METHOD FOR LYOPHILIZING LIVE VACCINE STRAINS OF FRANCISELLA TULARENSIS

Abstract

There are provided compositions and methods for lyophilization and/or storage of live vaccine strains of Francisella tularensis. More specifically, there are provided lyophilization media and uses thereof for the preparation and long-term storage of Francisella tularensis vaccines.

Claims

1.-62. (canceled)

63. A lyophilization medium for freeze-drying a Francisella tularensis (F. tularensis) strain, wherein the F. tularensis strain is a mutant strain in which the clpB gene is inactivated, wherein: the lyophilization medium comprises about 1% (w/v) of mannitol, about 1% (w/v) of a disaccharide, and about 0.25% (w/v) of gelatin in a phosphate buffer; wherein the disaccharide is selected from sucrose, trehalose, and a mixture of sucrose and trehalose; wherein initial post-lyophilization viability of the F. tularensis strain is at least about 35% of the F. tularensis strain's pre-lyophilization viability; and wherein the lyophilization medium is suitable for preserving viability of the F. tularensis strain during long-term storage in a lyophilized state.

64. The lyophilization medium of claim 63, wherein the lyophilization medium can preserve viability of the lyophilized F. tularensis strain at temperatures of about +4° C. or lower for at least 1 year.

65. The lyophilization medium of claim 63, wherein the lyophilization medium can preserve viability of the lyophilized F. tularensis strain at temperatures of about +4° C. or lower for at least 3 years.

66. The lyophilization medium of claim 63, wherein the lyophilization medium can preserve viability of the lyophilized F. tularensis strain at temperatures of about −20° C. for at least 1 year.

67. The lyophilization medium of claim 63, wherein the lyophilization medium can preserve viability of the lyophilized F. tularensis strain at temperatures of about −20° C. for at least 3 years.

68. The lyophilization medium of claim 63, wherein the lyophilization medium can preserve viability of the lyophilized F. tularensis strain for at least about 5×, at least about 10×, at least about 15×, or at least about 20× longer than a lyophilization medium consisting of 10% sucrose and 1.3% gelatin in a 10 mM potassium phosphate solution.

69. The lyophilization medium of claim 63, wherein said viability of the lyophilized F. tularensis strain after storage is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of said initial post-lyophilization viability of the F. tularensis strain.

70. The lyophilization medium of claim 63, wherein said viability of the lyophilized F. tularensis strain after storage is substantially unchanged compared to said initial post-lyophilization viability.

71. A method of inducing an immune response against and/or conferring immunity against F. tularensis in a subject, comprising administering to the subject a mutant F. tularensis strain in which the clpB gene is inactivated, wherein the mutant F. tularensis strain is a lyophilized vaccine strain produced using the lyophilization medium according to claim 63.

72. The method of claim 71, wherein the lyophilized vaccine strain is produced by the following method: 1) obtaining a live mutant F. tularensis strain suspended in a growth medium; 2) replacing the growth medium with the lyophilization medium of claim 63 to obtain a suspension of the live vaccine strain in the lyophilization medium; and 3) freeze-drying the suspension.

73. The method of claim 72, wherein the growth medium is replaced with the lyophilization medium using filtration.

74. A method of preventing or treating tularemia in a subject, comprising administering to the subject a mutant F. tularensis strain in which the clpB gene is inactivated, wherein the mutant F. tularensis strain is a lyophilized vaccine strain produced using the lyophilization medium according to claim 63.

75. The method of claim 74, wherein the lyophilized vaccine strain is produced by the following method: 1) obtaining a live mutant F. tularensis strain suspended in a growth medium; 2) replacing the growth medium with the lyophilization medium of claim 63 to obtain a suspension of the live vaccine strain in the lyophilization medium; and 3) freeze-drying the suspension.

76. The method of claim 75, wherein the growth medium is replaced with the lyophilization medium using filtration.

77. A vaccine for the prevention or treatment of F. tularensis infection and/or tularemia in a subject, the vaccine comprising a lyophilized vaccine strain produced using the lyophilization medium according to claim 63.

78. The vaccine of claim 77, wherein the lyophilized vaccine strain is produced by the following method: 1) obtaining a live mutant F. tularensis strain suspended in a growth medium; 2) replacing the growth medium with the lyophilization medium of claim 63 to obtain a suspension of the live vaccine strain in the lyophilization medium; and 3) freeze-drying the suspension.

79. The vaccine of claim 78, wherein the growth medium is replaced with the lyophilization medium using filtration.

80. A composition comprising a lyophilization medium and a strain of Francisella tularensis (F. tularensis), wherein the F. tularensis strain is a mutant strain in which the clpB gene is inactivated; wherein the lyophilization medium comprises about 1% (w/v) of mannitol, about 1% (w/v) of a disaccharide, and about 0.25% (w/v) of gelatin in a phosphate buffer; wherein the disaccharide is selected from sucrose, trehalose, and a mixture of sucrose and trehalose; and wherein initial post-lyophilization viability of the F. tularensis strain is at least about 35% of the F. tularensis strain's pre-lyophilization viability.

81. A lyophilizate of the composition of claim 80.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] For a better understanding of the invention and to show more clearly how it may be carried into effect, reference will now be made by way of example to the accompanying drawings, which illustrate aspects and features according to preferred embodiments of the present invention, and in which:

[0043] FIG. 1 shows viability of lyophilized samples stored at various temperatures for various amounts of time. SCHU S4ΔclpB bacteria were grown in modified casein partial hydrolysate (MCPH) broth, pelleted by centrifugation and resuspended and lyophilized in lyophilization medium (10 mM potassium phosphate buffer containing 1% mannitol, 1% sucrose and 0.25% gelatin, final pH7.2) at about 10.sup.10 colony forming units (CFU). These vials were stored at ambient (˜22° C.), refrigerator (+4° C.), or freezer (−20° C. or −80° C.) temperatures, as indicated. At various times, as indicated, sample vials were thawed, lyophilized material was solubilized in water, and plated in order to determine viability. The determined % viability (CFU count after reconstitution/CFU count pre-lyophilization×100) is shown in the figure.

