Patent Application
20150191510
Burkholderia is a genus of proteobacteria known for its pathogenic members: Burkholderia mallei, responsible for glanders, a disease that occurs mostly in horses and related animals; Burkholderia pseudomallei, causative agent of melioidosis; and the Burkholderia cepacia complex, which includes pathogens that are involved in pulmonary infections of people with cystic fibrosis (CF). The Burkholderia genus name refers to a group of common gram-negative, motile, obligately aerobic rod-shaped bacteria including animal, human, and plant pathogens. Due to their antibiotic resistance and the high mortality rate from their associated diseases, Burkholderia mallei and Burkholderia pseudomallei are considered to be potential biological warfare agents, with livestock and humans as potential targets.
Humans and animals are believed to acquire Burkholderia infection by inhalation of contaminated dust or water droplets, ingestion of contaminated water, and contact with contaminated soil, especially through skin abrasions.
Many non-human animal species can be susceptible to melioidosis caused by Burkholderia pseudomallei, including many livestock and/or companion animal species such as, for example, sheep, goats, horses, swine, cattle, dogs, and cats.
In the absence of treatment with appropriate antibiotics, the septicemic form of melioidosis has a mortality rate that exceeds 90%. With appropriate antibiotic treatment, the mortality rate is about 10% for uncomplicated cases but up to 80% for cases with bacteremia or severe sepsis. Because of its severe course of infection, aerosol infectivity, and worldwide availability, B. pseudomallei is identified as a potential agent of biological warfare or bioterrorism and is listed on the Centers for Disease Control list as a Category B bioterrorism agent. There is currently no vaccine and the organism is often refractory to antibiotic therapy, especially after it is established in a host.
In one aspect, this disclosure provides polypeptides and compositions including polypeptides. As used herein, “polypeptide” refers to a polymer of amino acids linked by peptide bonds. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes polypeptides that may include one or more post-expression modifications of the polypeptide such as, for example, a glycosylation, an acetylation, a phosphorylation, and the like. The term polypeptide does not connote a specific length of a polymer of amino acids. A polypeptide may be isolatable directly from a natural source or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. In the case of a polypeptide that is naturally occurring, such a polypeptide is typically isolated.
An “isolated” polypeptide is one that has been removed from its natural environment. For instance, an isolated polypeptide is a polypeptide that has been removed from the cytoplasm or from the membrane of a cell, and many of the polypeptides, nucleic acids, and other cellular material of its natural environment are no longer present.
A polypeptide characterized as “isolatable” from a particular source is a polypeptide that, under appropriate conditions, is produced by the identified source, although the polypeptide may be obtained from alternate sources using, for example, conventional recombinant, chemical, or enzymatic techniques. Thus, characterizing a polypeptide as “isolatable” from a particular source does not imply any specific source from which the polypeptide must be obtained or any particular conditions or processes under which the polypeptide must be obtained.
A “purified” polypeptide is one that is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. Polypeptides that are produced outside the organism in which they naturally occur, e.g., through chemical or recombinant means, are considered to be isolated and purified by definition, since they were never present in a natural environment.
As used herein, a “polypeptide fragment” refers to a portion of a polypeptide that results from digestion of a polypeptide with a protease.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one. The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Also, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
Generally, a polypeptide may be characterized by molecular weight, mass fingerprint, amino acid sequence, nucleic acid that encodes the polypeptide, immunological activity, or any combination of two or more such characteristics. The molecular weight of a polypeptide, typically expressed in kilodaltons (kDa), can be determined using routine methods including, for instance, gel filtration, gel electrophoresis including sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis, mass spectrometry, liquid chromatography (including HPLC), and calculating the molecular weight from an observed or predicted amino acid sequence. Unless indicated otherwise, molecular weight refers to molecular weight as determined by resolving a polypeptide using an SDS polyacrylamide gel having a stacking gel of about 4% and a resolving gel of about 10% under reducing and denaturing conditions.
As used herein, a “mass fingerprint” refers to a population of polypeptide fragments obtained from a polypeptide after digestion with a protease. Often, a mass fingerprint can be generated by digesting a polypeptide with trypsin. In principle, however, a mass fingerprint may be generated by digesting the polypeptide with any suitable protease. Typically, the polypeptide fragments resulting from a digestion are analyzed using a mass spectrometric method. Each polypeptide fragment is characterized by a mass, or by a mass (m) to charge (z) ratio, which is referred to as an “m/z ratio” or an “m/z value.” Methods for generating a mass fingerprint of a polypeptide are routine. An example of such a method is disclosed in Example 3.
The polypeptides described herein may be metal-regulated. As used herein, a “metal-regulated polypeptide” is a polypeptide that is expressed by a microbe at a greater level when the microbe is grown in low metal conditions compared to when the same microbe is grown in high metal conditions. Low metal and high metal conditions are described herein. For instance, certain metal-regulated polypeptides produced by Burkholderia spp. are not expressed at detectable levels during growth of the microbe in high metal conditions but are expressed at detectable levels during growth in low metal conditions.
Examples of metal-regulated polypeptides isolatable from B. thailandensis after growth in low iron conditions include metal-regulated polypeptides having molecular weights of 88 kDa, 84 kDa, 83 kDa, 81 kDa, 58 kDa, 56 kDa, 55 kDa, 44 kDa, 43 kDa, 42 kDa, 27 kDa, 24 kDa, or 19 kDa.
Additional examples of metal-regulated polypeptides include recombinantly-produced versions of polypeptides described herein. A recombinantly-produced polypeptide may include the entire amino acid sequence translatable from an mRNA transcript. Alternatively, a recombinantly-produced metal-regulated polypeptide can include a fragment or portion of the entire translatable amino acid sequence. For example, a recombinantly-produced metal-regulated polypeptide may lack a cleavable sequence at either terminal of the polypeptide—e.g., a cleavable signal sequence at the amino terminal of the polypeptide.
Thus, a metal-regulated polypeptide can be a polypeptide that includes the amino acid sequence depicted in, for example, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:21, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:10.
This disclosure also describes certain polypeptides that are not metal-regulated. Such polypeptides are expressed in the presence of a metal ion such as, for example, in the presence of ferric chloride, and also expressed when grown in low iron conditions. Examples of such polypeptides isolatable from B. thailandensis have molecular weights of 55 kDa, 40 kDa, 39 kDa, or 19 kDa.
Additional examples of polypeptides that are not metal-regulated include recombinantly-produced versions of polypeptides described herein. A recombinantly-produced polypeptide may include the entire amino acid sequence translatable from an mRNA transcript. Alternatively, a recombinantly-produced non-metal-regulated polypeptide can include a fragment or portion of the entire translatable amino acid sequence. For example, a recombinantly-produced non-metal-regulated polypeptide may lack a cleavable sequence at either terminal of the polypeptide—e.g., a cleavable signal sequence at the amino terminal of the polypeptide.
Thus, a polypeptide that is not metal-regulated can be a polypeptide that includes the amino acid sequence depicted in, for example, SEQ ID NO:15, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:18.
This disclosure also describes certain polypeptides whose metal-regulation has not been established with certainty. Such polypeptides may have been detected in the extract when grown in low iron or in the presence of iron. Alternatively, they may have been identified through bioinformatics analysis using structural similarity to known iron receptor proteins or functional roles in iron acquisition or storage. Examples of such polypeptides isolatable from B. thailandensis have molecular weights of 85 kDa, 81 kDa, 78 kDa, 55 kDa, 36 kDa, or 8.5 kDa.
Additional examples of polypeptides whose metal regulation is uncertain include recombinantly-produced versions of polypeptides described herein. A recombinantly-produced polypeptide may include the entire amino acid sequence translatable from an mRNA transcript. Alternatively, a recombinantly-produced polypeptide whose metal regulation is uncertain can include a fragment or portion of the entire translatable amino acid sequence. For example, a recombinantly-produced polypeptide whose metal regulation is uncertain may lack a cleavable sequence at either terminal of the polypeptide—e.g., a cleavable signal sequence at the amino terminal of the polypeptide.
Thus, a polypeptide whose metal regulation is uncertain can be a polypeptide that includes the amino acid sequence depicted in, for example, SEQ ID NO:23, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:19.
Whether a polypeptide is a metal-regulated polypeptide or a non-metal-regulated polypeptide can be determined by methods useful for comparing the presence of polypeptides, including, for example, gel filtration, gel electrophoresis including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis, mass spectrometry, isobaric tags for relative and absolute quantification (iTRAQ), and liquid chromatography including HPLC. Separate cultures of a microbe can be grown under high metal conditions and under low metal conditions, polypeptides may be isolated as described herein, and the polypeptides present in each culture can be resolved and compared. Typically, an equal amount of polypeptides from each culture is used. Preferably, the polypeptides can be resolved using an SDS polyacrylamide gel having a stacking gel of about 4% and a resolving gel of about 10% under reducing and denaturing conditions. For instance, 30 micrograms (μg) of total polypeptide from each culture may be used and loaded into wells of a gel. After running the gel and staining the polypeptides with Coomassie Brilliant Blue, the two lanes can be compared. When determining whether a polypeptide is or is not expressed at a detectable level, 30 μg of total polypeptide from a culture is resolved on an SDS-PAGE gel and stained with Coomassie Brilliant Blue using methods known in the art. A polypeptide that can be visualized by eye is considered to be expressed at a detectable level, while a polypeptide that cannot be visualized by eye is considered to not be expressed at a detectable level.
Alternatively, whether a polypeptide is a metal-regulated polypeptide or a non-metal-regulated polypeptide can be determined using microarray-based gene expression analysis. Separate cultures of a microbe can be grown under high metal conditions and under low metal conditions, RNA can be extracted from cells of each culture, and differences in RNA expression in cells grown in high metal conditions versus RNA expression in cells grown in low metal conditions can be detected and compared. For example, labeled cDNA can be prepared from 8-10 μg of bacterial RNA using established protocols. The labeled cDNA can be applied to a microarray of the Burkholderia spp. genome. Such microarrays are commercially available and gene expression using such arrays is routine.
The polypeptides described herein may have immunological activity. “Immunological activity” refers to the ability of a polypeptide to elicit an immunological response in an animal. An immunological response to a polypeptide is the development in an animal of a cellular and/or antibody-mediated immune response to the polypeptide. Usually, an immunological response includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed to an epitope or epitopes of the polypeptide. “Epitope” refers to the site on an antigen to which specific B cells and/or T cells respond so that antibody is produced. The immunological activity may be protective. “Protective immunological activity” refers to the ability of a polypeptide to elicit an immunological response in an animal that inhibits or limits infection by Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. Whether a polypeptide has protective immunological activity can be determined by methods known in the art such as, for example, methods described in Example 6. For example, a polypeptide, or combination of polypeptides, can protect a rodent such as a mouse against challenge with a Burkholderia spp. A polypeptide may have seroactive activity. As used herein, “seroactive activity” refers to the ability of a candidate polypeptide to react with antibody present in convalescent serum from an animal infected with a Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. In some aspects, the convalescent serum may be from an animal infected with B. thailandensis E264, B. pseudomallei K96243, B. mallei NCTC10229, B. cenocepacia AU1054, or B. multivorans ATCC17616.
A polypeptide may have immunoregulatory activity. As used herein, “immunoregulatory activity” refers to the ability of a polypeptide to act in a nonspecific manner to enhance an immune response to a particular antigen. Methods for determining whether a polypeptide has immunoregulatory activity are known in the art.
A polypeptide as described herein may have the characteristics of a polypeptide expressed by a reference microbe—i.e., a reference polypeptide. The characteristics can include, for example, molecular weight, mass fingerprint, amino acid sequence, or any combination thereof. The reference microbe can be a gram negative, preferably a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. Exemplary strains of Burkholderia spp. and representative strains are listed in
When the reference microbe is B. thailandensis E264, a candidate polypeptide can be considered to be a polypeptide as described herein if it has a molecular weight of 88 kDa, 84 kDa, 83 kDa, 81 kDa, 58 kDa, 56 kDa, 55 kDa, 44 kDa, 43 kDa, 42 kDa, 27 kDa, 24 kDa, or 19 kDa and has a mass fingerprint that is similar to the mass fingerprint of a metal-regulated polypeptide expressed by a reference microbe and having a molecular weight of—88 kDa, 84 kDa, 83 kDa, 81 kDa, 58 kDa, 56 kDa, 55 kDa, 44 kDa, 43 kDa, 42 kDa, 27 kDa, 24 kDa, or 19 kDa, respectively. Preferably, such polypeptides are metal-regulated. For instance, a candidate polypeptide can be a polypeptide as described herein if it has a molecular weight of 88 kDa and has a mass fingerprint similar to the mass fingerprint of an 88 kDa metal-regulated polypeptide produced by the reference strain B. thailandensis E264.
