# Patent

## Title
Vaccines comprising heat-sensitive transgenes

## Abstract

The present disclosure provides temperature sensitive essential nucleic acid molecules from a psychrophilic bacterium, proteins encoded by the nucleic acid molecules, as well as recombinant cells into which have been introduced such nucleic acid molecules. The disclosed recombinant cells containing one or more essential nucleic acid molecules from a psychrophilic bacterium are thereby made temperature sensitive, and can be administered to a mammal to induce an immune response in the mammal.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is a continuation application of U.S. application Ser. No. 13/496,723 filed Apr. 12, 2012, pending, which is the U.S. National Stage of International Application No. PCT/CA2010/001561, filed Oct. 7, 2010, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 61/249,385 filed Oct. 7, 2009, and U.S. Provisional Application No. 61/322,634 filed on Apr. 9, 2010, all herein incorporated by reference.


FIELD
The technology relates to genes derived from psychrophilic bacteria, for use in the development of heat-sensitive vaccines. In one example, the technology relates to recombinant pathogens harboring the heat-sensitive gene ligA from Colwellia psychrerythraea, Pseudoalteromonas haloplanktis, and Shewanella frigidimarina and to genes ligA, pyrG, hemC, ftsZ, cmk, murG, fmt, and dnaK from C. psychrerythraea. 
BACKGROUND
Vaccines against bacterial and viral diseases have played an important role in reducing infectious diseases in humans; however, there is still a need for innovative vaccines to reduce the current global burden of infectious diseases. Cold-adapted viruses have been used for decades as vaccines against human viral diseases. The best known example of such a vaccine is the Sabin polio virus vaccine. An alternate example is a cold adapted influenza vaccine called FluMist® (Medimmune LLC, Gaithersburg, Md., USA), which was introduced in the U.S. in 2003. FluMist® has been shown to be considerably more effective in certain demographic groups than influenza vaccines that practice the more common vaccination strategy of using inactivated virus to stimulate an immune response. Typically cold-adapted or “temperature-sensitive” (TS) viral strains have been developed by passing the virus repeatedly in eggs or cell culture at low temperatures and then testing the progeny for their inability to grow above about 37° C., generally thought of as the “normal” human body temperature.
The concept of a “normal” human body temperature takes into consideration anatomical sites, individual variations, gender, physiological conditions and ambient temperature. Despite the number of variables, the human body can function only in a very narrow temperature range, which is generally about 36° C.-39° C. If the human body core temperature falls to about 35° C., the body must be warmed or death will ensue. The skin temperature is always cooler than the body core regardless of the ambient temperature and clothing worn. At moderate temperatures (e.g., 21° C.), the temperature of the skin is about 32° C.-35° C.
Those skilled in these arts are of the view that bacteria generally have a set of about 100 to 150 genes, called “essential genes” that are absolutely required for maintenance of bacterial viability. Identifying essential genes is difficult due to their nature, as knockouts of these genes results in death of the organism. Essential genes encode proteins composed of amino acid sequences that are highly conserved among almost all bacterial genera and species. This conservation presumably reflects their common function and structure among the different species. A select number of essential genes have been shown to be competent in substituting for a homologue in another bacterial species and in some cases these substitutions were from distantly related bacterial species. The conservation of amino acid sequences is widespread among bacteria, the deduced amino acid sequences of essential genes from psychrophiles and thermophiles shows high identity with their mesophilic counterparts. Microbiologists have generally used conditional lethal mutations, such as TS mutations, to identify essential genes.
Many bacterial species play significant roles in the global burden of infectious diseases. However, the causative agent of tuberculosis is probably the most significant contributor to human morbidity and mortality caused by an infectious bacterial disease. Although the Bacille Calmette-Guérin (BCG) vaccine has been used for several decades to protect against tuberculosis, its low efficacy has failed to lower the incidence of tuberculosis to acceptable levels.
SUMMARY
The present disclosure provides methods for engineering, producing and using heat-sensitive host microbial cells. In one example, recombinant pathogens contain heat-sensitive essential genes, for example inserted using homologous recombination. “Psychrophile” is a term that is applied to organisms that function optimally at cold temperatures e.g., <20° C. Bacteria that live in cold ocean water, especially the Arctic and Antarctic oceans, are examples of psychrophilic bacteria. Enzymes and other proteins in psychrophilic bacteria function better in the cold than their homologous counterparts in mesophilic bacteria. Many of the enzymes from psychrophilic bacteria are also prone to denaturation at temperatures much lower than those that would affect their mesophilic counterpart. Presumably the pattern of temperature-sensitivity of psychrophilic enzymes extends to the products of essential genes.
Methods of identifying and manipulating psychrophilic essential genes with desired TS properties are provided. in vitro and in vivo recombinant technologies can be used. Francisella tularensis is the etiologic agent of the zoonotic disease, tularaemia. It can infect numerous animals by a variety of routes, and typically infects and grows in monocyte-derived cells in organs of the reticuloendothelial system. A closely related bacterium, Francisella novicida, has many of the properties of F. tularensis, and, in addition, is highly amenable to many genetic manipulations, including gene substitutions. The pathophysiology and genetic properties of F. novicida make it ideal for studying the effects of gene substitutions on a pathogenic bacterium. F. novicida is a mesophile with a maximal growth temperature of about 45° C.
This disclosure also provides methods to determine maximal growth temperature of both bacterial strains and their growth properties at restrictive temperatures. The recombinant bacterial strains tested grew below the restrictive temperature but not above the restrictive temperature. When a psychrophilic essential allele encoding an essential gene is inserted into an area of a mammalian body that is colder than the human body core, e.g., the skin, the recombinant pathogenic bacteria will have the ability to thrive thereby inducing an immune response. When the pathogenic recombinant bacteria migrate to organs in the human body core where the temperature is higher, they die and are unable to harm the host.
The present disclosure provides isolated temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium comprising at least 80%, at least 90%, or at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, 23, or 24. In some examples, the psychrophilic bacteria are operable at a temperature of about −10° C. to about 30° C., but inoperable at a temperature greater than about 30° C. Vectors and recombinant host cells (such as a recombinant bacterial host cell) that include such temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium are also provided. Immunogenic compositions that include such recombinant host bacteria (such as live or killed cells) are also disclosed. The disclosure also provides isolated proteins encoded by the disclosed isolated temperature-sensitive essential nucleic acid molecules, such as proteins having at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 26, 27, or 28.
Methods of making a temperature-sensitive microbial host cell, such as a recombinant host cell, are provided. In one example the method includes introducing (for example by inserting, substituting or replacing) a nucleic acid construct into the genome of a mesophilic bacterial strain, wherein the nucleic acid construct includes a temperature-sensitive essential nucleic acid molecule from a psychrophilic bacterial strain and one or more control sequences operably linked to the temperature-sensitive essential nucleic acid molecule, wherein the temperature-sensitive essential peptide encoded by the introduced temperature-sensitive essential nucleic acid molecule is operable (e.g., functional) at a temperature less than about 30° C. and inoperable (e.g., non-functional) at a temperature greater than about 30° C. In some examples the method also includes culturing the temperature-sensitive microbial host cell at a temperature wherein the temperature-sensitive peptide is operable, whereby said microbial host cell produces a plurality of peptides; increasing the culturing temperature to a temperature at which the temperature-sensitive peptide is inoperable; maintaining said culturing for a period of time sufficient to kill the temperature-sensitive microbial host cell; and harvesting the killed temperature-sensitive microbial host cells.
Methods for producing an immune response to a bacterium in a subject using the disclosed nucleic acid molecules, proteins, and recombinant host cells are provided. In one example the method includes administering to the subject a therapeutically effective amount of a temperature-sensitive bacterium, wherein the temperature-sensitive bacterium expresses a temperature-sensitive essential nucleic acid molecule from a psychrophilic bacterial strain, thereby inducing an immune response to the bacterium. Such methods can be used to prevent or treat a bacterial infection (such as a M. tuberculosis, Salmonella or Francisella infection).
The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a is a flowchart illustrating an exemplary method using polymerase chain reaction (PCR), FIG. 1b is a schematic chart illustrating an exemplary method showing DNA integration-excision events that result in a gene substitution.
FIG. 2a is a schematic illustrating the sequence of the wild type (wt) F. novicida ligA gene as it exists normally in a chromosome, FIG. 2b is a schematic illustrating ligACp gene substitutions into the F. novicida chromosome according to an exemplary method of the present disclosure, FIG. 2c is a schematic illustrating ligASf gene substitution into the F. novicida chromosome according to an exemplary method of the present disclosure, FIG. 2d is a schematic illustrating ligAPh gene substitutions into the F. novicida chromosome according to an exemplary method of the present disclosure, FIG. 2e is a schematic illustrating ligAPh2 gene substitutions into the F. novicida chromosome according to an exemplary method of the present disclosure.
FIG. 3a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the C. psychrerythraea ligACp gene substituted for the F. novicida homologue at 30° C., FIG. 3b is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligACp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 33° C. after 2 hours, FIG. 3c is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligACp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 34° C. after 3.5 hours, FIG. 3d is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligACp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 35° C. after 2 hours, FIG. 3e is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligACp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 37° C. after 2 hours.
FIG. 4a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the S. frigidimarina ligASf gene substituted for the F. novicida homologue at 30° C., FIG. 4b is a graph illustrating the growth curve of F. novicida with the S. frigidimarina ligASf gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 33° C. after 2 hours, FIG. 4c is a graph illustrating the growth curve of F. novicida with the S. frigidimarina ligASf gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 35° C. after 2 hours, FIG. 4d is a graph illustrating the growth curve of F. novicida with the S. frigidimarina ligASf gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 37° C. after 2 hours.
FIG. 5a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the P. haloplanktis ligAPh gene substituted for the F. novicida homologue at 30° C., FIG. 5b is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligAPh gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 33° C. after 2 hours, FIG. 5c is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligAPh gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 35° C. after 2 hours, FIG. 5d is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligAPh gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30° C. to 37° C. after 2 hours.
FIG. 6a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the P. haloplanktis ligAPh2 gene substituted for the F. novicida homologue at 21° C., FIG. 6b is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligAPh2 gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 21° C. to 26° C. after 2 hours, FIG. 6c is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligAPh2 gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 21° C. to 28° C. after 2 hours, FIG. 6d is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligAPh2 gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 21° C. to 30° C. after 2 hours.
FIG. 7 is a graph illustrating the decline in viability of wt F. novicida and F. novicida—ligACp cultures at 37° C. after being grown to late exponential phase at 33° C.
FIG. 8 is a digital image illustrating the growth of S. ser. Typhimurium-ligACP at 30° C. and the lack of growth at 37° C.
FIG. 9a is a graph illustrating the growth curve of wt Mycobacterium smegmatis and M. smegmatis-ligACp at 30° C., FIG. 9b is a graph illustrating the growth curve of M. smegmatis-ligACP and wt M. smegmatis with a temperature shift from 30° C. to 35° C. after 4 hours, FIG. 9c is a graph illustrating the growth curve of M. smegmatis-ligACP and wt M. smegmatis with a temperature shift from 30° C. to 37° C. after 4 hours.
FIGS. 10a-10d are a series of graphs showing the protective immunity induced by TS F. novicida strains.
FIGS. 11A-11L show sequences disclosed herein, with underlined portions being F. novicida sequence.



SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
SEQ ID NO: 1 is a full length nucleic acid coding sequence of the ligACp hybrid gene.
SEQ ID NO: 2 is the deduced 689 amino acid sequence of LigACp hybrid protein.
SEQ ID NO: 3 is a full length nucleic acid coding sequence of the ligAPh hybrid gene.
SEQ ID NO: 4 is the deduced 673 amino acid sequence of LigAPh hybrid protein.
SEQ ID NO: 5 is a full length nucleic acid coding sequence of the ligAPh2 hybrid gene.
SEQ ID NO: 6 is the deduced 673 amino acid sequence of LigAPh2 hybrid protein.
SEQ ID NO: 7 is a full length nucleic acid coding sequence of the ligASf hybrid gene.
SEQ ID NO: 8 is the deduced 670 amino acid sequence of LigASf hybrid protein.
SEQ ID NO: 9 is a full length nucleic acid coding sequence of the pyrGCp hybrid gene.
SEQ ID NO: 10 is the deduced 545 amino acid sequence of PyrGCp hybrid protein.
SEQ ID NO: 11 is a full length nucleic acid coding sequence of the hemCCp hybrid gene.
SEQ ID NO: 12 is the deduced 317 amino acid sequence of HemCCp hybrid protein.
SEQ ID NO: 13 is a full length nucleic acid coding sequence of the fmtCp hybrid gene.
SEQ ID NO: 14 is the deduced 327 amino acid sequence of FmtCp hybrid protein.
SEQ ID NO: 15 is a full length nucleic acid coding sequence of the murGCp hybrid gene.
SEQ ID NO: 16 is the deduced 387 amino acid sequence of MurGCp hybrid protein.
SEQ ID NO: 17 is a full length nucleic acid coding sequence of codon optimized ligACp optimized for M. tuberculosis. 
SEQ ID NO: 18 is the deduced 689 amino acid coding sequence of codon optimized LigACp hybrid protein with the first four codons changed to the M. tuberculosis form.
SEQ ID NO: 19 is a full length nucleic acid coding sequence of the dnaKCp hybrid gene.
SEQ ID NO: 20 is the deduced 638 amino acid coding sequence of DnaKCp hybrid protein.
SEQ ID NOS: 21 and 22 are a full length nucleic acid coding sequence of the essential gene tyrS from Colwellia psychrerythraea, and the corresponding amino acid sequence, respectively.
SEQ ID NO: 23 and 24 are a full length nucleic acid coding sequence of the essential gene cmk from Colwellia psychrerythraea and the corresponding amino acid sequence, respectively. As shown in FIG. 11J, F. novicida sequence is underlined. The underlined regions correspond to the F. novicida sequence in both the nucleotide and amino acid sequence. The “non-underlined” is Colwellia psychrerythraea sequence. In the amino acid sequence there is no underlined amino acids at the end since the F. novicida sequence starts at the stop codon.
SEQ ID NO: 25 and 26 are a full length nucleic acid coding sequence of the essential gene dnaKsf from Shewanella frigidimarina and the corresponding amino acid sequence, respectively. As shown in FIG. 11K, Francisella novicida sequence is underlined. The underlined regions correspond to the F. novicida sequence in both the nucleotide and amino acid sequence. The “non-underlined” shows the Shewanella frigidimarina sequence. In the amino acid sequence at the beginning (MGK) is identical between Shewanella and Francisella, so it is double underlined. The single underline at the end of the amino acid sequence corresponds to the F. novicida sequence.
SEQ ID NO: 27 and 28 are a full length nucleic acid coding sequence of the essential gene ftsZ from Colwellia psychrerythraea and the corresponding amino acid sequence, respectively. As shown in FIG. 11L, Francisella novicida sequence is underlined. The underlined regions correspond to the F. novicida sequence in both the nucleotide and amino acid sequence. The “non-underlined” regions are Colwellia psychrerythraea sequence. There is extensive F. novicida region at the 5′-end (N-terminus).
DETAILED DESCRIPTION
The following explanations of terms and methods are provided to better describe the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising a nucleic acid molecule” includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.
Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which a disclosed invention pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.
The references cited herein are incorporated by reference.
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided. Unless otherwise noted, technical terms are used according to conventional usage by those skilled in the arts
Adjuvant: A vehicle used to enhance antigenicity, for example antigenicity of a recombinant host bacterium containing a TS essential psychrophilic bacteria sequence disclosed herein. Adjuvants include a suspension of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.
Administration: The introduction of a composition (such as an immunogenic composition) into a subject (such as a mammal, for example a human) by a selected route. Exemplary routes of administration include, but are not limited to, topical, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Ameliorate: The improvement of a disease or pathological condition (such as a bacterial infection) with respect to the effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known to those skilled in the arts specific to the particular disease.
Animal: Living multi-cellular vertebrate organisms, a category that includes mammals and birds. The term “mammal” includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects (such as mice, rats, rabbits, dogs, cats, horses, and cattle).
Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
Antibodies include intact immunoglobulins and the variants and portions of antibodies well known in the art, such as Fab fragments, Fab′ fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
“Specifically binds” refers to the ability of individual antibodies to specifically immunoreact with an antigen, such as a bacterial antigen, relative to binding to unrelated proteins, such as non-bacterial proteins. The binding is a non-random binding reaction between an antibody molecule and an antigenic determinant of the T cell surface molecule. The desired binding specificity is typically determined from the reference point of the ability of the antibody to differentially bind the T cell surface molecule and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes. An antibody that specifically binds to a particular epitope is referred to as a “specific antibody”.
In some examples, an antibody specifically binds to a target (such as a bacterial protein) with a binding constant that is at least 103 M−1 greater, 104 M−1 greater or 105 M−1 greater than a binding constant for other molecules in a sample or subject. In some examples, an antibody or fragments thereof, has an equilibrium constant (Kd) of 1 nM or less. For example, an antibody binds to a target, such as a bacterial protein with a binding affinity of at least about 0.1×10−8M, at least about 0.3×10−8 M, at least about 0.5×10−8 M, at least about 0.75×10−8 M, at least about 1.0×10−8 M, at least about 1.3×10−8 Mat least about 1.5×10−8M, or at least about 2.0×10−8 M. Kd values can, for example, be determined by competitive ELISA (enzyme-linked immunosorbent assay) or using a surface-plasmon resonance device such as the Biacore T100, which is available from Biacore, Inc., Piscataway, N.J.
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term “antigen” includes all related antigenic epitopes. “Epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Generally, T cells recognize epitopes of continuous amino acids. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. An antigen can be a tissue-specific antigen, or a disease-specific antigen. These terms are not exclusive, as a tissue-specific antigen can also be a disease specific antigen. A tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. A tissue specific antigen may be expressed by more than one related type of tissue, such as alveolar and bronchial tissue. A disease-specific antigen is expressed coincidentally with a disease process. Specific non-limiting examples of a disease-specific antigen are an antigen whose expression correlates with, or is predictive of, a bacterial infection, such as tuberculosis. A disease-specific antigen can be an antigen recognized by T cells or B cells.
CD4: Cluster of differentiation factor 4, a T cell surface protein that mediates interaction with the MHC Class II molecule. CD4 also serves as the primary receptor site for HIV on T cells during HIV infection. Cells that express CD4 are often helper T cells.
CD8: Cluster of differentiation factor 8, a T cell surface protein that mediates interaction with the MHC Class I molecule. Cells that express CD8 are often cytotoxic T cells. “CD8+ T cell mediated immunity” is an immune response implemented by presentation of antigens to CD8+ T cells.
Contacting: The process of incubating one agent in the presence of another. Thus, when a cell is contacted with an agent (such as an immunogenic composition), the cell is incubated with the agent for a sufficient period of time for the agent and the cell to interact.
Cool parts of the body: Regions of a human or other mammalian body that generally have a lower temperature than other parts of the body. The concept of natural human (or other mammal) body temperature variation due to anatomical sites, gender, physiological and ambient temperature. Despite the number of variables, the human (or other mammalian) body can function only in a very narrow temperature range, hence, for example the human body core remains at about 36° C.-39° C. Cool parts of the body include skin, mouth and rectum. Skin temperature, for example, is about 32° C.-35° C. Thus, in some examples, cool parts of the body have temperatures that are at least 1° C. less, at least 2° C. less, at least 3° C. less, at least 4° C. less, at least 4° C. less, or at least 6° C. less, such as 1° C. to 8° C. less, 1° C. to 6° C. less, 2° C. to 6° C. less, or 2° C. to 4° C. less, than other parts of the body, such as the core.
Cytokine: Proteins made by cells that affect the behavior of other cells, such as lymphocytes. In one embodiment, a cytokine is a chemokine, a molecule that affects cellular trafficking. Specific, non-limiting examples of cytokines include the interleukins (IL-2, IL-4, IL-6, IL-10, IL-21, etc.), and IFN-γ.
Degenerate variant: A TS essential psychrophilic bacteria nucleic acid sequence that encodes a TS essential psychrophilic bacteria protein that includes a nucleic acid sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure as long as the amino acid sequence of the TS essential psychrophilic bacteria peptide encoded by the nucleotide sequence is unchanged.
EmR: Erythromycin resistance.
Essential gene: A gene that is necessary for the growth of the organism (such as a mesophilic bacterium) under all culturing conditions.
Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5′ or 3′ regions of the gene. Both constitutive and inducible promoters, are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. In one embodiment, the promoter is a cytomegalovirus promoter.
Heat-sensitive: An inability to perform an essential biological function at temperatures above about 28° C. Similarly, the term “heat-sensitive protein or polypeptide” refers to a non-functional mature protein resulting from heat-induced deactivation. An enzyme that does not catalyze its known reaction efficiently enough to support growth, development or life of the organism above about 28° C. is an example of such a protein.
Heat-sensitive allele: An allele comprising a gene encoding a heat-sensitive protein. Similarly the term, “heat-sensitive gene” refers to a gene encoding a heat-sensitive protein.
Host cells: Cells into which a heterologous nucleic acid molecule has been introduced. For example, such cells may include a nucleic acid vector that is propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The cell can be prokaryotic, such as a bacterial cell. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen or a particular TS recombinant microbial cell, such as mesophilic bacteria containing a psychrophile essential nucleic acid molecule provided herein. In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies. The development of an immune response following administration of mesophilic bacteria containing a psychrophile TS essential nucleic acid molecule can be measured using routine methods known in the art, for example by measuring cytokine production as an indication of a protective immune response.
Immunogenic composition: Compositions that include recombinant mesophilic bacteria containing a psychrophile TS essential nucleic acid molecule that induces a measurable CTL response against a recombinant mesophilic bacteria protein, or induces a measurable B cell response (such as production of antibodies that specifically bind a recombinant mesophilic bacteria-specific protein) against a recombinant mesophilic bacteria protein. For example, the immunogenic polypeptide or a nucleic acid encoding the immunogenic polypeptide can be present in a heat-sensitive mesophilic bacteria generated using the methods provided herein, wherein the bacteria is art of an immunogenic composition that can further include pharmaceutically acceptable carriers, and/or other therapeutic agents. An immunogenic composition can optionally include an adjuvant, a PD-1 antagonist, a co-stimulatory molecule, or a nucleic acid encoding a costimulatory molecule. An immunogenic composition can be readily tested for its ability to induce a CTL by art-recognized assays.
Immunogenic peptide: A peptide which comprises an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response (e.g. antibody production) against the antigen from which the immunogenic peptide is derived. Immunogenic peptides can also be identified by measuring their binding to a specific MHC protein and by their ability to stimulate CD4 and/or CD8 when presented in the context of the MHC protein.
Generally, immunogenic polypeptides can be used to induce an immune response in a subject, such as a B cell response or a T cell response. In one example, an immunogenic polypeptide, when bound to a MHC Class I molecule, activates cytotoxic T lymphocytes (CTLs) against the polypeptide. Induction of CTLs using synthetic peptides and CTL cytotoxicity assays are known in the art, see U.S. Pat. No. 5,662,907. In one example, an immunogenic peptide includes an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) response against the antigen from which the immunogenic peptide is derived.
Immunologically reactive conditions: Conditions that allow an antibody specific for a particular epitope to bind to that epitope to a greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. These conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. The immunologically reactive conditions employed in the disclosed methods are “physiological conditions” which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organ and intracellular environment is generally about pH 7 (e.g., from pH 6.0 to pH 8.0, or pH 6.5 to pH 7.5, such as pH 7.2), contains water as the predominant solvent, and exists at a temperature above 0° C. and below 50° C. Osmolarity is within the range that is supportive of cell viability and proliferation. These conditions are well known to those skilled in these arts.