[0044] FIG. 2 shows percent viability of SCHU S4 ΔclpB resuspended in lyophilization medium, serially diluted as indicated, and plated before lyophilization, and 63 days after storage at −80° C. A modest decrease in viability at 63 days was observed with the more dilute vials, but even at 1:1000 dilution there was still ˜70% retained viability after initial lyophilization.

[0045] FIG. 3 shows clinical scores observed in BALB/c mice immunized intradermally with 1×10.sup.5 CFU of ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C. Clinical scores are determined as follows: 1=healthy; 2=slight pilo-erection; 3=slight pilo-erection and decreased mobility; 4=slight pilo-erection, decreased mobility, and hunching; 5=slight pilo-erection, decreased mobility, hunching, and shivering; 6=dead. ΔclpB stored at +4° C. appears to show somewhat reduced toxicity, but overall there were no significant differences in clinical score between animals vaccinated with ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C. * indicates a time point that was not scored, so data is unavailable.

[0046] FIG. 4 shows levels of ΔclpB in tissues of BALB/c mice 4 days after intradermal immunization with 1×10.sup.5 CFU of ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C.

[0047] FIG. 5A shows skin reacogenicity at immunization site observed in BALB/c mice immunized intradermally with 1×10.sup.5 CFU of ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C. * denotes p<0.5 (Kruskal-Wallis with Tukey's correction for multiple comparisons). FIG. 5B shows the Francisella Skin Reaction Score Chart used for scoring.

[0048] FIG. 6 shows survival of mice challenged intranasally with SCHU S4 bacteria, 6 weeks after intranasal immunization with 1×10.sup.4 CFU of ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C. The differences between survival curves were not statistically significant by the Mantel-Cox test.

[0049] FIG. 7 shows survival of mice following intradermal immunization with 1×10.sup.5 CFU of ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C. and challenged intranasally (IN) with SCHU S4 six weeks later.

[0050] FIG. 8 shows the level of SCHU S4 bacteria remaining in the organs of mice following intradermal (ID) or intranasal (IN) immunization with 1×10.sup.5 CFU of ΔclpB lyophilized for 3 years and stored at +4° C., −20° C., or −80° C. and challenged with SCHU S4 six weeks later.

DETAILED DESCRIPTION

Definitions

[0051] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0052] As used herein, the term “about” refers to a value that is within the limits of error of experimental measurement or determination. For example, two values which are about 1%, about 2%, about 3%, about 5%, about 10%, about 15%, or about 20% apart from each other, after correcting for standard error, may be considered to be “about the same” or “similar”. In some embodiments, “about” refers to a variation of ±20%, ±10%, ±5%, ±3%, ±2% or ±1% from the specified value, as appropriate to perform the disclosed methods or to describe the disclosed compositions and methods, as will be understood by the person skilled in the art.

[0053] By the term “attenuation” or “attenuated”, it is meant that a pathogen is kept alive, but exhibits reduced virulence such that it does not cause the disease caused by the virulent pathogen. The attenuation of a particular strain may result from e.g. the inactivation of the clpB gene, or may be the result of other mechanisms for attenuation, for example and not limited to mutagenesis, deletion or inactivation of targeted genes, or natural attenuation, or a combination thereof.

[0054] By the term “virulent F. tularensis strain” is meant a strain that may cause any disease in the spectrum referred to as tularemia, whether mild or severe. Non-limiting examples of virulent F. tularensis strains include SCHU S4, FSC033, FSC108, and FSC200. In some embodiments, a mutant F. tularensis strain may have additional mutations introduced to further attenuate the pathogen (either partially or completely) or for other purpose.

[0055] It should be understood that the methods and compositions provided herein can be used for preparation and/or storage of F. tularensis mutants, including F. tularensis mutants with inactivated clpB. In some embodiments, F. tularensis mutants are strains in which the clpB gene has been deleted (ΔclpB mutants). In some embodiments, an F. tularensis ΔclpB mutant is a virulent type A strain (e.g., SCHU S4). In some embodiments, an F. tularensis ΔclpB mutant is a type B strain (e.g., FSC200).

[0056] In an embodiment, the F. tularensis mutant strain is SCHU S4 ΔclpB, such as without limitation the strain deposited with the Culture Collection University of Gothenburg (CCUG; Sahlgrenska Academy of the University of Gothenburg, Box 7193, SE-402 34, Gothenburg, Sweden) and granted accession number CCUG 59672.

[0057] A gene may be “inactivated” by any suitable manner known in the art. For example, and not wishing to be limiting, the clpB gene may be inactivated using methods known in the art, for example by its complete or partial deletion from the F. tularensis strain, by an inactivation mutation such as a multiple nucleotide substitution, or by an inactivating insertion such as a transposon insertion. Further, inactivation of the clpB gene may result in complete or partial attenuation of a mutant F. tularensis strain.

[0058] “Lyophilization”, also known as “freeze-drying”, is a process used for preserving biological material such as vaccines, bacteria, and proteins, by removing the water from the sample, which typically involves first freezing the sample and then drying it, under a vacuum, at very low temperatures. Lyophilization methods are well-known in the art. It should be understood that any standard lyophilization procedures known in the art may be used with the compositions and methods provided herein; it is within the capabilities of persons of skill in the art to select procedures for lyophilization.