Alternatively, when the reference microbe is B. thailandensis E264, a candidate polypeptide can be considered to be a polypeptide as described herein if it has an amino acid sequence that is structurally similar, as described in detail below, to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:21, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:10.
When the reference microbe is B. thailandensis E264, a candidate polypeptide can be considered to be a polypeptide as described herein if it has a molecular weight of 55 kDa, 40 kDa, 39 kDa, or 19 kDa and has a mass fingerprint that is similar to the mass fingerprint of a non-metal-regulated polypeptide expressed by a reference microbe and having a molecular weight of 55 kDa, 40 kDa, 39 kDa, or 19 kDa, respectively. Preferably, such polypeptides are non-metal-regulated. For instance, a candidate polypeptide can be a polypeptide as described herein if it has a molecular weight of 55 kDa and has a mass fingerprint similar to the mass fingerprint of a 55 kDa non-metal-regulated polypeptide produced by the reference strain B. thailandensis E264.
Alternatively, when the reference microbe is B. thailandensis E264, a candidate polypeptide can be considered to be a polypeptide as described herein if it has an amino acid sequence that is structurally similar, as described in detail below, to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:18.
When the reference microbe is B. thailandensis E264, a candidate polypeptide can be considered to be a polypeptide as described herein if it has a molecular weight of 85 kDa, 81 kDa, 78 kDa, 55 kDa, 36 kDa, or 8.5 kDa, respectively. Preferably, such polypeptides may or may not be metal-regulated. For instance, a candidate polypeptide can be a polypeptide as described herein if it has a molecular weight of 85 kDa and has a mass fingerprint similar to the mass fingerprint of an 85 kDa polypeptide produced by the reference strain B. thailandensis E264.
Alternatively, when the reference microbe is B. thailandensis E264, a candidate polypeptide can be considered to be a polypeptide as described herein if it has an amino acid sequence that is structurally similar, as described in detail below, to the amino acid sequence of SEQ ID NO:23, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:19.
As used herein, a polypeptide may be “structurally similar” to a reference polypeptide if the amino acid sequence of the polypeptide possesses a specified amount of sequence similarity and/or sequence identity compared to the reference polypeptide. A polypeptide also may be “structurally similar” to a reference polypeptide if the polypeptide exhibits a mass fingerprint possessing a specified amount of identity compared to a comparable mass fingerprint of the reference polypeptide. Thus, a polypeptide may be “structurally similar” to a reference polypeptide if, compared to the reference polypeptide, it possesses a sufficient level of amino acid sequence identity, amino acid sequence similarity, mass fingerprint similarity, or any combination thereof.
Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (for example, a candidate polypeptide and any appropriate reference polypeptide described herein) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A reference polypeptide may be a polypeptide described herein or any known metal-regulated polypeptide, as appropriate. A candidate polypeptide is the polypeptide being compared to the reference polypeptide. A candidate polypeptide can be isolated, for example, from a microbe, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.
Unless modified as otherwise described herein, a pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison Wis.). Alternatively, polypeptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al. (FEMS Microbiol Lett, 174:247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and filter on.
In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a polypeptide may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free —OH is maintained; and Gln for Asn to maintain a free —NH2. Likewise, biologically active analogs of a polypeptide containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity—such as, for example, immunological activity—of the polypeptide are also contemplated.
Thus, as used herein, reference to a polypeptide as described herein and/or reference to the amino acid sequence of one or more SEQ ID NOs can include a polypeptide with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference amino acid sequence.
Alternatively, as used herein, reference to a polypeptide as described herein and/or reference to the amino acid sequence of one or more SEQ ID NOs can include a polypeptide with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference amino acid sequence.
Consequently, a polypeptide as described herein can include certain variants including, for example, homologous polypeptides that originate-biologically and/or recombinantly—from microbial species or strains other than the microbial species or strain from which the polypeptide was originally isolated and/or identified.
For example, a polypeptide as described herein can include a polypeptide commonly known as a TonB-dependent siderophore receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:1. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:51, SEQ ID NO:101, SEQ ID NO:141, and SEQ ID NO:189.
For example, a polypeptide as described herein can include a polypeptide commonly known as resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:2. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:52, SEQ ID NO:102, SEQ ID NO:142, and SEQ ID NO:190.
For example, a polypeptide as described herein can include a polypeptide commonly known as outer membrane ferric siderophore receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:3. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:53, SEQ ID NO:143, and SEQ ID NO:191.
For example, a polypeptide as described herein can include a polypeptide commonly known as TonB-dependent heme/hemoglobin receptor family protein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:4. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:54, SEQ ID NO:103, SEQ ID NO:144, and SEQ ID NO:192.
For example, a polypeptide as described herein can include a polypeptide commonly known as Fe(III) pyochelin receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:5. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:55 and SEQ ID NO:145.
For example, a polypeptide as described herein can include a polypeptide commonly known as TonB-dependent siderophore receptor family protein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:6. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:56, SEQ ID NO:104, and SEQ ID NO:146.
For example, a polypeptide as described herein can include a polypeptide commonly known as TonB-dependent copper receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:7. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:57, SEQ ID NO:105, SEQ ID NO:147, and SEQ ID NO:193.
For example, a polypeptide as described herein can include a polypeptide commonly known as a TonB-dependent siderophore receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:8. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:58, SEQ ID NO:106, SEQ ID NO:148, and SEQ ID NO:194.
For example, a polypeptide as described herein can include a polypeptide commonly known as an OmpA family protein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:9. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:59, SEQ ID NO:107, SEQ ID NO:149, and SEQ ID NO:195.
For example, a polypeptide as described herein can include a polypeptide commonly known as OmpA family outer membrane protein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:10. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:60, SEQ ID NO:108, SEQ ID NO:150, and SEQ ID NO:196.
For example, a polypeptide as described herein can include a polypeptide commonly known as OmpA family protein that differs from the OmpA family protein described above. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:11. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:61, SEQ ID NO:109, SEQ ID NO:151, and SEQ ID NO:197.
For example, a polypeptide as described herein can include a polypeptide commonly known as an outer membrane porin. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:12. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:62, SEQ ID NO:152, and SEQ ID NO:198.
For example, a polypeptide as described herein can include a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porin described above. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:13. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:63, SEQ ID NO:110, SEQ ID NO:153, and SEQ ID NO:199.
For example, a polypeptide as described herein can include a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porins described above. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:14. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:64, SEQ ID NO:111, SEQ ID NO:154, and SEQ ID NO:200.
For example, a polypeptide as described herein can include a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porins described above. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:15. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:65, SEQ ID NO:112, SEQ ID NO:155, and SEQ ID NO:201.
For example, a polypeptide as described herein can include a polypeptide commonly known as a resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:16. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:66, SEQ ID NO:156, and SEQ ID NO:202.
For example, a polypeptide as described herein can include a polypeptide commonly known as a resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:17. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:67, SEQ ID NO:113, SEQ ID NO:157, and SEQ ID NO:203.
For example, a polypeptide as described herein can include a polypeptide commonly known as bacterioferritin (Bfr). One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:18. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:68, SEQ ID NO:114, SEQ ID NO:158, and SEQ ID NO:204.
For example, a polypeptide as described herein can include a polypeptide commonly known as bacterioferritin-associated ferredoxin (Bfd). One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:19. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:69, SEQ ID NO:115, SEQ ID NO:159, and SEQ ID NO:205.
For example, a polypeptide as described herein can include a polypeptide commonly known as dipeptide ABC transporter permease. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:20. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:70, SEQ ID NO:116, SEQ ID NO:160, and SEQ ID NO:206.
For example, a polypeptide as described herein can include a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porins described above. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:21. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:71, SEQ ID NO:161, and SEQ ID NO:207.
For example, a polypeptide as described herein can include a polypeptide commonly known as a resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:22. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:72, SEQ ID NO:117, and SEQ ID NO:208.
For example, a polypeptide as described herein can include a polypeptide commonly known as a TonB-dependent receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:23. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:73, SEQ ID NO:118, SEQ ID NO:162, and SEQ ID NO:209.
For example, a polypeptide as described herein can include a polypeptide commonly known as a TonB-dependent receptor. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:24. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:74, SEQ ID NO:119, SEQ ID NO:163, and SEQ ID NO:210.
For example, a polypeptide as described herein can include a polypeptide commonly known as resistance-nodulation-cell division (RND) superfamily efflux transporter MFP subunit. One embodiment of this polypeptide is reflected in the amino acid sequence of SEQ ID NO:25. Variant embodiments are reflected in the amino acid sequences of SEQ ID NO:75, SEQ ID NO:120, SEQ ID NO:164, and SEQ ID NO:211.
Table 1 summarizes identifying characteristics of reference polypeptides natively expressed by reference microbe B. thailandensis E264.
aID#, unique number assigned to each polypeptide for tracking purposes.
bMolecular weight (predicted).
cA protein is considered to be present (+) under iron restriction if detected by 2D gel/mass spectroscopy or in iTRAQ. ND, not detected.
dIron regulation was determined using criteria described in Example 5. IR, iron-regulated; NIR, non-iron regulated; U, iron regulation uncertain.
eImmunogenicity of a single protein was determined by antibody and/or T cell recall responses to a purified recombinant protein in mice immunized with either the iron-restricted extract or a mix of recombinant proteins.
fprotection is considered positive (+) if the protein was determined to be present in the immunizing extract for experiments shown in FIGS. 3 and 4, either by mass spectroscopy, in trial 3 by iTRAQ, or through production of antibodies to recombinant proteins from mice immunized with the protective extract vaccine.
A polypeptide as described herein also can be designed to provide one or more additional sequences such as, for example, the addition of coding sequences for added C-terminal and/or N-terminal amino acids that may facilitate purification by trapping on columns or use of antibodies. Such tags include, for example, histidine-rich tags that allow purification of polypeptides on nickel columns. Such gene modification techniques and suitable additional sequences are well known in the molecular biology arts.
A polypeptide as described herein also may be designed so that certain amino acids at the C-terminal and/or N-terminal are deleted. For example, one difference between the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:51 is that SEQ ID NO:51 possesses an N-terminal 17 amino acid addition that is not present in the reference polypeptide amino acid sequence of SEQ ID NO:1. Similar exemplary N-terminal additions, typically varying from about five amino acids to about 50 amino acids, are apparent when one compares, for example, the reference amino acid sequence of SEQ ID NO:101, SEQ ID NO:141, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:59, or SEQ ID NO:107 with certain variant embodiments of the respective amino acid sequence. Other amino acids additions and/or deletions, at either the N-terminal or the C-terminal, are possible.
A “modification” of a polypeptide as described herein includes a polypeptide (or an analog thereof such as, e.g., a fragment thereof) that is chemically or enzymatically derivatized at one or more constituent amino acids. Such a modification can include, for example, a side chain modification, a backbone modification, an N-terminal modification, and/or a C-terminal modification such as, for example, acetylation, hydroxylation, methylation, amidation, and the attachment of a carbohydrate and/or lipid moiety, a cofactor, and the like, and combinations thereof. Modified polypeptides as described herein may retain the biological activity—such as, for example, immunological activity—of the unmodified polypeptide or may exhibit a reduced or increased biological activity compared to the unmodified polypeptide.
A polypeptide as described herein (including a biologically active analog thereof and/or a modification thereof) can include a native (naturally occurring), a recombinant, a chemically synthesized, or an enzymatically synthesized polypeptide. For example, a polypeptide as described herein may be prepared by isolating the polypeptide from a natural source or may be prepared recombinantly by conventional methods including, for example, preparation as fusion proteins in bacteria or other host cells.
