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Wo 2010/096640 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 26 August 2010 (26.08.2010) WO 2010/096640 A2 (51) International Patent Classification: partment of health and human services [US/US]; Office C12N 1/20 (2006.0 1) C12N 1/38 (2006.0 1) of Technology Transfer, 601 1 Executive Blvd., Suite 325, Rockville, Maryland 20852-3804 (US). (21) International Application Number: PCT/US20 10/024706 (72) Inventors; and (75) Inventors/Applicants (for US only): HEINZEN, Robert (22) International Filing Date: [US/US]; 128 Tawney View, Hamilton, Montana 59840 19 February 2010 (19.02.2010) (US). OMSLAND, Anders [NO/US]; 120 Pintail Loop, (25) Filing Language: English #8, Hamilton, Montana 59840 (US). COCKRELL, Di¬ ane C. [US/US]; 458 Saddle Ridge Rd., Hamilton, Mon (26) Publication Language: English tana 59840 (US). HOWE, Dale [US/US]; 800 Hollibaugh (30) Priority Data: Rd., Stevensville, Montana 59870 (US). 61/154,330 20 February 2009 (20.02.2009) US (74) Agents: HINSCH, Matthew E. et al; Townsend and (71) Applicant (for all designated States except US): THE Townsend and Crew LLP, Two Embarcadero Center, 8th GOVERNMENT OF THE UNITED STATES OF Floor, San Francisco, California 941 11-3834 (US). AMERICA as represented by the secretary of the de¬ [Continued on next page] (54) Title: METHOD AND COMPOSITIONS FOR HOST CELL-FREE GROWTH OF PATHOGENS (57) Abstract: The disclosure provides compositions comprising Coxiella burnetii substantially free of eukaryotic host cells, and methods of making and using such compositions. FIG. 1 pH 4 5 pH 7 0 FIG. 1B (81) Designated States (unless otherwise indicated, for every GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, kind of national protection available): AE, AG, AL, AM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ML, MR, NE, SN, TD, TG). ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, Published: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, — without international search report and to be republished TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. upon receipt of that report (Rule 48.2(g)) (84) Designated States (unless otherwise indicated, for every — with sequence listing part of description (Rule 5.2(a)) kind of regional protection available): ARIPO (BW, GH, METHOD AND COMPOSITIONS FOR HOST CELL-FREE GROWTH OFPATHOGENS CROSS-REFERENCES TO RELATED APPLICATIONS [0001] The present application claims benefit of priority to US Provisional Patent Application No. 61,154,330, filed February 20, 2009, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] The work performed during the development of this disclosure utilized intramural support from the National Institutes of Health. The United States government has certain rights in the disclosure. BACKGROUND OF THE INVENTION [0003] Coxiella burnetii is an obligate intracellular bacterial pathogen that causes human Q (Query) fever, a debilitating flu-like illness. Q fever is a zoonotic disease and farm animals, pets, and rodents are the primary reservoirs for C. burnetii. Typically humans are exposed to Q fever by the inhalation of the bacterium deposited with animal waste such as urine, feces, and placental tissue and fluid. C. burnetii is highly resistant to environmental conditions; therefore, it persists in soil for a long period of time. C. burnetii has a cell wall similar to gram negative bacteria and is characterized by a phase transition. Phase 1 bacteria are isolated form infected humans and animals and has a smooth hydrophilic lipopolysaccharide (LPS) surface structure. Phase II bacteria lose the smooth LPS structure upon culture in tissue culture or embryonated eggs to truncated rough LPS molecules exhibiting a hydrophobic surface. [0004] Q fever causes acute and chronic illness and is a reportable disease in the U.S. C. burnetii can establish a persistent latent infection that can reactivate to cause chronic disease. Q fever causes a high fever and around 50% of those who become symptomatically infected develop pneumom a. Abnormal liver function and hepatitis can be a complication of acute infection. Additionally, a majority of those infected have abnormal liver function and some develop hepatitis. It is believed that as many as 50% of those infected are asymptomatic. A small percentage of those infected develop chronic Q fever that can be fatal in as many as 5% of cases. There is no vaccine approved in the U.S. for Q fever. [0005] The pathogen is a recognized category B biothreat with potential for illegitimate use due to low infectious dose, stability in the environment, and an aerosol route of transmission. Outbreaks of Q