Patent Grant
7815911
The presently-disclosed subject matter relates to compositions and methods for preventing and/or treating plague, including compositions and methods for preventing and/or treating infection by Yersinia pestis.
Yersinia pestis is a highly virulent extracellular pathogen, which causes lethal infection in humans and other animals. Y. pestis infection causes plague, including bubonic, pneumonic, and septicemic plague. The bacterium poses an ominous threat as an agent of bioterrorism. It is highly infectious and contagious by aerosol and can kill in as little as two days after exposure.
Y. pestis can be categorized into sub-types, including: Antigua, Medievalis, and Orientalis. Y. pestis pathogenicity can be blocked by antibodies to two protein antigens, the capsule subunit Caf1 (also known as F1 antigen, or Fraction 1 antigen), and a surface protein called V antigen (also known as LcrV). In this regard, compositions including the F1 antigen and the V antigen have been studied for use in treating and preventing Y. pestis infection. Although such compositions have been shown to offer modest protection, against some strains of Y. pestis, there are many virulent strains of Y. pestis. Examples of such strains include, for example, strain KIM of the Mediaevalis sub-type, and strain CO92 of the Orientalis sub-type. Additionally, modifications to Y. pestis antigens can be made without destroying virulence. For example, the V-antigen is variable, such that known compositions containing the V-antigen may not be fully protective against certain modified strains. Furthermore, the F1 antigen is not required for virulence in pneumonic plague, and its gene is easily deleted, posing the potential of a weaponized F1-lacking Y. pestis against which known compositions containing F1 antigen are not effective.
Accordingly, there remains a need in the art for compositions and methods for preventing and/or treating various types and strains of Y. pestis infection.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
The presently-disclosed subject matter includes compositions and methods for treating a Yersinia pestis (Y. pestis) infection (or plague resulting from such an infection). Compositions and methods of the presently-disclosed subject matter can be used for inducing an immune response in a subject.
In some embodiments, compositions include an isolated polypeptide molecule comprising a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, the isolated polypeptide molecule comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3. In some embodiments, the isolated polypeptide molecule comprises the sequence of SEQ ID NO: 3.
In some embodiments, compositions include an isolated polypeptide molecule comprising a sequence at least 80%, 85%, 90%, or 95% identical to an amino acid sequence including residues 137-345 of SEQ ID NO: 1. In some embodiments, compositions include an isolated polypeptide molecule comprising a sequence at least 80%, 85%, 90%, or 95% identical to an amino acid sequence including residues 137-345 of SEQ ID NO: 1. In some embodiments, compositions include an isolated polypeptide molecule comprising a sequence at least 80%, 85%, 90%, or 95% identical to an amino acid sequence including residues 32-345 of SEQ ID NO: 1.
In some embodiments, compositions include an isolated polypeptide molecule comprising an amino acid sequence including residues 32-345 of SEQ ID NO: 1. In some embodiments, compositions include an isolated polypeptide molecule comprising an amino acid sequence including residues 32-409 of SEQ ID NO: 1. In some embodiments, compositions include an isolated polypeptide molecule comprising an amino acid sequence including residues 32-409 of SEQ ID NO: 1.
In some embodiments, compositions include an isolated polypeptide molecule comprising a sequence at least about 80%, 85%, 90%, or 95% identical to an amino acid sequence of SEQ ID NO: 1, wherein up to about 5, 10, 15, 20, 25, 30, 50, 125, or 150 amino acids are truncated from the N-terminus of SEQ ID NO: 1. In some embodiments, the isolated polypeptide molecule comprises at least about 200, 210, 225, 235, 245, 255, 265, 275, 285, 295, 300, 325, 350 or 375 amino acids of SEQ ID NO: 1.
In some embodiments, compositions include an isolated polypeptide molecule comprising a sequence at least about 80%, 85%, 90%, or 95% identical to an amino acid sequence of SEQ ID NO: 1, wherein up to about 10, 50, 75, 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, or 400 amino acids are truncated from the C-terminus of SEQ ID NO: 1. In some embodiments, the isolated polypeptide molecule comprises at least about 200, 210, 225, 235, 245, 255, 265, 275, 285, 295, 300, 325, 350 or 375 amino acids of SEQ ID NO: 1.
In some embodiments, compositions comprise an polypeptide molecule encoded by a nucleotide sequence at least about 80, 85, 90, or 95% identical to SEQ ID NO: 2, or a subsequence thereof. The encoded polypeptide has improved immune response activity relative to, or has all or some of the immune response activity of, a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 2. In some embodiments, the polypeptide molecule is encoded by a subsequence of the nucleotide sequence of SEQ ID NO: 2, wherein up to about 15, 30, 45, 60, 75, 90, 150, 375, or 450 nucleotide residues are truncated from the 5′ terminus of SEQ ID NO: 2. In some embodiments, the polypeptide molecule is encoded by a subsequence of the nucleotide sequence of SEQ ID NO: 2, wherein up to about 30, 150, 225, 300, 450, 525, 600, 675, 750, 825, 900, 975, 1050, or 1200 nucleotide residues are truncated from the 3′ terminus of SEQ ID NO: 2.