A polypeptide expressed by a reference microbe can be obtained by growing the reference microbe under low metal conditions as described herein and the subsequent isolation of a polypeptide by the processes disclosed herein. Alternatively, a polypeptide expressed by a reference microbe can be obtained by identifying coding regions expressed at higher levels when the microbe is grown in low metal conditions—i.e., metal-regulated. A metal-regulated coding region can be cloned and expressed, and the expressed metal-regulated polypeptide may be identified by the processes described herein. A candidate polypeptide can be isolatable from a microbe or identified from a microbe, preferably a gram negative microbe, more preferably, a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. A candidate polypeptide may also be produced using enzymatic or chemical techniques.
A candidate polypeptide may be evaluated by mass spectrometric analysis to determine whether the candidate polypeptide has a mass fingerprint similar to one of the polypeptides expressed by a reference microbe and referred to above by molecular weight. Typically, the candidate polypeptide can be isolated, for instance by resolving the candidate polypeptide by gel electrophoresis and excising the portion of the gel containing the candidate polypeptide. Any gel electrophoresis method that separates polypeptides based on differing characteristics can be used, including one-dimensional or two-dimensional gel electrophoresis, as well as liquid chromatographic separation based on, for instance, hydrophobicity, pI, or size. The candidate polypeptide can be fragmented, for instance by digestion with a protease. Preferably, the protease can cleave the peptide bond on the carboxy-terminal side of the amino acid lysine and the amino acid arginine, except when the amino acid following the lysine or the arginine is a proline. An example of such a protease is trypsin. Methods for digesting a polypeptide with trypsin are routine and known in the art. An example of such a method is disclosed in Example 3.
Methods for the mass spectrometric analysis of polypeptides are routine and known in the art and include, but are not limited to, nano high-pressure liquid chromatography electrospray tandem mass spectrometry (nanoLC-EDI-MS/MS). Often, when a candidate polypeptide is analyzed by mass spectroscopy, both the candidate polypeptide and the reference polypeptide—i.e., the polypeptide from the reference microbe—are prepared and analyzed together, thereby decreasing any potential artifacts resulting from differences in sample handling and running conditions. Preferably, all reagents used to prepare and analyze the two polypeptides are the same. For instance, the polypeptide from the reference microbe and the candidate polypeptide are isolated under substantially the same conditions, fragmented under substantially the same conditions, and analyzed by nanoLC-EDI-MS/MS on the same machine under substantially the same conditions. A candidate polypeptide may be considered to be “structurally similar” to a reference polypeptide if it exhibits a mass fingerprint possessing at least 80%, at least 90%, at least 95%, or substantially all of the m/z values present in the spectrum of the reference microbe polypeptide and above the background level of noise are also present in the spectrum of the candidate polypeptide. (See, e.g., United States Patent Application Publication No. 2006/0233824 A1).
In another aspect, a polypeptide can be considered to be a polypeptide as described herein if it has a molecular weight of a reference polypeptide described herein and has a mass fingerprint that includes a subpopulation including at least a specified percentage of the polypeptide fragments of the mass fingerprint of the reference polypeptide.
The mass fingerprint of a candidate polypeptide can be determined by a mass spectrometric method, for instance by nanoLC-EDI-MS/MS. The mass fingerprint of a candidate polypeptide will generally have additional polypeptide fragments and, therefore, can have additional m/z values other than those identified in any particular analysis. When the candidate polypeptide is being compared to a polypeptide as described herein, the candidate polypeptide can be isolatable from a microbe, preferably a gram negative microbe, more preferably, a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans.
Metal-regulated candidate polypeptides can be obtained by growing a microbe under low metal conditions and subsequently isolating a polypeptide by the processes described herein. Non-metal-regulated candidate polypeptides can be obtained by growing a microbe under low metal conditions or high metal conditions and subsequently isolating a polypeptide by the processes described herein. Alternatively, a candidate polypeptide can be obtained by recombinant expression of a polynucleotide that encodes the candidate polypeptide.
Polypeptides as described herein also may be identified in terms of the polynucleotide that encodes the polypeptide. Thus, this disclosure provides polynucleotides that encode a polypeptide as described herein or hybridize, under standard hybridization conditions, to a polynucleotide that encodes a polypeptide as described herein, and the complements of such polynucleotide sequences.
As used herein, reference to a polynucleotide as described herein and/or reference to the nucleic acid sequence of one or more SEQ ID NOs can include polynucleotides having a sequence identity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an identified reference polynucleotide sequence.
In this context, “sequence identity” refers to the identity between two polynucleotide sequences. Sequence identity is generally determined by aligning the bases of the two polynucleotides (for example, aligning the nucleotide sequence of the candidate sequence and a nucleotide sequence that includes, for example, the nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:27) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. A candidate sequence is the sequence being compared to a known sequence—e.g., a nucleotide sequence that includes the nucleotide sequence of, for example, SEQ ID NO:76 or SEQ ID NO:77. For example, two polynucleotide sequences can be compared using the Blastn program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett., 174:247-250 (1999)), and available on the world wide web at ncbi.nlm.nih.gov/BLAST/. The default values for all BLAST 2 search parameters may be used, including reward for match=1, penalty for mismatch=−2, open gap penalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, and filter on.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a TonB-dependent siderophore receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:26. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:76, SEQ ID NO:121, SEQ ID NO:165, and SEQ ID NO:212.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:27. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:77, SEQ ID NO:122, SEQ ID NO:166, and SEQ ID NO:213.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as outer membrane ferric siderophore receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:28. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:78, SEQ ID NO:167, and SEQ ID NO:214.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as TonB-dependent heme/hemoglobin receptor family protein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:29. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:79, SEQ ID NO:123, SEQ ID NO:168, and SEQ ID NO:215.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as Fe(III) pyochelin receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:30. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:80 and SEQ ID NO:169.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as TonB-dependent siderophore receptor family protein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:31. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:81, SEQ ID NO:124, and SEQ ID NO:170.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as TonB-dependent copper receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:32. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:82, SEQ ID NO:125, SEQ ID NO:171, and SEQ ID NO:216.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a TonB-dependent siderophore receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:33. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:83, SEQ ID NO:126, SEQ ID NO:172, and SEQ ID NO:217.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as an OmpA family protein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:34. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:84, SEQ ID NO:127, SEQ ID NO:173, and SEQ ID NO:218.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as OmpA family outer membrane protein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:35. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:85, SEQ ID NO:128, SEQ ID NO:174, and SEQ ID NO:219.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as OmpA family protein that differs from the OmpA family protein described above. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:36. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:86, SEQ ID NO:129, SEQ ID NO:175, and SEQ ID NO:220.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as an outer membrane porin. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:37. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:87, SEQ ID NO:176, and SEQ ID NO:221.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porin described above. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:38. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:88, SEQ ID NO:130, SEQ ID NO:177, and SEQ ID NO:222.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porins described above. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:39. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:89, SEQ ID NO:131, SEQ ID NO:178, and SEQ ID NO:223.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porins described above. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:40. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:90, SEQ ID NO:132, SEQ ID NO:179, and SEQ ID NO:224.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:41. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:91, SEQ ID NO:180, and SEQ ID NO:225.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:42. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:92, SEQ ID NO:133, SEQ ID NO:181, and SEQ ID NO:226.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as bacterioferritin (Bfr). One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:43. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:93, SEQ ID NO:134, SEQ ID NO:182, and SEQ ID NO:227.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as bacterioferritin-associated ferredoxin (Bfd). One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:44. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:94, SEQ ID NO:135, SEQ ID NO:183, and SEQ ID NO:228.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as dipeptide ABC transporter permease. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:45. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:95, SEQ ID NO:136, SEQ ID NO:184, and SEQ ID NO:229.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as an outer membrane porin that differs from the outer membrane porins described above. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:46. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:96, SEQ ID NO:185, and SEQ ID NO:230.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a resistance-nodulation-cell division (RND) superfamily efflux system outer membrane lipoprotein. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:47. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:97, SEQ ID NO:137, and SEQ ID NO:231.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a TonB-dependent receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:48. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:98, SEQ ID NO:138, SEQ ID NO:186, and SEQ ID NO:232.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as a TonB-dependent receptor. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:49. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:99, SEQ ID NO:139, SEQ ID NO:187, and SEQ ID NO:233.
For example, a polynucleotide as described herein can include a polynucleotide that encodes a polypeptide commonly known as resistance-nodulation-cell division (RND) superfamily efflux transporter MFP subunit. One embodiment of this polynucleotide is reflected in the polynucleotide sequence of SEQ ID NO:50. Variant embodiments are reflected in the polynucleotide sequences of SEQ ID NO:100, SEQ ID NO:140, SEQ ID NO:188, and SEQ ID NO:234.
Finally, a polynucleotide as described herein can include any polynucleotide that encodes a polypeptide as described herein. Thus, the nucleotide sequence of the polynucleotide may be deduced from the amino acid sequence that is to be encoded by the polynucleotide.
This disclosure also provides whole cell preparations of a microbe, where the microbe expresses one or more of the polypeptides described herein. The cells present in a whole cell preparation may be inactivated such that the cells cannot replicate but the immunological activity of the polypeptides as described herein expressed by the microbe is maintained. Typically, the cells may be killed by exposure to agents such as glutaraldehyde, formalin, or formaldehyde.
A composition as described herein may include at least one isolated polypeptide described herein, or a number of polypeptides that is an integer greater than one (e.g., at least two, at least three, at least four). Unless a specific level of sequence similarity and/or identity is expressly indicated herein (e.g., at least 80% sequence similarity, at least 90% sequence identity, etc.), reference to the amino acid sequence of an identified SEQ ID NO includes variants having the levels of sequence similarity and/or the levels of sequence identity described herein in the section headed “Polypeptide sequence similarity and polypeptide sequence identity.
A recombinantly-produced polypeptide may be expressed from a vector that permits expression of the polypeptide when the vector is introduced into an appropriate host cell. A host cell may be constructed to produce one or more recombinantly-produced polypeptides as described herein and, therefore, can include one more vectors that include at least one polynucleotide that encodes a polypeptide as described herein. Thus, each vector can include one or more polynucleotides as described herein—i.e., a polynucleotide that encodes a polypeptide as described herein.
Certain compositions such as, for example, those including recombinantly-produced polypeptides, can include a maximum number of polypeptides. In some embodiments, the maximum number of polypeptides can refer to the maximum total number of polypeptides. Certain compositions can include, for example, no more than 50 polypeptides such as, for example, no more than 40 polypeptides, no more than 30 polypeptides, no more than 25 polypeptides, no more than 20 polypeptides, no more than 15 polypeptides, no more than 10 polypeptides, no more than eight polypeptides, no more than seven polypeptides, no more than six polypeptides, no more than five polypeptides, no more than four polypeptides, no more than three polypeptides, no more than two polypeptides, or no more than one polypeptide. In other embodiments, a maximum number of recombinantly-produced polypeptides may be specified in a similar manner. In still other embodiments, a maximum number of nonrecombinantly-produced polypeptides may be specified in a similar manner.
A composition can include polypeptides isolatable from one microbe, or can be isolatable from a combination of two or more microbes. For instance, a composition can include polypeptides isolatable from two or more Burkholderia spp., or from a Burkholderia spp. and a different microbe that is not a member of the genus Burkholderia. In certain embodiments, a composition can include a whole cell preparation in which the whole cell expresses one or more of the polypeptides as described herein. In some of these embodiments, the whole cell can be a Burkholderia spp. In some embodiments, a composition can include whole cell preparations from two, three, four, five, or six strains.
Optionally, a polypeptide as described herein can be covalently bound or conjugated to a carrier polypeptide to improve the immunological properties of the polypeptide. Useful carrier polypeptides are known in the art. The chemical coupling of polypeptides as described herein can be carried out using known and routine methods. For instance, various homobifunctional and/or heterobifunctional cross-linker reagents such as bis(sulfosuccinimidyl) suberate, bis(diazobenzidine), dimethyl adipimidate, dimethyl pimelimidate, dimethyl superimidate, disuccinimidyl suberate, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide, sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl 4-(N-maleimidomethyl) cycloheane-1-carboxylate, sulfosuccinimidyl 4-(p-maleimido-phenyl) butyrate and (1-ethyl-3-(dimethyl-aminopropyl) carbodiimide can be used (see, for instance, Harlow and Lane, Antibodies, A Laboratory Manual, generally and Chapter 5, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, N.Y. (1988)).