Interferon gamma (IFN-γ): IFN-γ is a dimeric protein with subunits of 146 amino acids. The protein is glycosylated at two sites, and the pI is 8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acids including a secretory signal sequence of 23 amino acids. Two molecular forms of the biologically active protein of 20 and 25 kDa have been described. Both of them are glycosylated at position 25. The 25 kDa form is also glycosylated at position 97. The observed differences of natural IFN-γ with respect to molecular mass and charge are due to variable glycosylation patterns. 40-60 kDa forms observed under non-denaturing conditions are dimers and tetramers of IFN-γ. The human gene has a length of approximately 6 kb. It contains four exons and maps to chromosome 12q24.1.
IFN-γ can be detected by sensitive immunoassays, such as an ELISPOT test that allows detection of individual cells producing IFN-γ. Minute amounts of IFN-γ can be detected indirectly by measuring IFN-induced proteins such as Mx protein. The induction of the synthesis of IP-10 has been used also to measure IFN-γ concentrations. In addition, bioassays can be used to detect IFN-γ, such as an assay that employs induction of indoleamine 2,3-dioxygenase activity in 2D9 cells. The production of IFN-γ can be used to assess T cell activation, such as activation of a T cell by bacterial antigen.
Isolated: A biological component (such as a nucleic acid molecule, protein or organelle) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acid molecules and proteins that have been “isolated” include nucleic acid molecules and proteins purified by standard purification methods. In another embodiment, “isolated” refers to nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
ligA: A wt allele of the gene encoding NAD-dependent DNA ligase found in mesophilic bacteria such as F. novicida, M. smegmatis or E. coli. Furthermore, ligA with a subscript, such as ligACp, ligASf, or ligAPh, refers to a wt allele of the gene encoding NAD-dependent DNA ligase found in psychrophilic bacteria. For example ligACp refers to the wt allele of ligA found in the Arctic bacterium C. psychrerythraea strain 34H which has a maximal growth temperature below 18° C. The ligA sequences from psychrophilic bacteria can be introduced into mesophilic bacteria, to confer temperature sensitivity to the mesophilic bacteria.
Mesophile: An organism naturally found in environments at temperatures between about 20° C. and 50° C. A bacterial mesophile refers to a bacterium that is normally associated with a mammal and thus is normally functioning at temperatures between about 32° C. and 45° C.
Psychrophile: An organism naturally found in environments that are permanently below 20° C., often permanently below 10° C. and sometimes below 0° C. Such permanently cold environments include most ocean environments, permafrost soils, Arctic and Antarctic environments. Those skilled in these arts will understand that “psychrophile” and “psychrotroph” are commonly used to describe bacteria that grow in cold environments.
Psychrophilic: Features found in psychrophiles. For example, a “psychrophilic enzyme” is an enzyme isolated from a psychrophile.
Peptide modifications: Analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) of proteins that can be used in the methods and compositions provided herein. Peptides are comprised of amino acids, which may be either L- and/or D-amino acids, naturally occurring and otherwise. The peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. Modifications are well known to those skilled in these arts.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic composition, such as an immunogenic composition.
The disclosed purified active compositions can be administered alone or combined with an acceptable carrier. Preparations can contain one type of therapeutic molecule, or can be composed of a combination of several types of therapeutic molecules. The nature of the carrier will depend on the particular mode of administration being utilized.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Preventing or treating a disease: “Preventing” a disease refers to inhibiting the full development of a disease, for example in a person who is known to be at risk of infection with M. tuberculosis, or M. leprae. An example of a person with a known predisposition is someone living with a person diagnosed with tuberculosis, health care professionals, or someone otherwise known to have been exposed to M. tuberculosis. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as tuberculosis, after it has begun to develop.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein is more pure than the protein in its originating environment within a cell. A preparation of a protein is typically purified such that the protein represents at least 50% of the total protein content of the preparation. However, more highly purified preparations may be required for certain applications. For example, for such applications, preparations in which the protein includes at least 75% or at least 90% of the total protein content may be employed.
Recombinant: A nucleic acid molecule that has a sequence not naturally occurring or a sequence that is made by an artificial combination of two naturally separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, by genetic engineering techniques, for example. Also refers to cells into which a non-native nucleic acid molecule has been introduced.
Resistant to infection: Animals (e.g., mammals) that demonstrate decreased symptoms of infection compared to non-resistant animals. Evidence of resistance to infection can appear as, for example, lower rates of mortality, increased life spans measured after exposure to the infective agent, fewer or less intense physiological symptoms, such as fewer lesions, or decreased cellular or tissue concentrations of the infective agent. In one embodiment, resistance to infection is demonstrated by a heightened immune response.
Restrictive temperature: The lowest temperature at which an organism is unable to grow. For example, in Table 1 “restrictive temperature” specifically refers to the lowest temperature at which the F. novicida strain with a psychrophilic gene integrated is unable to form an isolated colony on agar media. Due to the variation in the temperature of incubators, these temperatures are interpreted as being about ±1° C.
sacB cassette: A modular DNA sequence encoding the enzyme levansucrase from Bacillus subtilus. Expression of this gene is lethal in the presence of sucrose to many bacteria and can thus be used as a counter-selection agent to help select for the loss of gene sequences.
Selective hybridization: Hybridization under moderately or highly stringent conditions that exclude non-related nucleotide sequences, the techniques of hybridization are known to those skilled in these arts.
Sequence identity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1154 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 15-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15÷20*100=75).
For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 30% sequence identity or more counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a protein disclosed herein. Thus in one example, a protein that can be used in the disclosed methods and compositions has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 and retains the ability to confer TS (such as heat-sensitivity) to a mesophilic bacteria.
One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with a disclosed nucleic acid sequence as determined by this method. Thus in one example, a nucleic acid sequence that can be used in disclosed methods and compositions has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and retains the ability to encode a protein that can confer TS (such as heat-sensitivity) to a mesophilic bacteria. An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the peptide which the first nucleic acid encodes is immunologically cross reactive with the peptide encoded by the second nucleic acid.
Temperature-sensitive (TS)” or “heat-sensitive (HS): A bacterial component (such as a protein) or bacterium that is active up to about 30° C. and inactivated at a temperature that is normally found in the human body, e.g., above about 30° C.
Tester strain: A mesophilic bacterium that is amenable to gene replacement allowing the substitution of a psychrophilic essential gene for the homologue naturally found in the tester strain.
Therapeutically effective amount: An amount of a composition that alone, or together with an additional therapeutic agent(s) sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent (such as an immunogenic composition provided herein) can be dependent on several factors, including, but not limited to the subject or cells being treated, the particular therapeutic agent, and the manner of administration of the therapeutic composition. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease, such as a bacterial infection (e.g., tuberculosis).
In one example, a desired response is to reduce or inhibit one or more symptoms associated with a bacterial infection. The one or more symptoms do not have to be completely eliminated for the composition to be effective. The effective amount of an agent that includes one of the disclosed immunogenic compositions that is administered to a human or veterinary subject will vary depending upon a number of factors associated with that subject, for example the overall health of the subject. An effective amount of an agent can be determined by varying the dosage of the product and measuring the resulting therapeutic response, such as the prevention of bacterial infection. Effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays. The disclosed agents can be administered in a single dose, or in several doses, as needed to obtain the desired response.
In particular examples, a therapeutically effective dose of an immunogenic composition includes at least 102 colony forming units (CFU), such as at least 103, at least 104, at least 105, at least 106, at least 107, or at least 108 CFU, for example 102 to 108 CFU. In one example, 102 to 108 CFU of live bacteria are administered intradermally or intranasally. However, one skilled in the art will recognize that higher or lower dosages also could be used, for example depending on the particular immunogenic composition. In particular examples, such daily dosages are administered in one or more divided doses (such as 2, 3, or 4 doses) or in a single formulation. The disclosed immunogenic composition can be administered alone, in the presence of a pharmaceutically acceptable carrier, in the presence of other therapeutic agents.
Treatment: A therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. In one example, the immunogenic compositions disclosed herein following administration to a mammal achieves a reduction in one or more signs of a bacterial infection.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transduced or transformed host cell, referred to herein as a recombinant cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker gene and other genetic elements known in the art. Vectors include plasmid vectors, including plasmids for expression in gram negative and gram positive bacterial cells. Exemplary vectors include those for expression in E. coli and Salmonella. Vectors also include viral vectors, such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.
Temperature-Sensitive Essential Genes from Psychrophilic Bacteria
It is disclosed herein that several nucleic acid molecules, and their corresponding peptides, can be introduced into a bacteria to confer temperature sensitivity (TS), such as heat-sensitivity, to the host bacteria. The resulting bacteria can be used to induce an immune response to the temperature-sensitive bacteria, such as a T cell response. Exemplary psychrophilic essential genes with desired temperature sensitivity, and their corresponding peptides, are provided herein. For example, host mesophilic bacteria can be transformed with one or more psychrophile TS essential nucleic acid molecules, thereby conferring TS to the mesophilic bacteria. The resulting recombinant mesophilic bacteria can be formulated into an immunogenic composition, to treat or prevent infection by the meosophilic bacteria. For example, recombinant mesophilic M. tuburculosis bacterium containing one or more psychrophile TS essential nucleic acid molecules can be used to treat or prevent tuberculosis. The same approach can be used to make TS forms of Bacillus anthracis, Brucella abortus, Burkholderia pseudomallei, Haemophilus influenzae, Mycobacterium bovis, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus, Streptococcus pneumoniae, and Yersinia pestis which cause anthrax, brucellosis, melioidosis, meningitis, bovine tuberculosis, typhoid fever, dysentery, numerous types of nosocomial infections, pneumonia, and plague. Thus, such TS bacteria can be used to treat or prevent such conditions.
Temperature-sensitive essential proteins from a psychrophilic bacterium are provided herein, such as those from Colwellia sp., Psuedoalteromonas sp., or Shewanella sp. Exemplarily proteins include ligA, pyrG, hemC, ftsZ, cmk, murG, fmt, and dnaK. Exemplary sequences are provided in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. However, one skilled in the art will appreciate that variant sequences can also be used. For example, a peptide having a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 is encompassed by the present disclosure, and can be used in the methods provided herein. Variant sequences retain the biological activity of the native temperature-sensitive essential protein from a psychrophilic bacterium, such as conferring the ability to make a bacterium TS (such as heat sensitivity), for example operable at a temperature of −10° C. to about 30° C. (such as 0° C. to 30° C.), but inoperable at a temperature greater than about 30° C. (for example 4° C. to 30° C.), such as greater than 35° C. Exemplary sequences can be obtained using computer programs that are readily available on the internet and the amino acid sequences set forth herein. In one example, the variant peptide retains a function of the native protein, such as the ability to confer temperature sensitivity to a bacterium.
A specific, non-limiting example of a variant protein is a conservative variant of the native protein (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28). Substitutions of the amino acids sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 can be made based on this table, as long as the pathogenic mesophilic bacteria are rendered TS and are able to initiate an immune response to its pathogenic antigens. For example, protein sequences can be altered without significantly altering their biological properties, for example by introducing one or more conservative amino acid substitutions. Therefore, any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 can be modified by making 1 to 20, 1 to 15, 1 to 12, 1 to 10, or 1 to 5 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 50 conservative amino acid substitutions, while retaining the ability to render a mesophilic bacteria temperature sensitive (TS). Examples of conservative substitutions are shown below:













Original Residue
Conservative Substitutions








Ala
Ser



Arg
Lys



Asn
Gln, His



Asp
Glu



Cys
Ser



Gln
Asn



Glu
Asp



His
Asn; Gln



Ile
Leu, Val



Leu
Ile; Val



Lys
Arg; Gln; Glu



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr



Thr
Ser



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu









Minor modifications to the disclosed protein sequences can result in peptides which have substantially equivalent activity as compared to the unmodified counterpart protein described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the proteins produced by these modifications are included herein.
Temperature-sensitive essential proteins (and nucleic acid molecules) from a psychrophilic bacterium are disclosed herein that can be used to induce temperature sensitivity in a desired bacterial host, wherein the resulting recombinant bacteria can be used to induce an immune response (for example in a mammal). These peptides can include fragments of the full-length native protein, as long as the ability to confer temperature sensitivity in the host cell is retained. In these examples, the peptide does not include the full-length amino acid sequences set forth as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. For example no more than 10%, no more than 5%, or no more than 1% of the amino acids can be deleted, such as 1% to 5% of the amino acids.
The isolated temperature-sensitive essential proteins can be part of a fusion protein. Thus, the fusion protein can include the temperature-sensitive essential protein (see above) and a second heterologous moiety, such as a myc protein, an enzyme or a carrier (such as a hepatitis carrier protein or bovine serum albumin) covalently linked to the temperature-sensitive essential protein. In additional examples, the temperature-sensitive essential protein includes six sequential histidine residues, a f3-galactosidase amino acid sequence, or an immunoglobulin amino acid sequence, for example at the C- or N-terminus of the temperature-sensitive essential protein. The temperature-sensitive essential protein can also be covalently linked to a carrier. Suitable carriers include, but are not limited to, a hepatitis B small envelope protein HBsAg.
The temperature-sensitive essential proteins disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly. An exemplary process for polypeptide production is described in Lu et al., Federation of European Biochemical Societies Letters. 429:31-35, 1998. Proteins can also be produced using molecular genetic techniques, such as by inserting a nucleic acid encoding a temperature-sensitive essential protein into an expression vector, introducing the expression vector into a host cell. They can also be isolated by methods including preparative chromatography and immunological separations.
Temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium are provided herein. Exemplary sequences are provided in the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. However, one skilled in the art will appreciate that variant sequences can also be used. For example, a nucleic acid molecule having a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence set forth in one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence set forth in nucleotides 10-2067 of SEQ ID NO: 1, nucleotides 10-2019 of SEQ ID NO: 3, nucleotides 10-2019 of SEQ ID NO: 5, nucleotides 10-2010 of SEQ ID NO: 7) is encompassed by the present disclosure, and can be used in the methods provided herein. In some examples, the codons of a nucleic acid molecule are optimized for the bacterium into which it is introduced. In some examples, such optimization does not alter the amino acid sequence encoded thereby. For example, the psychrophilic bacterium TS essential nucleic acid can be modified to optimize codon usage for the mesophilic bacterium (e.g., M. tuberculosis or F. novicida) into which the psychrophilic bacterium TS essential nucleic acid is introduced. Exemplary sequences can be obtained using computer programs that are readily available on the internet and the nucleic acid sequences set forth herein. In one example, the variant nucleic acid sequence retains the ability to encode a protein having the function of the native protein, such as the ability to confer temperature sensitivity (e.g., heat sensitivity) to a mesophilic bacterium.
The disclosed temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium include DNA, cDNA and RNA sequences which encode the temperature-sensitive essential peptide. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (e.g., L. Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H. 5 Freeman and Co., NY).
A nucleic acid molecule encoding a temperature-sensitive essential peptide from a psychrophilic bacterium can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Qβ replicase amplification system (QB). For example, a polynucleotide encoding the protein can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art. PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent hybridization conditions.
The nucleic acid molecules encoding a temperature-sensitive essential peptide from a psychrophilic bacterium include a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleic acid molecules disclosed herein can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.
The nucleic acid molecules encoding a temperature-sensitive essential peptide from a psychrophilic bacterium can be part of a vector, such as a plasmid or viral vector. Suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors and poliovirus vectors. Specific exemplary vectors are poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MVA), adenovirus, baculovirus and the like. Other viral vectors that can be used include other DNA viruses such as herpes virus and adenoviruses, and RNA viruses such as retroviruses and polio.
The nucleic acid molecules encoding a temperature-sensitive essential peptide from a psychrophilic bacterium can be operably linked to at least one expression control element. The expression control elements are inserted in the vector or plasmid to control and regulate the expression of the nucleic acid sequence. For example, an expression control sequence operatively linked to a temperature-sensitive essential peptide coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. Specific examples of expression control elements include, but are not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40. Additional operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the temperature-sensitive essential peptide from a psychrophilic bacterium in the host system. The expression vector can contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods (Ausubel et al., (1987) in “Current Protocols in Molecular Biology,” John Wiley and Sons, New York, N.Y.) and are commercially available.
In one example, vector introduced into a host bacterium includes one or more of the following elements: (i) a prokaryotic origin of replication, so that the vector may be amplified in a prokaryotic host; (ii) a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector (e.g., a gene encoding antibiotic resistance); (iii) at least one DNA sequence encoding one or more temperature-sensitive essential peptides from a psychrophilic bacterium located adjacent to a transcriptional promoter capable of directing the expression of the sequence; and (iv) DNA sequences homologous to the region of the parent virus genome where the foreign gene(s) will be inserted, flanking the construct of element (iii).
The vector can contain an additional gene that encodes a marker that will allow identification of recombinant cells containing inserted foreign DNA. These include genes that encode antibiotic or chemical resistance (e.g., see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988, J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), as well as genes such as the E. coli lacZ gene, that permits identification of recombinant plaques by colorimetric assay.
Methods of introducing nucleic acid molecules, such as those that encode a temperature-sensitive essential peptide from a psychrophilic bacterium, are well known to those skilled in the art. Where the host is prokaryotic, such as, a bacterium, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbC1 can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation. Hosts cells can include bacterial cells, such as bacteria that cause disease. Examples of such bacteria that can be used as host cells for temperature-sensitive essential nucleic acids/peptides from a psychrophilic bacterium include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella bumetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaminogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia among others.
Following transformation of bacterial cells, recombinant host cells can be identified by one of several techniques. For example, expression of a gene encoding a marker or indicator gene with the temperature-sensitive gene, as described above, can be used to identify recombinant progeny. One specific non-limiting example of an indicator gene is the E. coli lacZ gene. Recombinant bacterial cells expressing beta-galactosidase can be selected using a chromogenic substrate for the enzyme. Once a recombinant bacterium has been identified, it can be selected and amplified for use in an immunogenic composition provided herein.
Methods of Making Temperature-Sensitive Bacterial Strains
The exemplary embodiments relate to methods for generating recombinant TS bacteria for use in stimulating an immune response to the TS bacteria. In one aspect, an exemplary TS immunogenic composition is suitable for immunoprophylaxis to prevent infectious disease or alternatively immunotherapy to treat an infectious disease. Such TS bacteria are generated by the introduction of one or more TS essential genes from psychrophilic bacteria into a target bacteria (such as a mesophilic bacteria that causes a disease that one wants to treat or prevent). Thus, the disclosure provides safe immunogenic compositions based on live genetically altered bacterial microorganisms. This was accomplished by taking advantage of essential genes from psychrophilic bacteria, by creating a fusion of the psychrophilic structural genes with the transcriptional and translational control elements of the “host” genome or by making fusions between the host gene and the psychrophilic gene. The exemplary embodiments provide live vaccines and immunogenic compositions that mimic a number of cold adapted viral vaccines and are unable to grow at the normal body temperature.
According to another exemplary embodiment it is suitable for mass production purposes, specifically of antigen; due to the TS strain's non-virulent nature the aerosols produced are rendered harmless and therefore, this methods and compositions disclosed herein can significantly reduce or eliminate human risk of infection.
Another aspect, the methods and compositions provided herein has value as a research diagnostic, or as a research/educational tool because it allows for experimentation to be performed on organisms that are normally highly pathogenic in their viable state without posing threats to the researcher.
The methods and compositions provided herein can be employed to stimulate the immune system with TS organisms with the intention of prevention or treatment of a disease.
A large number of psychrophilic bacteria contain TS genes, which can be used to generate TS mesophilic bacteria of the present disclosure. For example, one or more TS essential genes from psychrophilic bacteria can be introduced into a mesophilic bacterium (for example into a chromosome of a mesophilic bacteria), thereby generating a TS strain that can be used to induce an immune response in a subject into whom it is administered. Recombinant methods for introducing a nucleic acid into bacteria are routine in the art. Appropriate TS essential genes from psychrophilic bacteria can be identified using the methods provided herein. As shown in Tables 1 and 2, nine of the twenty one essential genes from the psychrophilic C. psychrerhraea were introduced into F. novicida and substituted for an essential host gene to generate TS strains of F. novicida (“Group I”). Group I genes generated a range of TS phenotypes with the restrictive temperatures of about 33° C. to 44° C. Thus, the genes of Group I can be used to generate TS strains of the present disclosure. Group II in Table 1 consists of the C. psychrerhraea genes that either functioned poorly or not at all in the exemplary bacterial strain F. novicida. F. novicida strains carrying an integrate with the psychrophilic essential gene resolve the integrate under counter selection pressure generated by the presence of sacB and sucrose. However, the resolved strains retain copies of both the psychrophilic gene and the F. novicida homologue and the strains are not TS (“Group III” in Table 1); indicating that these psychrophilic essential genes do not function in the mesophilic host. Alleles of the same gene from different psychrophilic bacteria can be selected to identify those that generate hybrid strains with the same TS properties when substituted into the chromosomes of mesophilic bacteria. The ligA alleles from three different psychrophilic bacteria generated three different TS phenotypes when substituted into the mesophile F. novicida. The pyrGCp allele from C. psychrerhraea created a TS strain when substituted into F. novicida but the pyrGSf allele from S. frigidimarina (SF) did not. PH refers to P. haloplanktis.