In some embodiments, compositions include a conservatively-substituted variant of the polypeptide molecule of SEQ ID NO: 1, or functional fragments thereof. In some embodiments, compositions include a conservatively-substituted variant of the polypeptide molecule of SEQ ID NO: 3, or functional fragments thereof.
In some embodiments, the polypeptide molecule includes the amino acid sequence of SEQ ID NO: 1, or a subsequence thereof, with up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 conservative amino acid substitutions. In some embodiments, the polypeptide molecule includes the amino acid sequence of SEQ ID NO: 3, or a subsequence thereof, with up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 conservative amino acid substitutions. In some embodiments, the conservative amino acid substitutions are at or between residues 346-622 of the YadC polypeptide of SEQ ID NO: 1, or a subsequence thereof. In some embodiments, the polypeptide molecule includes the amino acid sequence of SEQ ID NO: 1, or a subsequence thereof, with up to about 1, 2, 3, 4, or 5 conservative amino acid substitutions are at or between residues 32-345 of the YadC polypeptide of SEQ ID NO: 1, or a subsequence thereof.
In some embodiments, compositions induce an immune response in a subject against F1-positive and F1-negative strains of Y. pestis. In some embodiments, compositions induce an immune response in a subject against F1-negative strains of Y. pestis. In some embodiments, the subject is in need of treatment for a Y. pestis infection. In some embodiments, the subject is susceptible to Y. pestis infection.
In some embodiments, methods include administering to a subject a composition including a YadC polypeptide, as described herein. The composition can be administered in an amount sufficient to induce an immune response in the subject. The presently-disclosed subject matter includes methods for treating a Yersinia pestis (Y. pestis) infection and/or treating a plague resulting from such an infection, including administering to the subject an effective amount of a composition including a YadC polypeptide, as described herein. In some embodiments, methods include introducing into a subject a composition comprising an isolated polypeptide molecule comprising an amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, methods include introducing into a subject a composition comprising an isolated polypeptide molecule comprising an amino acid sequence of SEQ ID NO: 3, or a functional fragment thereof. In some embodiments, methods include introducing into a subject a composition comprising a sequence at least 80, 85, 90, or 95% identical to SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, methods include introducing into a subject a composition comprising a sequence at least 80, 85, 90, or 95% identical to SEQ ID NO: 3, or a functional fragment thereof. In some embodiments, methods include introducing into the subject a composition comprising an isolated polypeptide molecule comprising the sequence of SEQ ID NO: 1 with up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 conservative amino acid substitutions, or a functional fragment thereof. In some embodiments, methods include introducing into the subject a composition comprising an isolated polypeptide molecule comprising the sequence of SEQ ID NO: 3 with up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 conservative amino acid substitutions, or a functional fragment thereof.
SEQ ID NO: 1 is an amino acid sequence for a YadC polypeptide;
SEQ ID NO: 2 is a nucleotide sequence encoding the YadC polypeptide of SEQ ID NO: 1;
SEQ ID NO: 3 is an amino acid sequence for an exemplary fragment of the YadC polypeptide of SEQ ID NO: 1; and
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs. Although any methods, compositions, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, compositions, and materials are now described.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, 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 indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
The presently-disclosed subject matter includes compositions and methods for inducing an immune response in a subject. The presently-disclosed subject matter further includes antibodies, such as humanized monoclonal antibodies, that recognize epitopes of YadC, and active antibody fragments. As used herein, the term “immune response” refers to a humoral and/or cellular immune response, in a subject, to an introduction of a composition described herein, which response interferes with or inhibits the virulence, activity, spread, and/or growth of a Yersinia pestis (Y. pestis). Examples of immune responses include the stimulation of antibodies, T-cells, macrophages, B-cells, dendritic cells, etc. These responses can be measured routinely, as will be understood by those of ordinary skill in the art. As used herein, an immune response activity refers to the ability of a compound to elicit an immune response.
As used herein, the term “subject” refers to both human and animal subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently-disclosed subject matter. As such, the presently disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
The compositions and methods of the presently-disclosed subject matter can be used for treating a Yersinia pestis (Y. pestis) infection and/or treating a plague resulting from such an infection. As used herein, the terms “treatment” and “treating” relate to any treatment of a Y. pestis, including but not limited to prophylactic treatment (i.e., pre-infection), and therapeutic treatment (i.e., post-infection). The terms “treatment” or “treating” include: preventing the development of a Y. pestis infection and/or plague occurring in a subject; reducing the severity of a Y. pestis infection and/or plague occurring in a subject; and/or ameliorating or relieving the symptoms associated with a Y. pestis infection and/or plague occurring in a subject.
