP. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

[0049] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

[0050] Preferably, the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 150 pg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 200 pg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 250 pg per dose.

[0051] Additionally, the composition can include one or more pharmaceutical-acceptable or veterinary-acceptable carriers. As used herein, “a pharmaceutical-acceptable carrier’’ or "‘veterinary-acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.

[0052] The compositions and methods of the present disclosure can also comprise the addition of any stabilizing agent, such as for example saccharides, trehalose, mannitol, saccharose and the like, to increase and / or maintain product shelflife and / or to enhance stability.

[0053] “Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position- by-position basis, e g., the sequences are “identical” at a particular position if at thatposition, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press. New York (1993); Computer Analysis of Sequence Data. Part I. Griffin, A M., and Griffin, H. G., eds, Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D.. SIAM J. Applied Math.. 48: 1073 (1988). the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al, J. Molec. Biol, 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al, NCVI NLM NIH Bethesda, MD 20894, Altschul. S. F. et al, J. Molec. Biol, 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of thenucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%. preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity7to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity7.

[0054] "‘Sequence homology”, as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology7, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity ”, conserv ative amino acid substitutions are counted as a match when determining sequence homology7. In other words, to obtain apolypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homologous sequence comprises at least a stretch of 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.In some forms, the avirulent strain used in the immunological composition or vaccine will have at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9, or even 100% sequence homology or identity with any publicly available Iowa strain.

[0055] A “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

[0056] Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.

[0057] Those of skill in the art will understand that the composition herein may incorporate known injectable, physiologically acceptable, sterile solutions. For preparing a ready -to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions are readily available. In addition, the immunogenic and vaccine compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents caninclude sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others. Suitable adjuvants, are those described above.

[0058] In one aspect of the present invention, the immunogenic composition or vaccine includes at least one immunological active component from one or more disease-causing organism in canines or humans in addition to Rickettsia rickettsii. Preferably the other disease-causing organism in canine is selected from the group consisting of: rabies, canine parvovirus, canine coronavirus, canine distemper, canine influenza, infectious canine hepatitis, canine herpesvirus, pseudorabies, canine minute virus, brucellosis, leptospirosis, spirochaete, Borrelia burgdorferi, Rhipicephalus sanguineus, Clostridium perfringens, Clostridium difficile, Bordetella bronchiseptica, Blastomycosis dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neofromans, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Sporothris schenckii, Aspergillus fumigatus, Phythium insidiosum, Mucomycosis, or any combination thereof.

[0059] An “immunological active component” as used herein means a component that induces or stimulates the immune response in an animal to which said component is administered. According to a preferred embodiment, said immune response is directed to said component or to a microorganism comprising said component. According to a further preferred embodiment, the immunological active component is an attenuated microorganism, including modified live bacteria, a killed- microorganism or at least an immunological active part of a microorganism.

[0060] Beyond protecting dogs, the vaccine described herein has significant public health implications. By preventing clinical disease and reducing the bacterial load in vaccinated dogs, the described vaccine also reduces the probability of ticks acquiring the R. rickettsii pathogen from an infected canine individual or population. Intermption of the transmission cycle is important because the RMSF pathogen is readily transmitted from dogs to people via infected ticks. Furthermore, direct exposure to an infectious lesion on a dog can also pose a risk to humans. Therefore, widespread vaccination of dogs would significantly reduce the RMSF disease infection and diseaseburden risk in humans, functioning in a manner analogous to canine Rabies vaccination programs, which have proven highly successful in reducing the risk of human infection.

[0061] It must be understood that the present disclosure does not relate to the genomic nucleotide sequences taken in their natural environment, that is to say in the natural state. It concerns sequences which it has been possible to isolate, purify or partially purify, starting from separation methods such as, for example, ion-exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively fractionation techniques based on solubility7in different solvents, or starting from methods of genetic engineering such as amplification, cloning and subcloning, it being possible for the sequences of the disclosure to be carried by vectors.