The compositions as described herein optionally further include a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” refers to a diluent, carrier, excipient, salt, etc., that is compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Typically, the composition includes a pharmaceutically acceptable carrier when the composition is used as described herein. Exemplary pharmaceutically acceptable carriers include buffer solutions and generally exclude blood products such as, for example, whole blood and/or plasma. The compositions as described herein may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration, including routes suitable for stimulating an immune response to an antigen. Thus, a composition as described herein can be administered via known routes including, for example, oral; parenteral including intradermal, transcutaneous and subcutaneous; intramuscular, intravenous, intraperitoneal, etc. and topically, such as, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous and rectally, etc. It is foreseen that a composition can be administered to a mucosal surface, such as by administration to the nasal or respiratory mucosa (e.g., via a spray or aerosol), in order to stimulate mucosal immunity, such as production of secretory IgA antibodies, throughout the animal's body.
A composition as described herein can also be administered via a sustained or delayed release implant. Implants suitable for use according to the invention are known and include, for example, those disclosed in International Publication No. WO 2001/037810 and/or International Publication No. WO 1996/001620. Implants can be produced at sizes small enough to be administered by aerosol or spray. Implants also can include nanospheres and microspheres.
A composition as described herein may be administered in an amount sufficient to treat certain conditions as described herein. The amount of polypeptides or whole cells present in a composition as described herein can vary. For instance, the dosage of polypeptides can be between 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10 mg. When the composition is a whole cell preparation, the cells can be present at a concentration of, for instance, 102 bacteria/ml, 103 bacteria/ml, 104 bacteria/ml, 105 bacteria/ml, 106 bacteria/ml, 107 bacteria/ml, 108 bacteria/ml, or 109 bacteria/ml. For an injectable composition (e.g. subcutaneous, intramuscular, etc.) the polypeptides may be present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0 to 2.0 ml. When the composition is a whole cell preparation, the cells are preferably present in the composition in an amount that the total volume of the composition administered is 0.5 ml to 5.0 ml, typically 1.0 to 2.0 ml. The amount administered may vary depending on various factors including, but not limited to, the specific polypeptides chosen, the weight, physical condition and age of the animal, and the route of administration. Thus, the absolute weight of the polypeptide included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight and physical condition of the animal, as well as the method of administration. Such factors can be determined by one of skill in the art. Other examples of dosages suitable for the invention are disclosed in U.S. Pat. No. 6,027,736.
The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the active compound (e.g., a polypeptide or whole cell as described herein) into association with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.
A composition including a pharmaceutically acceptable carrier also can include an adjuvant. An “adjuvant” refers to an agent that can act in a nonspecific manner to enhance an immune response to a particular antigen, thus potentially reducing the quantity of antigen necessary in any given immunizing composition, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Adjuvants may include, for example, IL-1, IL-2, emulsifiers, muramyl dipeptides, dimethyl dioctadecyl ammonium bromide (DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins (including, for instance, those available from under the tradename EMULSIGEN from MVP Laboratories, Ralston, Nebr.), ISA-70, RIBI and other substances known in the art. It is expected that polypeptides as described herein will have immunoregulatory activity and that such polypeptides may be used as adjuvants that directly act as T cell and/or B cell activators or act on specific cell types that enhance the synthesis of various cytokines or activate intracellular signaling pathways. Such polypeptides are expected to augment the immune response to increase the protective index of the existing composition.
In another embodiment, a composition as described herein including a pharmaceutically acceptable carrier can include a biological response modifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-α, IFN-γ, and other cytokines that effect immune cells. An immunizing composition can also include other components known in the art such as an antibiotic, a preservative, an anti-oxidant, or a chelating agent.
This disclosure also provides methods for obtaining the polypeptides described herein. The polypeptides and whole cells as described herein may be isolatable from a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. Other gram negative microbes from which polypeptides can be isolated include, for example, Achromobacter spp., Ralstonia spp., Pseudomonas spp., Bordetella spp., and Acinetobacter spp. Microbes useful for obtaining polypeptides as described herein and making whole cell preparations are commercially available from a depository such as American Type Culture Collection (ATCC). In addition, such microbes are readily obtainable by techniques routine and known to the art. The microbes may be derived from an infected animal as a field isolate, and used to obtain the polypeptides and/or the whole cell preparations as described herein, or stored for future use, for example, in a frozen repository at from −20° C. to −95° C., or from −40° C. to −50° C., in bacteriological media containing 20% glycerol, and other like media.
When a polypeptide as described herein is to be obtained from a microbe, the microbe can be incubated under low metal conditions. As used herein, the phrase “low metal conditions” refers to an environment, typically bacteriological media, that contains an amount of a free metal that causes a microbe to express metal-regulated polypeptides at a detectable level. As used herein, the phrase “high metal conditions” refers to an environment that contains an amount of a free metal that causes a microbe to express a metal-regulated polypeptide at a decreased level compared to expression of the metal-regulated polypeptide under low metal conditions. In some cases, “high metal conditions” can refer to an environment that causes a cell to fail to express one or more of the metal-regulated polypeptides described herein at a detectable level.
In some cases, “high metal conditions” can include a metal-rich natural environment and/or culture in a metal-rich medium without a metal chelator. In contrast, in some cases, “low metal conditions” can include culture in a medium that includes a metal chelator, as described in more detail below. Metals are those present in the periodic table under Groups 1 through 17 (IUPAC notation; also referred to as Groups I-A, II-A, III-B, IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B, III-A, IV-A, V-A, VI-A, and VII-A, respectively, under CAS notation). Preferably, metals are those in Groups 2 through 12, more preferably, Groups 3-12. Even more preferably, the metal is iron, zinc, copper, magnesium, nickel, cobalt, manganese, molybdenum, or selenium, most preferably, iron.
Low metal conditions are generally the result of the addition of a metal chelating compound to a bacteriological medium, the use of a bacteriological medium that contains low amounts of a metal, or the combination thereof. High metal conditions are generally present when a chelator is not present in the medium, when a metal is added to the medium, or the combination thereof. Examples of metal chelators include natural and synthetic compounds. Examples of natural compounds include plant phenolic compounds, such as flavenoids. Examples of flavenoids include the copper chelators catechin and naringenin, and the iron chelators myricetin and quercetin. Examples of synthetic copper chelators include, for instance, tetrathiomolybdate, and examples of synthetic zinc chelators include, for instance, N,N,N′,N′-Tetrakis (2-pyridylmethyl)-ethylene diamine Examples of synthetic iron chelators include 2,2′-dipyridyl (also referred to in the art as θ,θ′-bipyridyl), 8-hydroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxamine methanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin, biological siderophores, such as, the catecholates and hydroxamates, and citrate. An example of a general divalent cation chelator is CHELEX resin. Preferably, 2,2′-dipyridyl is used for the chelation of iron. Typically, 2,2′-dipyridyl is added to the media at a concentration of at least 300 micrograms/milliliter (μg/ml), at least 600 μg/ml, or at least 900 μg/ml. High levels of 2,2′-dipyridyl can be, for example, 1200 μg/ml, 1500 μg/ml, or 1800 μg/ml.
The B. thailandensis genome encodes a ferric uptake regulator (Fur) homolog. The Fur protein controls the uptake of metal ions by regulating the expression of iron receptors such as the siderophore receptor proteins in response to iron limitation. It also plays a role in oxidative stress resistance and virulence. It is expected that a gram negative organism, preferably, a Burkholderia spp., with a mutation in the fur coding region will result in the constitutive expression of many, if not all, of the metal-regulated polypeptides as described herein. The production of a fur mutation in a gram negative, preferably, a Burkholderia spp., can be produced using routine methods including, for instance, transposon, chemical, or site-directed mutagenesis useful for generating gene missense or knock-out mutations in gram negative bacteria.
The medium used to incubate the microbe and the volume of media used to incubate the microbe can vary. When a microbe is being evaluated for the ability to produce one or more of the polypeptides described herein, the microbe can be grown in a suitable volume, for instance, 10 milliliters to 1 liter of medium. When a microbe is being grown to obtain polypeptides for use in, for instance, administration to animals, the microbe may be grown in a fermenter to allow the isolation of larger amounts of polypeptides. Methods for growing microbes in a fermenter are routine and known to the art. The conditions used for growing a microbe preferably include a metal chelator, more preferably an iron chelator, for instance 2,2′-dipyridyl, a pH of between 6.5 and 7.5, preferably between 6.9 and 7.1, and a temperature of 37° C.
In some aspects of the invention, a microbe may be harvested after growth. Harvesting includes concentrating the microbe into a smaller volume and suspending in a media different than the growth media. Methods for concentrating a microbe are routine and known in the art, and include, for example, filtration or centrifugation. Typically, the concentrated microbe is suspended in an appropriate buffer. An example of a buffer that can be used contains Tris-base (7.3 grams/liter), at a pH of 8.5. Optionally, the final buffer also minimizes proteolytic degradation. This can be accomplished by having the final buffer at a pH of greater than 8.0, preferably, at least 8.5, and/or including one or more proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride). Optionally, the concentrated microbe is frozen at −20° C. or below until disrupted.
When the microbe is to be used as a whole cell preparation, the harvested cells may be processed using routine and known methods to inactivate the cells. Alternatively, when a microbe is to be used to prepare polypeptides as described herein, the microbe may be disrupted using chemical, physical, or mechanical methods routine and known to the art, including, for example, boiling, French press, sonication, digestion of peptidoglycan (for instance, by digestion with lysozyme), or homogenization. An example of a suitable device useful for homogenization is a model C500-B AVESTIN homogenizer, (Avestin Inc., Ottawa, Canada). As used herein, “disruption” refers to the breaking up of the cell. Disruption of a microbe can be measured by methods that are routine and known to the art, including, for instance, changes in optical density. Typically, a microbe is subjected to disruption until the percent transmittance is increased by 20% when a 1:100 dilution is measured. When physical or mechanical methods are used, the temperature during disruption is typically kept low, preferably at 4° C., to further minimize proteolytic degradation. When chemical methods are used the temperature may be increased to optimize for the cell disruption. A combination of chemical, physical, and mechanical methods may also be used to solubilize the cell wall of microbe. As used herein, the term “solubilize” refers to dissolving cellular materials (e.g., polypeptides, nucleic acids, carbohydrates) into the aqueous phase of the buffer in which the microbe was disrupted, and the formation of aggregates of insoluble cellular materials. Without intending to be limited by theory, the conditions for solubilization are believed to result in the aggregation of polypeptides as described herein into insoluble aggregates that are large enough to allow easy isolation by, for instance, centrifugation.
The insoluble aggregates that include one or more of the polypeptides as described herein may be isolated by methods that are routine and known to the art. Preferably, the insoluble aggregates are isolated by centrifugation. Typically, centrifugation of polypeptides, such as membrane polypeptides, can be accomplished by centrifugal forces of 100,000×g. The use of such centrifugal forces requires the use of ultracentrifuges, and scale-up to process large volumes of sample is often difficult and not economical with these types of centrifuges. The methods described herein provide for the production of insoluble aggregates large enough to allow the use of continuous flow centrifuges, for instance T-1 Sharples (Alfa Laval, Inc., Richmond, Va.), which can be used with a flow rate of 250 ml/minute at 17 psi at a centrifugal force of 46,000×g to 60,000×g. Other large scale centrifuges can be used, such as the tubular bowl, chamber, and disc configurations. Such centrifuges are routinely used and known in the art, and are commercially available from such manufactures as Pennwalt, Ltd., GEA Westfalia Separator Division (GEA Mechanical Equipment US, Inc.), or Alpha Laval, Inc.
The final harvested proteins can be washed and/or dialyzed against an appropriate buffer using conventional methods such as, for example, diafiltration, precipitation, hydrophobic chromatography, ion-exchange chromatography, affinity chromatography, or ultra-filtration, followed by washing the polypeptides, for instance, in alcohol, by diafiltration. After isolation, the polypeptides suspended in buffer and stored at low temperature, for instance, −20° C. or below.