In some embodiments, the compositions and methods of the presently-disclosed subject matter can be used for treating an F1-positive (F1+) or an F1-negative (F1−) Y. pestis strain. In some embodiments, the compositions and methods can be used for treating a Y. pestis CO92 strain. In some embodiments, the compositions and methods can be used for treating an F1− Y. pestis strain. In some embodiments, the compositions and methods can be used for treating an F1−Y. pestis CO92 strain. Embodiments of the compositions and methods can be used for treating any Y. pestis strain having a yadC gene.
In some embodiments, compositions include an isolated YadC polypeptide. (YadC is also known as YPO1388 of Y. pestis CO92). In some embodiments, compositions include at least two isolated YadC polypeptides. In some embodiments, compositions can include a YadC polypeptide, and at least one additional compounds for treating Y. pestis infection, for example, one or more of the compounds identified in one of the following references, each of which is incorporated herein by this reference: U.S. Pat. Nos. 3,137,629; 5,985,285; 6,638,510; 6,706,522; and 6,964,770; and U.S. Patent Publication Nos: 2004/0151727; 2005/0136075; 2005/0220797; and 2005/0232940; and PCT Publication Nos: WO95/18231; WO95/24475; WO2004/019980; WO2005/023205; and WO2005/120561.
The terms “polypeptide,” “protein,” and “peptide” refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size or function. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. Therefore, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein, unless otherwise noted. Exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. The term “isolated”, when used in the context of an isolated polypeptide, is a polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated polypeptide can exist in a purified form or can exist in a non-native environment.
As used herein, the term “fragment” refers to a sequence that comprises a subset of another sequence. When used in the context of an amino acid sequence, the terms “fragment” and “subsequence” are used interchangeably. A fragment or subsequence of an amino acid sequence can be any number of residues that is less than that found in a reference polypeptide, and can include, but is not limited to, domains, features, repeats, etc. Also, it is understood that a fragment or subsequence of an amino acid sequence need not comprise the entirety of the amino acid sequence of the domain, feature, repeat, etc. A fragment can also be a “functional fragment,” in which the fragment retains some or all of the activity of the reference polypeptide as described herein. For example, a functional fragment of a reference polypeptide that is a Y. pestis antigen can retain some or all of the immune response activity of the reference polypeptide.
The terms “variant” and “modified,” when used to describe polypeptide refer to a polypeptide that is different from a reference polypeptide as a result of an intentional manipulation of the amino acid sequence of the polypeptide, or the nucleic acid sequence encoding the amino acid sequence of the polypeptide. For example, a modified polypeptide or polypeptide variant can contain at least one amino acid substitution relative to a reference polypeptide. In some embodiments, a modified polypeptide can include one or more additions and/or deletions or relative to the amino acid sequence of the reference polypeptide, so long as the modified polypeptide maintains some or all of the activity of the reference polypeptide as described herein.
The term “conservatively substituted variant” refers to a polypeptide comprising an amino acid sequence that differs from a reference polypeptide by one or more conservative amino acid substitution, and maintains some or all of the activity of the reference polypeptide as described herein. A “conservative amino acid substitution” is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine or arginine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan for another. The phrase “conservatively substituted variant” also includes polypeptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting polypeptide maintains some or all of the activity of the reference polypeptide as described herein.
“Percent identity,” or “percent homology” when used herein to describe to an amino acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul, et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST) are used.
In some embodiments of the presently-disclosed subject matter, a composition for inducing an immune response in a subject comprises an isolated polypeptide molecule comprising an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 2. In some embodiments, the composition for inducing an immune response in a subject comprises an isolated polypeptide molecule comprising an amino acid sequence encoded by a subsequence of the nucleotide sequence of SEQ ID NO: 2, so long as the encoded polypeptide has improved immune response activity relative to, or has all or some of the immune response activity of, a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 2. In some embodiments, the isolated polypeptide molecule comprises an amino acid sequence encoded by a fragment of the nucleotide sequence of SEQ ID NO: 2, wherein up to about 15, 30, 45, 60, 75, 90, 150, 375, or 450 nucleotide residues are truncated from the 5′ terminus of SEQ ID NO: 2, and/or up to about 30, 150, 225, 300, 450, 525, 600, 675, 750, 825, 900, 975, 1050, or 1200 nucleotide residues are truncated from the 3′ terminus of SEQ ID NO: 2.
