WO2010096640A2 - Method and compositions for host cell-free growth of pathogens - Google Patents

Abstract

The disclosure provides compositions comprising Coxiella burnetii substantially free of eukaryotic host cells, and methods of making and using such compositions.

Description

METHOD AND COMPOSITIONS FOR HOST CELL-FREE GROWTH

OF PATHOGENS

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(11):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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1. Supplemented CCM supports enhanced C. burnetii metabolic activity. Effects of casamino acids and L-cysteine on C. burnetii catabolic capacity were determined by incubating organisms in CCM or CCM supplemented with 2.5 mg/ml casamino acids, 1.5 raM L-cysteine or both (ACCM). Bacteria were preincubated in the respective media for 24 h, then labeled with [35S]Cys/Met in labeling buffer (pH 4.5) for 3 h. (A) De novo protein synthesis by C. burnetii was measured by quantification of radiolabel incorporation by scintillation counting. Results are expressed as fold increase in incorporation when compared incorporation of bacteria preincubated in CCM, then labeled in labeling buffer (pH 7.0) (negative control). Casamino acids and L-cysteine significantly improved C. burnetii metabolic activity. (B) SDS-PAGE and autoradiography confirmed incorporation of radiolabel into bacterial proteins. Values are mean ± SEM (n = 3). The level of radiolabel incorporation in CCM (pH 7.0) is normalized to 1.

[0025] Figure 2. The number of substrates oxidized by C. burnetii increases with decreasing oxygen availability. The ability of C. burnetii to oxidize substrates in different oxygen environments was assessed using Phenotype Microarrays (PM). (A) C. burnetii genes encoding terminal oxidases associated with aerobic (cytochrome o) and microaerobic (cytochrome bd) metabolism suggested C. burnetii can respire under microaerophilic conditions. (B) Purified C. burnetii was added to PM-I plates and incubated for 24 h in 20%, 5% and 2.5% oxygen. The number of metabolites oxidized increased with decreasing oxygen tension, consistent with microaerophilic metabolism. Representative PM plate images are shown. (C) Seventeen substrates were efficiently oxidized by C. burnetii in 2.5% oxygen. Signal intensities were measured using an OmniLog detection system and expressed as relative OmniLog units (OLU). Quantitative analysis is representative of at least 3 independent experiments. Substrate key (rows A-H, columns 1-12): Al - no substrate control, A5 - succinate, A8 - L-proline, Al 1 - D-mannose, B12 - L-glutamate, C2 - D- galactonic acid-7-lactone, C9 - α-D-glucose, D6 - α-ketoglutarate, El - L-glutamine, El 2 - adenosine, F5 - fumarate, F6 - bromo succinate, G4 - L-threonine, G5 - L-alanine, G9 - mono methyl succinate, H8 - pyruvate, H9 - L-galactonic acid-7-lactone, HI l - phenylethylamine.

[0026] Figure 3. ACCM supports axenic cell division of infectious C. burnetii under microaerobic conditions. (A) C. burnetii GE were assessed by QPCR daily for 6 d. Incubation in 20% (■) oxygen did not support C. burnetii replication while incubation in 2.5% (•) oxygen resulted in considerable C. burnetii replication. (B) Increases in C. burnetii GE during incubation in ACCM correlated with production of infectious bacteria as determined by a quantitative FFU assay. Values are mean ± SEM (n = 3). (C) Representative staining of FFUs contained in equal aliquots of ACCM harvested at 2, 4, and 6 d post inoculation. A magnified view of the inset in the 6 d post inoculation panel in also shown. Bars, 30 μm.

[0027] Figure 4. C. burnetii SCV to LCV development occurs in ACCM. To determine whether C. burnetii transitions between non-replicative SCV and replicative LCV developmental forms during incubation in ACCM, medium was inoculated with purified SCVs and TEM used to assess developmental transitions. (A and B) TEM showed the inoculum had ultrastructural characteristics of the SCV including small cell size (average diameter: 0.188 ± 0.0044 μm) and condensed chromatin. Organisms incubated in ACCM for 3 d exhibited ultrastructural characteristics of the LCV including increased cell size (average diameter: 0.456 ± 0.0078 μm) and dispersed chromatin. Following 6 d of incubation, a mixed population of SCVs and LCVs was observed, resulting in an overall reduction in cell size (average diameter: 0.290 ± 0.0087 μm). Values are mean ± SEM (n = 60 cells). Bar, 0.5 μm. (C) Immunoblot demonstrating the presence of ScvA (3.5 kDa), a SCV19 specific DNA- binding protein, only in the SCV inoculum and the LCV/SCV mixture present in stationary phase.

[0028] Figure 5. C. burnetii forms colonies on solid ACCM medium. C. burnetii was spread on a 23 1% ACCM-agarose base and covered with 0.25% ACCM-agarose. Colonies (0.05 - 0.1 mm) were present after a 14 d incubation in a 2.5% oxygen environment. A representative image is shown. [0029] Figure 6. C. burnetii SCVs can initiate growth in ACCM. ACCM was inoculated with purified C. burnetii SCVs and culture aliquots analyzed for increases in C. burnetii GE every 24 h for 6 d by QPCR. Values are mean ± SEM (n = 3). Cultures exhibited an approximate 3 log increase in GE over 6 days.

[0030] Figure 7. ACCM-2 supports substantial growth of C. burnetii. C. burnetii was used to inoculate ACCM-2 and growth of the organism in a micropaerobic environment (2.5% oxygen) measured by optical density every 24 h over 9 days. C. burnetii was cultivated with moderate shaking. A substantial increase in culture optical density was observed over time.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The disclosure provides compositions of isolated C. burnetii substantially free of a eukaryotic host cell as well as methods of culturing C. burnetii. .