[0059] As used herein, the terms “lyophilization medium” and “lyophilization matrix” are used interchangeably to refer to a composition in which samples (e.g., bacteria) are suspended before being subjected to the freeze drying process. Bacteria are generally resuspended in a lyophilization medium prior to lyophilization using standard procedures, as are known in the art. Typically a suitable lyophilization medium for bacteria will help maintain their viability through the freeze drying process and subsequent storage, for example by stabilizing the cells and/or helping to retain structure of biomolecules.

[0060] As used herein, “viability” refers to the number of live bacteria or the percentage of live vs. dead bacteria in a sample prior to freezing, after freezing but prior to lyophilization, or after lyophilization and storage for periods of up to 3 years. Viability is often provided as a percentage, referring to the percentage of bacteria in a sample that are live after treatment (e.g., freezing or lyophilizing) compared to the number of viable bacteria before any such treatment. Alternatively, viability may be provided herein as the number of colony forming units (CFU) in a treated sample divided by the CFU prior to any such treatment. Many methods for determining viability of a bacterial sample are known in the art and any art-recognized technique or assay may be used. In some embodiments, post-lyophilization viability is determined as the CFU count after reconstitution/CFU count pre-lyophilization×100.

[0061] The terms “preservation of viability” and “stability of viability” are used herein to mean that viability is substantially unchanged. As used herein, “substantially unchanged” viability means that the viability is approximately the same, e.g., within about 10% or about 20% of starting values, or that there is a loss of no more than about 0.5 log.sub.10 CFU, about 1 log.sub.10 CFU, or about 2 log.sub.10 CFU. In some embodiments, in the context of a vaccine, “substantially unchanged” viability means that viability is maintained at a sufficient level for usefulness as a vaccine, e.g., that immunogenicity is maintained. Preservation of a certain level of viability means that a certain level of viable cells is maintained in a sample relative to the level of viable cells present at a starting point, for example before a treatment (e.g. lyophilization) and/or before the passage of time. For example, preservation of 50% viability means that at least 50% of the cells in the sample remain alive, relative to the number of cells alive at a starting point. Preservation of viability after lyophilization may be assessed by assessing the viability of the culture after lyophilization in comparison to the viability of the culture immediately prior to lyophilization. Preservation of viability after lyophilization means that the viability of the culture is a given percentage, or higher, of the viability of the culture immediately prior to lyophilization. For example, preservation of viability of 35% means that 35% of cells remain alive after lyophilization relative to the starting population of viable cells immediately prior to lyophilization. As another example, if the initial viability of a sample immediately after lyophilization is 80%, relative to the viability of the sample immediately prior to lyophilization, then preservation of 50% viability at a given timepoint means that viability of at least 40%, relative to the viability of the sample immediately prior to lyophilization, is maintained in the sample at that timepoint.

[0062] In the context of an F. tularensis strain or a vaccine, “stability” means that immunogenicity is maintained and/or that viability is substantially unchanged, e.g., that the bacterial titer is sufficient for effective use as a vaccine against tularemia in a subject.

[0063] As used herein, “long-term” storage is used to refer to storage for an extended period of time. Without intending to limit the term, it generally refers to storage for at least several months (e.g., about or at least 2 months, about or at least 3 months, about or at least 6 months), or about or at least one year, about or at least two years, about or at least three years, or longer.

[0064] Pharmaceutical Compositions and Methods

[0065] There are provided herein compositions and methods for the prevention or amelioration of tularemia in a subject comprising F. tularensis strains prepared and/or stored as described herein. Compositions and methods for inducing an immune response to F. tularensis are also provided. Methods provided herein comprise administration of a live F. tularensis strain prepared and/or stored as described herein to a subject in an amount effective to induce an immune response against F. tularensis, thereby reducing, eliminating, ameliorating or preventing tularemia. Compositions and methods provided may also be used for the generation of antibodies for use in passive immunization against tularemia and treatment thereof.

[0066] A F. tularensis strain prepared and/or stored using the compositions and methods provided herein may thus be used to vaccinate a subject, administered to a subject to prevent or treat tularemia, etc., as described further herein. It will be understood by the person skilled in the art that a lyophilized strain provided herein will generally be reconstituted to liquid form prior to administration to a subject. For example, a lyophilized strain may be reconstituted in a pharmaceutically acceptable carrier, diluent or excipient, and/or in lyophilization medium, suitable for administration. It should be understood that reference to administration or use of a lyophilized strain herein is meant to include administration or use of a strain that was lyophilized and/or stored in a lyophilized state as described herein, and has been reconstituted in a form suitable for administration. In an embodiment, a lyophilized strain provided herein is reconstituted in water prior to administration to a subject.

[0067] Similarly, the dosage for administration will be dependent on various factors, including the size of the subject and the specifics of the composition formulated, and as discussed further below. Based on experience with LVS, and without wishing to be limiting in any manner, a dose of approximately 10.sup.7 CFU may in some cases be used for administration to humans. It would be within the capabilities of persons of skill in the art to determine appropriate dosages for vaccination.

[0068] The terms “subject” and “patient” are used interchangeably herein to refer to a subject in need of prevention for tularemia. A subject may be a vertebrate, such as a mammal, e.g., a human, a non-human primate, a rabbit, a rat, a mouse, a cow, a horse, a goat, or another animal. Animals include all vertebrates, e.g., mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles. In an embodiment, a subject is a human.