In some embodiments, the YadC polypeptide of the compositions includes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, or a functional fragment thereof. For example, in some embodiments, the polypeptide molecule comprises the amino acid sequence including amino acid residues 137-409 of YadC, which fragment is set forth in SEQ ID NO: 3. For another example, in some embodiments, the polypeptide molecule comprises the amino acid sequence including amino acid residues 137-345 of YadC. For another example, in some embodiments, the polypeptide molecule comprises the amino acid sequence including amino acid residues 32-345 of YadC. For another example, in some embodiments, the polypeptide molecule comprises the amino acid sequence including amino acid residues 32-409 of YadC. In some embodiments the polypeptide molecule comprises a fragment of SEQ ID NO: 1, wherein up to about 5, 10, 15, 20, 25, 30, 50, 125, or 150 amino acids are truncated from the N-terminus of SEQ ID NO: 1 and/or wherein up to about 10, 50, 75, 100, 150, 175, 200, 225, 250, 275, 300, 325, 350, or 400 amino acids are truncated from the C-terminus of SEQ ID NO: 1. In some embodiments, the polypeptide molecule comprises at least about 200, 210, 225, 235, 245, 255, 265, 275, 285, 295, 300, 325, 350 or 375 amino acids of SEQ ID NO: 1.
In some embodiments, the YadC polypeptide is a variant, such as a conservatively-substituted variant, of a polypeptide having the amino acid sequence of SEQ ID NO: 1, or a subsequence thereof. For example, in some embodiments, the YadC polypeptide is a conservatively-substituted variant having the amino acid sequence of SEQ ID NO: 1, or a subsequence thereof, with up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 conservative amino acid substitutions. In some embodiments, the YadC polypeptide is a conservatively-substituted variant of a polypeptide having the amino acid sequence of SEQ ID NO: 3. In some embodiments, up to about 1, 2, 3, 4, or 5 amino acid substitutions are between amino acid residues 32-345 of the YadC polypeptide of SEQ ID NO: 1, or a subsequence thereof. In some embodiments, the amino acid substitutions are primarily between amino acid residues 346-622 of the YadC polypeptide of SEQ ID NO: 1, or a subsequence thereof. In some embodiments, all of the amino acid substitutions are between amino acid residues 346-622 of the YadC polypeptide of SEQ ID NO: 1, or a subsequence thereof. Variants can have improved immune response activity relative to, or can maintain some or all of the immune response activity of the polypeptide having the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the YadC polypeptide is a modified polypeptide, relative to the polypeptide having the amino acid sequence of SEQ ID NO: 1, or a subsequence thereof. Such modified polypeptides can have improved immune response activity relative to, or maintain some or all of the immune response activity of, the polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the YadC polypeptide includes a polypeptide having 80%, 85%, 90%, or 95% identity to a polypeptide having the amino acid sequence of SEQ ID NO: 1, or a subsequence thereof, so long as the polypeptide has improved immune response activity relative to, or maintain some or all of the immune response activity of, the polypeptide having the amino acid sequence of SEQ ID NO: 1.
In some embodiments of the presently-disclosed subject matter, the compositions can be provided in a formulation appropriate for administration to a subject, as will be understood by those of ordinary skill in the art. For example, in some embodiments, the compositions can be provided in as a liquid solution or a suspension, suitable for injection. In some embodiments, solid forms of the compositions that are suitable for solution in, or suspension in, liquid prior to injection can be provided. In some embodiments, compositions can be mixed with excipients, such as, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. In some embodiments, formulations can include one or more adjuvants. In some embodiments, the adjuvant is a aluminum hydroxide, e.g., aluminum hydroxide gel known as ALHYDROGEL® (E. M. Sergeant Pulp & Chemical Co. (Clifton, N.J.)). Other suitable adjuvants for use in the practice of the present subject matter include, but are not limited to polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units, cation lipids containing a quaternary ammonium salt, cytokines, and aluminum phosphate.
The presently-disclosed subject matter includes methods for inducing an immune response in a subject, including administering to a subject a composition including a YadC polypeptide, as described above. The composition can be administered in an amount sufficient to induce an immune response in the subject. The presently-disclosed subject matter includes methods for treating a Yersinia pestis (Y. pestis) infection and/or treating a plague resulting from such an infection, including administering to the subject an effective amount of a composition including a YadC polypeptide, as described above.
As used herein, the term “effective amount” refers to a dosage sufficient to elicit an immune response. The exact amount that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the particular carrier or adjuvant being used, mode of administration, and the like. As such, the effective amount will vary based on the particular circumstances, and an appropriate effective amount can be determined in a particular case by one of ordinary skill in the art using only routine experimentation.
For additional guidance regarding formulation and dose, see Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, N.J.; Goodman et al., (2006) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 11th ed. McGraw-Hill Health Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Fla.; Katzung, (2007) Basic & Clinical Pharmacology, 10th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New York; Remington et al., (1990) Remington's Pharmaceutical Sciences, 18th ed. Mack Pub. Co., Easton, Pa.; and Speight et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland/Philadelphia.