[0032] In some embodiments, the disclosure provides a method of culturing C. burnetii without an eukaryotic host cell comprising: 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. The C. burnetii can be recovered from the after a suitable incubation period if desired. [0033] The disclosure also provides a culture medium for culturing C. burnetii or other microorganisms. In some embodiments, the disclosure provides a medium comprising a complex coxiella medium (CCM) as described in Omsland et al., J Bacteriol. 190:3203 (2008). CCM comprises the contents of Table 1 except it does not include L-cysteine or casamino acids. CCM provides for metabolic activity of C. burnetii for about 24 hours and provides for morphological differentiation. CCM has not been shown to provide for obvious growth of C. burnetii. Metabolic testing shows that C. burnetii metabolism is optimal in a citrate based buffer. The strong buffering capacity of citrate in the metabolically permissive pH range of 4 to 6 increases the utility of this buffer. Increased metabolic activity of C. burnetii was observed between pH 4.5-5.5. Table 1: Components of Acidified Citrate Cysteine Medium (ACCM) Ions Final Concentration ^% Nutrients Final Concentration

Na⁺ 190 Neopeptone 0.1 mg/ml

SO₄ ^" 0.06 FBS 1%

Fe₂ ⁺ 0.01 RPMI 1640 1/8X cr 140 Citrate 29

Ca⁺ 0.15 Casamino acids ^c 2.5 mg/ml

Mg₂ ⁺ 1.0 L-cysteine ^c 1.5

K⁺ 4.3

PO₄ ^' 4.5

NO₃ ^" 0.1

HCO₃ ^" 3.0 a Concentrations are in mM unless otherwise specified. b Excluding contribution from neopeptone and FBS. c Absent in CCM.

[0034] In some embodiments, the media provides for replication of C. burnetii. This can be achieved through inclusion of one, two, or all three of casamino acids, peptone (e.g., as described for ACCM), and methyl- β-cyclodextrin (e.g., as described for ACCM-2, for instance in Example 2).

[0035] hi some embodiments, the medium comprises peptone. Peptone provides for a high quality source of a spectrum of nutrients including nucleotides, peptides, vitamins, minerals, and amino acids. As noted in the examples, the inventors have successfully used neopeptone in their media. However, it is believed that other types of peptone (e.g., Bacto™ peptone, etc.) could also be used.

[0036] The inventors have found that inclusion of methyl-β-cyclodextrin in the medium can result in a 10-fold increase in C. burnetii growth. Moreover, in some embodiments, methyl-β-cyclodextrin is used in the absence of peptone. One advantage of eliminating peptone from the medium is that the resulting medium is completely chemically-defined, which under certain circumstances (e.g., vaccine development) can be advantageous. [0037] The presence of chloride ions independent of cation was demonstrated to provide for increased axenic metabolic activity. In some embodiments, about 125mM chloride ion is included in to the medium, hi some embodiments, preferably about 10OmM to IM chloride ion is included in the cell culture, hi an embodiment about 125 to about 15OmM chloride ion is included. Chloride ion may help to maintain cytoplasmic pH homeostasis, hi some embodiments, 1% or less of fetal bovine serum (FBS) is present. In other embodiments, FBS is not present.

[0038] A listing of ranges of concentrations the inventors have found to support C. burnetii are disclosed below and are applicable for media otherwise comprising the components of ACCM-I and ACCM-2:

14.5 - 58 mM citrate

1/16 - 1/4 dilution of RPMI cell culture medium

70 - 280 mM chloride (Cl^") ion

95 - 380 mM sodium (Na+) ion 2.15 - 8.6 mM potassium (K+) ion

0.5 - 2.0 mM magnesium (Mg++) ion

0.075 - 0.30 mM calcium (Ca+) ion

0.005 - 0.02 mM ferrous iron (Fe++) ion

1.25 - 5 mg/ml casamino acids 0.75 - 3.0 mM cysteine

0.03 - 0.12 mM sulfate (SO₄-)

0.5 to 2.0 mg/ml or more methyl- β-cyclodextrin (ACCM-2)

[0039] Accordingly, the present invention provides for media capable of supporting axenic C. burnetii growth comprising casamino acids, L-cysteine, chloride ion, and citrate, further comprising one or more of the above-described components, optionally at a pH of less than 6 and in an oxygen concentration of less than about 10%. While the above concentrations are provided as tested by the inventors, in some embodiments, one or more ingredient can be used at a concentration outside the above-listed ranges. Moreover, in some embodiments, the invention provides for a concentrated media intended for later dilution as needed. Concentrated media can be formulated in, e.g., at least 5X, 10X, 50X, 10OX, 100OX, for example. Concentrated media can be packaged for sale, for example, in media containers of one liter, 500 ml, 250 ml, or other volumes convenient for commerce.

[0040] When providing for cell growth, the medium further comprises L-cysteine and casamino acids. L-cysteine can act as an anti-oxidant and as a nutrient source. In some embodiments, L-cystiene is present in the medium at about 0.75 mM to about 3 mM, e.g., 1.OmM to about IM. hi an embodiment, L-cysteine is present at 1.5 mM. Casamino acids provide for an increase in metabolism of C. burnetii. Casamino acids are present in the medium at about 1 to 50 mg/ml, or about 1 to 10 mg/ml. hi an embodiment, a medium comprises the components of Table 1. This medium is described as Acidified Citrate Cysteine Medium (ACCM) and has a pH of about 4-6, or about 4.5 to 5.5. The medium can be either solid or liquid. When the medium is solid it further comprises agarose.

[0041] As noted above, in some embodiments, the medium is chemically-defined and comprises methyl-β-cyclodextrin, citrate, casamino acids, chloride ions, and L-cysteine, optionally in an oxygen concentration of less than about 10%. hi some embodiments, this medium has a pH of about 4-6, or about 4.5 to 5.5. The medium can be either solid or liquid. When the medium is solid it further comprises agarose. An example of this medium is ACCM-2. In some embodiments, the medium comprise the components of ACCM-2 as described in Example 2 and can optionally either include, or lack peptone.