[0062] Complementary7nucleotide sequence of a sequence of the disclosure is understood as meaning any DNA whose nucleotides are complementary7to those of the sequence of the disclosure, and whose orientation is reversed (antiparallel sequence).

[0063] All ranges provided herein include each and every7value in the range as well as all sub-ranges there-in-between as if each such value or sub-range was disclosed. Further, all aspects and embodiments of the disclosure comprise, consist essentially of, or consist of any aspect or embodiment, or combination of aspects and embodiments disclosed herein.

[0064] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.EXAMPLES

[0065] Materials and Methods

[0066] Propagation of R. rickettsii in Vero cells for preparation of stocks: R. rickettsii (Sheila Smith strain) was grown in Vero (African green monkey kidney) cells (clone E6: ATCC CRL-1586) as previously described (Rydkina, E., D.J. Silverman, and S.K. Sahni, (2005), Ammerman, N.C., M. Beier-Sexton, and A.F. Azad, (2008)). Briefly, confluent monolayers of Vero cells grown in Dulbecco’s modified Eagle’s medium DMEM supplemented with 2% fetal bovine serum and 2 mM L-glutamine, were infected w ith R. rickettsii at a multiplicity of infection (MOI) of 0. 1 and incubated at 35°C incubator set at 5% CO2 until 50% of the monolayer was disrupted due to infection. The rickettsial stocks were prepared by differential centrifugation of lysates from infected cells (Rydkina, E., D.J. Silverman, and S.K. Sahni, (2005)), suspended in K-36 buffer (0. 1 M potassium chloride, 0.015 M sodium chloride, 0.05 M potassium phosphate buffer (pH 7.0)), and the numbers of viable R. rickettsii organisms were determined by plaque titration assay (Rydkina. E., L.C. Turpin, and S.K. Sahni, (2010), Sahni, S.K., D.J. Van Antwerp, M.E. Eremeeva, et al., (1998)).

[0067] Whole-cell antigen preparation for WCA vaccine: The WCA was prepared using Iowa strain bacteria (Rocky Mountain National Labs. Hamilton, Montana) by incubating the organisms in a 56°C water bath for 30 min, with mixing once every 10 min. The protein concentration w as estimated according to the Bradford protein method (Bio- Rad, Hercules, CA). In the primary' and booster vaccinations, 70 pg each of the WCA in a formulation containing 250 pg Quil A was given to all vaccinated dogs. Unvaccinated dogs received only Quil A injections. All vaccines were administered subcutaneously. Infection challenges were performed with approximate doses of 106viable bacteria per dog injected intravenously.

[0068] Experimental infections in dogs: Experiments with dogs complied with the Public Health Service (PHS) Policy on the Humane Care and Use of Laboratory' Animals, the US Department of Agriculture’s (USDA) Animal Welfare Act & Regulations (9CFR Chapter 1, 2.31). Dogs were provided a commercially available dry dog food and water ad libitum and were also provided adequate space allowing them to freely move about for regular exercise activity. All groups of dogs independentof vaccinated or not, or infected or non-infected controls were monitored daily for health and behavioral changes and twice weekly for body temperature and hematological changes. Dogs were infected with R. rickettsii Sheila Smith strain, Morgan strain, or Iowa strain. Dogs were monitored daily for fever, and clinical signs of the disease. Body weights were also measured.

[0069] Evaluated vaccines were prepared with heterologous strains for defining protection. The adjuvant used was 250 pg / dose of Quil A. Five vaccinated groups (n=5) were included in the study: one group vaccinated with a virulent WCA Sheila Smith strain and challenged with a Morgan strain; one group vaccinated with a virulent Morgan strain and challenged with a Morgan strain; one group vaccinated with a virulent Morgan strain and challenged with a Sheila Smith strain; one group vaccinated with an avirulent Iowa strain and challenged with a Morgan strain; and one group vaccinated with an avirulent Iowa strain and challenged with a Sheila Smith strain. Three unvaccinated groups (n=5) were included in the study: one challenged with a Morgan strain; one challenged with a Sheila Smith strain; and one challenged with an inactivated avirulent Iowa strain. Groups were provided a primary vaccination on day 0 followed by a boost on day 21. The infection challenge took place 42 days after vaccination. For a subsequent 40 days, each group was monitored for various measures of vaccine protection.