In some embodiments, compositions can further include additional compounds for treating Y. pestis infection, for example, one or more of the compounds identified in one of the following references, each of which is incorporated herein by this reference: U.S. Pat. Nos. 3,137,629; 5,985,285; 6,638,510; 6,706,522; and 6,964,770; and U.S. Patent Publication Nos: 2004/0151727; 2005/0136075; 2005/0220797; and 2005/0232940; and PCT Publication Nos: WO95/18231; WO95/24475; WO2004/019980; WO2005/023205; and WO2005/120561.
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the presently-disclosed subject matter.
Bacterial strains and plasmids. Y. pestis CO99-3015 strains were grown in Heart Infusion Broth (Difco laboratories, Detroit, Mich.) supplemented with 2.5 mM CaCl2 and 0.2% xylose (sHIB) on sHIB agar. E. coli strains were grown in Luria-Bertani broth (LB) or on LB-based agar. The bacterial strains and plasmids used in studies described herein are set forth in Table 1. Antibiotics were used at the following concentrations during genetic constructions: carbenicillin (Cb), 100 μg/ml; kanamycin, 25 μg/ml chloramphenicol, 25 μg/ml. Cb was used at 50 ug/ml for growth of Y. pestis strains containing pCD2Ap. The presence of the pigmentation locus (Perry 1997) was confirmed by the formation of red colonies on Congo Red agar (Surgalla 1969). The presence of a functional Lcr virulence plasmid was confirmed by absence of growth at 37° C. on sHIB agar containing 20 mM MgCl2 and 20 mM sodium oxalate (MgOx plates) and by assaying expression and secretion of Yops during growth in the defined medium TMH (Straley 1986) with and without 2.5 mM CaCl2.
Y. pestis
E. coli
aFull virulence is conditional on intravenous route of infection (Perry 1997).
bFull virulence is conditional on intranasal route of infection (Davis 1996).
Production of the protein capsule F1 was abolished by deleting the caf1 gene. Primers were used to generate a PCR fragment containing the FRP site-flanked cat gene from pKD3 flanked by caf1 sequence, and caf1 was deleted by allelic exchange mediated by the Red and F1p recombinases as described in Datsenko, 2000. The resulting deletion started at −12 by upstream of the caf1 translation initiation codon through the last translated codon. The absence of F1 expression was verified by immunoblot of 37° C.-grown yersiniae using the monoclonal antibody YPF1 (Research Diagnostics, Inc., Flanders, N.J.), and the absence of cat in the final strain was confirmed by PCR and by inability to grow on chloramphenicol-containing plates.
Virulence was reconstituted in BSL3 containment with select-agent security by introducing pCD2Ap by electroporation. Briefly, Y. pestis CO99-3015.S2 was electroporated with plasmid DNA from Y. pestis CO99-3015.S6, that contained the Y. pestis plasmids pFra (110 kb) and pPst (9 kb) (Perry 1997) in addition to pCD2Ap. Their presence in the transformation would not pose a problem since the recipient strains already contained copies of these two plasmids.
Transformed strains were selected for Cb-resistance and screened for the Pgm+ and Lcr+ phenotypes on agar media. Individual isolates that passed these tests were then subcultured two times in 3 ml TMH+Cb at 26° C. for a total of 24 hours prior to the experiment. The cultures were subcultured a third time into TMH with or without 2.5 mM CaCl2 and allowed to grow for an additional two hours at 26° C. Following this, each culture was shifted to 37° C. and permitted to grow another four hours. A 500-μl sample of each culture was removed and briefly placed on ice to aid cellular aggregation. Cells were pelleted in a microcentrifuge at room temperature (RT) for 10 min. The top 250 μl of supernatant was removed, transferred to a microcentrifuge tube containing trichloroacetic acid to a final concentration of 5%, and incubated overnight at 4° C. to precipitate secreted proteins. The rest of the supernatant was discarded. The precipitated proteins were collected by centrifugation at RT. Pellets of whole cells and of secreted proteins were suspended in lysis loading buffer (400 mM TrisCl, pH 6.8, 25% glycerol, 8% SDS, 5% dithiothreitol, 0.4% bromophenol blue) to a concentration corresponding to a culture OD620 of 2.0 and heated to 95° C. for 15 minutes. Half of each heat-treated sample was plated and incubated for 5 days at 28° C. to ensure that no viable cells remained prior to removing the samples from the containment facility.
The resultant cellular extracts and supernatant proteins were then analyzed by immunoblot for the presence of secreted LcrV and YopM, as described in Wulff-Strobel 2001, to determine whether the type three secretion system was fully functional. Several isolated colonies were then pooled and stocked.