[0042] Growth of C. burnetii is increased under an environment of low oxygen, hi some embodiments, the culture is incubated under conditions of 10% or less oxygen, 1 to 10% oxygen, or 1 to 5% oxygen. In an embodiment, C. burnetii was able to oxidize 10 to 17 substrates when the cultures were incubated at 2.5% and 5% oxygen. Substrates include those shown in Figure 2c. Cultures are grown for a suitable period of time depending on the organism, hi some embodiments, the cells are cultured for about 3 to 15 days, or 3-6 days. [0043] Growth of C. burnetii can be measured by determining growth of colonies or by measuring an increase in the genomes of the pathogens using methods known to those of skill in the art. hi an embodiment, the culture conditions provide for an increase in genomes of the C. burnetii of at least 1.5 to 5 logs or 2 to 3 logs.

[0044] hi some embodiments, the medium is sterile. Alternatively, in some embodiments, the medium of the invention comprises C. burnetii or another microorganism, hi some aspects, the disclosure also provides for compositions including C. burnetii, substantially free of any eukaryotic host cells, hi some embodiments, the compositions are also substantially free of eukaryotic host cell contaminants. Contaminants of eukaryotic host cells can include other eukaryotic proteins, eukaryotic nucleic acids, embryonated egg components, and eukaryotic viruses. "Substantially free" indicates that eukaryotic host cell or host cell contaminants are 2 % or less of the composition, e.g., less than 1%, 0.1%, 0.001%, or completely free of host cells or contaminants. [0045] In some embodiments, the medium comprises a replicating C. burnetii free of other host cells. This is in contrast to, for example, the media described in Omsland, et al, J. Bacteriol, 190(9):3203-3212 (2008), which described viable C. burnetii in media in the absence of host cells, but wherein obvious growth of the C. burnetii was not observed.

[0046] In some embodiments, the C. burnetii is isolated from the culture medium. In some embodiments, the isolated C. burnetii can be used to generate a vaccine (e.g., as an attenuated or killed vaccine). In some embodiments, the isolated C. burnetii is combined with a physiological acceptable excipient.

[0047] The isolated C. burnetii may be further fractioned into subunits including one or more of antigenic components. Antigenic components of C. burnetii may include O antigen, phase I LPS, Pl, P28, Com 1, and Cb-mip as described in Shannon et al, Immunological Res. 2008. Such components may be useful in immunogenic compositions such as may be used in vaccine compositions. Sequences of the genomes and components of the bacterial or protozoal pathogens described herein are available in publicly available databases such as Genbank. [0048] hi addition, the isolated bacterial or protozoal composition may be heat inactivated or extracted with a solvent in order to prevent the organism from further replication.

[0049] The isolated bacterial or protozoal composition may be passaged several times in host cells such as embryonated egg cells, primary macrophages or monocytes, macrophage or monocyte cell lines in order to attenuate the virulence of the bacteria. The attenuated strain may then be grown in the medium as described herein that is free of any eukaryotic cells.

[0050] Another aspect of the disclosure provides a method of preparing an immunogenic composition comprising: culturing a sample containing the C. burnetii with a medium as described herein (including but not limited to comprising casamino acids, L-cysteine, chloride ion, and citrate at a pH of 6 or less, under conditions of less than 10% oxygen) in the absence of any eukaryotic host cell; isolating the C. burnetii from the culture after a suitable incubation period; and combining an immunogenic effective amount of the isolated pathogen with a physiologically acceptable excipient. In embodiments, the method further comprises inactivating the C. burnetii, either before or after the combining step.

[0051] Immunologic compositions can comprise an isolated bacterial or protozoal pathogen substantially free of any eukaryotic host cell in admixture with a physiological excipient. In some embodiments, the excipient is one that provides for aerosol administration. Other modes of administration however are also contemplated. Immunologic compositions also may further comprise, for example, an adjuvant or a cytokine. Adjuvants useful in immunogenic compositions are known to those of skill in the art. Immunogenic compositions are useful to make therapeutic antibodies, antibodies for diagnostics and in vaccine formulations.

Example 1

[0052] Preparation of Acidified Citrate Cysteine medium (ACCM) for growth of C. burnetii without a eukaryotic host cell.

Materials and Methods Cultivation and purification of C. burnetii from Vero cells.

[0053] C. burnetii Nine Mile phase II (RSA439, clone 4) was propagated in African green monkey kidney (Vero) fibroblasts (CCL-81; American Type Culture Collection) grown in RPMI medium (Invitrogen Corp., CA) supplemented with 2% fetal bovine serum (FBS). At 7 d post infection, host cells were disrupted by sonication and C. burnetii purified by differential centrifugation as described (Shannon JG & Heinzen RA, Methods MoI Biol

431:189-200 (2008); Cockrell DC et al., J Microbiol Methods 72:321-325 (2008)). At this time point post infection, infected Vero cells contain roughly equal numbers of SCV and LCV morphological forms (Coleman SA et al., JBacteriol 186:7344-7352 (2004)). C. burnetii SCVs were generated by prolonged culture in Vero cells as previously reported (Coleman SA et al., JBacteriol 186:7344-7352 (2004)) and purified as described above. Purified bacteria were resuspended in K-36 buffer (0.05 M K2HPO4 and KH2PO4, 0.1 M KCl, 0.15 M NaCl, pH 7.0) (Weiss E, JBacteriol 90:243-253 (1965)) and stored at -80°C until further use. With the exception of experiments examining C. burnetii biphasic development (Figs. 4 and 6), all experiments utilized C. burnetii purified from Vero cells at 7 d post infection.