[0070] Rickettsial bacterial presence assessed by culture recovery: Whole blood samples from dogs collected frequently into sterile blood collection tubes. The blood samples were used to culture recover the presence of bacteria in V ero cell cultures as described above; the analysis was performed as a blind study where the person performing the culture recovery7experiments did not know the dogs receiving or not receiving the vaccine. Similarly, the information pertaining to infections with avirulent and virulent strains was kept confidential from the person conducting the experiment until all data analysis was completed.

[0071] Enzy me linked immune-sorbent assays (ELISAs) for R. rickettsii specific IgG: The ELISAs were performed using R. rickettsii inactivated whole cell antigens. Serum samples from all dogs collected prior to infection and several daysfollowing vaccinations and infection challenges were assessed by ELISA for the presence of the R. rickettsii-specific IgG antibodies. Briefly, the 96-well Immulon 2HB ELISA plates were coated with the inactivated whole cell R. rickettsii antigen at a concentration of 20 ng / well prepared in 50 mM sodium carbonate buffer, pH 9.6. Serum samples were diluted 1 :50 in PBS, added to triplicate antigen-coated wells and incubated for 2 h at room temperature. The wells were then washed thrice with PBS containing 0.05% Tween 20 (PBST) and incubated with HRP -conjugated goat anti-dog total IgG at a dilution of 1:40,000. Unbound secondary antibodies were removed by washing with PBST three times, and the specific interactions were assessed by color development using TMB (3, 30, 5, 50-tetramethyl benzidine) as the substrate.

[0072] ELISA for canine IFNy: Peripheral blood mononuclear cells (PBMC) were collected on the indicated days before or after RMSF pathogen infection challenges. Cells were isolated by density centrifugation from buffy coat fractions of peripheral blood collected into 2x acid citrate dextrose. Cells were washed and resuspended in complete RPMI composed of RPML1640 (Gibco, Carlsbad, CA) supplemented with 2 mM L-glutamine, 25 mM HEPES buffer, 1% antibiotic- antimycotic solution, 50 mg / mL gentamicin sulfate, 1% nonessential amino acids, 2% essential amino acids, 1% sodium pyruvate, 50 pM 2-mercaptoethanol. and 10% (v / v) fetal bovine serum. Cells were cultured at 37°C with 4xl05cells / well in 96-well plates and were stimulated with 10 pg / mL whole cell antigen. As a positive control, cells were stimulated with 5 pg / mL Concanavalin A (Sigma-Aldrich). Negative control wells remained unstimulated. PBMC culture supernatants were collected after 5 days of stimulation and the presence of canine IFNy in the supernatants was assessed by commercial ELISA kit per manufacturer’s instructions.

[0073] Histopathology analysis: Selected tissues, including cerebrum, cerebellum, brainstem, lung, liver, testicle and epididymis, were fixed in 10% neutral buffered formalin and processed routinely with hematoxylin and eosin at 4 pm sections prepared. All slides were then reviewed by two pathologists where they did not know sample assignments during the analysis (a blind study). A comprehensive numeric score chart was developed to assign grades ranging from 1 to 5 to define the low-grade severity to high-grade severity and distribution of inflammation in all organs examined.The overall predominant cell types were also assessed in each inflammatory foci for all organs assessed to distinguish perivascular from non-perivascular inflammations. After completing the analysis, mean values were calculated for all tissues of each animal and the animals’ identities were assigned to the respective experimental groups. Subsequently, organ-based inflammatory7assessment scores were generated based on the average values for each animal group.