In those aspects as described herein where a whole cell preparation is to be made, after growth a microbe can be killed with the addition of an agent such as glutaraldehyde, formalin, or formaldehyde, at a concentration sufficient to inactivate the cells in the culture. For instance, formalin can be added at a concentration of 0.3% (vol:vol). After a period of time sufficient to inactivate the cells, the cells can be harvested by, for instance, diafiltration and/or centrifugation, and washed.
In other aspects, an isolated polypeptide as described herein may be prepared recombinantly. When prepared recombinantly, a polynucleotide encoding the polypeptide may be identified and cloned into an appropriate expression host as described below in Example 6. The recombinant expression host may be grown in an appropriate medium, disrupted, and the polypeptides isolated as described above.
In another aspect, this disclosure further provides methods of using the compositions as described herein. The methods include administering to an animal an effective amount of a composition as described herein. The animal can be, for instance, avian (including, for instance, chickens or turkeys), bovine (including, for instance, cattle), caprine (including, for instance, goats), ovine (including, for instance, sheep), porcine (including, for instance, swine), bison (including, for instance, buffalo), equine (including, for instance, horses), a companion animal (including, for instance, dogs or cats), members of the family Cervidae (including, for instance, deer, elk, moose, caribou and reindeer), or human.
In some aspects, the methods may further include additional administrations (e.g., one or more booster administrations) of the composition to the animal to enhance or stimulate a secondary immune response. A booster can be administered at a time after the first administration, for instance, one to eight weeks, preferably two to four weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually. Without intending to be limited by theory, it is expected that in some aspects as described herein annual boosters will not be necessary, as an animal will be challenged in the field by exposure to microbes expressing polypeptides present in the compositions having epitopes that are identical to or structurally related to epitopes present on polypeptides of the composition administered to the animal.
In one embodiment, the method can involve making antibody, for instance by inducing the production of antibody in an animal or by recombinant techniques. As used herein, the term “antibody”—when not preceded by a definite or indefinite article—can be used generically to refer to any preparation that includes at least one molecular species of immunoglobulin or a fragment (e.g., scFv, Fab, F(ab′)2 or Fv or other modified fragment) thereof. Therefore, “antibody” can generically include one or more monoclonal antibodies and/or a polyclonal antibody preparation. Antibody produced by the method can include antibody that specifically binds at least one polypeptide present in the composition. In this context, an “effective amount” is an amount effective to result in the production of antibody in the animal. Methods for determining whether an animal has produced antibody that specifically binds a polypeptide present in a composition can be determined as described herein. This disclosure therefore further provides antibody that specifically binds to a polypeptide as described herein, and compositions including such antibody.
The method may be used to produce antibody that specifically binds to a polypeptide expressed by a microbe other than the microbe from which the polypeptide of the composition were isolated. As used herein, antibody that can “specifically bind” a polypeptide is antibody that interacts with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. At least some of the polypeptides present in the compositions as described herein typically include epitopes that are conserved in the polypeptides of different species and different genera of microbes. Accordingly, antibody produced using a composition derived from one microbe is expected to bind to polypeptides expressed by other microbes and provide broad spectrum protection against gram negative organisms. Examples of gram negative microbes to which the antibody may specifically bind are Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. Therefore, antibody produced using a composition of polypeptides as described herein may be used to identify and characterize polypeptides as described herein independent of the origin, source, and/or manner of obtaining the polypeptide.
In another aspect, this disclosure provides the use of such antibody to target a microbe expressing a polypeptide as described herein or a polypeptide having an epitope structurally related to an epitope present on a polypeptide as described herein. A compound can be covalently bound to an antibody, where the compound can be, for instance, a toxin. Likewise, such compounds can be covalently bound to a bacterial siderophore to target the microbe. The chemical coupling or conjugation of an antibody as described herein, or a portion thereof (such as a Fab fragment), can be carried out using known and routine methods.
In another aspect, this disclosure provides methods for treating an infection in an animal, including a human, caused by a gram negative microbe, preferably by a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. As used herein, the term “infection” refers to the presence of a gram negative microbe in an animal's body, which may or may not be clinically apparent. An animal with an infection by a member of the genus Burkholderia that is not clinically apparent is often referred to as an asymptomatic carrier.
Treating an infection can be prophylactic or, alternatively, therapeutic—in this context, treatment after a subject manifests one or more indication of infection by a microbe. Generally, treatment that is prophylactic—in this context, initiated before a subject is infected by a microbe or while an infection remains subclinical—is referred to herein as treatment of a subject that is “at risk” of infection. As used herein, the term “at risk” refers to an animal that may or may not actually possess the described risk—in this context, an animal that may or may not be infected by a particular microbe. Thus, typically, an animal “at risk” of infection by a microbe is an animal present in an area where animals have been identified as infected by the microbe and/or is likely to be exposed to the microbe even if the animal has not yet manifested any detectable indication of infection by the microbe and regardless of whether the animal may harbor a subclinical amount of the microbe. Accordingly, administration of a composition can be performed before, during, or after the animal has first contact with the microbe. Treatment initiated after the animal's first contact with the microbe may result in decreasing the severity of symptoms and/or clinical signs of infection by the microbe, completely removing the microbe, and/or decreasing the likelihood of experiencing a clinically evident infection compared to an animal to which the composition is not administered. The method includes administering an effective amount of the composition as described herein to an animal having, or at risk of having, an infection caused by a gram negative microbe, and determining whether the number of microbes causing the infection has decreased. In this context, an “effective amount” is an amount effective to reduce the number of the specified microbes in an animal or reduce the likelihood that the animal experiences a clinically-evident infection compared to an animal to which the composition is not administered. Methods for determining whether an infection is caused by a gram negative microbe are routine and known in the art, as are methods for determining whether the infection has decreased.
In another aspect, the present invention is directed to methods for treating one or more symptoms or clinical signs of certain conditions in an animal that may be caused by infection by a gram negative microbe, preferably by a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. The method includes administering an effective amount of a composition as described herein to an animal having or at risk of having a condition, or exhibiting symptoms and/or clinical signs of a condition, and determining whether at least one symptom and/or clinical sign of the condition is changed, preferably, reduced.
Melioidosis caused by B. pseudomallei tends to be more prevalent in people during the fourth and fifth decades of life, especially among those who have other comorbidities such as diabetes, alcoholism, immunosuppression, and renal failure. Melioidosis may be transmitted through direct skin contact with contaminated soil or water, ingestion of contaminated water, inhalation of dust contaminated with the bacterium, sexual contact, or intravenous drug use. Human-to-human transmission is rare but has been documented. The cutaneous form results in local infection with ulceration and swollen lymph glands. General symptoms include fever, rigors, night sweats, myalgia, anorexia, and headache. Additional symptoms are dependent on the route of exposure but may include chest pain, cough, photophobia, lacrimation, and diarrhea. Physical findings may include fever, cervical adenopathy, pustular skin lesions, hepatomegaly, or splenomegaly. During primary melioidosis, patients may experience severe urticaria. The chronic form may involve multiple abscesses that affect the liver, spleen, skin, or muscles, and may reactivate many years after the primary infection.
The pulmonary form of melioidosis may be manifest as pneumonia, pulmonary abscesses, and pleural effusions. Cutaneous abscesses may also develop and can take months to appear. Those who develop septicemia may develop respiratory distress, headaches, fever, diarrhea, pus-filled skin lesions, and abscesses throughout the body. Pustules often occur in association with regional lymphadenitis, cellulitis, or lymphangitis. Specifically, septicemia, high fever, and rigor are often present and may be accompanied by confusion, dyspnea, abdominal pain, muscle tenderness, pharyngitis, diarrhea, and jaundice. Although the foci of infection may be lungs or skin, once septicemia has developed, the disease spreads to the liver, spleen, kidney, brainstem, and parotid gland, leading to acidosis and shock with a high mortality rate exceeding 90% and death occurring within 24-48 hours.
B. mallei is the causative agent of glanders, which occurs primarily in horses and other solipeds. B. mallei is highly virulent and exhibits a pathophysiology in humans that is similar to glanders, where the clinical symptoms are similar to melioidosis as described above. Transmission is through direct skin or mucous membrane contact with infected animal tissues. Human-to-human transmission is possible and has been reported. Septicemia may include cutaneous, hepatic, and splenic involvement and is usually fatal within 7-10 days. The chronic form may involve multiple abscesses that affect the liver, spleen, skin, or muscles.
The Burkholderia cepacia complex is a group of at least 17 species responsible for opportunistic infections that are particularly problematic in diseases that cause impaired pulmonary function such as cystic fibrosis or chronic granulomatous disease. These organisms have also been a source of catheter-related infections in cancer patients and in those who are on hemodialysis. They have also been a source of skin and soft tissue infection, surgical wound infection, and genitourinary infection. The symptoms of pulmonary infection vary widely, ranging from asymptomatic infection to serious respiratory infections, especially in individuals that are immunocompromised, the young, the elderly, and people with lung disease. Symptoms are similar to other lung infections, with cough, wheezing shortness of breath, congestion, and fever. Thus, infection may be difficult to diagnose. Bacteria can persist in the lungs for years without symptoms.
Treatment of symptoms and/or clinical signs associated with these conditions can be prophylactic or, alternatively, therapeutic—in this context, treatment initiated after the subject exhibits one or more symptoms or clinical signs associated with a condition caused by infection by a gram negative microbe. As used herein, the term “symptom” refers to subjective evidence of disease or condition experienced by the patient and caused by infection by a microbe. As used herein, the term “clinical sign” or, simply, “sign” refers to objective evidence of disease or condition caused by infection by a microbe. Symptoms and/or clinical signs associated with conditions referred to herein and the evaluations of such symptoms are routine and known in the art. Treatment that is prophylactic—in this context, treatment that is initiated before a subject manifests symptoms or signs of a condition caused by a microbe—is referred to herein as treatment of a subject that is “at risk” of developing the condition. Thus, typically, an animal “at risk” of developing a condition is an animal present in an area where animals having the condition have been diagnosed and/or is likely to be exposed to a microbe causing the condition even if the animal has not yet manifested symptoms or signs of any condition caused by the microbe. Accordingly, administration of a composition can be performed before, during, or after the occurrence of the conditions described herein. Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms. In this aspect of the invention, an “effective amount” is an amount effective to prevent the manifestation of symptoms of a disease, decrease the severity of the symptoms of a disease, and/or completely remove the symptoms. The successful treatment of a gram negative microbial infection in an animal is disclosed in Example 6, which demonstrates the protection against disease caused by B. thailandensis in a mouse model by administering a composition as described herein. These mouse models are a commonly accepted model for the study of human disease caused by these microbes.
This disclosure also provides methods for decreasing colonization by gram negative microbes, for instance blocking the attachment sites of gram negative microbe, including tissues of the skeletal system (for instance, bones, cartilage, tendons and ligaments), muscular system, (for instance, skeletal and smooth muscles), circulatory system (for instance, heart, blood vessels, capillaries and blood), nervous system (for instance, brain, spinal cord, and peripheral nerves), respiratory system (for instance, nose, trachea lungs, bronchi, bronchoceles, alveoli), digestive system (for instance, mouth, salivary glands esophagus, liver, stomach, large intestine, or small intestine), excretory system (for instance, kidneys, ureters, bladder and urethra), endocrine system (for instance, hypothalamus, pituitary, thyroid, pancreas and adrenal glands), reproductive system (for instance, ovaries, oviduct, uterus, vagina, mammary glands, testes, and seminal vesicles), lymphatic/immune systems (for instance, lymph, lymph nodes and vessels, mononuclear or white blood cells, such as macrophages, neutrophils, monocytes, eosinophils, basophils, and lymphocytes, including T cells and B cells), and specific cell lineages (for instance, precursor cells, epithelial cells, stem cells), and the like. Preferably, the gram negative microbe is a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. Protein conservation suggests that the Burkholderia vaccines may also decrease colonization by other gram negative microbes such as Achromobacter spp., Ralstonia spp., Pseudomonas spp., Bordetella spp., and Acinetobacter spp.