[0069] “Treating” or “treatment” refers to either (i) the prevention of infection or reinfection, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the disease of interest, e.g., therapy. “Treating” or “treatment” can refer to the administration of a composition comprising a F. tularensis vaccine strain prepared and/or stored as described herein, or to the administration of antibodies raised against these F. tularensis vaccine strains. Treating a subject can prevent or reduce the risk of infection and/or induce an immune response to F. tularensis.

[0070] Treatment can be prophylactic (e.g., to prevent or delay the onset of the disease, to prevent the manifestation of clinical or subclinical symptoms thereof, or to prevent recurrence of the disease) or therapeutic (e.g., suppression or alleviation of symptoms after the manifestation of the disease). “Preventing” or “prevention” refers to prophylactic administration or vaccination with a F. tularensis vaccine strain prepared and/or stored as described herein or compositions thereof in a subject who has not been infected or who is symptom-free and/or at risk of infection.

[0071] As used herein, the term “immune response” refers to the response of immune system cells to external or internal stimuli (e.g., antigens, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, production of soluble effectors of the immune response, and the like. An “immunogenic” molecule is one that is capable of producing an immune response in a subject after administration.

[0072] “Active immunization” refers to the process of administering an antigen (e.g., an immunogenic molecule, e.g., a F. tularensis vaccine strain described herein) to a subject in order to induce an immune response. In contrast, “passive immunization” refers to the administration of active humoral immunity, usually in the form of pre-made antibodies, to a subject. Passive immunization is a form of short-term immunization that can be achieved by the administration of an antibody or an antigen-binding fragment thereof. Antibodies can be administered in several possible forms, for example as human or animal blood plasma or serum, as pooled animal or human immunoglobulin, as high-titer animal or human antibodies from immunized subjects or from donors recovering from a disease, as polyclonal antibodies, or as monoclonal antibodies. Typically, immunity derived from passive immunization provides immediate protection or treatment but may last for only a short period of time.

[0073] As used herein, “tularemia” refers to all F. tularensis-associated diseases, i.e., diseases caused by F. tularensis infection.

[0074] In some embodiments, there are provided compositions and methods for active immunization against tularemia. Compositions and methods are provided for inducing an immune response to F. tularensis bacteria in a subject, comprising administering to the subject a F. tularensis vaccine strain prepared and/or stored as described herein, optionally in the presence of an adjuvant, in an amount effective to induce an immune response in the subject. In one embodiment, there is provided a composition comprising an effective immunizing amount of a F. tularensis vaccine strain as provided herein and an adjuvant, wherein the composition is effective to prevent or treat tularemia in a subject in need thereof. In an embodiment, an adjuvant is not required, i.e., compositions and methods are provided for inducing an immune response to F. tularensis bacteria in a subject, comprising administering to the subject an F. tularensis vaccine strain as provided herein and a pharmaceutically acceptable carrier, excipient, or diluent, in an amount effective to induce an immune response in the subject.

[0075] Adjuvants generally increase the specificity and/or the level of immune response. An adjuvant may thus reduce the quantity of antigen necessary to induce an immune response, and/or the frequency of injection necessary in order to generate a sufficient immune response to benefit the subject. Any compound or compounds that act to increase an immune response to an antigen and are suitable for use in a subject (e.g., pharmaceutically-acceptable) may be used as an adjuvant in compositions, vaccines, and methods of the invention. In some embodiments, the adjuvant may be the carrier molecule (for example, but not limited to, cholera toxin B subunit, liposome, etc.) in a conjugated or recombinant antigen. In alternative embodiments, the adjuvant may be an unrelated molecule known to increase the response of the immune system (for example, but not limited to attenuated bacterial or viral vectors, AMVAD, etc.). In one embodiment, the adjuvant may be one that generates a strong mucosal immune response such as an attenuated virus or bacteria, or aluminum salts.

[0076] Examples of an adjuvant include, but are not limited to, cholera toxin, E. coli heat-labile enterotoxin, liposome, immune-stimulating complex (ISCOM), immunostimulatory sequences oligodeoxynucleotide, and aluminum hydroxide. The composition can also include a polymer that facilitates in vivo delivery (See, e.g., Audran R. et al. Vaccine 21:1250-5, 2003; and Denis-Mize et al., Cell Immunol., 225:12-20, 2003). Other suitable adjuvants are well-known to those of skill in the art. Alternatively, in some embodiments, F. tularensis vaccine strains as provided herein are used in vaccines against tularemia without additional adjuvant.

[0077] F. tularensis vaccine strains as provided herein may be combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical composition suitable for administration to a subject. Pharmaceutically acceptable carriers can include a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rate of a pharmaceutical composition. Generally, a pharmaceutically acceptable carrier must be compatible with the active ingredient of the composition, optionally capable of stabilizing the active ingredient, and not deleterious to the subject to be treated. Physiologically acceptable compounds can include, e.g., water, phosphate buffered saline, a bicarbonate solution, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of glycopeptides, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (“Remington's”). One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier, diluent or excipient including a physiologically acceptable compound depends, for example, on the mode and route of administration of the vaccine, composition, or bacterial strain of the invention, and on its particular physio-chemical characteristics. In some embodiments, lyophilized F. tularensis vaccine strains as provided herein are combined with water to form a pharmaceutical composition suitable for administration to a subject.

[0078] Compositions and vaccines of the present invention may be administered by any suitable means, for example, orally, such as in the form of pills, tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intradermal, intranasal, intravenous, intramuscular, intraperitoneal or intrasternal injection or using infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, such as by inhalation spray, aerosol, mist, or nebulizer; topically, such as in the form of a cream, ointment, salve, powder, or gel; transdermally, such as in the form of a patch; transmucosally; or rectally, such as in the form of suppositories. The present compositions may also be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.