Transcription microarray analysis.

[0054] For analysis of C. burnetii transcript profiles during intracellular growth, Vero cells at 90-95% confluence in T-75 cell culture flasks were infected with C. burnetii at a multiplicity of infection of 100 for 2 h at room temperature. The inoculum was removed, cell cultures washed to remove non-internalized bacteria, then 10 ml RPMI supplemented with 2% FBS added to the culture flasks. At 5 d post infection, host cell cultures were washed with 10 ml of Hank's buffered saline solution, then lysed with 3 ml of Trizol reagent (Invitrogen). For analysis of C. burnetii transcript profiles in CCM, 2.5 x 10⁹ GE of C. burnetii were incubated in triplicate in 0.5 ml CCM in 24-well plates for 24 h. Bacteria were transferred to a 1.5 ml Microfuge tube, pelleted by centrifugation, then Trizol reagent (1.0 ml) added to each tube.

[0055] The contents of CCM are shown in Table 1 except CCM does not include L- cysteine or casamino acids. CCM provides for metabolic activity of C. burnetii for about 24 hours and provides for morphological differentiation. CCM has not been shown to provide for obvious growth of C. burnetii. Metabolic testing shows that C burnetti metabolism is optimal in a citrate based buffer, (data not shown) the strong buffering capacity of citrate in the metabolically permissive pH range of 4 to 6 increases the utility of this buffer. Increased metabolic activity of C. burnetii was observed in between pH 4.5-5.5. Neopeptone provides for a high quality source of a spectrum of nutrients including nucleotides, peptides, vitamins, minerals, and amino acids. The presence of chloride ions independent of cation was demonstrated to provide for increased axenic metabolic activity; preferably about 125mM chloride ion is added to the medium. Chloride ion may help to maintain cytoplasmic pH homeostasis.

[0056] Total RNA in Trizol samples was purified and processed as previously described (Virtaneva K et al, Proc Natl Acad Sd USA 102:9014-9019 (2005)). A MicrobEnrich kit (Applied Biosystems, Foster City, CA) was used to increase the relative level of C. burnetii RNA derived from Vero cell-propagated organisms. Enriched RNA (about 1 μg) was amplified using a MessageAmp II Bacteria kit (Applied Biosystem, Foster City, CA).

Briefly, double stranded (ds) cDNA was synthesized, product was purified using a QiaQuick 96-well system, and biotin-labeled complimentary RNA was in vitro transcribed using ds cDNA as a template. RNA samples from C. burnetii cultured in CCM were treated identically to facilitate comparisons with RNA samples derived from Vero cell-propagated C. burnetii.

[0057] All cRNA originating from Vero cell-propagated C. burnetii and 3 μg cRNA from CCM-cultivated organisms were hybridized to a custom Affymetrix GeneChip designed as previously described (Beare PA et al., J Bacteriol 188:2309- 2324 (2006)). Microarray data were analyzed using Partek Genomics Suite software (Partek Inc., St. Louis, MO) essentially as described (Li M et al, Proc Natl Acad Sd USA 104:9469-9474 (2007)).

Radiolabeling with [³⁵S] cysteine/methionine.

[0058] Radiolabeling of C. burnetii proteins was conducted using 2.5 x 10⁹ GE of freshly thawed organisms. Following preincubations in 6-well plates containing 2.0 ml medium per well, bacteria were pelleted (20,000 x g for 9 min) and washed in 200 μl citrate salts buffer (Omsland A et al., JBacteriol 190:3203-3212 (2008)) supplemented with 1.0 mM glutamate (labeling buffer) to remove excess nutrients. Bacteria were then resuspended in 500 μl labeling buffer containing 25-50 μCi [35S]Cys/Met protein labeling mix (Perkin Elmer, Waltham, MA) and incubated for 3 h in a screw-cap tube to allow incorporation of the radionuclide. Following radiolabeling of bacterial proteins, bacteria were pelleted for 14 min at 20,000 x g and washed in 100 μl of phosphate-buffered saline (PBS; 10 mM Na2HPO4, 10 mM NaH2PO4, 150 mM NaCl, pH 7.8) to remove unincorporated [35S]Cys/Met. Bacterial pellets were lysed in equal volumes of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 10 min. Equal volumes of each sample were analyzed by scintillation counting to determine counts per min (CPM). Twelve percent SDS-PAGE followed by autoradiography using CL-Xposure (Pierce, Rockford, IL) film was used to visualize radiolabeled proteins (3 h exposure). Precision Plus Protein Dual Color Standards (Biorad, Hercules, CA) were used as molecular mass markers. Phenotype Microarrays.

[0059] C. burnetii oxidation of substrates under different oxygen concentrations was tested using Phenotype Microarrays (PM-I) (Biolog Inc., Hayward, CA) containing 95 substrates including amino acids and carbohydrates. Purified C. burnetii were suspended in CCM (7) (5 x 10⁹ GE/ml) supplemented with 0.65 mM of the reporter dye tetrazolium violet (Sigma- Aldrich, St. Louis, MO) (37). CCM (7) was used as the inoculation fluid rather than more nutritionally rich ACCM to promote oxidation of individual PM substrates. Bacterial suspension (100 μl) was added to each well of the 96-well PM plate and plates incubated for 24 h in an Innova CO-48 incubator (New Brunswick Scientific, Edison, NJ) at 37 °C adjusted to 5% CO2 and 20%, 5%, or 2.5% 02. Atmospheric oxygen was displaced by nitrogen gas. At the end of the incubation period, reduction of reporter dye was quantified using an

OmniLog detection system (Biolog Inc., Hayward, CA) and expressed as OmniLog Units (OLU). Preparation of ACCM medium and incubation conditions.