TABLE 1






Restrictive




Gene
Temp.(° C.)




symbol
Source
Product Function
Group







ligAPh2
28/PH
NAD-dependent DNA ligase
I


ligASf
33/SF
NAD-dependent DNA ligase
I


ligACp
34/CP
NAD-dependent DNA ligase
I


ligAPh
36.8/PH
NAD-dependent DNA ligase
I


hemCCp
36.8/CP
Porphobilinogen deaminase
I




(Hydroxymethylbilane synthase)



pyrGCp
37.2/CP
CTP synthetase
I


dnaKCp
38.2/CP
Molecular chaperone DnaK
I


murGCp
38.2/CP
UDP-N-acetylglucosamine-N-
I




acetylmuramyl-(pentapeptide) 





pyrophosphoryl-undecaprenol N-





acetylglucosamine transferase



fmtCp
41/CP
Methionyl-tRNA formyltransferase
I


ftsZCp
42/CP
Cell division protein
I


cmkCp
43/CP
Cytidylate kinase
I


tyrSCp
44/CP
Aminoacyl tRNA synthetases for Tyr
I


adkCp
>44/CP
Adenylate kinase (proved resolution)
II


accDCp
>44/CP
AcetylCoA carboxylase. The F. novicida
II




integrate containing accDCp fails to resolve.



murICp
>44/CP
Glutamate racemase. The CP version of
II




MurI appears to function poorly at all





temperatures in F. novicida.