[0074] RESULTS

[0075] FIGS. 1A and IB shows cellular immune responses (antigen-specific proliferation) in vaccinated and control dogs on day 0 (prior to infection). In each group, the first of three bars in the graph represents the Iowa strain; the second of three bars represents the Morgan strain; the third of three bars represents the Sheila Smith strain. Four out of 5 groups had detectable RMSF-specific CD4 T cell responses compared to nonvaccinated controls. Although there is some variability, there is cross reactivity in the cellular response between all three RMSF strains (i.e. dogs immunized with M mount a proliferative response to IA antigens). Group 5 (IA immunized dogs) have the least amount of cross reactivity and do not differ from controls before infection. *p<0.05, compared to nonvaccinated controls (Groups 6-8) by 2-way ANOVA. Negative control (mock) values were subtracted. Values represent change over mock.

[0076] FIGS. 2A and 2B show7cellular immune responses (antigen-specific proliferation) in vaccinated and control dogs on day 7 after infection. Overall proliferative responses are lower for this week - I suspect this is due to the infection, and background proliferation was higher than preinfection samples. 3 out of 5 groups had detectable RMSF-specific CD4 T cell responses compared to nonvaccinated controls. CD8 T cell responses were low and did not differ across treatment groups. As with the preinfection samples, there is some cross reactivity to antigens from all three strains. *p<0.05, compared to nonvaccinated controls (Groups 6-8) by 2-way ANOVA. Negative control (mock) values were subtracted. Values represent change over mock.

[0077] FIGS. 3A provides data on average temperatures of dogs in groups 4. 5, 6, 7 and 8 (unvaccinated control groups) measured daily from zero to seven dayspost-infection. FIG. 3A shows that significant fevers were observed in the control groups receiving Sheila Smith and Morgan strain infections (groups 6 and 7, respectively), but not in the control group receiving the Iowa strain (group 8). FIG. 3B compares the temperature data from Iowa control group 8 to temperature data from groups 4 and 5, which were vaccinated with Iowa and infected with Morgan and Sheila Smith, respectively. Groups 4 and 5, vaccinated with the WCA Iowa vaccine, also did not manifest fevers over the period shown.

[0078] Weight changes w ere also measured in groups 4, 5, 6, 7 and 8. Results are provided in Table 1 below. Weight loss was observed in unvaccinated groups 6 and 7 particularly, but no significant weight loss was observed in unvaccinated group 8. which was challenged with the Iowa strain. Groups 4 and 5, which were vaccinated with the WCA Iowa vaccine, showed no weight loss. Instead, group 4, which was infected with Sheila Smith, maintained an essentially even weight, while group 5, which was infected with Morgan, showed slight weight gain.Table 1 — Weight Changes

[0079] Infection culture positives were also observed only with unvaccinated dogs that had been infected with virulent strains, as show in Table 2 below. All vaccinated groups including those vaccinated with the avirulent Iowa strain tested negative for the presence of bacteria in the blood.Table 2 - Infection Culture Positives

[0080] Severe disease including fatalities was observed in the unvaccinated groups, but not in the groups that were vaccinated, whether they were vaccinated withthe virulent Sheila Smith or Morgan strains or with the avirulent Iowa strain. As shown in Table 3 below, fatalities were only observed in unvaccinated control groups infected with either Sheila Smith or Morgan.Table 3 — Severe Disease Fatalities

[0081] As show n in Table 4 below, the Morgan and Sheila smith control dogs also suffered fatalities due to severe disease, while the vaccinated Morgan and Sheila smith dogs did not.Table 4 - Fatalities in Unvaccinated Control Dogs

[0082] In addition to preventing serious infection, including significant weight or temperature changes, the Iowa WCA vaccine prevented visible clinical signs in all Iowa vaccine groups that were infection challenged with Sheila Smith or Morgan strains. A visual comparison is provided in FIGS. 4A and 4B. FIG. 4A shows a control dog 3 days after infection with a Sheila Smith strain, showing visible clinical signs of disease on the ear. By contrast, the ear of the dog in FIG. 4B. which was vaccinated with the Iowa WCA vaccine, shows no visible clinical signs 3 days post-infection.