[0060] The ingredients of ACCM are listed in Table 1. Casamino acids were prepared fresh at the time of medium preparation while other components were kept as refrigerated (citrate buffer, salt solution, RPMI cell culture medium) or frozen (neopeptone, FBS, L- cysteine, FeSO4) stocks. The pH of ACCM was adjusted to 4.75 using 6 N NaOH and the medium filtered through a 0.22 μm filter to sterilize. C. burnetii cultures were established in T-25 and T-75 polystyrene cell culture flasks containing 7 ml and 20 ml of ACCM, respectively. Flasks were inoculated with C. burnetii (1.0 x 10⁶ GE/ml) purified from Vero host cells. Cultures were incubated in an Innova CO-48 incubator as described for PM analysis. Growth of C. burnetii on solid medium was conducted using a soft agarose overlay method. A 2X solution of ACCM nutrients was adjusted to pH 4.75, sterilized by filtration and 7.5 ml added to an equal volume of 2% (w/v in water) molten UltraPure Agarose (Invitrogen). The 1% ACCM-agarose was poured into 100 x 20 mm petri dishes to create a solid medium base. Purified C. burnetii was spread on the ACCM-agarose base and 10 ml 0.25% ACCM-agarose added as top agarose. Plates were incubated for 6 d in an Innova CO- 48 incubator adjusted to 5% CO2 and 2.5% 02. Colonies were imaged using a Axiovert 40 C microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) equipped with an AxioCam ICc digital camera (Carl Zeiss).

Quantification of C. burnetii cell division and infectivity. [0061] C. burnetii replication during incubation in ACCM was quantified by QPCR of C. burnetii GE using a primer and probe set specific to dotA of C. burnetii (Coleman SA et al, J Bacteriol 186:7344-7352 (2004)).

QdotA-F GCGCAATACGCTCAATCACA (SEQ ID NO: 1) QdotA-R CCATGGCCCCAATTCTCTT (SEQ ID NO:2) GENE PROBE

CCGGAGATACCGGCGGTGGG (SEQ ID NO:3)

Other probes and primers are known to those of skill in the art and are described in Coleman et al. (Coleman SA et al., J Bacteriol 186:7344-7352 (2004)).

[0062] ACCM culture aliquots (50 μl) were diluted in 150 μ\ sterile PBS, diluted 5-fold further with sterile water, then mechanically disrupted to release bacterial DNA using a FastPrep homogenizer (Q-Biogene Inc., CA) and 0.1 mm zerconia/silica beads (Biospec Products Inc., Bartlesville, OK) as lysing matrix. Samples were centrifuged for 1 min at 20,000 x g to pellet the lysing matrix, and equal volumes of supernatant containing chromosomal DNA was used as template DNAfor PCR reactions. QPCRs were performed using TaqMan Universal PCR Master Mix 19 and a Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Infectivity of C. burnetii cultivated in ACCM was determined by a fluorescent focus forming unit (FFU) assay (Coleman SA et al, J Bacteriol 186:7344-7352 (2004)). Ten microliter aliquots of C. burnetii cultures in ACCM were collected at the indicated times and added to 240 μl RPMI cell culture medium which was then frozen at -80 ⁰C until needed. Equal volumes of thawed culture aliquots were used to infect confluent Vero cell cultures in 24-well plates. Following a 2 h incubation at room temperature with rocking, 1 ml of fresh RPMI medium supplemented with 2% fetal bovine serum was added to each well. After a 5-day incubation, infected cells were fixed with 100% cold methanol and FFUs stained by indirect immunofluorescence employing polyclonal guinea pig antiserum generated against formalin-killed C. burnetii and Alexa Fluor 448- conjugated goat anti-guinea pig immunoglobulin G serum (Molecular Probes, Eugene, Oreg.). FFUs were enumerated by fluorescence microscopy using a Ziess Axiovert 25 inverted microscope. Color saturation was adjusted equally for all images using Adobe PhotoShop.

Transmission electron microscopy.

[0063] Specimens were fixed as previously described (Coleman SA et al., J Bacteriol

186:7344-7352 (2004)). Thin sections were cut with an RMC MT-7000 ultramicrotome (Ventana, Tucson, AZ), stained with 1% uranyl acetate and Reynold's lead citrate prior to viewing at 80 kV on a Philips CM-10 transmission electron microscope (FEI, Eindhoven, Netherlands). Digital images were acquired with a Hammamatsu XR-100 bottom mount CCD system (Advanced Microscopy Techniques, Danvers, MA). Cross section diameters were measured with the AMT software measuring tool and data analyzed using Prism software (GraphPad Software Inc., CA). Final images were processed with Adobe PhotoShop (Adobe Systems, Inc., San Jose, CA).

Immunoblotting.

[0064] C. burnetii was pelleted by centrifugation and lysed by boiling in a solution of 1% SDS. The protein concentration of each sample was determined using a DC Protein Assay kit (Biorad, Hercules, CA). Samples were diluted in SDS-PAGE sample buffer and 10 μg total protein separated by SDS-PAGE on a 10-20% Tris-HC Ready Gel (Biorad, Hercules, CA). Proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) that was blocked overnight at 4°C in PBS containing 0.1% Tween-20 and 3% nonfat milk (PBST). Membranes were then incubated for 1 h at room temperature in PBST containing anti-ScvA rabbit polyclonal antibody (15). Membranes were washed, then incubated for 1 h at room temperature in PBST containing anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Pierce, Rockford, IL). Reacting proteins were detected via enhanced chemiluminescence using ECL Pico reagent (Pierce, Rockford, IL) and CL-XPosure film (Pierce, Rockford, IL).

Statistical analysis.