Decreasing colonization in an animal may be performed prophylactically or, alternatively, therapeutically—in this context, initiated after the animal is colonized by the microbe. Treatment that is prophylactic—in this context, initiated before a subject is colonized by a microbe or while any colonization remains undetected—is referred to herein as treatment of a subject that is “at risk” of colonization by the microbe. Thus, typically, an animal “at risk” of colonization by a microbe is an animal present in an area where animals have been identified as colonized by the microbe and/or is likely to be exposed to the microbe even if the animal has not yet manifested any detectable indication of colonization by the microbe and regardless of whether the animal may harbor a subcolonization number of the microbe. Accordingly, administration of a composition can be performed before, during, or after the animal has first contact with the microbe. Treatment initiated after the animal's first contact with the microbe may result in decreasing the extent of colonization by the microbe, completely removing the microbe, and/or decreasing the likelihood that the animal becomes colonized by the microbe compared to an animal to which the composition is not administered. Thus, the method includes administering an effective amount of a composition as described herein to an animal colonized by, or at risk of being colonized by, a gram negative microbe. In this context, an “effective amount” is an amount sufficient to decrease colonization of the animal by the microbe, where decreasing colonization refers to one or more of: decreasing the extent of colonization by the microbe, completely removing the microbe, and/or decreasing the likelihood that the animal becomes colonized by the microbe compared to an animal to which the composition is not administered. Methods for evaluating the colonization of an animal by a microbe are routine and known in the art. For instance, colonization of an animal's intestinal tract by a microbe can be determined by measuring the presence of the microbe in the animal's feces. It is expected that decreasing the colonization of an animal by a microbe will reduce transmission of the microbe to humans.
A composition as described herein can be used to provide for active or passive immunization against bacterial infection. Generally, the composition can be administered to an animal to provide active immunization. However, the composition also can be used to induce production of immune products, such as antibody—e.g., a polyclonal antibody preparation or a monoclonal antibody—that can be collected from the producing animal and administered to another animal to provide passive immunity Immune components such as, for example, antibody can be collected from serum, plasma, blood, colostrum, etc. to prepare compositions (preferably containing the collected antibody) for passive immunization therapies. Antibody compositions including monoclonal antibodies and/or anti-idiotypes can also be prepared using known methods. Chimeric antibodies include human-derived constant regions of both heavy and light chains and murine-derived variable regions that are antigen-specific (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81(21):6851-6855; LoBuglio et al., 1989, Proc. Natl. Acad. Sci. USA 86(11):4220-4224; Boulianne et al., 1984, Nature 312(5995):643-646.). Humanized antibodies substitute the murine constant and framework (FR) (of the variable region) with the human counterparts (Jones et al., 1986, Nature 321(6069):522-525; Riechmann et al., 1988, Nature 332(6162):323-327; Verhoeyen et al., 1988, Science 239(4847):1534-1536; Queen et al., 1989, Proc. Natl. Acad. Sci. USA 86(24):10029-10033; Daugherty et al., 1991, Nucleic Acids Res. 19(9): 2471-2476.). Alternatively, certain mouse strains can be used that have been genetically engineered to produce antibodies that are almost completely of human origin; following immunization the B cells of these mice are harvested and immortalized for the production of human monoclonal antibodies (Bruggeman et al. 1997, Curr. Opin. Biotechnol. 8(4):455-458; Lonberg et al., 1995, Int. Rev. Immunol. 13(1):65-93; Lonberg et al., 1994, Nature 368:856-859; Taylor et al., 1992, Nucleic Acids Res. 20:6287-6295.).
Passive antibody compositions and fragments thereof, e.g., scFv, Fab, F(ab′)2 or Fv or other modified forms thereof, may be administered to a recipient in the form of serum, plasma, blood, colostrum, and the like. Antibody may, however, also be isolated from serum, plasma, blood, colostrum, and the like, using known methods for later use in a concentrated or reconstituted form such as, for instance, lavage solutions, impregnated dressings and/or topical agents and the like. Passive immunization preparations may be particularly advantageous for the treatment of acute systemic illness, or passive immunization of young animals that failed to receive adequate levels of passive immunity through maternal colostrum. Antibody useful for passive immunization also may be useful to conjugate to various drugs or antibiotics that could be directly targeted to bacteria expressing during a systemic or localized infection a polypeptide as described herein or a polypeptide having an epitope structurally related to an epitope present on a polypeptide as described herein.
Animal models, in particular mouse models, are available for experimentally evaluating the compositions as described herein. These mouse models are commonly accepted models for the study of human disease caused by members of the genus Burkholderia, and, in particular B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans. In those cases where a member of the genus Burkholderia causes disease in an animal, the natural host can be used to experimentally evaluate the compositions as described herein.
However, protection in a mouse model is not the only way to assess whether a composition can confer protection to an animal against infection by a Burkholderia spp. The adaptive immune response consists of two primary divisions: the humoral (antibody) response and the cellular (T cell) response. Following infection by a bacterial pathogen, dendritic cells at the infection site encounter microbial antigens and produce signaling molecules such as, for example, surface receptors and cytokines in response to conserved molecular patterns associated with the specific bacterium. These signals are shaped by the nature of the pathogen and ideally lead to the appropriate antibody and T cell responses that protect the host from disease. While some bacterial diseases are controlled primarily through antibody functions, others require T cell responses or both antibody and T cell responses for protection. The goal of vaccine biology is to identify the immune responses that provide protection and then design a vaccine to reproduce one or more of these responses in humans.
Antibodies can have many different functions in the conferring protection against infection such as, for example, complement fixation, opsonization, neutralization, and/or agglutination. Moreover, some subclasses of antibodies are better than others at specific functions; for example, for complement fixation the following hierarchy exists for human IgG subclasses: IgG3>IgG1>IgG2>IgG4.
Antibody immunological functions can be studied in a variety of ways. For instance, Western blots are used to identify antigen-specific binding based on size of separated proteins, while the standard enzyme-linked immunosorbent assay (ELISA) is used to produce quantitative information about antibody titers within serum. Antibody surface binding studies are used to determine whether antibody in serum are able to recognize antigens on the surface of intact bacteria, an important indicator of whether the antibodies have the potential to work in vivo. Thus, one skilled in the art recognizes that antibody binding assays such as a Western blot, ELISA (e.g., using human antisera), and/or surface binding correlate positively with the specifically-bound antigens providing immunological activity against infection by Burkholderia spp. However, one skilled in the art further recognizes that a lack of antibody binding in an assay such as, for example, a Western blot, ELISA, or surface binding assay does not mean that the assayed antigen fails to provide immunological activity against infection by Burkholderia spp.
For example,
Techniques such as opsonophagocytosis assays (OPA), in which antibody and complement-bound bacteria are combined with human or mouse phagocytes to determine levels of bacterial killing, are useful for studying antibody function. Positive OPA results correlate with vaccine-induced protection in a mouse model. (Stranger-Jones et al., 2006, Proc. Nati. Acad. Sci. 103(45):16942-16947). A similar oxidative burst assay can be used to assess the level of reactive oxygen species (ROS) by fresh human or mouse neutrophils following interaction with antibody and complement-bound bacteria.
In some cases, one can determine that a candidate polypeptide possesses cell-mediated immunological activity and, therefore, the candidate polypeptide may exhibit immunological activity in the absence of inducing the production of antibodies. (Spellberg et al., 2008, Infect. Immun. 76(10):4575-4580). Cytotoxic or CD8+ T cells primarily kill infected cells directly through various effector mechanisms, while helper CD4+ T cells function to provide important signaling in the way of cytokines. These T cell classes can be further subdivided based on the cytokines they produce, and different subclasses are effective against different bacterial pathogens. T cells are often studied by assessing their phenotypes with flow cytometry, where antibodies are used to visualize the levels of specific surface markers that enable classification of the T cells as, for example, a recently activated CD4+ T cell, a memory CD8+ T cell, etc. In addition, cytokines and other products of T cells can be studied by isolating the T cells from lymphoid tissue and re-stimulating them with cognate antigen. Following antigen stimulation the T cells produce cytokines that may be visualized by, for example, intracellular cytokine staining coupled with flow cytometry, or collecting the cell supernatants and using Luminex bead technology to measure 15-25 cytokines simultaneously.
For example, Table 9 provides cytokine recall response of spleen cells from mice immunized with representative Burkholderia iron-regulated polypeptides.
Thus, in addition to mouse models, those of ordinary skill in the art recognize that immunological activity commensurate with the methods described herein may correlate with any one or more of the following: Western blot data showing that serum from animals exposed to a Burkholderia spp. contains antibody that specifically binds to a candidate polypeptide, cell surface binding assays demonstrating that antibody that specifically binds to a candidate polypeptide specifically binds to a Burkholderia spp., opsonophagocytosis data, and cytokine induction.
Another aspect of the present invention provides methods for detecting antibody that specifically binds polypeptides as described herein. These methods are useful in, for instance, detecting whether an animal has antibody that specifically binds polypeptides as described herein, and diagnosing whether an animal may have a condition caused by a microbe expressing polypeptides described herein, or expressing polypeptides that share epitopes with the polypeptides described herein. Such diagnostic systems may be in kit form. The methods include contacting an antibody with a preparation that includes a polypeptide as described herein to result in a mixture. The antibody may be present in a biological sample, for instance, blood, milk, or colostrum. The method further includes incubating the mixture under conditions to allow the antibody to specifically bind the polypeptide to form a polypeptide:antibody complex. As used herein, the term “polypeptide:antibody complex” refers to the complex that results when an antibody specifically binds to a polypeptide. The preparation that includes the polypeptides as described herein may also include reagents, for instance a buffer, that provide conditions appropriate for the formation of the polypeptide:antibody complex. The polypeptide:antibody complex is then detected. The detection of antibodies is known in the art and can include, for instance, immunofluorescence or peroxidase. The methods for detecting the presence of antibodies that specifically bind to polypeptides as described herein can be used in various formats that have been used to detect antibody, including radioimmunoassay and enzyme-linked immunosorbent assay.
In another aspect, this disclosure provides a kit for detecting antibody that specifically binds polypeptides as described herein. The antibody detected may be obtained from an animal suspected to have an infection caused by a gram negative microbe, more preferably, a member of the family Burkholderiaceae, preferably, Burkholderia spp. such as, for example, B. thailandensis, B. mallei, B. pseudomallei, B. cenocepacia, or B. multivorans.
The kit can include at least one of the polypeptides as described herein (e.g., one, at least two, at least three, etc.), in a suitable packaging material in an amount sufficient for at least one assay. Optionally, other reagents such as buffers and solutions needed to practice the invention are also included. For instance, a kit may also include a reagent to permit detection of an antibody that specifically binds to a polypeptide as described herein, such as a detectably labeled secondary antibody designed to specifically bind to an antibody obtained from an animal. Instructions for use of the packaged polypeptides are also typically included. As used herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by conventional methods, generally to provide a sterile, contaminant-free environment. The packaging material may have a label which indicates that the polypeptides can be used for detecting antibody that specifically binds polypeptides as described herein. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to detect the antibody. As used herein, the term “package” refers to a container such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the polypeptides, and other reagents, for instance a secondary antibody. Thus, for example, a package can be a microtiter plate well to which microgram quantities of polypeptides have been affixed. A package can also contain a secondary antibody. “Instructions for use” typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Compositions derived from B. thailandensis E264 included novel polypeptides expressed under iron restricted growth. Master seed stocks were prepared by inoculating a single colony of strain E264 into 100 mL Tryptic Soy Broth (TSB, Difco Laboratories, Detroit, Mich.) followed by incubation in a shaking incubator at 37° C. and 400 rpm overnight. The culture was expanded by a 1/100 dilution into fresh TSB and incubated as before until it reached the mid-log phase of growth. The bacteria were pelleted by centrifugation at 5000×g, 4° C., for 10 minutes. The supernatant was decanted, and an equal volume of PBS was added to resuspend the pellet. The bacteria were pelleted by centrifugation as before and the pellet was resuspended in TSB containing 15-50% glycerol at one-tenth of the original culture volume. Stocks were frozen in aliquots of 100-1000 μL and stored at −80° C. Working seed stocks were prepared using the same procedure but with the initial inoculum obtained from a frozen master stock vial.