Preparation of YadC Polypeptide. The YadC polypeptide selected for testing in the present study was a fragment of the YadC polypeptide of SEQ ID NO: 1. The fragment includes amino acid residues 137-409 of the amino acid sequence of SEQ ID NO:1, which are set forth in SEQ ID NO: 3. To produce GST-YadC137-409 (Forman 2006), E. coli DH5α containing plasmid pGEX-YadC137-409 was grown to an OD620 of 0.5, IPTG was added to a final concentration of 1.0 mM and the bacteria were incubated for an additional 3 hours. Cells were harvested by centrifugation at 4° C., and resuspended at 0.05 the original volume in column buffer (20 mM Tris, 200 mM NaCl, 1 mM EDTA pH 7.4). Samples were passed twice through a French press to lyse bacteria, cleared by centrifugation at 9000×g for 30 minutes at 4° C., diluted 1:4 with column buffer and passed over a glutathione sepharose column. Bound protein was eluted using column buffer containing 10 mM glutathione and collected in 1 mL aliquots. Purity was assessed on SDS-PAGE gels stained with Coomasie brilliant blue. Protein-containing fractions were pooled and dialyzed, and the protein concentration measured by the bicinchoninic acid assay (Pierce Chemical Co., Rockford, Ill.).
N-terminally His6-tagged LcrV (HT-LcrV) was expressed in E. coli DH5α from plasmid pHT-V and purified on Talon metal-affinity resin (Clonetech Laboratories, Inc, Palo Alto, Calif.) as described by Fields, 1999.
Protection studies. Sets of 5 female outbred Swiss-Webster mice were immunized subcutaneously with 40 μg amounts of purified fusion proteins (GST-YadC137-409, or HT-LcrV) or buffer emulsified with ALHYDROGEL® (aluminum hydroxide gel) (Sigma Chemical Company, St. Louis, Mo.; 6.25% vol/vol final concentration) on days 0, 8, 14, and 22. On day 35, they were challenged intravenously with 10,000 CFU (5 LD50 doses) of Y. pestis CO99-3015.S7.
Similarly, groups of 8 C57BL/6 mice were immunized with 80 μg of HT-LcrV, GST-YadC137-409, or GST (Sigma Chemical Company, St. Louis, Mo.) adjuvanted with ALHYDROGEL® as in the previous experiment. Two weeks after the 3rd immunization, the mice were challenged intranasally with 3000 CFU of CO92.S2 (6 times the LD50 dose) in BSL3 conditions.
Determination of IgG isotypes. Measurement of antibody titer and isotype response was performed by a modified ELISA on serum samples taken 1 week prior to bacterial challenge. 96-well ELISA plates were coated with 10 μg/ml of GST-YadC137-409, HT-LcrV, GST or with buffer alone. Mouse sera were then used at serial dilutions to a maximum dilution of 1:5,120,000. HRP conjugates against mouse IgG, IgG1, IgG2a, IgG2b, and IgG3 (BD Pharmingen, San Diego, Calif.) were used as secondary antibodies. Protein-specific antibody titer was estimated as the maximum dilution of serum giving an absorbance at 450 nm reading 0.1 units over the non-immunized mouse background sera (pre-immunization sera). Mean titers were determined per treatment group.
Human macrophages and cytokine analysis. The cytokine response of human peripheral blood monocytes (PBMs) to HT-LcrV, GST-YadC137-409, and the negative control GST was evaluated. Blood was obtained from two healthy volunteers. Red blood cells were lysed with ACK buffer (155 mM NH4Cl/l mM KHCO3/0.5 mM EDTA, pH 7.2), the samples were washed, and PBMs were isolated by Ficoll-Paque density gradient. PBMs were cultured for one week in RPMI with 12.5% human serum and 10 μg/ml of penicillin, streptomycin, and neomycin at a concentration of 1×106 cells/ml in 5% CO2 to differentiate into macrophages.
Differentiated PBMs were stimulated with 10 μg of F1, HT-LcrV, GST, or GST-YadC137-409 in 96-well ELISA plates. 100 μL of supernatant was removed at 3 hours and then at 24 hours. Supernatants were kept at −80° C. until processed for cytokine analysis. All analyses were done in triplicate in 2 different experiments. Cytokine levels were measured by Cytokine Bead Analysis (BDBiosciences) and evaluated by flow cytometry.