[0065] Statistical analyses were performed by unpaired Student's t-test using Prism software (GraphPad Software Inc., CA). Differences between data sets where P < 0.05 were considered statistically significant. Results

[0066] C. burnetii exhibits reduced ribosomal gene expression in CCM. As an initial step to identify nutritional deficiencies of CCM that could preclude C. burnetii cell division, a comparison of genome wide transcript profiles between organisms replicating in Vero cells and incubated in CCM for 24 h was conducted. This analysis showed substantially reduced expression of ribosomal genes during incubation in CCM (Table 2), suggesting that protein synthesis was insufficient to support C. burnetii replication in this axenic medium. Supplementation of CCM with pyruvate, succinate or glutamate, efficiently oxidized energy sources of C. burnetii (Hackstadt T & Williams JC, J Bacteriol 148:419-425(1981)), did not improve C. burnetii de novo protein synthesis in CCM (Omsland A et al., J Bacteriol 190:3203-3212 (2008)), suggesting energy starvation was not the reason for reduced ribosomal gene expression.

Table 2: Ribosomal gene expression of C. burnetii during incubation in CCM

[0067] Supplementation of CCM with protein precursors improves C. burnetii catabolic activity. Amino acid deficiencies in CCM could also explain reduced ribosomal gene expression. C. burnetii has multiple amino acid auxotrophies that appear compensated forl by amino acid and peptide transporters (Seshadri R, et al., Proc Natl Acad Sci USA 100:5455- 5460 (2003)). Moreover, intracellular bacteria frequently use amino acids as carbon sources (Appelberg R, JLeukoc Biol 79:1117-1128 (2006)), with an exceptionally high concentration of L-cysteine required for axenic growth by some (Ewann F & Hoffman PS, Appl Environ Microbiol 72:3993-4000 (2006)).

[0068] To evaluate whether supplementation of CCM with amino acids and peptides improves C. burnetii metabolic activity, casamino acids (a mixture of amino acids and peptides) and/or L-cysteine were added to the medium (Table 1). Following 24 h preincubations in media, C. burnetii was subjected to a 3 h [35S]Cys/Met pulse and the fold increase in radiolabel incorporation over the negative control (i.e., organisms labeled in labeling buffer at pH 7) used to assess the catabolic capacity (Omsland A et al., JBacteriol 190:3203-3212 (2008)) of the organism. CCM supplemented with casamino acids or L- cysteine supported statistically significant increases in C. burnetii radiolabel incorporation of (39.1 ± 5.1)-fold and (134.5 ± 23.4)-fold, respectively (Fig. IA). The effect of supplementing CCM with both casamino acids and L-cysteine was additive, resulting in a (232.7 ± 33.5)- fold increase in incorporation (Fig. IA). Overall, this medium termed Acidified Citrate Cysteine Medium (ACCM) (Table 1) supported an approximately 13-fold increase in protein synthesis compared to CCM. [0069] SDS-PAGE and autoradiography confirmed that radiolabel was incorporated into C. burnetii de novo synthesized protein (Fig. IB).

[0070] C. burnetii substrate oxidation increases under microaerobic conditions. Terminal oxidases containing either cytochrome o or cytochrome bd, associated with aerobic and microaerobic respiration, respectively, are encoded by the C. burnetii genome (Fig. 2A). This observation suggested that C. burnetii responds to alterations in oxygen tension during intracellular growth. Therefore, we assessed the effect of oxygen tension on C. burnetii metabolism of a wide variety of metabolites, including amino acids and carbohydrates, using Phenotype Microarrays (PM).

[0071] C. burnetii oxidation of 95 substrates in PM-I arrays was tested under oxygen tensions of 20%, 5% and 2.5%. Incubation in 20% oxygen resulted in efficient oxidation of only succinate (Fig. 2B). However, incubations in 5% or 2.5% oxygen showed oxidation of 10 and 17 substrates, respectively (Fig. 2B). Intermediates of major metabolic pathways including the tricarboxylic acid cycle and glycolysis were most efficiently oxidized (Fig. 2C). Several substrates efficiently oxidized by C. burnetii in PMs have also been identified as substrates for the organism in independent studies (e.g., succinate, glutamate, and proline) (Hackstadt T & Williams JC, JBacteriol 148:419-425(1981); Hendrix L & Mallavia LP, J Gen Microbiol 130:2857-2863 (1984)). [0072] ACCM supports C. burnetii replication in a microaerobic environment. The characterization of C. burnetii as a potential microaerophile suggested that incubation in ACCM in an optimal oxygen environment might support growth. Therefore, ACCM was inoculated with C. burnetii and the cultures incubated in a 20% or 2.5% oxygen environment. Cultures were monitored for C. burnetii replication by measuring bacterial genome equivalents (GE) by quantitative Taqman PCR (QPCR) every 24 h over 6 d.

[0073] Incubation of C. burnetii in ACCM in 20% oxygen did not result in an increase in C. burnetii GE (Fig. 3A). However, substantial replication of C. burnetii occurred in 2.5% oxygen (8.71 x 104 to 5.49 x 107 GEImX) (Fig. 3A). C. burnetii replication in ACCM in 1% or 5% oxygen was similar to that observed in 2.5% while no replication was observed in 10% oxygen (data not shown). The growth cycle of organisms incubated in ACCM in 2.5% oxygen consisted of a lag phase of approximately 1 d, followed by 3 d of exponential growth and the onset of stationary phase thereafter. The generation time of C. burnetii during exponential growth in ACCM was 9.1 h which is 1-2 h less than the generation time in Vero cells (Coleman SA et al., JBacteriol 186:7344-7352 (2004)). C. burnetii also formed colonies (about 0.05 - 0.1 mm in diameter) after a 14 d incubation in an ACCM-based solid agarose medium (Fig. 5).