[0079] In some embodiments, pharmaceutical compositions described herein may be administered parenterally, e.g., by subcutaneous or intradermal or intramuscular injection, or using other modes of administration such as suppositories and oral formulations. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients such as pharmaceutical grades of saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. Compositions may be prepared as final products for injections, as liquid solutions, or emulsions, for example (See, e.g., U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and 4,596,792).

[0080] It is often advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic or immunogenic effect in association with the required pharmaceutical carrier. The dosage unit may be housed within a container, such as a vial, bottle, or ampoule.

[0081] In an embodiment, a composition or vaccine is prepared as an injectable, either as a liquid solution or suspension, or as a solid form which is suitable for solution or suspension in a liquid vehicle prior to injection. In another embodiment, a composition or vaccine is prepared in solid form, emulsified or encapsulated in a liposome vehicle or other particulate carrier used for sustained delivery. For example, a vaccine can be in the form of an oil emulsion, a water-in-oil emulsion, a water-in-oil-in-water emulsion, a site-specific emulsion, a long-residence emulsion, a sticky emulsion, a microemulsion, a nanoemulsion, a liposome, a microparticle, a microsphere, a nanosphere, or a nanoparticle. A vaccine may include a swellable polymer such as a hydrogel, a resorbable polymer such as collagen, or certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of a vaccine.

[0082] It will be understood that the specific dose level and frequency of dosage for any particular subject may be varied and may depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion and clearance, drug combinations, and severity of the particular condition.

Kits

[0083] Kits are provided for preventing or treating tularemia, comprising one or more F. tularensis vaccine strain, composition, and/or vaccine as described herein. Instructions for use or for carrying out the methods described herein may also be provided in a kit. A kit may further include additional reagents, solvents, buffers, adjuvants, etc., required for carrying out the methods described herein. For example, a kit may comprise a lyophilization medium comprising: mannitol, a disaccharide selected from sucrose, trehalose, and a mixture of sucrose and trehalose, and gelatin in a weight ratio of about 1 mannitol:about 1 disaccharide:about 0.25 gelatin; a lyophilization medium comprising about 1% mannitol, about 1% disaccharide selected from sucrose, trehalose, and a mixture of sucrose and trehalose, and about 0.25% gelatin in phosphate buffer; a lyophilization medium comprising 1% mannitol, 1% sucrose, and 0.25% gelatin in 10 mM phosphate buffer pH 7.2; and/or the reagents mannitol, sucrose and/or trehalose, and gelatin in a weight ratio of about 1 mannitol:about 1 disaccharide (sucrose, trehalose, or a mixture of sucrose and trehalose):about 0.25 gelatin.

[0084] The technology described herein is not meant to be limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It should also be understood that terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

EXAMPLES

[0085] The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner.

[0086] Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology.

[0087] General Procedures for Testing of Lyophilization Matrices.

[0088] For testing of lyophilization matrices in the Examples below, small volumes of F. tularensis ΔclpB mutants were grown in either Chamberlain's defined medium (CDM) (Chamberlain, R. E., Appl. Microbiol. 1965, 13: 232-5) or modified casein partial hydrolysate (MCPH) liquid medium (Karlsson, J. et al., Microb. Comp. Genomics 2000, 5(1): 25-39; Chamberlain, R. E., Appl. Microbiol. 1965, 13: 232-5). Cultures were then pelleted by centrifugation and re-suspended in an equal volume of various lyophilization matrices. For larger volumes (e.g., from 25 litres of SCHU S4 ΔclpB grown in a fermenter), tangential flow filtration could be used to exchange growth medium with lyophilization matrix. Thereafter, ΔclpB strains were lyophilized as follows:

[0089] Cultures of ΔclpB were pelleted and resuspended in (an equal volume of) lyophilization matrix. Two mL aliquots were dispensed into 10 mL glass vials, and slotted rubber stoppers were placed loosely on top. The vials were placed on the shelves of the top chamber of a Lyph-Lock 6 Liter lyophilizer (Labconco, Kansas City, MO) at ambient temperature. Next, the temperature of the upper chamber was ramped down slowly from ambient temperature to −40° C. over 60-90 minutes. The temperature was held at −40° C. for 1 more hour to let the samples freeze. The vacuum pump was then turned on until the reading for the system stabilized (usually 5-10 M BAR×10-3). The temperature of the upper chamber was then raised to −10° C. while the lower chamber remained at −40° C. The system was then run overnight for 18-19 hours under these conditions (primary drying). The next day the temperature of the upper chamber was adjusted to +20° C. over an approximately 45 minute period, then the samples were left to dry for another two hours (secondary drying). After secondary drying, the vials were sealed under vacuum by raising the bladder underneath the shelves of the upper chamber. The vacuum was turned off and vials to be stored were capped with metal caps. Samples were re-constituted with 2 mL of sterile water and plated on appropriate media to determine the viable bacterial count after lyophilization and storage for up to 3 years. This was compared to the viable bacterial count for the original re-suspension to determine the percentage loss after lyophilization.

[0090] As reported further below, the tested bacteria lost approximately 50% viability after lyophilization in 1% mannitol, 1% sucrose, 0.25% gelatin in 10 mM phosphate buffer, but thereafter remained stable for at least 1 year if stored at +4° C. or below, and for at least 3 years if stored at −20° C. or below. In contrast, the same lyophilized bacteria held at ambient temperature (22° C.) had lost essentially all viability by day 105 of storage. Additionally, we showed that diluting the bacteria by 100-fold preserved viability of ˜50% immediately post-lyophilization and for at least 3 years thereafter.