[0083] Measurements of IgG response in groups vaccinated with the WCA Sheila Smith vaccine, the WCA Morgan vaccine, and the WCA Iowa vaccine are provided in FIGS. 5A, 5B, and 5C, respectively. In each of the provided plots, primary vaccination occurred on day 0, a booster was provided on day 21, and the challenge period began on day 42. Each plot contains six data series: three series representing vaccinated groups challenged by either the Sheila Smith strain, the Morgan strain, orthe Iowa strain, and three series representing control groups challenged by either the Sheila Smith strain, the Morgan strain, or the Iowa strain.

[0084] FIG. 5A shows that all unvaccinated control groups show no significant IgG response until after the start of the challenge period. However, a relatively low-level IgG response is shown in groups vaccinated with the WCA Sheila Smith vaccine in the 20 days after primary vaccination, followed by a rise in IgG levels after the day 21 booster and a peak at 28 days. By day 45, IgG levels have declined to close to their pre-boost levels, although they remain higher than in the unvaccinated control groups.

[0085] FIG. 5B shows that all unvaccinated control groups show no significant IgG response until after the start of the challenge period. However, a relatively low-level IgG response is shown in groups vaccinated with the WCA Morgan vaccine in the 20 days after primary vaccination, followed by a rise in IgG levels after the day 21 booster and a peak at 28 days. IgG levels maintain a relative plateau afterday 36, although they remain higher than in the unvaccinated control groups.

[0086] FIG. 5C shows that all unvaccinated control groups show no significant IgG response until after the start of the challenge period. However, an IgG response is shown in groups vaccinated with the WCA Iowa vaccine in the 20 days after primary vaccination that is surprisingly higher than that seen in either the WCA Sheila Smith or WCA Morgan vaccinated groups. Vaccination with the WCA Iowa vaccine appears to produce a higher immediate IgG response than either Sheila Smith or Morgan despite Iowa being an avirulent strain. Furthermore, while groups vaccinated with the WCA Iowa vaccine also show an IgG peak at day 28, IgG levels are maintained at a relatively high level throughout the monitoring period and are superior to those measured in the control groups.

[0087] FIGS. 6A-6G provide microscopy photos comparing various bodily tissues across control groups. FIG. 6A shows microscopy images of the thymus. FIG. 6B shows microscopy images of the testes, FIG. 6C shows microscopy images of the lungs, FIG. 6D shows microscopy images of the liver, FIG. 6E shows microscopyimages of the kidneys, FIG. 6F shows microscopy images of the heart, and FIG. 6G shows microscopy images of the brain. In each of FIGS. 6A-6G, an image is provided of the corresponding tissue from an unvaccinated dog infected with the avirulent Iowa strain, another image is provided from an unvaccinated dog infected with a virulent strain, and a third image is provided from a dog vaccinated with the WC A Iowa vaccine and subsequently challenged with a virulent strain. In all of FIGS. 6A-6G, images corresponding to a dog vaccinated with the WCA Iowa vaccine comprising an avirulent inactivated Iowa strain and subsequently challenged with a virulent strain show grater similarity to images taken from an unvaccinated dog infected with the avirulent Iowa strain. By contrast, in each of FIGS. 6A-6G, images taken from unvaccinated dogs challenged by virulent strains show marked changes indicative of disease, including pathological lesions.

[0088] DISCUSSION

[0089] Rocky Mountain spotted fever is a life-threatening disease in dogs and people resulting from tick-home infections with Rickettsia rickettsii (a bacterial pathogen). If untreated, the disease can cause high fatalities ranging from 40-60% in parts of the USA, Mexico, and many countries in central and south Americas. While the disease caused by highly virulent strains of the pathogen, an avirulent strain highly homologous to Rickettsia rickettsii virulent strains also exist in nature. Namely, the ‘Iowa’ strain of Rickettsia rickettsii is previously identified as nonpathogenic in guinea big model infection studies. To develop a safe and efficacious vaccine, we tested the Iowa strain for its infectivity in dogs. Secondly, we performed vaccine studies using a whole cell inactivated antigens prepared from the Iowa strain in the vaccine formulation and tested its ability to prevent the classical RMSF disease in dogs caused by two different virulent strains: Sheila Smith and Morgan. The Iowa strain vaccine was prepared by heat inactivating the culture derived purified bacteria and mixing with Quil A as the adjuvant.