[0074] To confirm that the increase in C. burnetii genome equivalents corresponded to an increase in C. burnetii infective units, organisms incubated in ACCM in 2.5% oxygen were assayed for Vero cell infectivity using a fluorescent infectious focus-forming unit (FFU) assay that employs a Coxiella-specific antibody. ACCM culture aliquots taken every 24 h for 6 d post-inoculation showed an increase in FFUs of 2.19 logs (Fig. 3B-C) with the kinetics of FFU development directly corresponding to increases in GE (Fig.3A). Coxiella containing vacuoles showed typical staining for the lysosomal marker CD63 (data not shown). Ten to fifteen genomes were required for development of each vacuole, equivalent to published GE/FFU ratios (Coleman SA et al., JBacteriol 186:7344-7352 (2004)). Collectively, these data show that ACCM supports robust cell division of infectious C. burnetii that are antigenically similar to cell culture-cultivated organisms. [0075] C. burnetii developmental transitions occur in ACCM. During infection of eukaryotic host cells, C. burnetii undergoes a biphasic developmental cycle characterized by transition of metabolically dormant, non-replicative small cell variants (SCV) to metabolically active, replicative large cell variants (LCV) (Coleman SA et al., JBacteriol 186:7344-7352 (2004)). To determine whether this developmental program occurs during replication in ACCM, medium was inoculated with purified SCVs and morphological differentiation assessed by transmission electron microscopy (TEM). Ultrastructural features of the inoculum were characteristic of the SCV, most notably small cell size (< 0.2 jum) and electron-dense chromatin (Fig. 4A-B)(Coleman SA et al., JBacteriol 186:7344-7352 (2004)). Organisms incubated in ACCM for 3 d and in exponential phase displayed ultrastructural characteristics of the LCV including increased cell size (> 0.2 μm) and dispersed chromatin (Fig. 4A-B) (Coleman SA et al., JBacteriol 186:7344-7352 (2004)). Organisms incubated in ACCM for 6 d and in early stationary phase were a mixture of SCVs and LCVs (Fig. 4A-B). ACCM cultures initiated with purified SCVs also showed approximately 3 logs of growth after 6 d of incubation (Fig. 6). Developmental transitions were confirmed by immunoblotting for ScvA, a DNA-binding protein specific to the SCV (Coleman SA et al., J Bacteriol 186:7344-7352 (2004); Heinzen RA et al., MoI Microbiol 22:9-19 (1996)). ScvA was detected only in the SCV2 inoculum and the LCV/SCV mixture present at early stationary phase (Fig. 4C). Thus, C. burnetii undergoes a developmental program in ACCM that is similar to in vivo propagated organisms.

Discussion

[0076] The systematic approach described herein allowed identification of nutritional and biophysical conditions that support C. burnetii host cell-free growth. Based on a 2.5 - 3 log increase in C. burnetii GE after 6 d of culture in ACCM in 2.5% oxygen, we estimate that 100 ml of ACCM can yield the same number of organisms as 7 x 10⁸ infected Vero cells.

Growth of C. burnetii in ACCM was also established with an inoculum as low as 100 GE/ml (data not shown), suggesting ACCM can be used to isolate C. burnetii from the small number of organisms typically contained in clinical samples. Axenic culture of C. burnetii in ACCM will improve our ability to define factors required for intracellular growth and pathogenesis. Moreover, growth of C. burnetii on solid ACCM medium will facilitate clonal isolation and development of genetic tools for this organism.

[0077] The substantial increase in C. burnetii metabolic activity in ACCM compared to CCM is largely an effect of L-cysteine. An elevated level of L-cysteine is also required for in vitro culture of Legionella pneumophila (George JR et al., J Clin Microbiol 11 :286-291 (1980); Barker J et al., J Med Microbiol 22:97-100 (1986) where the amino acid may serve as an anti-oxidant in scavenging hydrogen peroxide (Hoffman PS et al., Appl Environ Microbiol 45:784-791 (1983)). While L-cysteine is bioaccessible to L. pneumophila, the oxidized form L-cystine is not, making it necessary to supplement nutrient media with L-cysteine in great excess of what is consumed by the organism (Ewann F & Hoffman PS, Appl Environ Microbiol 72:3993-4000 (2006)). ACCM containing the alternative antioxidant L- glutathione instead of L- cysteine did not support C. burnetii growth (data not shown), suggesting that C. burnetii' s requirement for L-cysteine in ACCM is nutritional, hi addition to serving as a precursor in protein synthesis, L-cysteine may also be a source of sulfur. [0078] C. burnetii replication in ACCM is optimal in a 2.5% oxygen environment and the presence of genes encoding cytochrome bd (i.e., cydAB) with high affinity for oxygen provides a biochemical/physiological explanation for the observed growth phenotype. Interestingly, the intracellular bacteria Mycobacterium tuberculosis (Kana BD, et al., J Bacteriol 183:7076-7086 (2001)), Chlamydia trachomatis (Stephens RS, et al., Science 282:754-759 (1998)) and Rickettsia rickettsii all encode cydAB suggesting adaptation to microaerobic metabolism in intracellular bacteria may be under-appreciated. Indeed, transcriptional analysis of M. tuberculosis during infection of macrophages indicates the organism adapts to a reduced oxygen environment (Schnappinger D, et al., J Exp Med 198:693-704 (2003)), and improved growth of Chlamydia pneumoniae is observed under low oxygen conditions (Juul N et al., J Bacteriol 189:6723- 6726 (2007)). Like M. tuberculosis, C. burnetii can occupy tissue granulomas (La Scola B et al., Infect Immun 65:2443-2447 (1997)), a defined low oxygen environment (Via LE, et al., Infect Immun 76:2333-2340 (2008)). Mammalian cell intracellular oxygen tension can be significantly lower than the extracellular oxygen tension (Hu H et al., Biochim Biophys Acta 1112:161-166 (1992)). Moreover, the membrane of the C. burnetii replicative vacuole is enriched in cholesterol