pyrGSf
>44/SF
CTP synthetase
III


trxACp
>44/CP
Thioredoxin
III


g/mSCp
>44/CP
Glucosamine—fructose-6-phosphate
III




aminotransferase



argSCp
>44/CP
Aminoacyl tRNA synthetases for Arg
III


cdsCp
>44/CP
phosphatidate cytidylyltransferase
III


murCpC
>44/CP
UDP-N-acetylmuramate—alanine ligase
III


valSCp
>44/CP
Aminoacyl tRNA synthetases for Val
III


proSCp
>44/CP
Aminoacyl tRNA synthetases for Pro
III


metKCp
≦44/CP
S-adenosylmethionine synthetase
III


ftsWCp
>44/CP
Cell division protein
III

















TABLE 2








Mutation rate in F. novicida to



temperature resistance













Re-
Chal-






stricted
lenge






Temp
Temp





Gene
(° C.)
(° C.)
Trial #1
Trial #2
Trial #3















ligASf
33
37
 4.0 × 10−6
 3.3 × 10−7
 9.7 × 10−7


ligACp
34
37
<1.2 × 10−10
<7.93 × 10−11 
<1.1 × 10−10


ligAPh
36.8
39
<1.5 × 10−10
<7.8 × 10−11
<6.2 × 10−11


dnaKCp
38.2
39.5
<3.2 × 10−10
<1.9 × 10−10
<3.2 × 10−10


hemCCp
36.8
43
<2.5 × 10−10
<3.6 × 10−11
<3.7 × 10−11


pyrGCp
37.2
40
 8.5 × 10−8
 1.0 × 10−9
 6.5 × 10−8


murGCp
38.2
43
 2.6 × 10−4
 3.0 × 10−5
 8.5 × 10−5


dnaKSf
39
42
 3.1 × 10−10
 8.5 × 10−10









To make a TS bacterial pathogen, an essential gene from an Arctic psychrophile bacterium was substituted into the genome of a mesophilic pathogenic bacterium. The Arctic bacterial essential gene ligASf rendered F. novicida unable to grow at a temperature of 33° C. or higher. Table 2 outlines the restrictive temperature properties imposed on F. novicida following the replacement of the mesophilic essential gene for its psychrophilic counterpart. Any of the genes in Table 2 may be introduced into a pathogenic bacteria strain to create live heat-sensitive vaccines. Exemplary pathogenic bacteria include but are not limited to: Mycobacterium sp., Haemophilus sp., Vibrio sp., Escherichia sp., Salmonella sp., Streptococcus sp., Burkholderia sp., Campylobacter sp., Neisseria sp., and Francisella sp.
The disclosure relates to genes derived from psychrophilic bacteria for use in the development of heat-sensitive immunogenic compositions, and methods of using these compositions to stimulate an immune response in a subject. In a specific example, the disclosure provides recombinant pathogens (such as Mycobacterium sp., Haemophilus sp., Vibrio sp., Escherichia sp., Salmonella sp., Streptococcus sp., Burkholderia sp., Campylobacter sp., Neisseria sp., and Francisella sp.) containing one or more heat-sensitive genes, exemplified by ligA, pyrG, hemC, ftsZ, cmk, dnaK, and fmt, that can be administered to a subject to provide a prophylactic immune response against diseases caused by such bacteria.
Methods of making a recombinant temperature-sensitive (TS) bacterial cell are provided. In one example the method includes introducing into the genome of a mesophilic bacterial strain a nucleic acid construct that includes a TS essential nucleic acid molecule from a psychrophilic bacteria (such as one that encodes a peptide that is operable at a temperature of about −10° C. to about 30° C., and/or inoperable at a temperature greater than about 30° C., for example Colwellia sp., Psuedoalteromonas sp., or Shewanella sp) and one or more control sequences operably linked to the TS essential nucleic acid molecule. The temperature-sensitive essential polynucleotide renders the mesophilic bacteria operable at a temperature less than about 30° C. and inoperable at a temperature greater than about 30° C. In some examples, the temperature-sensitive essential nucleic acid molecule includes a nucleotide sequence having at least 80%, at least 90%, or at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In some examples the method also includes isolating the TS essential nucleic acid molecule from the genome of the psychrophilic bacterial strain. The method can also include constructing or generating the nucleic acid construct comprising the TS essential nucleic acid molecule and one or more control sequences operably linked to the TS essential nucleic acid molecule.
In some examples, the method further includes culturing the recombinant TS bacterial host cell at a temperature wherein the temperature-sensitive peptide is operable, whereby said recombinant TS bacterial host cell produces a plurality of peptides; increasing the culturing temperature to a temperature at which the temperature-sensitive peptide is inoperable; maintaining said culturing for a period of time sufficient to kill the recombinant TS bacterial host cell; and harvesting the killed recombinant TS bacterial host cells.
Methods of making a recombinant TS bacterial host cell can also include the following. A psychrophilic microbial genome is screened for detection of a TS essential polynucleotide that encodes a peptide that is inactivated at about greater than 30° C.; isolating said TS essential polynucleotide; constructing a nucleic acid construct comprising the TS essential polynucleotide and one or more control sequences operably linked to the TS polynucleotide; inserting the nucleic acid construct into the genome of a selected mesophilic bacterial host cell (such as Francisella novicida) thereby functionally replacing the host cell's homologue of the TS essential polynucleotide whereby the TS peptide (and thus the bacteria in which it is expressed) is operable at a temperature less than about 30° C., and inoperable at a temperature greater than about 30° C. and mimics the temperature sensitivity of the original designated host bacterium. The resulting recombinant mesophilic bacterial host cell comprising the TS polynucleotide is cultured or grown at a temperature less than about 30° C. to confirm the viability of the recombinant mesophilic bacterial host cell; further culturing the recombinant mesophilic bacterial host cell comprising the TS polynucleotide at a temperature greater than about 30° C. to determine if the mesophilic bacterial host cell is killed. If the mesophilic bacterial host cell is killed, the nucleic acid construct is introduced into the genome of a selected destination mesophilic bacterial host cell (such as Salmonella sp. or Mycobacterium sp.) thereby functionally replacing the host cell's homologue of the temperature-sensitive essential polynucleotide whereby the temperature-sensitive peptide (and thus the bacteria in which it is expressed) is operable at a temperature less than about 30° C., and inoperable at a temperature greater than about 30° C. and mimics the temperature sensitivity of the original tester host bacterium.
In some examples, the mesophilic bacteria is one that is operable at a temperature selected from the range of about 10° C. to about 50° C. prior to introduction of the TS essential nucleic acid molecule from a psychrophilic bacteria. Examples of such mesophilic bacteria include strains of fermentative bacteria or bioremediation bacteria. Other exemplary bacteria are provided above.
In some examples, the TS essential nucleic acid molecule expresses a peptide during a culturing of the recombinant TS bacteria, such as a peptide having at least 80%, at least 90%, or at least 95% sequence identity to an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.
Recombinant TS bacteria generated by these methods, as well as compositions that include such bacteria, are also provided herein.
Temperature-Sensitive Bacterial Strain Compositions
Compositions are provided that include recombinant TS bacteria provided herein. In some examples, the compositions include more than one type of recombinant TS bacteria, such as 2, 3, 4 or 5 different recombinant TS bacteria. In some examples, the recombinant TS bacteria contain two or more different TS essential psychrophilic coding sequences (such as two or more of the Group I genes listed in Table 1, such as ligA and another Group I gene). In particular examples, the recombinant TS bacteria is a Francisella sp., Salmonella sp., or Mycobacterium sp. (other particular examples are provided above).
In some examples, such compositions are immunogenic, in that they can stimulate an immune response in a mammal. The compositions can include other components, such as pharmaceutically acceptable carriers (such as saline), adjuvants, preservatives, combinations thereof, and the like.
Methods of Stimulating an Immune Response Using Temperature-Sensitive Bacterial Strains
The TS recombinant bacteria disclosed herein can be used to generate an immune response in a subject. In some examples, the subject is infected with a bacterium, or as at risk of being infected with a bacterium (such as a health care worker), such as Mycobacterium tuberculosis. Thus, in several embodiments, the methods include administering to a subject a therapeutically effective amount of one or more of the TS recombinant bacteria disclosed herein in order to generate an immune response, such as, but not limited to, a protective immune response. For example, two or more different TS recombinant bacteria (such as those expressing different TS essential peptides from psychrophilic bacteria) can be used to generate an immune response in a subject. In some examples, the recombinant bacterium used to generate an immune response in a subject expresses two or more different temperature-sensitive essential peptides from a psychrophilic bacterium or the same temperature-sensitive essential peptide from two or more different psychrophilic bacteria.
The TS recombinant bacterium administered is selected based on the bacterial infection to be prevented or treated. For example, if the bacterial infection to be prevented or treated in the subject is tuberculosis, then the TS recombinant bacteria is Mycobacterium tuberculosis expressing at least one TS essential peptide from a psychrophilic bacterium. In another example, if the bacterial infection to be prevented or treated in the subject is tularemia, then the TS recombinant bacteria is F. tularensis expressing at least one TS essential peptide from a psychrophilic bacterium.
In exemplary applications, compositions are administered to a subject having in an amount sufficient to produce an immune response to the TS recombinant bacteria. These TS recombinant bacteria are of use to prevent a bacterial infection (such as Mycobacterium tuberculosis) prevent progression to disease in a subject having a latent bacterial infection, or to treat a disease resulting from the bacterial infection (such as tuberculosis). In several examples, administration of a therapeutically effective amount of a composition including the TS recombinant bacteria disclosed herein induces a sufficient immune response to decrease a symptom of a disease due to bacterial infection, to prevent the development of one or more symptoms of the disease associated with the infection, or to prevent infection with the bacteria.
In some examples, the compositions are of use in preventing a future bacterial infection. Thus, a therapeutically effective amount of the composition is administered to a subject at risk of becoming infected with a bacterium, such as Mycobacterium tuberculosis. For example the disclosed compositions can be used to prevent the development of tuberculosis, such as latent or active tuberculosis in the subject upon subsequent exposure to Mycobacterium tuberculosis. In one example, the compositions are administered to a subject with a latent Mycobacterium tuberculosis infection, and prevent the development of symptoms of tuberculosis. Thus the compositions are of use in treating a subject with latent tuberculosis, such that the subject does not develop active tuberculosis.
Amounts effective for these uses will depend upon the severity of the disease, the general state of the patient's health, and the robustness of the patient's immune system. In one example, a therapeutically effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In other examples, a therapeutically effective amount is an amount sufficient to prevent an infection with the bacterium in a subject upon subsequent exposure of the subject to the bacterium. In additional examples, a therapeutically effective amount is an amount sufficient to prevent development of symptom in a subject infected with a bacterium.
The TS recombinant bacteria-containing composition can be administered by any means known to one of skill in the art either locally or systemically, such as by intramuscular injection, subcutaneous injection, intraperitoneal infection, intravenous injection, oral administration, nasal administration, transdermal administration or even anal administration. In one embodiment, administration is by oral, subcutaneous injection or intramuscular injection. To extend the time during which the TS recombinant bacteria is available to stimulate a response, the TS recombinant bacteria can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle. A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. Aluminum salts can also be used as adjuvants to produce an immune response.
In one specific, non-limiting example, the TS recombinant bacteria are administered in a manner to direct the immune response to a cellular response (that is, a cytotoxic T lymphocyte (CTL) response), rather than a humoral (antibody) response.
Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF or G-CSF; one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7 related molecules; one or more molecules such as OX-40L or 41 BBL, or combinations of these molecules, can be used as biological adjuvants (see, for example, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or locally) to the subject. In some examples, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B7-2, OX-40L, 41 BBL and ICAM-1 are administered. In various embodiments, the nucleic acid encoding the biological adjuvant can be cloned into same vector as the psychrophilic TS essential peptide coding sequence, or the nucleic acid can be cloned into one or more separate vectors for co-administration into the bacteria.
A pharmaceutical composition including TS recombinant bacteria is thus provided. These compositions are of use to promote an immune response to a particular bacterium. In one embodiment, TS recombinant bacteria are mixed with an adjuvant containing two or more of a stabilizing detergent, a micelle-forming agent, and an oil. Suitable stabilizing detergents, micelle-forming agents, and oils are detailed in U.S. Pat. Nos. 5,585,103; 5,709,860; 5,270,202; and 5,695,770, all of which are incorporated by reference. A stabilizing detergent is any detergent that allows the components of the emulsion to remain as a stable emulsion. Such detergents include polysorbate, 80 (TWEEN) (Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured by ICI Americas, Wilmington, Del.), TWEEN 40TM, TWEEN 20TM, TWEEN 60TM, ZWITTERGENTTM 3-12, TEEPOL HB7TM, and SPAN 85TM. These detergents are usually provided in an amount of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle forming agent is an agent which is able to stabilize the emulsion formed with the other components such that a micelle-like structure is formed. Such agents generally cause some irritation at the site of injection in order to recruit macrophages to enhance the cellular response. Examples of such agents include polymer surfactants described by BASF Wyandotte publications, e.g., Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol. 129:1244, 1981, PLURONICTM L62LF, L101, and L64, PEG1000, and TETRONICTM 1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of such agents are well known in the art. In one embodiment, the agent is chosen to have a hydrophile-lipophile balance (HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be provided in an effective amount, for example between 0.5 and 10%, or in an amount between 1.25 and 5%.
In one example oil is included in the composition. Examples of such oils include squalene, Squalane, EICOSANETM, tetratetracontane, glycerol, and peanut oil or other vegetable oils. In one specific, non-limiting example, the oil is provided in an amount between 1 and 10%, or between 2.5 and 5%. The oil should be both biodegradable and biocompatible so that the body can break down the oil over time, and so that no adverse affects, such as granulomas, are evident upon use of the oil.
In one embodiment, the adjuvant in the composition is a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.). An adjuvant can also be an immunostimulatory nucleic acid, such as a nucleic acid including a CpG motif, or a biological adjuvant (see above).
Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μm in diameter and are administered subcutaneously or intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, 1992).
In particular examples, at least 102 CFU of the TS bacteria disclosed herein are administered per dose, such as at least 103 CFU, at least 104 CFU, at least 105 CFU, at least 106 CFU, at least 107 CFU, at least 108 CFU, such as102 to 108 CFU or 104 to 108 CFU. In particular examples, such dosages are administered intradermal or intranasal.
Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result is achieved. In one embodiment, the dose is sufficient to treat or ameliorate symptoms or signs of bacterial infection without producing unacceptable toxicity to the subject. In another embodiment, the dose is sufficient to prevent infection with a bacterium upon subsequent exposure to the bacterium (such as M. tuberculosis). In a further embodiment, the dose is sufficient to prevent a symptom of bacterial infection (e.g., tuberculosis) in a subject with a latent bacterial infection. Systemic or local administration can be utilized.
Thus the disclosure provides methods for producing an immune response to a bacterium in a subject. The method can include administering to the subject a therapeutically effective amount of a TS bacterium, wherein the temperature-sensitive bacterium expresses a psychrophilic TS essential protein or nucleic acid molecule provided herein (such as a nucleic acid coding sequence in a vector), thereby inducing an immune response to the bacterium. The method can further include administering other agents, such as an adjuvant or antimicrobial agent (such as an antibiotic). In some examples, the immune response is a protective immune response. The subject may have a bacterial infection, be at risk for acquiring a bacterial infection, or have a latent bacterial infection. Exemplary bacterial infections include infections with is M. tuberculosis, Salmonella or Francisella. 
Methods of measuring an immune response following stimulation with a bacterial antigen, such as a cytokine response, are known in the art. In some examples, the method further includes measuring an immune response following administration of the therapeutic compositions provided herein. In one example, a cytokine response is increased following administration of the composition provided herein, such as an increase relative to the absence of administration of the composition. In one example, cytokine production increases by at least 20%, such as at least 40%, at least 50%, at least 75%, at least 90%, or at least 95% following administration of the composition, relative to the cytokine response in the absence of administration of the composition.
The disclosure is illustrated by the following non-limiting Examples.
EXAMPLE 1
This example pertains to an exemplary method to create recombinant psychrophilic genes joined to flanking DNA of a mesophilic host.
FIG. 1a exemplifies the fusion PCR (also known as, “extension overlap PCR”, “overlap PCR” or “splice overlap PCR”) strategy used to incorporate the C. psychrerythraea essential gene (C2) into the wt F. novicida genome. The C. psychrerythraea genes were engineered with overlap PCR to contain the ribosome binding site (RBS) and the initial three codons and stop codon of the surrounding F. novicida genes (F1 and F3) to promote translation of the C2 gene at normal levels for F. novicida. The fusion PCR product was ligated to an erythromycin resistant sacB cassette (EmR-sacB) prior to its transformation into F. novicida. EmR colonies containing the fusion PCR product were grown in the presence of sucrose and colonies were screened for the loss of EmR, the F. novicida essential gene (F2), and the presence of C2.
FIG. 1b illustrates the introduction of the psychrophilic gene fusion construct into the target organism's chromosome via a single crossover event. Furthermore, it illustrates that the excision can be enhanced using the counter-selectable sacB marker. For genes that are not a part of a multi-cistronic operon the upstream pathogen genomic region was fused to the psychrophilic structural gene from codon 4 through to the stop codon. A similar approach was used when substituting a psychrophilic allele into the middle of an operon. However, as one skilled in the art can appreciate, depending on the nature of the operon, some of the codons at the C terminus of the host homologue remained if they were important for translation of the downstream cistron.
EXAMPLE 2
This example pertains to an exemplary method to insert the psychrophilic allele into the mesophilic bacteria.
FIG. 2a illustrates the substitution region for the psychrophilic ligA gene, corresponding to SEQ ID NO: 1. Additionally, it illustrates its incorporation into the wt F. novicida chromosome. FIGS. 2b-e illustrate the integration point for the psychrophilic ligA genes of C. psychrerythraea, S. frigidmarina, P. haloplanktis I, and P. haloplanktis 2 respectively. The first three codons for F. novicida were retained in order to maximize the potential for ligA expression levels. FIG. 2a-e illustrates that in most cases the integration and excision events result in a simple substitution of the psychrophilic gene for the mesophilic host homologue. However, the integration and excision events may also lead to the formation of a hybrid gene as illustrated in FIG. 1b. 
EXAMPLE 3
This example pertains to an exemplary method to determine the maximal growth temperature of each bacterial strain and to show its growth properties at restrictive temperatures.
Each bacterial strain was tested on agar plates placed in a highly stable (±1° C.) incubator; the restrictive temperatures were defined as the lowest temperatures that did not permit the formation of isolated colonies on an agar streak plate. The growth properties at different temperatures of four different transgenic strains of F. novicida carrying psychrophilic ligA substitutions and the growth properties of wt F. novicida are shown in FIGS. 3-6. The psychrophilic ligA genes are ligACp, ligASf, ligAPh and ligAPh2, as represented by SEQ ID NOs: 1, 7, 3, and 5 respectively. In the first panels in FIGS. 3-6, growth is shown at a permissive temperature, i.e., a temperature below the restrictive temperature. In subsequent panels, growth of both the transgenic and wt strains is shown before and after a shift to the restrictive temperature or higher.
Extended growth curves of both the F. novicida transgenic and wt strains are shown as inserts in select panels of FIG. 3-6. These curves were generated by taking a fully grown culture, diluting it, and monitoring its growth in fresh growth media. More specifically, the wt and TS transgenic F. novicida cultures were grown at restrictive temperatures until they reached stationary phase at which point, they were diluted and re-incubated for growth at the restrictive temperatures again. The additional growth curves demonstrate that the cessation of growth exhibited by the transgenic strain is a real phenomenon, as opposed to a temporary adjustment to the temperature shift.
EXAMPLE 4
This pertains to an exemplary method used to determine the frequency of mutations that permit bacterial growth at temperatures higher than the restrictive temperature of TS F. novicida transgenic strains.
Cultures were grown to late logarithmic phase at the permissive growth temperature, they were then diluted in a series of 109-105 cells/plate on agar and incubated at temperatures about 3° C. above the restrictive temperature, as well as at temperatures about 3° C. below the restrictive temperature. From this dilution series the rate of mutations that allow for growth at higher temperatures were calculated, Table 2 exemplifies the frequency of mutation to temperature resistance in F. novicida. Remarkably, some of the psychrophilic genes are unable to mutate to forms that will function above their restrictive temperature. One skilled in the art may hypothesize that the millions of years required to adapt to a cold climate renders some of the psychrophilic essential gene products unable to adopt simple changes allowing them to function in temperatures typical to their mesophilic counterparts. These include ligACp, ligAPh, hemCCp, dnaKCp, fmtCp, and dnaKSf.
EXAMPLE 5
This example pertains to an exemplary method to determine the duration of viability of the recombinant TS bacterial strains at the restrictive temperature.
An exemplary culture of a TS transgenic strain that has a maximal growth temperature of about 33° C., was grown at about 30° C. and a sample of the culture was incubated at about 37° C. to mimic the typical temperature of human body core tissues. Samples were taken at varying time points between 0-24 hours, and the individual samples were re-diluted, plated on to growth media, and then cultured at about 30° C. to determine the death rate above the restrictive temperature. As a control, the same experiment was carried out with the wt bacterium.
The persistence of F. tularensis strains carrying the psychrophilic essential genes within their macrophages was determined. Transgenic strains were cultured at about 30° C. and used to infect macrophages at about 37° C. in 24 well tissue culture plates using standard methods known to those skilled in these arts. For several days monitoring the infected macrophages a subset of cells were lysed and the bacteria were plated onto agar medium and incubated at about 30° C. The data generated in these experiments showed the lifespan of transgene strains during an infection with macrophages at a restrictive temperature and helped to predict the persistence of TS strains during infections.
This example can be extrapolated to provide an in vitro correlation for what can occur in a mammal. A TS transgenic strain will grow in a cool part of the body such as the skin. Replication of the strain at and about this cool site will constantly cause the TS transgenic strain progeny to be moved into the draining lymph nodes. Depending on the locations of the lymph nodes and the restrictive temperature of the TS transgenic strain, the TS progeny will die over a period of several hours. The presence of the TS transgenic strain both in its live and dead states will stimulate an immune response.
EXAMPLE 6
This pertains to an exemplary method to determine the ability of a TS essential gene from a psychrophile to impart its TS phenotype on a mesophilic bacterium. Specifically, it provides a method for transferring a psychrophilic essential gene encoding a TS product to a variety of bacteria as well as the transfer of the TS essential gene between mesophiles.
Several psychrophilic essential genes were substituted into the genome of the mesophilic bacterium F. novicida. Multiple approaches can be used to inserting a psychrophilic essential gene into a given bacterium in place of its mesophilic homologue. Furthermore, it can be appreciated that one can substitute a given psychrophilic essential gene into many different bacteria. The following three methods exemplify various ways of substituting ligACp into three different bacteria. A common approach to gene substitution is illustrated in FIG. 1b, and involves the integration of a foreign gene in a bacterium that is in close proximity to the hosts' homologous gene through PCR. Following integration, a counter selective marker, such as sacB, can be used to help identify the results of the integration and excision events. Specifically this approach was used to replace the F. novicida ligA gene with the psychrophilic ligACp gene.
An alternate approach was used to replace the S. enterica ligA. The strain of S. enterica used had a bacteriophage Mu insertion in the chromosomal copy of ligA (Park et al., 1989. J. Bacteriol. 171: 2173-80). A wt copy of the bacteriophage T4 DNA ligase was carried on the ampicillin resistant plasmid, pBR313. The ligACp gene was introduced on the compatible chloramphenicol resistant plasmid, pSUP2716, and the recombinant S. enterica strain was cultured in the absence of ampicillin and the presence of chloramphenicol. These growth conditions allow the pBR313:T4 DNA ligase recombinant plasmid to be lost. S. enterica strains that had lost the plasmid encoding the T4 DNA ligase, rendering them ampicillin sensitive, were dependant on the ligACp for viability and were TS.
Another alternate approach can be employed when introducing a psychrophilic essential gene into Gram-positive bacteria. The method of insertion of ligACp into M. smegmatis described herein exemplifies this method. A version of ligACp (SEQ ID NOS: 17 and 18) designed with optimal codons was cloned into the mycobacterial plasmid, pSM1; this a precautionary step due to the low G+C content in the ligACp gene when compared to that of the M. smegmatis and M. tuberculosis ligA genes. The recombinant pSMT3:ligACp was electroporated into M. smegmatis. Subsequently a large fragment of the M. smegmatis ligA gene was deleted resulting in a strain dependent on ligACp for viability. This strain was TS at about 34° C. This temperature is reflective of the TS nature of the F. novicida transgene strain encoding ligACp.
This example illustrates the use of a mesophilic tester strain which contains a psychrophilic essential gene to predict the TS phenotype when said psychrophilic essential gene is used to construct a transgene strain of another mesophilic bacterium. In this example, the tester strain was F. novicida. The substitution of ligACp for the F. novicida ligA homologue showed that ligACp functioned in the mesophile and imparted a TS phenotype having a restrictive temperature of about 34° C. The phenotype of the transgenic strain of F. novicida carrying ligACp predicted that substitution of the ligACp gene into other mesophiles (destination hosts) would results in viable bacteria that had a restrictive temperature of 34° C. The phenotype of the Salmonella and Mycobacteria transgene strains carrying ligACp showed that the inter-genus transfer of a TS psychrophilic essential gene could result in a phenotype seen in the tester strain.
EXAMPLE 7
This example describes an exemplary method to combine psychrophilic genes or fragments thereof (as represented by SEQ ID NO 1-24) or mutant essential psychrophilic genes to create gene products with desired TS properties.
Combining about 30%, at the 5′-end, of the novicida pyrG gene with about 2⁄3 of the 3′-end of the C. psychrerythraea pyrG gene (pyrGCp) in the region of codon 157-159 created a recombinant gene that was TS at 37° C. The F. novicida and C. psychrerythraea pyrG genes are identical at codons 157-159 inclusive. Additionally, the single point mutation at amino acid residue 149 in ligAPh from an asparagine (“N”) residue to a lysine (“K”) residue changes the restrictive temperature from 37° C. to 28° C.
This approach could be applied to different psychrophilic genes by using either in vitro or in vivo recombinant technologies to combine two or more homologues of the same gene.
EXAMPLE 8
This example pertains to an exemplary method to determine the distribution of a transgenic strain from a site of infection in a mammal.
F. novicida (a.k.a. F. tularensis subspecies novicida) carrying a psychrophilic transgene was used. One skilled in the art will appreciate that similar methods can be used to generate and examine TS strains of F. tularensis. F. novicida is highly virulent in mice. The infection of mice by F. novicida serves as a model for the infection of larger mammals with F. tularensis. Most strains of F. tularensis are highly virulent in most mammals.
The distribution of F. novicida transgenic strains from the site of infection was assessed either by injecting the recombinant strains through the skin, or by introduction via the nose, and measuring the amount of viable F. novicida cells in internal organs such as the lung, liver and spleen about three to ten days after the inoculation. It was found that TS F. novicida transgenic strains did not spread significantly from the site of inoculation. A direct correlation between the inactivation temperature of the psychrophilic essential gene and the level of distribution throughout the system was observed; the dissemination of TS F. novicida strains is Lewis Rats is outlined in Table 3.