[0090] Seventy micrograms (pg) of vaccine in a formulation containing 250 pg of Quil A each was administered on day zero, then boosted similarly on day 21, and subsequently on day 42, all vaccinated dogs (n=5 per group) and unvaccinated dogs(n=5 per group) were challenged with virulent strains; Sheila Smith or Morgan. Similarly, the three unvaccinated groups were challenged with Sheila Smith, Morgan or Iowa strain. All dogs were then followed for the disease progression for 40 days. Severe form of the disease was observed when dogs did not receive the vaccine and challenged with both the virulent strains; the parameters of the disease included high fever, visible clinical signs, multiple severe disease observations leading to 60% of the dogs requiring euthanasia in less than two weeks. These animals also tested positive for the bacteria by culture recovery method and exhibited pathological lesions in multiple organs. Contrary7to these observations, all avirulent Iowa strain infected dogs stayed healthy and so did not develop any of the clinical, hematological or pathological signs. These results demonstrated that the avirulent Iowa strain infections are safe in dogs as the infection did not result in inducing the classical RMSF disease.

[0001] Dogs receiving the Iowa strain-derived vaccine and challenged with either one of the virulent strains also stayed healthy ad they did not develop any signs of the clinical disease and remained completely free of the bacterial presence assessed by culture recovery7, similar to unvaccinated dogs receiving Iowa strain infection.

[0002] WCA vaccines to prevent RMSF that are prepared from virulent strains of R. rickettsii pose a potential nsk, particularly if antigen preparation is not performed properly. By contrast, preparing a R. rickettsii WCA vaccine using avirulent strains, particularly the Iowa strain, obviate this risk because the Iowa strain poses no risk of causing disease in the patient. Surprisingly, the Iowa-based WCA vaccine demonstrated and described above provided 100% protection against virulent strains Sheila Smith and Morgan, which are both geographically and genetically distinct and are known to cause high fatalities, as seen in the demonstrate fatality7rates of 60% described above. Accordingly, an avirulent-strain-based WCA vaccine such as the Iowa-based WCA vaccine advantageously provides protection against other virulent strains.

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition comprising: an inactivated whole cell bacteria selected from the group consisting of an avirulent strain of Rickettsia rickettsir. and an adjuvant.

2. The composition of claim 1, wherein the avirulent strain of Rickettsia rickettsii is the Iowa strain.

3. The composition of claim 1, wherein the adjuvant is Quil A.

4. The composition of claim 1, wherein the adjuvant is included in an amount between about 100 pg to about 10 mg per dose.

5. The composition of claim 1, wherein the inactivated whole cell bacteria comprises at least 103bacteria per dose.

6. The composition of claim 1, further comprising an additional component selected from the group consisting of veterinary-acceptable carriers, solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, compositions know n to increase immunity, and any combination thereof.

7. The composition of claim 1, further comprising at least one antigen from a diseasecausing organism in canine selected from the group consisting of: rabies, canine parvovirus, canine coronavirus, canine distemper, canine influenza, infectious canine hepatitis, canine herpesvirus, pseudorabies, canine minute virus, brucellosis, leptospirosis, spirochaete, Borrelia burgdorferi, Rhipicephalus sanguineus, Clostridium perfringens, Clostridium difficile, Bordetella bronchiseptica, Blastomycosis dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neofromans, Microsporum canis, Microsporum gypseum. Trichophyton mentagrophytes, Sporothris schenckii, Aspergillus fumigatus, Phythium insidiosum, Mucomycosis, or any combination thereof.