The iron-regulated polypeptide (IRP) composition was prepared by inoculating B. thailandensis from a frozen stock into 10 mL tryptic soy broth (TSB) supplemented with 200 μM 2,2′-dipyridyl (DP) (Sigma-Aldrich, St. Louis, Mo.). Iron-replete cultures contained 300 μM FeCl3. Cultures were incubated at 37° C. on a shaker at 400 rpm. After 16-24 hours incubation, 10 mL of culture was transferred into 90 mL prewarmed TSB supplemented with either 200 μM DP or 300 μM FeCl3 and incubated at 37° C. on a shaker at 400 rpm. After 16-24 hours, 90 mL of culture was transferred into 900 mL prewarmed TSB supplemented with either 300 μM FeCl3 or 400 μM DP and incubated at 37° C. on a shaker at 400 rpm. After 16-20 hours the cells were harvested by centrifugation at 10,000×g for 20 minutes at 4° C., resuspended in PBS and centrifuged to obtain the final cell pellet. The cell pellets were weighed and stored frozen at −80° C.
The frozen cell pellet was thawed at room temperature and resuspended by the addition of 25 mL Tris-EDTA buffer (15 mM Tris-HCl, 3 mM EDTA, pH 8.5) per gram of pellet. The cell suspension was distributed into sterile 50 mL conical tubes at a volume of 35 mL/tube. Tubes were placed in a −80° C. freezer for a minimum of 30 minutes after which they were removed and thawed at 25° C.-37° C. The cells were disrupted by sonication (Branson, Danbury, Conn.) for 90 seconds on ice. The disrupted cell suspension was transferred to a sterile 40 mL round bottom centrifuge tube and centrifuged at 39,800×g for 20 minutes at 4° C. The soluble membrane fraction in the supernatant was transferred to a sterile 40 mL round bottom centrifuge tube, and 3 mL of 30% sarcosine (N-lauroylsarcosine sodium salt, Sigma-Aldrich, St. Louis, Mo.) was added to each tube. Tubes were incubated for 16-24 hours at 4° C. with rocking. The detergent-insoluble membrane fraction was pelleted by centrifugation at 39,800×g for two hours at 4° C. followed by removal of the supernatant. Pellets were dried by inverting the tubes for a minimum of five minutes. The pellets were resuspended in 75 μl PBS (pH 7.2).
A sample of the pellet was evaluated by denaturing SDS-PAGE using 10% gels stained with Coomassie Blue and imaged using a LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebr.)(
Two-dimensional PAGE separation of membrane extracts from B. thailandensis grown under either iron-rich or iron-depleted conditions was performed using an acidic polyacrylamide system with cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and sodium dodecyl sulfate (SDS) for the second dimension (Hartinger et al., 1996, Anal Biochem 240:126-133). A 50 μg sample of membrane extract was solublized in 7.5 M urea, 10% 16-BAC (w/v), 75 mM DTT, and 0.05% pyronin Y, and electrophoresis was conducted using an 8.7% acrylamide gel with a 50 mM phosphoric acid running buffer. Electrophoretic separation in the 16-BAC phase was carried out at a current of 15 mA, from anode to cathode, at 4° C. overnight until the dye front migrated out of the gel. The gel was stained with 8250 Coomassie Blue. Each lane was excised and equilibrated through four changes of 0.1 M Tris, pH 6.8, with further equilibration in reducing buffer (75 mM Tris, 576 mM glycine, 0.3% SDS, 5% β-mercaptoethanol) for five minutes. Gel strips were overlaid onto the second dimension gel and fixed into place with 0.1% agarose. SDS-PAGE (5-16% gradient) separation was performed using a PROTEAN plus dodeca cell (Bio-Rad Laboratories, Inc., Hercules, Calif.) at 25 mA/gel until the dye front migrated out of the gel.
Separation in the first and second dimensions is dependent on molecular weight, hence, larger polypeptides appear in the upper right portion of the second dimension gel, and polypeptides of decreasing molecular weight appear on a diagonal toward the lower left portion of the gel (
Extracts of B. thailandensis grown under iron depleted conditions were subjected to 2D gel electrophoresis as described in Example 2. Regions of the gel that stained positive for polypeptides were excised from the second dimension gel and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) according to the following procedure (Wilm et al., 1996, Nature 379:466-469). The excised gel spot was cut into smaller pieces and washed twice with water for 10 minutes each. All wash volumes were approximately equal to twice the volume of the gel pieces. The gel pieces were washed with a 1:1 mix of acetonitrile and 100 mM ammonium bicarbonate, pH 7.4, for 15-30 minutes. The wash was repeated once or twice as needed to remove the stain. The last wash was replaced with sufficient 100% acetonitrile (ACN) to cover the gel pieces until they turned opaque and sticky, whereupon they were removed and dried in a SAVANT SPEEDVAC (Thermo Fisher Scientific Inc., Waltham, Mass.) at 30° C. for 30 minutes. The dried gel pieces were placed into 25-30 μl of 50 mM ammonium bicarbonate containing trypsin at 110 ng/μl and digested for 16-18 hours at 37° C. Following digestion, the mixture was separated by centrifugation, the supernatant was removed, and a volume of 25-30 μl 0.1% trifluoroacetic acid (TFA) was added to extract the peptides. The samples were sonicated intermittently for 30 minutes, and supernatants containing the peptides were transferred into new tubes. The gel extraction was repeated using a solution of 0.1% TFA/30% ACN followed by 0.1% TFA/70% ACN. The pooled supernatants were concentrated in a SPEEDVAC to a final volume of 30-70 μl.
MALDI-MS analysis was performed using nano high-pressure liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) methods with LTQ ORBITRAPS (Thermo Fisher Scientific Inc., Waltham, Mass.) coupled with NanoLC-2D pumps (Eksigent Technologies, LLC, AB SCIEX, Framingham, Mass.) for data acquisition and Scaffold analysis tool (Proteome Software, Portland, Oreg.) to compile the outputs from multiple search algorithms.
A polypeptide was considered to be present if at least two unique peptides for that polypeptide were identified in an excised gel spot. Fifteen polypeptides of interest were identified in the iron restricted extract (Table 2). Eight of the fifteen were detected only in the iron restricted extract and not in extracts from B. thailandensis grown in the presence of iron. These eight polypeptides are reflected in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:25.
B. thailandensis grown under iron restricted conditions.
aMolecular weight (predicted).
The isobaric tags for relative and absolute quantification (iTRAQ) method was used to evaluate changes in expression of outer membrane proteins associated with growth of B. thailandensis in iron-depleted compared with iron-replete medium. Cultures were grown as described in Example 1, and 40 μg of outer membrane extract was evaluated. The primary amines of peptides and polypeptides were labeled with isobaric agents using ITRAQ-8plex reagents (Applied Biosystems, Life Technologies Corp., Carlsbad, Calif.) according to the manufacturer's instructions. Cation exchange chromatography was applied using an MCX column (Waters Corp., Milford, Mass.), and the peptides were separated using an UltiMate 3000 nano LC system (Dionex Corp., Sunnyvale, Calif.) coupled to ESI mode using a QSTAR XL mass spectrometer (Applied Biosystems, Life Technologies Corp., Carlsbad, Calif.).
The results are shown as the fold increase for a given protein in the iron restricted extract relative to the iron-replete extract (Table 3). A polypeptide was considered to be iron-regulated if it displayed a fold increase greater than 1 in at least two trials. Polypeptides identified as iron-regulated using iTRAQ analysis include those reflected in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:16.
aMolecular weight (predicted).
bFold upregulation of polypeptides in extract vaccine from B. thailandensis E264 grown under iron-restricted compared with iron-replete conditions. A value of “ND” indicates that no peptides were detected.
A bioinformatics approach was undertaken to identify additional polypeptides that might be involved in iron uptake or utilization in B. thailandensis E264. Polypeptides of interest that had not already been identified as described in Examples 3 and 4 were downselected further to those shown in Table 4 based on structural similarity to known iron receptor proteins, localization in the bacterial outer membrane (computationally predicted by PSORTb V3.0), and additional information implicating them as iron receptors through comparison with B. pseudomallei orthologs. In addition, the polypeptide reflected in SEQ ID NO:19, which is involved in the acquisition and release of iron from intracellular storage by bacterioferritin, was also identified as a potential vaccine target.
aMolecular weight (predicted).
The final determination of metal regulation for candidate proteins was based on mass spectroscopy, iTRAQ analysis, and bioinformatics described in Examples 3, 4, and 5, respectively. Each of the 25 polypeptides was determined to be either iron-regulated, non-iron regulated, or had iron regulation that was uncertain (Table 5). This determination was made using the following, step-wise inclusion criteria. First, any protein that was detected in mass spectroscopy of iron-restricted but not iron replete extracts was considered to be iron-regulated. Second, any protein that demonstrated a fold increase in expression of greater than 1 in iTRAQ analysis was considered to be iron-regulated. This resulted in a total of 14 polypeptides being classified as iron-regulated. Of the remaining proteins, four were considered to be non-metal-regulated if they were detected in at least two iTRAQ trials but did not demonstrate a fold increase greater than 1. Seven polypeptides could not be definitively categorized as either iron-regulated or non-iron regulated, and these were classified as polypeptides whose iron regulation was uncertain.
aMolecular weight (predicted).
bIR, iron regulated; NIR, non-iron regulated; U, iron regulation is uncertain
Vaccine efficacy studies were conducted in BALB/c and A/J mice using 15 mice per group. Vaccines were formulated to deliver doses of 50 ng, 100 ng, and 300 ng of extract vaccine containing 10 ng/dose of CpG and emulsified in 50% incomplete Freund's adjuvant (IFA). The placebo group consisted of IFA/CpG alone. Mice were immunized subcutaneously in a volume of 100 μl at Day 0 with a booster at Day 28.
On Day 42, mice were challenged intratracheally with a previously determined LD80 of B. thailandensis E264, and survival was monitored for 14 days. The challenge dose of B. thailandensis E264 was prepared as follows. A loop of bacteria was taken from a frozen glycerol stock, placed into 10 mL Luria Bertani (LB) broth, and incubated at 37° C. overnight. The 10 mL culture was inoculated into 100 ml of LB, incubated at 37° C. and monitored for growth by optical density at a wavelength of 600 nm (OD600). When the culture reached an OD600 of 1.9 (approximately 1×109 cfu/ml), the suspension was serially diluted with PBS to the predetermined challenge dose. All suspensions and dilutions were plated on LB agar to verify the actual concentration of bacteria administered.
Aerosolized challenge of mice was performed by intratracheal instillation according to an established protocol. In brief, mice were lightly anesthetized with a mixture of ketamine and xylazine (80 mg/kg ketamine and 20 mg/kg xylazine). The animal was manually restrained in an upright position and a padded forceps was used to gently open the mouth and hold the tongue down to the lower jaw to prevent swallowing. A second investigator then carefully administered 30 μl fluid to the back of the mouth using a sterile pipette tip and a p100 PIPETMAN (Gilson, Inc., Middleton, Wis.). This was followed by placing a gloved finger over the mouse nostrils to prevent obligate nasal breathing. The combination of holding the tongue to prevent swallowing and closing off the nostrils to prevent nasal breathing causes the mouse to inhale through the mouth and aspirate the instilled fluid. An immediate cough by the mouse, which can be detected both audibly and visibly, was used to verify that the procedure was performed correctly.
The results are shown in
Because iron-regulated polypeptides tend to be evolutionarily conserved, the B. thailandensis extract vaccine would be expected to protect against infection by two of the most highly virulent species of Burkholderia, namely, B. pseudomallei and B. mallei. It may also protect against other Burkholderia such as those in the B. cepacia complex (BCC) that cause opportunistic infections in people with cystic fibrosis and chronic granulomatous disease. In this regard, B. cenocepacia (formerly BCC genomovar III) and B. multivorans (formerly BCC genomovar II) are two of the most common isolates from cystic fibrosis patients and are associated with increases in morbidity and mortality.