Example 1. Viability of Various F. tularensis Strains Following Lyophilization

[0091] First, a commercial lyophilization matrix called “Microbial Freeze Drying Buffer” (MFDB) from OPS Diagnostics LLC, NJ, was tested. MFDB is a general purpose lyophilization solution for use where high viability is not needed (e.g., for long-term storage of starter cultures), and is not suitable for clinical use. Nevertheless, it was tested to provide insight into the conditions needed to lyophilize live F. tularensis vaccine strains with high viability after prolonged storage. MFDB was used with several attenuated strains of F. tularensis and two different growth media (Table 1). As shown in Table 1, only SCHUS4ΔclpB grown in modified casein partial hydrolysate medium (MCPH) and lyophilized in MFDB resulted in prolonged viability when stored at −80° C. All strains grown in Chamberlain's Defined Medium (CDM) survived lyophilization with unacceptably low viability.

TABLE-US-00001 TABLE 1 Viability of various F. tularensis strains following growth in CDM or MCPH and lyophilization in MFDB. % viability Culture Lyophilization post- Storage Storage % Bacterium medium matrix lyophilization.sup.4 temperature period viability.sup.4 LVS.sup.1 CDM.sup.2 MFDB.sup.3    6% −80° C. 28 days  3.3% 18 h 210  3.4% days LVS CDM 24 h MFDB 14.61% −80° C. N/A NA SCHU S4 CDM 24 h MFDB  1.57% −80° C. N/A NA ΔclpB FSC043.sup.5 CDM 24 h MFDB  1.38% −80° C. N/A N/A LVS CDM 24 h MFDB  6.46% −80° C. N/A N/A FSC043 CDM 24 h MFDB  1.48% −80° C. N/A N/A SCHU S4 MCPH.sup.6 MFDB 51.92% −80° C. 11 days 46.15% ΔclpB 24 h 444 31.92% days SCHU S4 MCPH MFDB 48.28% −80° C. N/A N/A ΔclpB 18 h −80° C. .sup.1F. tularensis live vaccine strain. .sup.2CDM: Chamberlain’s defined medium (liquid). .sup.3MFDB: Commercial lyophilization matrix for bacteria in general. .sup.4CFU count after reconstitution/CFU count pre-lyophilization × 100. .sup.5FSC043: naturally attenuated strain of SCHU S4. .sup.6MPCH: modified casein partial hydrolysate broth.

[0092] Since a lyophilization matrix not approved for human use could preserve the viability of SCHU S4ΔclpB, alternative matrices were examined using only clinically acceptable excipients. First 10% sucrose, 1.3% gelatin, 10 mM potassium phosphate was tested, as this mix was previously used for production of clinical LVS (Table 2). However, 10% sucrose, 1.3% gelatin did not give satisfactory survival of SCHU S4ΔclpB, nor did substituting sucrose with 5% mannitol (another common lyophilization excipient).

TABLE-US-00002 TABLE 2 Lyophilization of SCHU S4 ΔclpB in sucrose or mannitol. % viability Culture Lyophilization post- Storage Storage % Bacterium medium matrix lyophilization.sup.1 temperature period viability SCHU S4 MCPH  10% sucrose, 8.89% −80° C. N/A N/A ΔclpB 18 h 1.3% gelatin   5% mannitol 0.06% 1.3% gelatin   5% mannitol 1.72% .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0093] Next we examined whether adding trehalose to the growth medium itself would improve survival after lyophilization (Table 3). Trehalose did not affect survival, although survival of bacteria grown solely in MCPH prior to lyophilization showed a 2-fold increase in viability compared to previous tests.

TABLE-US-00003 TABLE 3 Effect of adding trehalose to the growth medium on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization % viability Culture matrix 10 mM post- Storage Storage % Bacterium medium phosphate lyophilization.sup.1 temperature period viability.sup.1 SCHU S4 MCPH  10% sucrose, 22.86 −80° C. N/A N/A ΔclpB MPCH/ 1.3% gelatin 16.15% 3 mM trehelose MCPH 10% sucrose, 14.12% MPCH/  1.3% gelatin, 12.35% 3 mM  3 mM trehalose trehelose MCPH  10% sucrose, 14.19% MPCH/ 1.3% gelatin, 3 mM 10 mM trehelose trehalose 11.62% .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0094] Next, various ratios of sucrose and mannitol were tested (Table 4). Altering the ratio of either sugar from 1% caused a noticeable decline in viability after lyophilization, with no obvious pattern.

TABLE-US-00004 TABLE 4 Effect of mannitol:sucrose ratio on viability of SCHU S4 ΔclpB following lyophilization. Lyphophilization matrix 10 mM % phosphate + viability Culture % mannitol:% post- Storage Storage % Bacterium medium sucrose lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH 1:1 27.08 −80° C. NA NA ΔclpB 1:3 10.00 1:5 13.33 3:1 8.21 3:3 7.10 3:5 5.42 5:1 5.71 5:3 18.10 5:5 10.80 .sup.1CFU count after reconstitution/CFU count pre-lyphophilization × 100.

[0095] Next, sucrose was replaced by trehalose (Table 5), as trehalose had been used by others for lyophilization and short-term storage of LVS. Altering either sugar to greater than 1% was detrimental to survival after lyophilization. Survival of SCHU S4ΔclpB with either 1% mannitol+1% sucrose or +1% trehalose was similar.