Results
yadC Operon and Predicted Properties of YadC. The adhesin YadA of the enteropathogenic yersiniae is encoded by the same Lcr plasmid that encodes LcrV. YadA contributes to colonization through binding to extracellular matrix components such as fibronectin, collagen, and laminin that in turn are bound to β1 integrins (Tahir E Y 2001; Hudson 2005). YadA belongs to a family of oligomeric coiled-coil (Oca) adhesins that are a subgroup of autotransporters composed of trimers with short outer membrane-anchor domains. The trimeric character may provide increased avidity for tight adherence (Hoiczyk 2000; Cotter 2005). Each YadA monomer is composed of an N-terminal head that contains a collagen binding motif, a neck that serves as a platform for the head and contributes to stability of the complex, a stalk composed of seven 15-mer units, a coiled-coil linker of two to three 7-mer units that is necessary for transport of the head, neck, and stalk through the outer membrane, and a membrane anchor (Hoiczyk 2000; Tahir E Y 2001; Roggenkamp A 2003; Nummelin H 2004).
In Y. pestis, the yadA gene contains a frameshift mutation that prevents the expression of a functional YadA protein. To identify surface proteins of potential pathogenic importance for Y. pestis, we examined the Y. pestis CO92 genome for genes encoding proteins similar to YadA and identified the 1869 by ORF YPO1388 that was subsequently named yadC. yadC is likely part of a bicistronic operon with the upstream 1095 by ORF encoding another protein with homology to YadA and called yadB (YPO1387). yadBC is fairly isolated on the chromosome with the nearest gene upstream gene 300 base pairs away and the nearest downstream gene 366 base pairs away.
The yadB and yadC genes in the chromosome of Y. pestis CO92 also encode members of the Oca family (denoted as 653a/b in ref (Hoiczyk 2000)) and share the structural arrangement seen in YadA, although the amino-acid sequence similarity is not strong (
Importance of YadC for virulence in bubonic plague. Applicants have found that some human plague convalescent sera recognize YadC, and a yadBC mutant Y. pestis has been found to show decreased adherence to epithelioid cells (Forman 2006), suggesting that YadB and YadC might have a virulence role in plague. Further, the yadBC mutant was attenuated from the subcutaneous route (mimicking bubonic plague) as compared to the virulent Y. pestis parent strain. This finding shows that yadBC encodes a new virulence determinant for bubonic plague. This attenuation was not demonstrated when mice were challenged intranasally (mimicking pneumonic plague).
Protection studies. One goal of the studies described herein is to obtain information useful for improving subunit plague treatment by identifying components that will provide protection against pneumonic plague due to an F1− Y. pestis strain or against bubonic and systemic plague due to an F1+ strain. Since YadC was determined to be a virulence factor for bubonic plague and is detectable by antibody. it likely is displayed on the bacterial surface where it would be accessible to antibody. Therefore, its role as a protective antigen was tested. Two tests were made for protective efficacy, using different challenge strains and infection routes. Mice were immunized and challenged either intravenously or intranasally, as outlined above.
Negative-control mice received either ALHYDROGEL® only or GST-ALHYDROGEL® immunization. HT-LcrV-immunized mice were used as the positive control. From an intravenous route, a test with Swiss-Webster mice challenged with 5 LD50 doses (10,000 CFU) of Y. pestis CO99-3015.S7 revealed no protection for those immunized with buffer alone, full protection for HT-LcrV-immunized mice, and partial protection for GST-YadC137-409-immunized mice (with 40% of mice surviving) (
IgG isotypes. Protein-specific IgG1, IgG2A, and IgG2B, and IgG3 antibodies were measured by ELISA in the sera of the mice immunized with GST-ALHYDROGEL®, GST-YadC137-409 or HT-LcrV. As has been reported, protection with the F1-LcrV antigen is associated with predominantly a Th2 IgG1 response (Williamson 1999). Likewise, in experiments described herein, mice immunized with HT-LcrV demonstrated a similar IgG isotype distribution (IgG1>IgG2a˜IgG2b˜IgG3, p<0.005 when IgG1 compared to other isotypes). Mice immunized with YadC demonstrated a more balanced Th1/Th2 response (IgG1=IgG3>IgG2a>IgG2b, p<0.005 when IgG1 and IgG3 compared to other isotypes). The total IgG titers to GST-YadC137-409 (1:5.6×106) were significantly higher than those for HT-LcrV (1:1×106) and total IgG titers to the GST component in the negative control mice (1:6.4×105).
Anti-inflammatory/pro-inflammatory cytokines. In response to stimulation with either GST-YadC137-409, HT-LcrV, or GST, PBMs produced little or no IL-10. PBMs stimulated with YadC showed a significant TNF-α and IL-6 production that was much higher than that due to LPS or HT-LcrV (
Discussion. It is clear from previous studies (Cowan 2000) that Y. pestis has multiple adhesins and invasins to substitute for the absence of proteins such as YadA. These and other Y. pestis surface proteins could alter the current plague treatment compositions to elicit a more balanced Th1/Th2 phenotypic profile. Antigens, such as those identified herein, are desirable for their ability to induce production of antibodies that can block crucial surface function such as epithelial adhesion and invasion. Ideally, genes for such crucial proteins could not be deleted from a potential bioterroristic weapon without loss of virulence, unlike F1. This study examined Y. pestis YadC which, as an acidic protein, could have a significant impact on the bacterial surface and be important in host-pathogen interactions.