fever occur worldwide and the disease is primarily associated with the presence of infected farm animals. Outbreaks occur in agricultural areas and in urban areas. Outbreaks in urban areas have been linked to windborne Coxiella burnetii. The epidemiology of Q fever is diverse and the disease does not discriminate between developed and developing countries. [0006] Methods for culturing this pathogen have required the presence of a eukaryotic host cell. Common culture methods include using fertilized eggs or mammalian cell culture. The inability to grow obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental and production constraints that have negatively impacted progress in understanding pathogen virulence and producing vaccine and diagnostic antigens free of host cell contaminants. BRIEF SUMMARY OF THE INVENTION [0007] Prior to this invention, there are no means for growing Coxiella burnetii extracellularly. A preparation of host-cell free Coxiella burnetii is useful, for example, for the development of a whole-cell Q fever vaccine that is free host-cell related impurities, for example, allergens from eggs, endogenous viruses, or other host-cells used to grow Coxiella burnetii. [0008] Current methods of growing and purifying C. burnetii are expensive, time consuming, and technically challenging. Moreover, mechanical breakage of host cells to release intracellular C. burnetii can result in hazardous, and highly infectious, aerosols. Bacteria prepared in this fashion are contaminated with host material that can be potentially harmful in a clinical setting. Contaminants can include egg material, which can cause allergic reactions in sensitized individuals, and/or endogenous viruses that are harbored by mammalian cell lines. Thus, host cell-free growth of C. burnetii will dramatically aid in the production of highly pure preparations of bacterial antigens for use in diagnostics and vaccines. [0009] We have developed a complex nutrient medium that supports substantial growth of C. burnetii in a 2.5% oxygen environment. Components of media that supports axenic C. burnetii, as well as axenic C. burnetii growth, include a high concentration of chloride (e.g., 70-280 mM), casamino acids, a L-cysteine, and citrate buffer (pH < 6.0). Complex Coxiella Medium (CCM) is described in Omsland et al. J. Bacteriol 190(9):3203-3212 (2008). Acidified Citrate Cysteine Media (ACCM) is described in Omsland et al., Proc. Natl. Acad. ScL USA 106(1 1):4430-4434 (2009). [0010] The present invention provides for a method of culturing Coxiella burnetii, optionally without an eukaryotic host cell. In some embodiments, the method comprises: a. culturing a sample containing the bacterial pathogen with a medium comprising casamino acids, L-cysteine, chloride ion, and citrate at a pH of 6 or less, under conditions of less than 10% oxygen and in the absence of any eukaryotic host cell; b. recovering the bacterial pathogen from the culture after a suitable incubation period. [0011] In some embodiments, the medium comprises peptone (e.g., neopeptone or another type of peptone). [0012] In some embodiments, the medium comprises methyl- β-cyclodextrin. In some embodiments, the medium comprises methyl-β-cyclodextrin and lacks peptone. [0013] In some embodiments, the chloride ion is present in the medium at a concentration ofat least l25mM. [0014] In some embodiments, the L-cysteine is present at least 1.0 mM. [0015] In some embodiments, the pH of the medium is between 4.5 to 5.5. [0016] In some embodiments, the concentration of oxygen is 1 to 5%. [0017] In some embodiments, the method further comprises isolating the Coxiella burnetii from the culture after an incubation period. In some embodiments, the incubation period is at least 3 days. [0018] In some embodiments, the method further comprises combining an immunogenically-effective amount of the isolated Coxiella burnetii with a physiologically acceptable excipient. [0019] In some embodiments, the method further comprises inactivating the Coxiella burnetii. [0020] The present invention also provides for Coxiella burnetii prepared by the method described above or elsewhere herein. [0021] The present invention also provides for a composition comprising Coxiella burnetii, substantially free of any eukaryotic host cell contaminants in a physiological acceptable medium. [0022] In some embodiments, the Coxiella burnetii is replicating. [0023] The present invention also provides for an immunogenic composition comprising an isolated Coxiella burnetii substantially free of any eukaryotic host cell in an admixture with a physiological excipient.
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