TABLE 3






Restrictive





Temp.
CFU/Tail



F. novicida strain
(° C.)
injection site
CFU/Spleen


















wt−
45
9.7 × 103/7.1 × 103
3.7 × 106/2.2 × 106


ligACp
34
5 × 102/3 × 102
0/0


ligAPh
36.8
3 × 102/2 × 102
0/0


dnaKCp
38.2
1.5 × 104/7.6 × 104
5.0 × 102/0    


fmtCp
41
5.2 × 103/2.4 × 103
3.5 × 105/2.1 × 105









As a further example, one of the psychrophilic essential genes (ligACp) was substituted into the genome of M. tuberculosis to create a transgenic strain. Some psychrophilic essential genes originate in bacteria with DNA with low G+C content. Thus the genes were optimized with codons for M. tuberculosis prior to inserting the psychrophilic genes into the pathogenic bacteria (SEQ ID NOS: 17 and 18 provide the optimized sequences). Codon optimization is a method well known to those skilled in these arts and can be accomplished using freely available bioinformatic tools. The codon optimized psychrophilic essential genes were inserted into M. tuberculosis by methods that are well described in Examples 1 and 2. M. tuberculosis, like M. smegmatis, are Gram-positive bacteria.
Another exemplary method pertains to an exemplary method the distribution of a Gram-negative pathogenic strain. A psychrophilic essential gene was introduced into S. enterica. Upon introduction of the ligACp psycrophilic essential gene into S. enterica, the result was a transgenic strain that was unable to grow at 37° C., as illustrated in FIG. 8. Furthermore, this strain was unable to disperse from the site of inoculation in infected mice, as evidenced by the inability of the strain to migrate to the lungs, liver or spleen.
EXAMPLE 9
This example pertains to an exemplary method to determine the level of protective immune response generated from the inoculation of a mammal with a TS transgenic bacterial strain. Methods of inoculation are known in the art, and can include i.v., i.m., s.c., or i.p injection, as well as inhalation, oral, and transdermal routes of delivery. One skilled in the art will appreciate that methods similar to those described in this example can be used to test any transgenic TS bacterial strain that includes one or more psychrophilic essential nucleic acid sequences.
Inoculation of mice with a TS F. novicida transgenic strain (Fn-ligAPh,Fn-ligACp or Fn-dnaKCp) caused the cells of their immune systems to be stimulated (as measured by reduced bacterial organ burdens) resulting in protection against infection with wt F. novicida (FIGS. 10a-d). Mice were initially inoculated with the TS transgenic strain and then challenged with an inoculation three weeks later of the wt F. novicida strain. This resulted in reduced growth in the livers and spleens of mice infected with the wt strains as compared to mice that had not been inoculated with recombinant F. novicida. Furthermore, decreases in the morbidity and mortalities were observed among the inoculated group of mice resulting in the conclusion that immune protection was achieved.
Similarly, mice vaccinated with M. tuberculosis and S. enterica transgeneic strains (ligAPh) were shown to be more resistant to infections with the wt pathogens than were un-vaccinated mice.
EXAMPLE 10
This example pertains to an exemplary method of discovering novel psychrophilic essential genes.
Psychrophilic bacterium can be isolated from a cold environment, for example ocean waters near the Earth's poles. Essential genes can be identified by using degenerate PCR or other standard techniques to find highly conserved genes, such as bacterial essential genes. Once these genes have been identified, they can be substituted into the genome of a mesophile using the methods provided herein or known in the art, displacing the host homologue of the gene. The resulting strain can then be tested for temperature sensitivity as described herein.
EXAMPLE 11
This example pertains to an exemplary method of using TS transgene strains in drug discovery research. Although a TS F. tularensis strain is exemplified, one skilled in the art will appreciate that similar methods can be used for other TS strains generated using the methods provided herein.
A TS transgenic strain of F. tularensis (ligAPh) that was inoperable above about 37° C. was used to infect cell line macrophages grown in microtiter plates at 34° C. A library of antimicrobial drug candidates was introduced to individual wells that contained the infected macrophages, and the effect of the drug candidates on the killing of F. tularensis was measured by lysing the macrophages at various time points and determining the number of viable TS transgenic F. tularensis by plating on agar plates. Wt F. tularensis is extremely infectious and causes a deadly disease. The use of the TS transgenic F. tularensis strain allowed one to use greatly relaxed biological containment conditions because the strain is incapable of causing disease in humans.
EXAMPLE 12
This example pertains to an exemplary method of generating and using TS strains of Mycobacterium containing temperature-sensitive essential nucleic acid molecules from psychrophilic bacteria to develop an immunogenic composition, which for example can be used to stimulate an immune response in a mammal, to protect or treat an M. tuberculosis infection in the mammal.
The ligAPh and pryGCp genes will separately be introduced into M. tuberculosis H37Rv using an integration/excision approach. The counter-selectable marker sacB will be used to enhance the generation of excision events that can be detected. C57BL/6 mice will be vaccinated by introducing 10,000 bacteria subcutaneously at the base of the tail. Negative controls mice injected with PBS and positive control mice injected with the BCG strain will processed at the same time. The mice will be rested for 30 days. Following this period all of the mice will be exposed to an aerosol of M. tuberculosis H37Rv that deposits 150 bacteria into the lungs. At weeks 0, 4, 8, 16 and 32 following exposure to M. tuberculosis H37Rv, the mice will be euthanized and the number of M. tuberculosis H37Rv in the lungs and spleens determined. If the transgenes TS M. tuberculosis strains are successful at inducing a protective immune response, the number of bacteria in the mice organs will be less than that of the negative control. Subsequent experiments will be performed in a guinea pig model of tuberculosis.
In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

## Claims

1. A method of making a recombinant mesophilic bacterium that is temperature-sensitive (TS), comprising:
introducing into the genome of a mesophilic bacterium by homologous recombination a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium flanked on both sides by a nucleic acid molecule homologous to a region of the mesophilic bacterium genome where the TS essential nucleic acid molecule from the psychrophilic bacterium will be inserted into the mesophilic bacterium genome, and
functionally replacing the mesophilic bacterium's homolog of the TS essential nucleic acid molecule, thereby making a recombinant mesophilic bacterium that is TS,
wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C., and
wherein the recombinant mesophilic bacterium that is TS has a restrictive temperature between 33° C. and 44° C.

2. A method of making a killed recombinant mesophilic bacterium that is temperature-sensitive (TS), comprising:
introducing into the genome of a mesophilic bacterium a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium flanked on both sides by a nucleic acid molecule homologous to a region of the mesophilic bacterium genome where the TS essential nucleic acid molecule from the psychrophilic bacterium will be inserted into the mesophilic bacterium genome,
wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C., and
wherein the recombinant mesophilic bacterium that is TS is viable at a temperature of 0° C. to 30° C., nonviable at a temperature greater than 30° C., and has a restrictive temperature between 33° C. and 44° C.;

culturing the recombinant mesophilic bacterium that is TS at a temperature wherein a protein encoded by the TS essential nucleic acid molecule is operable, whereby the recombinant mesophilic bacterium that is TS produces a plurality of peptides;
increasing the culturing temperature to a temperature at which a protein encoded by the TS nucleic acid molecule is inoperable;
maintaining said culturing for a period of time sufficient to kill the recombinant mesophilic bacterium that is TS; and
harvesting the killed recombinant mesophilic bacterium that is TS.

3. A method of making a recombinant mesophilic bacterium that is temperature-sensitive (TS), comprising:
introducing into the genome of a mesophilic bacterium a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium, and
functionally replacing the mesophilic bacterium's homolog of the TS essential nucleic acid molecule, thereby making a recombinant mesophilic bacterium that is TS,
wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C., and
wherein the recombinant mesophilic bacterium that is TS has a restrictive temperature between 33° C. and 44° C.

4. The method of claim 3, wherein the psychrophilic bacterium is Colwellia sp., Pseudoalteromonas sp., or Shewanella sp.

5. The method of claim 3, wherein the protein encoded by the TS essential nucleic acid molecule comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.

6. The method of claim 3, wherein one or more control sequences are operably linked to the TS essential nucleic acid molecule.

7. The method of claim 3, wherein introducing into the genome of the mesophilic bacterium the nucleic acid construct comprising a TS essential nucleic acid molecule from the psychrophilic bacterium comprises introducing into the genome of the mesophilic bacterium by homologous recombination one or more TS essential nucleic acid molecules from the psychrophilic bacterium.

8. A recombinant mesophilic bacterium that is temperature-sensitive (TS) made by the method of claim 3.

9. The recombinant mesophilic bacterium that is TS of claim 8, wherein the recombinant mesophilic bacterium that is TS is a Francisella novicida cell.

10. The recombinant mesophilic bacterium that is TS of claim 8, wherein the recombinant mesophilic bacterium that is TS is a fermentative microbial strain or a bioremediation strain.

11. The recombinant mesophilic bacterium that is TS of claim 8, wherein the psychrophilic bacterium is Colwellia sp., Pseudoalteromonas sp., or Shewanella sp.

12. The recombinant mesophilic bacterium that is TS of claim 8, wherein the psychrophilic bacterium is Pseudoalteromonas haloplanktis or C. psychrerhraea.

13. The recombinant mesophilic bacterium that is TS of claim 8, wherein the mesophilic bacterium that is TS is Haemophilus sp., Vibrio sp., Escherichia sp., Streptococcus sp., Burkholderia sp., Campylobacter sp., Neisseria sp., Francisella sp., Salmonella sp. or Mycobacterium sp.

14. The recombinant mesophilic bacterium that is TS of claim 8, wherein the TS essential nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, wherein expression of the TS essential nucleic acid molecule confers temperature sensitivity.

15. The recombinant mesophilic bacterium that is TS of claim 8, wherein the protein encoded by the TS essential nucleic acid molecule comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.

16. The recombinant mesophilic bacterium that is TS of claim 8, wherein the protein encoded by the TS essential nucleic acid molecule is operable at a temperature above of 4° C. and inoperable at a temperature greater than 30° C.

17. The recombinant mesophilic bacterium that is TS of claim 8, wherein the nucleic acid construct comprising the TS essential nucleic acid molecule comprises a vector.

18. A composition comprising the recombinant mesophilic bacterium that is TS of claim 8.

19. The composition of claim 18, wherein the composition is an immunogenic or therapeutic composition.

20. The composition of claim 18, further comprising an adjuvant.

21. The composition of claim 18, further comprising a pharmaceutically acceptable carrier.

22. The composition of claim 18, comprising more than one type of recombinant mesophilic bacterium that is TS.

23. The composition of claim 18, wherein the recombinant mesophilic bacterium that is TS is a Francisella sp., Salmonella sp., or Mycobacterium sp.