In the Example 6 lethal challenge studies, B. thailandensis was selected as a seed strain for the extract vaccine due to safety considerations associated with employing a BSL3 agent in manufacturing. However, recombinant polypeptide vaccines represent an alternative approach that is not subject to this limitation. Thus, a recombinant Burkholderia vaccine could employ polypeptides cloned directly from B. pseudomallei, which is highly lethal and difficult to treat, recognized as a potential bioweapon, endemic in certain parts of Asia and Australia, and considered to be an emerging infectious agent in other parts of the world. To address the possibility of whether a broad spectrum vaccine could be created using extract or recombinants, a bioinformatics approach was undertaken to compare the percent identity of the polypeptides identified in Examples 3, 4, and 5 across a variety of Burkholderia species and strains. Targeted vaccine polypeptides were compared by standard protein BLAST (blastp, NCBI) using a database of non-redundant polypeptide sequences and default parameters. To provide information on both the B. thailandensis extract vaccine and the B. pseudomallei recombinant vaccine, the analysis was performed using B. thailandensis E264 or B. pseudomallei K96243 polypeptides as the query against a subset of the sequenced Burkholderia strains available through GenBank. This panel of targeted strains was selected to achieve diversity based on geographic variability and differences in clinical disease and/or outcomes. Strains of particular interest to the Defense Threat Reduction Agency, as indicated in Broad Agency Announcements, were also included. Information on the selected strains and their sources is shown in Table 6.
B.
pseudomallei
B.
mallei
B.
thailandensis
B.
cenocepacia
B.
multivorans
Amino acid sequence comparisons for the polypeptides reflected in SEQ ID NO:1 through SEQ ID NO:25 are shown in
To further evaluate the conservation of the iron-regulated polypeptides across multiple species, a cross-species alignment was performed for the polypeptides reflected in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:9 using Clustal Omega (v1.2.0, European Molecular Biology Laboratory-European Bioinformatics Institute) and one strain from each of the five species. These three polypeptides were selected because they are representative of three different types of iron-regulated polypeptides, namely, a siderophore receptor (SEQ ID NO:1), an RND efflux system polypeptide (SEQ ID NO:2), and an OmpA family outer membrane polypeptide (SEQ ID NO:9), and they showed varying levels of identity across species. The strains used for comparison were B. thailandensis E264, B. pseudomallei K96243, B. mallei NCTC10229, B. cenocepacia AU1054, and B. multivorans ATCC17616.
The alignments indicate that certain regions of these three polypeptides are highly conserved across strains selected from all five Burkholderia species of interest (
A comparative immunogenicity study was performed to evaluate the humoral and cellular immune responses elicited by the B. thailandensis IRP (iron-regulated polypeptide) extract vaccine, a purified subunit of the IRP extract vaccine, and a mixture of eight recombinant polypeptides cloned from B. pseudomallei K96243 (eight recombinant Burkholderia IRPs, rBIRP8, Table 7).
aMolecular weight (predicted).
The extract vaccine used in these studies was prepared as described in Example 1, and the purified subunit vaccine was prepared with the following modifications. After breaking the cells, the soluble fraction was diluted 1:1 with 0.1 M sodium carbonate and mixed at 4° C. for one hour before continuing with centrifugation of the membrane fraction and sarcosine extraction. The insoluble extract product was subjected to a hot phenol extraction (Westphal et al., 1965, Methods in Carbohydrate Chemistry 5:83-91). In brief, the insoluble extract was resuspended in water and heated to 70° C. An equal volume of phenol was added, the temperature was re-equilibrated to 70° C., and the mixture was maintained at this temperature for 30 minutes. The phases were separated at room temperature using a glass separatory funnel. The phenol fraction and interface were combined and precipitated by the addition of methanol at a 3:1 v:v ratio. The polypeptides were recovered by centrifugation for one hour at 30,000×g and the pellet was washed with methanol. Methanol was removed through two sequential washes with a 50 mM Tris/1 mM EDTA buffer, and the final pellet was recovered by centrifugation for one hour at 45,000×g. The pellet was resuspended in a buffer containing 50 mM Tris, 1 mM EDTA, 7 M urea, 2 M thiourea, and 1% zwittergent by adding a volume of 1 ml per mg of pellet. The pellet was further solubilized by sonication at 60° C.-65° C. for 10 minute cycles interspersed with a 10 minute rest, followed by overnight incubation at room temperature on a rocking platform. The resulting product had an endotoxin level of <100 EU/mg as determined by a kinetic-turbidimetric Limulus amoebocyte lysate (LAL) assay (Charles River Laboratories, Wilmington, Mass.).
Eight IRP coding regions were cloned from B. pseudomallei K96243 DNA and expressed in E. coli (Table 7). The signal peptide sequence was predicted using SignalP (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark) and, in some cases, PrediSi (Institute for Microbiology, Technical University of Braunschweig, Braunschweig, Germany) or Phobius (Stockholm Bioinformatics Centre, Stockholm University, Stockholm, Sweden). Gene-specific primers were designed to express the polypeptide without the signal sequence. PCR was performed using DNA Herculase II (Agilent Technologies, Santa Clara, Calif.), and the PCR products were ligated into the pQE30Xa expression vector (Qiagen, Valencia, Calif.), which adds a 6× Histidine tag to the N-terminus Ligation reactions were used to transform E. coli XL-1 blue. Clones were selected and verified by DNA sequencing (ACGT, Inc., Wheeling, Ill.). The recombinant B. pseudomallei iron-regulated polypeptides (rBIRPs) were expressed and purified using standard methods. In brief, frozen bacterial stocks (100 μl) were used to inoculate 20 ml of Luria-Bertani Broth containing 100 μg/ml of ampicillin, and the culture was grown at 37° C. in a shaking incubator at 250 rpm. After 16 hours, the culture was diluted added to 1 L of Luria-Bertani Broth containing 100 μg/ml of ampicillin, grown to an optical density (600 nm) of 0.6, and induced by the addition of 1 mM IPTG to a final concentration of 1 mM. Cultures were incubated for an additional 4-20 hours, depending on the optimum time for expression as previously determined for each clone. Bacterial cell pellets were harvested by centrifugation at 4,000 rcf for 20 minutes at 4° C., washed in PBS, and resuspended in 20 mM Tris buffer containing 100 μg/ml lysozyme. The cells were disrupted by sonication at 50% duty cycle and output (Branson Sonifier, Danbury, Conn.) for eight minutes on ice. The lysate was subjected to centrifugation for 10 minutes at 40,000×g at 4° C. to remove insoluble material. The soluble supernatants were processed by immobilized metal affinity chromatography (HisTrap FF 5 ml, GE Healthcare) to purify the histidine-tagged recombinant polypeptide, followed by anion exchange chromatography to increase the purity and remove endotoxin. Polypeptide concentration was determined using the BCA method (Thermo Scientific, Rockford, Ill.) and polypeptide purity was measured by SDS-PAGE and densitometry.
BALB/c mice were vaccinated with 100 μg B. thailandensis E264 extract vaccine, 100 μg B. thailandensis E264 purified subunit vaccine (Example 6), or a mixture of eight rBIRPs (rBIRP8, polypeptides of SEQ ID NO:1-SEQ ID NO:8 at 10 μg each). Vaccines were formulated with 10 μg CpG per dose and emulsified in 50% IFA. The placebo group consisted of IFA/CpG alone. Each group contained a total of 5 mice. Vaccines were administered subcutaneously in a volume of 100 μl at Day 0 with a booster at Day 28. Mice were bled on Day −1 (one day before the initial vaccination) and Day 27 with a terminal bleed performed on Day 56. Blood was processed to obtain serum, which was stored at −80° C. Equal volumes of individual serum samples were pooled and evaluated for antibody production.
IgG antibody titers to individual rBIRPs were determined by ELISA. In brief, 100 μl of polypeptide at 2 μg/ml, solubilized in 8 M urea, was added to each well of a 96-well EIA/RIA plate (Corning Inc., Tewksbuty, Mass.) and incubated overnight at 4° C. All remaining steps were performed at room temperature. The plate was washed three times with PBS wash buffer (PBS containing 0.05% Tween 20) followed by the addition of 200 μl/well sample buffer consisting of PBS containing 0.05% Tween 20 and 1% bovine serum albumin. After 90 minutes, the sample buffer was replaced with 100 μl/well PBS sample buffer. Serial ⅓ dilutions of the primary antisera were performed in the plate by the addition of 50 μl to the first row, mixing 10 times, and transfer of 50 μl to the next row. The plate was incubated for 90 minutes followed by three washes and addition of 100 μl/well of an HRP conjugated goat anti-mouse IgG, heavy chain specific antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). After a 90 minute incubation, the plate was washed four times followed by the addition of 100 μl TMB/well (BioFX, SurModics, Inc., Eden Prairie, Minn.). Color was allowed to develop for 30 minutes, and the reaction was stopped by the addition of 100 μl stop reagent (BioFX, SurModics, Inc., Eden Prairie, Minn.). The absorbance was measured at a wavelength of 450 nm, and the titer was calculated as the inverse of the dilution corresponding to an absorbance of 1.0. Controls included a standardized primary serum included on each plate to monitor assay variability and wells that were uncoated to subtract background. The limit of detection for the assay was the inverse of the initial serum dilution (indicated as a dotted line on
All eight rBIRPs were highly and equivalently immunogenic, eliciting antibody titers greater than 100,000 by ELISA (
Antibody specificity was further evaluated by western blotting. Each rBIRP was electrophoresed on a 4-15% TGX gradient gel and the gel was blotted onto a nitrocellulose membrane. All membrane incubations were performed on a rocking platform at room temperature. Membranes were incubated for 1 hour in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, Nebr.) diluted 1:1 in Tris buffered saline. The blocking buffer was decanted and replaced with the primary antiserum diluted in Odyssey blocking buffer as above with the addition of 0.05% Tween 20. After one hour, the primary antiserum was decanted and the membrane was washed three times for 10 minutes each with TBS containing 0.05% Tween. The final wash was decanted and goat anti-mouse IgG (IR dye conjugate, LI-COR), diluted in the same buffer as the primary antibody, was added at the dilution recommended by the manufacturer. After one hour the membrane was washed three times as before, rinsed with TBS, and dried overnight in the dark. Blots were imaged on a LI-COR Odyssey imaging system.
The results from western blots paralleled those for the ELISA (Table 8). Antibodies to all eight rBIRPs were detected in serum from mice vaccinated with either rBIRP8 or the PS vaccine. The polypeptide of SEQ ID NO:2 was detected by serum from mice immunized with the extract vaccine, and similar to what was seen in the ELISA, this polypeptide appears to be highly immunogenic for eliciting an antibody response.
aReactivity was assessed as: − (not detected), + (low), ++ (moderate), or +++ (strong).
At termination of the experiment on Day 56, spleens were harvested, processed into mononuclear cell preparations, and cultured individually with 10 μg/ml of individual rBIRPs or the purified subunit. After 48 hours, the cell supernatants were harvested and frozen at −80° C. until assessment for cytokine production using a cytometric bead array kit (BD Biosciences, San Jose, Calif.) and flow cytometer (FACSCanto2, BD Biosciences, San Jose, Calif.), performed according to the manufacturer's protocol. The net production of cytokine for each polypeptide stimulus was calculated by subtracting the corresponding value for the placebo group.
—b
a5 mice were tested individually in each vaccine group
b—, below the level of the placebo group (background) or below the level of detection for the assay.
cPS, purified subunit
Recall responses to individual rBIRP polypeptides and the PS vaccine polypeptide mixture were observed in all three vaccine groups (extract, PS, and rBIRP8, Table 9). Negative control spleen cell cultures incubated with PBS did not exceed background levels in the placebo control and therefore are not shown. Mice immunized with the extract vaccine demonstrated low but measurable production of IL-2 and IL-17 in response to four of the eight rBIRPS (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7). In addition, the polypeptides reflected in SEQ ID NO:2 and SEQ ID NO:7 stimulated production of IFN-γ. The pattern of responses was similar though not identical for mice vaccinated with the extract or PS vaccines. Most notable was that both groups exhibited a strong IL-6 response to PS antigens. Mice immunized with rBIRP8 tended to exhibit stronger cytokine recall responses to individual rBIRPs than mice immunized with extract or PS vaccines, suggesting that responses to IRPs can be boosted by decreasing the complexity of the vaccine composition and increasing the amounts of targeted IRP antigens. Moreover, the Thl (IFN-γ, TNF-α)/Th17 (IL-17, TNF-α) type of response observed for rBIRP8, coupled with IL-2 production to promote proliferation and an undetectable IL-10 (regulatory) response, is intriguing since IFN-γ is generally associated with protection to intracellular pathogens, and IFN-γ,TNF-α, and IL-17 have been implicated in protective responses to Burkholderia infections.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/922,504, filed Dec. 31, 2013, which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61922504 | Dec 2013 | US |