8. A method of reducing the incidence of or severity of at least one clinical sign or symptom of infection by Rickettsia rickettsii comprising the step of administering at least one dose of an immunogenic composition or vaccine comprising one or more inactivated whole cell bacteria selected from the group consisting of an avirluent Rickettsia rickettsii together with an adjuvant to an animal in need thereof.

9. The method of claim 8. wherein the avirulent Rickettsia rickettsii is the Iowa strain.

10. The method of claim 8, wherein the administration is provided by a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous route, or oral.

11. The method of claim 8, wherein the at least one clinical symptom or clinical sign is caused by infection by R. rickettsia and is selected from the group consisting of fever, headache, nausea, vomiting, muscle pain, lack of appetite, rash, and death.

12. The method of claim 8, wherein the incidence of clinical signs or clinical symptoms is reduced by at least 10% in comparison to an animal that has been subjected to the same challenge conditions but did not receive an administration of the immunogenic composition or vaccine.

13. The method of claim 8, wherein the adjuvant is Quil A.

14. The method of claim 8, wherein the adjuvant is included in an amount between about 100 pg to about 10 mg per dose.

15. The method of claim 8, wherein the inactivated whole cell bacteria comprises at least 103bacteria per dose.

16. The method of claim 8, wherein the immunogenic composition or vaccine further comprises an additional component selected from the group consisting of veterinary- acceptable carriers, solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, compositions known to increase immunity, and any combination thereof.

17. The method of claim 8, wherein the immunogenic composition or vaccine further comprises at least one antigen from a disease-causing organism in canine selected from the group consisting of: rabies, canine parvovirus, canine coronavirus, canine distemper, canine influenza, infectious canine hepatitis, canine herpesvirus, pseudorabies, canine minute virus, brucellosis, leptospirosis, spirochaete, Borrelia burgdorferi, Rhipicephalus sanguineus, Clostridium perfringens, Clostridium difficile, Bordetella bronchiseptica, Blastomycosis dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neofromans, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Sporothris schenckii, Aspergillus fumigatus, Phythium insidiosum, Mucomycosis, or any combination thereof.

18. The method of claim 8, wherein at least one administration occurs when the animal is between 2 and 8 weeks of age.

19. The method of claim 8, wherein more than one administration is provided and there is between 2 and 4 weeks between administrations.

20. A method of reducing the bacterial load of at least one tick-bome pathogen of the disclosure in a canine or human, wherein the method comprises the step of administering an immunogenic composition or vaccine comprising an avirulent strain of Rickettsia rickettsii, and an adjuvant to a canine or human.

21. The method of claim 20, wherein the avirulent strain of Rickettsia rickettsii is the Iowa strain.

22. The method of claim 20. wherein the administration is provided by a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous route, or oral.

23. The method of claim 20, wherein the adjuvant is Quil A.

24. The method of claim 20, wherein the adjuvant is included in an amount between about 100 pg to about 10 mg per dose.

25. The method of claim 20, wherein the inactivated whole cell bacteria comprises at least 103bacteria per dose.

26. The method of claim 20, wherein the immunogenic composition or vaccine further comprises an additional component selected from the group consisting of veterinary - acceptable carriers, solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, compositions known to increase immunity, and any combination thereof.

27. The method of claim 20, wherein the immunogenic composition or vaccine further comprises at least one antigen from a disease-causing organism in canine selected from the group consisting of: rabies, canine parvovirus, canine coronavirus, canine distemper, canine influenza, infectious canine hepatitis, canine herpesvirus, pseudorabies, canine minute virus, brucellosis, leptospirosis, spirochaete, Borrelia burgdorferi, Rhipicephalus sanguineus, Clostridium perfringens, Clostridium difficile, Bor detell a bronchiseptica. Blastomycosis dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neofromans, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Sporothris schenckii, Aspergillus fumigatus, Phythium insidiosum, Mucomycosis, or any combination thereof.

28. The method of claim 20, wherein at least one administration occurs when the animal is between 2 and 8 weeks of age.

29. The method of claim 20, wherein more than one administration is provided and there is between 2 and 4 weeks between administrations.

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