(Howe D & Heinzen RA, Cell Microbiol 8:496-507 (2006)), and cholesterol-rich membranes are known to impede oxygen diffusion (Khan N, et al., Biochemistry 42:23-29 (2003)). Collectively, these observations suggest the vacuolar compartment of C. burnetii is a low oxygen environment, hi addition to potentially serving as a terminal oxidase in low oxygen conditions, CydAB may also protect C. burnetii against oxidative agents of the phagolysosome. In support of this hypothesis, cydB mutants of Brucella abortus and Escherichia coli are hypersensitive to hydrogen peroxide (Endley S et al., J Bacteriol 183:2454-2462 (2001); Goldman BS et al., J Bacteriol 178:6348-6351 (1996)). [0079] The debilitating nature of acute Q fever, along with C. burnetii 's environmental stability and aerosol route of transmission, have raised concerns over potential illegitimate use of this microorganism (Madariaga MG et al., Lancet Infect Dis 3:709-721 (2003)). In this regard, axenic cultivation of C. burnetii will aid molecular characterization of the organism to enable development of protective measures against Q fever including improved diagnostic tools and efficacious vaccines. Moreover, the strategy employed here to establish culture conditions for C. burnetii may be broadly applicable to identifying media formulations and biophysical conditions that support growth of other currently obligate intracellular bacterial pathogens of humans within the genera Anaplasma, Ehrlichia, Treponema, Chlamydia and Rickettsia.

Example 2

[0080] Preparation of a completely chemically defined Acidified Citrate Cysteine medium- 2("ACMM-2") for growth of C. burnetii without a eukaryotic host cell. [0081] An improved medium, called ACCM-2, in which fetal bovine serum has been replaced with methyl-β-cyclodextrin, has been formulated. ACCM-2 supports a faster generation time and approximately 10-fold more Coxiella growth than ACCM. See, Figure 7. We have also discovered that neopeptone can be removed from ACCM-2 without affecting growth, thereby resulting in a completely chemically defined medium. [0082] The recipe for ACCM-2 as used to generate the above-described data is as follows: ACIDIFIED CITRATE CYSTEINE MEDIUM-2 (ACCM-2)

Component Amt / L Final concentr

Citric acid 2568 mg 13.40 mM

Sodium citrate 4740 mg 16.11 mM

Potassium phosphate 500 mg 3.67 mM

Magnesium chloride 200 mg 0.98 mM calcium chloride 13.2 mg 0.09 mM

Iron sulfate 2.78 mg 0.01 mM sodium chloride 7280 mg 125 mM

L-cysteine 263.4 mg 1.50 mM

Bacto

Neopeptone 100 mg 0.1 mg/ml casamino acids 2500 mg 2.5 mg/ml

Methyl-β-

Cyclodextrin 1000 mg 1 mg/ml

RPMI w/glutamax 125 ml 1/8 X Deionized H2O 865 n/a

Adjust pH to 4.75 with 6 N NaOH and sterilize by filtration

[0083] We also increased or decreased the components list below by factors of 2, 4, 8, etc., to determine ranges of concentrations that support Coxiella growth. The following concentration ranges for the components of ACCM and/or ACCM-2 were determined:

14.5 - 58 mM citrate

1/16 - 1/4 dilution of RPMI cell culture medium

70 - 280 mM chloride (Cl^") ion 95 - 380 mM sodium (Na+) ion

2.15 - 8.6 mM potassium (K+) ion

0.5 - 2.0 mM magnesium (Mg++) ion

0.075 - 0.30 mM calcium (Ca+) ion

0.005 - 0.02 mM ferrous iron (Fe++) ion 1.25 - 5 mg/ml casamino acids

0.75 - 3.0 mM cysteine

0.03 - 0.12 mM sulfate (SO₄-)

0.5 to 2.0 mg/ml or more methyl-β-cyclodextrin (ACCM-2)

[0084] Any patent publications and references referred to herein are herby incorporated by reference. Examples as described herein are illustrative only and other modifications may be made to the disclosed methods and compositions without departing from the spirit of the invention.

Claims

WHAT IS CLAIMED IS:

L A method of culturing Coxiella burnetii without an eukaryotic host cell comprising: 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.

2. The method of claim 1, wherein the medium comprises peptone.

3. The method of claim 1 or 2, wherein the medium comprises methyl-β- cyclodextrin.

4. The method of claim 3, wherein the medium lacks peptone.

5. The method of any one of claims 1 -4, wherein the chloride ion is present in the medium at a concentration of at least 125mM.

6. The method of any one of claims 1-5, wherein the L-cysteine is present at least LO mM.

7. The method of any one of claims 1 -6, wherein the pH of the medium is between 4.5 to 5.5.

8. The method of any one of claims 1 to 7, wherein the concentration of oxygen is 1 to 5%.

9. The method of any of claims 1 to 8, further comprising isolating the Coxiella burnetii from the culture after an incubation period.

10. The method of any one of claims 1 to 9, wherein the incubation period is at least 3 days.

11. The method of any of claim 9, further comprising combining an immunogenically-effective amount of the isolated Coxiella burnetii with a physiologically acceptable excipient.

12. The method of claim 9 or 11, further comprising inactivating the Coxiella burnetii.

13. Coxiella burnetii prepared by the method of any one of claims 1 to 10.

14. A composition comprising Coxiella burnetii, substantially free of any eukaryotic host cell contaminants in a physiological acceptable medium.

15. The composition of claim 14, wherein the Coxiella burnetii is replicating.

16. An immunogenic composition comprising an isolated Coxiella burnetii substantially free of any eukaryotic host cell in an admixture with a physiological excipient.