A critical step in the clearance of pulmonary infections is the ability of the lung antigen-presenting cell to migrate to regional lymph nodes where it can present the antigen to T cells to initiate an adaptive immune response. Previous studies have demonstrated a role for macrophage-lineage cells in mediating protection by anti-LcrV antibody in at least the liver (Philipovskiy A V 2004; Cowan C 2005; Philipovskiy 2005). Recently it has been shown that when Lcr− Y. pestis was given to mice intratracheally, the bacteria were taken up by a characteristic population of DCs (CD11c+DEC20530 CD11b−) (Bosio C 2005). Data presented here suggest that PBMs when stimulated by a more favorable antigen (i.e., YadC) can participate in the induction of cytokines such as IL-1, TNF-α, and IL-6 that would favor development of adaptive Th1 cellular immunity in the lung and increased local inflammatory response. Additional pro-inflammatory cytokines from AMs suggest that YadC may play a role in recruiting additional innate immune cells such as natural killer cells to the lung environment.
Finally, as YadC has shown to be required for virulence from a subcutaneous infection and is partially protective against pneumonic and systemic plague, it is believed that YadC polypeptides can be included in useful compositions for treating Y. Pestis infection.
F1+ Subcutaneous Challenge and F1− Intranasal Challenge of Subjects Treated with Composition Including Full-Length YadC Polypeptide.
The following is a prophetic example. Studies are conducted to determine whether subjects treated with a composition including the YadC polypeptide of SEQ ID NO: 1 are protected from: subcutaneous challenge with Y. Pestis CO92 pCD2 Ap(res) F1+, and intranasal challenge with Y. Pestis CO92 pCD2 Ap(res) F1−.
For the study involving subcutaneous challenge, three groups of mice are provided. A test group is immunized with the composition including the YadC polypeptide of SEQ ID NO: 1, a positive control group is immunized with an HT-LcrV composition, and a negative control group receives adjuvant alone (ALHYDROGEL®). Y. Pestis CO99 F1+ cells are plated on HIB agar plates containing 2.5 mM CaCl2, 0.10% xylose containing 50 μg/ml Cb (sHIBCb50 agar), and Congo Red containing 50 μg/ml Cb (Congo Redcb50). Pigmentation (Pgm+) colonies are picked and placed into media. The sample is then vortexed and the concentration is determined by obtaining the OD620 of a 1:20 dilution. Mice are anesthetized, and subcutaneously challenged with about 100 cells/100 μl of CO99 F1+Y. pestis.
For the study involving intranasal challenge, three groups of mice are provided. A test group is immunized with the composition including the YadC polypeptide of SEQ ID NO: 1, a positive control group is immunized with an HT-LcrV composition, and a negative control group receives adjuvant alone (ALHYDROGEL®). Y. Pestis CO99 F1+ cells are plated on HIB agar plates containing 2.5 mM CaCl2, 0.10% xylose containing 50 μg/ml Cb (sHIBcb50 agar), and Congo Red containing 50 μg/ml Cb (Congo Redcb50). Pigmentation (Pgm+) colonies are picked and placed into media. The sample is then vortexed and the concentration is determined by obtaining the OD620 of a 1:20 dilution. Mice are anesthetized, and intranasally challenged with about 5000 cells/20 μl of CO99 F1−Y. pestis.
For the HT-LcrV-treated mice receiving subcutaneous challenge, no symptoms are observed, and there is an overall survival of 100%. For the mice treated with adjuvant alone, after subcutaneous challenge, there is a low overall survival. For YadC (SEQ ID NO:1)-treated mice receiving subcutaneous challenge with CO99 F1+Y. pestis, there is a protective effect, as compared to the mice receiving adjuvant alone.
For the HT-LcrV-treated mice receiving intranasal challenge with CO99 F1−Y. pestis, no symptoms are observed, and there is an overall survival of 100%. For the mice treated with adjuvant alone, after intranasal challenge, there are no mice that survive. For YadC (SEQ ID NO:1)-treated mice receiving intranasal challenge with CO99 F1−Y. pestis, there is a protective effect, as compared to the mice receiving adjuvant alone.
Throughout this document, various references are mentioned. All such references are incorporated herein by reference, including the references set forth in the following list:
This application claims priority from U.S. Provisional Application Ser. No. 60/828,895 filed Oct. 10, 2006, the entire disclosure of which is incorporated herein by this reference.
Subject matter described herein was made with U.S. Government support under Grant Number AI48491, IUF4 AI057175, and 5K30HL004163-05 awarded by the National Institute of Allergy and Infectious Diseases of the National Institute of Health. The United States government has certain rights in the subject matter.
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