TABLE-US-00005 TABLE 5 Effect of mannitol:trehalose ratios on viability of SCHU S4 ΔclpB following lyophilization. Lyophilization matrix 10 mM phosphate % viability Culture % mannitol:% post- Storage Storage % Bacterium medium trehalose lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH 1:1 30.48 −80° C. NA N/A ΔclpB 1:3 17.20 1:5 9.33 3:1 11.25 3:3 11.43 3:5 7.41 5:1 4.75 5:3 10.43 5:5 9.50 .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0096] The effect of adding gelatin to a lyophilization mixture of 1% mannitol:% sucrose was tested, with varying amounts of gelatin. Gelatin had been used previously in two distinct preparations of LVS for human vaccination (Table 6).

TABLE-US-00006 TABLE 6 Effect of different gelatin concentrations on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization matrix 10 mM phosphate 1% mannitol:1% % viability Culture sucrose + post- Storage Storage % Bacterium medium gelatin at: lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH    0% 23.33 −80° C. NA NA ΔclpB  0.5% 26.92  1.0% 16.67 1.25% 20.00  1.5% 15.38 .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0097] Since lower amounts of gelatin appeared to preserve viability better, gelatin was tested in a lower range next (Table 7).

TABLE-US-00007 TABLE 7 Effect of very low concentrations of gelatin on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization matrix 10 mM phosphate 1% % mannitol:1% viability Culture sucrose + post- Storage Storage % Bacterium medium gelatin at: lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH   0% 41.82 −80° C. N/A N/A ΔclpB  0.1% 38.15 0.25% 51.18  0.5% 31.54 0.75% 20.50 .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0098] Next, it was determined whether time of harvest of SCHU S4 cpB from flask growth in MCPH had an effect on viability following lyophilization and resuspension. In all cases, viability was greater in the presence than the absence of 0.25% gelatin (Table 8). This experiment was partially repeated using the 18h and 21 h time points to harvest the ΔclpB from MCPH broth, with similar results of ≥55% viability (Table 9). The lyophilized material was at a concentration of ˜1×10.sup.10 CFU/ml, and it had to be diluted through 6-8 log.sub.10 for manageable colony counts. Thus, the reported viabilities could be conservatively underestimated, given the potential for pipetting errors over such a large dilution range.

TABLE-US-00008 TABLE 8 Effect of gelatin and time of harvest on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization matrix 10 mM phosphate 1% mannitol:1% % viability Culture sucrose +/− post- Storage Storage % Bacterium medium 0.25% gelatin lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH 18 h w/o gelatin 25.0 −80° C. N/A N/A ΔclpB 18 h + gelatin 41.4 21 h w/o gelatin 34.6 21 h + gelatin 60.1 24 h w/o gelatin 28.9 24 h + gelatin 45.3 .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

TABLE-US-00009 TABLE 9 Effect of gelatin and time of harvest on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization matrix 10 mM phosphate 1% mannitol:1% sucrose + % viability Culture 0.25% gelatin post- Storage Storage % Bacterium medium at: lyophilization1 temperature period viability SCHUS4 MCPH 18 h w/o gelatin 15.7 −80° C. N/A N/A ΔclpB 18 h + gelatin 50.0 21 h w/o gelatin 28.2 21 h + gelatin 55.3 .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0099] It is known that cold shocking suspensions of bacteria can enhance viability after lyophilization. Therefore, we determined whether this would make a difference in the presence or absence of gelatin or trehalose (Table 10). In this case, cold shocking was achieved by cooling for 1 h at +400 with stirring. Again, the addition of 0.25% gelatin to non-cold shocked bacteria re-suspended in 10 mM phosphate buffer containing 1% mannitol and 1% sucrose resulted in approximately 50% viability following lyophilization.

TABLE-US-00010 TABLE 10 Effect of cold shocking and addition of gelatin or trehalose on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization matrix: 10 mM phosphate 1% mannitol:1% sucrose +/− % viability Culture 0.25% gelatin and/or post- Bacterium medium trehalose and/or cold shock lyophilization.sup.1 SCHUS4 MCHP, No additives or cold shock 34.3 ΔclpB 21 h No cold shock + 0.25% gelatin 50.7 cold shock but no additives 26.7 Cold shock 0.25% gelatin 43.9 Cold shock 3 mM trehalose 19.0 Cold shock + 3 mM trehalose + 40.0 0.25% gelatin .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0100] Next, the need for 10 mM phosphate buffer in the lyophilization matrix was tested. As can be clearly seen, 10 mM phosphate buffer had a significant effect to preserve the viability of ΔclpB at all dilutions (Table 11).

TABLE-US-00011 TABLE 11 Effect of 10 mM phosphate buffer on viability of SCHU S4 ΔclpB after lyophilization. Lyophilization matrix 1% mannitol:1% sucrose + % viability Culture 0.25% gelatin post- Storage Storage % Bacterium medium at: lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH, 10 mM 46.8 −80° C. N/A N/A ΔclpB 21 h phosphate neat water 7.6 1:100 10 mM 38.3 phosphate water <1% 1:1000 10 mM 22.0 phosphate water <1% .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0101] Next, lyophilized bacteria were grown in MCPH, pelleted by centrifugation and resuspended in 10 mM phosphate buffer containing 1% mannitol, 1% sucrose and 0.25% gelatin. These vials were stored at ambient (˜22° C.), refrigerator (+4° C.), or freezer (−20° C. or −80° C.) temperatures. At various times, sample vials were thawed and viability determined as shown in FIG. 1. This experiment showed that SCHU S4ΔclpB could be stored for at least one year at +4° C. or colder and still retain essentially 100% of its original post-lyophilization viability when stored below freezing, or ˜70% of its original post-lyophilization viability when stored at refrigeration temperature. The concentration of bacteria in this experiment was 2.0×10.sup.10 CFU/ml, which represents about 400 human equivalent doses of LVS, an amount that is potentially acceptable for a vaccine developed for emergency use.

[0102] It was then determined whether the aforementioned lyophilization procedure would work when the bacteria were diluted to a single dose/vial. The results are shown in FIG. 2. A modest decrease in viability at 63 days was observed with the more dilute vials, but even at 1:1000 dilution there was still ˜70% retained viability after initial lyophilization.

[0103] It was next determined whether the optimized lyophilization matrix could be used for other strains of F. tularensis, beginning with F. tularensis strain LVS (Table 12). A side-by-side comparison of commercial lyophilization media (MFDB), lyophilization media having 1% mannitol, 1% sucrose, and 0.25% gelatin, and one of the original lyophilization mediums used for LVS (higher sucrose (10%) and gelatin (1.3%) but no mannitol) was performed (Table 12). Compared to LVS grown in Chamberlain's defined medium (Table 1), LVS grown in MCPH survived lyophilization much better in MFDB. However, its viability was lower than for SCHU S4 ΔclpB using the optimized lyophilization matrix.

TABLE-US-00012 TABLE 12 Ability of optimized lyophilization matrix to preserve viability of LVS. Lyophilization % viability Culture matrix 10 mM post- Storage Storage % Bacterium medium phosphate+: lyophilization.sup.1 temperature period viability.sup.1 LVS MCPH MFDB 63.3 −80° C. 364 94.5 21 h 1% 17.22 days 18.8 mannitol:1% sucrose + 0.25% gelatin   10% sucrose, 16.15 16.7  1.3% gelatin .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0104] To determine whether the same results would be obtained with other strains of F. tularensis subspecies holarctica, we repeated the above experiment with FSC200ΔclpB. For this strain, time of harvest appeared to have a major impact on survival of bacteria after lyophilization and resuspension (Table 13). Moreover, in this case MFDB performed poorly compared to the results obtained with LVS or FSC237ΔclpB.

TABLE-US-00013 TABLE 13 Ability of optimized lyophilization matrix to preserve viability of FSC200 ΔclpB. Lyophilization % viability Culture matrix 10 mM post- Storage Storage % Bacterium medium phosphate+: lyophilization.sup.1 temperature period viability.sup.1 FSC200 MCPH MFDB 7.1 −80° C. NA NA ΔclpB 18 h 1% 2.04 mannitol:1% sucrose + 0.25% gelatin MCPH MFDB 26.7 21 h 1% 33.0 mannitol:1% sucrose + 0.25% gelatin .sup.1CFU count after reconstitution/CFU count pre-lyophilization × 100.

[0105] Finally, it was determined whether the 10 mM phosphate lyophilization medium with 1% mannitol, 1% sucrose, and 0.25% gelatin, could be used on SCHU S4ΔclpB generated under standard manufacturing conditions. To this end, a 2.0 ml frozen stock was thawed and used to inoculate 1 litre of fresh MCPH broth in a 4 litre baffle flask. The flask was incubated for 18h at 37° C. with shaking, and then 16.5 mL was transferred to a 25 litre fermenter containing 22 litres of fresh MCPH broth. After 22h of controlled growth, 200 mL of SCHU S4ΔclpB was removed from the fermenter. 50 mL of this sample was pelleted by centrifugation, re-suspended in lyophilization matrix and freeze dried neat or at 1:100 dilution and held at +4° C., −20° C. or −80° C. for 3 years. At the highest storage temperature there was about a 50% loss in viability compared to the initial post-lyophilization viability, but at −20° C. and −80° C. storage temperatures, essentially 100% viability was retained (Table 14).

[0106] Moreover, all three vaccine preparations were identical in their safety and ability to protect mice from intranasal challenge with virulent strain SCHU S4 (FIGS. 3-8). FIG. 3 shows that storage of the vaccine for 3 years at any of the aforementioned three temperatures yielded similar clinical scores in BALB/c mice immunized ID with 1×105 CFU. FIG. 4 shows the bacterial burdens of ΔclpB 4 days after ID vaccination with 1×10.sup.5 CFU. FIGS. 5A and 5B show the skin reactogenicity score at the site of injection of the mice depicted in FIG. 3. FIG. 6 shows that all three preparations of the vaccine stored for 3 years had similar lethality for BALB/c mice when given in a dose of 1×10.sup.4 CFU by the IN route. FIG. 7 shows that mice immunized ID with 1×10.sup.5 CFU of any one of the three vaccine preparations were equally protected from a subsequent IN challenge with 100 CFU of virulent strain SCHU S4 42 days later, while FIG. 8 shows that by 28 days after challenge the residual numbers of SCHU S4 in the target organs of the pathogen were also similar.

TABLE-US-00014 TABLE 14 Ability of optimized lyophilization matrix to preserve viability long-term. % viability Culture Lyophilization post- Storage Storage % Bacterium medium matrix lyophilization.sup.1 temperature period viability.sup.1 SCHUS4 MCPH, 1% 48.6  +4° C. 3 years 41.9 ΔclpB fermenter mannitol:1% −20° C. 123 22 h neat sucrose + −80° C. 138 MCPH 0.25% gelatin 24.5  +4° C. 3 years 50.3 fermenter in 10 mM −20° C. 138 22 h phosphate −80° C. 133 1:100 dilution .sup.1CFU count after storage/CFU count immediately post-lyophilization × 100.

[0107] Although this invention is described in detail with reference to preferred embodiments thereof, these embodiments are offered to illustrate but not to limit the invention. It is possible to make other embodiments that employ the principles of the invention and that fall within its spirit and scope as defined by the claims appended hereto.

[0108] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0109] The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.