Patent Application
20210023194
This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled “29300540201 SequenceListing_ST25.txt” having a size of 339 kilobytes and created on Nov. 9, 2016. The information contained in the Sequence Listing is incorporated by reference herein.
Fusobacterium spp. are gram-negative, obligately anaerobic and pleomorphically rod shaped bacterium responsible for a variety of necrotic infections in animals and in humans (Langworth, Bacteriol. Rev., 41, 373-390 (1977)). Pathogenic species in the Genus Fusobacterium include F. necrophorum, F. nucleatum, F. canifelinum, F. gonidiaformans, F. mortiferum, F. naviforme, F. necrogenes, F. russii, F. ulcerans, and F. varium. Fusobacterium necrophorum is the most pathogenic and is classified into two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme and are responsible for a number of clinical manifestations in various species of animals, such as cattle, horses, goats, sheep, fowl, and swine, including hepatic abscesses, foot rot, laminitis, purulent and interdigital dermatitis, contagious ecthyma, necrotic rhinitis, and necrotic laryngitis. Taxa formally in the genus Fusobacterium include Fillfactor alocis, commonly found in the periodontal pockets of patients having periodontitis (Kumar et al., 2003, J Dent Res., 82(5):338-44); Faecalibacterium prausnitzii, and Eubacterium sulci, also associated with odontogenic infections (Munson et al., 2002, J. Dent Res., 81:761, Paster et al., 2006, Periodontology 2000, 42:80). Although the primary etiologic agent of liver abscesses has been shown to be Fusobacterium necrophorum abscessation has been associated with other bacteriological agents such as Arcanobacterium pyogenes. Bacteroides spp., Salmonella spp., Clostridium spp., Pasteurella spp., E. coli spp., and Peptostreptococcus spp.
In humans, F. necrophorum and F. nucleatum are considered to be the most pathogenic and is the causative agent of skin ulcers, peritonsillar abscesses, septic arthritis, Lemierre's syndrome, periodontal diseases, endocarditis and metastatic abscesses in the lungs, liver, joints, and pleural spaces. On a population-based perspective Fusobacterium bacteremia is relatively uncommon in humans with an overall annual incidence of 0.55 per 100,000 population. The incidence of F. nucleatum was found to be 0.34/100,000 and F. necrophorum was 0.14/100,000 with a median age of 53.5 years while F. necrophorum cases had a median age of 21 years. Overall mortality due to bacteremia was 11 percent. F. necrophorum affects mostly young health adults. In contrast, F. nucleatum affects older individuals with seemingly compromised healthy conditions (Afra, Infectious Diseases, 13: 264 (2013)). A number of other species of fusobacteria have been implicated as the etiological agent in a variety of diseases, for example, F. ulcercans (skin ulcers), F. russi (animal bite infections), and F. varium (eye infections) (Smith et al., Epidemiol Infect., 110, 499-506 (1993)).
In beef-breed and Holstein steers, the incidence of liver abscesses average from 12 to 32% in most feedlots, and has been shown to be influenced by a number of dietary and management factors. Liver abscesses are categorized as mild, moderate or severe. Severe liver lesions are most often associated with high economic losses to producers, packers, and ultimately consumers. Besides liver condemnation, economic impacts include reduced feed intake, reduced weight gain, decreased feed efficiency, and decreased carcass yield.
F. necrophorum possesses a number of virulence factors that participate in the penetration and colonization of the ruminal epithelium and subsequent entry and establishment of infection in the liver, including a potent secreted leukotoxin which has been shown to be specifically toxic to ruminant polymorphonuclear leukocytes (Tan et al., Vet. Res. Commun. 20, 113-140 (1996)). The role of leukotoxin as a virulence factor has been documented. For instance, experiments have indicated a correlation between toxin production and the ability of F. necrophorum to induce abscesses in laboratory animals (Coyle et al., Am. J. Vet. Res., 40, 274-276. (1979), and Tan et al., Am. J. Vet. Res., 55, 515. (1994)). Experiments have also shown that non-leukotoxin producing strains are unable to induce foot abscesses in cattle following challenge. It has also been shown that neutralizing antibody produced by an inactivated toxoid derived from leukotoxin reduced infection and liver abscesses in vaccinated cattle.
Control of liver abscesses in feedlot cattle generally has depended on the use of antimicrobial compounds. Five antibiotics (i.e., bacitracin methylene disalicylate, chlortetracycline, oxytetracycline, tylosin, and virginiamycin) are approved for prevention of liver abscesses in feedlot cattle. Tylosin is the most effective and the most commonly used feed additive.
A number of commercial killed whole cell bacterins have been used to control necrotic infection in farm animals incorporating multiple strains including the most prevalent serotypes such as biotype A (F. necrophorum subsp. necrophorum). Another approach to vaccine development has been the incorporation of leukotoxin as a toxoid to prevent the pathological effect of the secreted toxin (Saginala et al., J. Anim. Sci., 75, 11601166 (1997)).
Divalent metal ions such as iron, cobalt, copper, magnesium, manganese, molybdenum, nickel, selenium, and zinc and are trace elements often required for the survival of bacteria infecting both animal and human hosts. These trace metal elements are used by bacteria as cofactors for enzymes that catalyze biochemical reactions for various metabolic pathways required by the organism. The impact of iron on the pathogenesis of bacteria has been studied extensively. Iron is essential for nearly all life and is required for enzymatic and metabolic pathways of cells at all phylogenic levels. It has been well-documented that during bacterial sepsis there is an alteration in the concentration of a number of metal ions in serum such as, iron, copper, and zinc. For instance, serum levels of zinc decrease from 10 percent to 60 percent with the onset of infection. Following the onset of infection, zinc is then redistributed from plasma to liver where it is bound to metallothionein. Decreases in serum iron of up to 50 percent have been described during infectious illness, whereas serum copper has been shown to increase in response to inflammatory stimuli. The alteration of these trace metal ions in serum may directly affect the severity or progression of any bacterial infection.
The ability of Fusobacterium to evade the natural defense mechanisms of the vertebrate host depends in part on its ability to obtain host iron, which in turn, directly influences the host-pathogen interaction. Because of iron's essential nature, vertebrate hosts have developed elaborate mechanisms to bind iron in body fluids (e.g., transferrin in blood and lymph fluids and lactoferrin in external secretions). These high affinity iron binding proteins create an iron restricted environment within the host, reducing the level of iron to approximately 10−18 molar, a concentration too low to support the growth of nearly all bacteria. These iron sequestering mechanisms of the host act as a natural defense mechanism to combat bacterial invasion. To circumvent these iron-restrictive conditions many bacterial species have evolved mechanisms for obtaining iron. The most common mechanisms include the diffusion of soluble iron through porins and specialized transport systems that mediate the uptake of iron by siderophores. This latter system is one of the most common and well-studied mechanisms for iron acquisition and involves the specific chelation of ferric iron by siderophores and the synthesis of their cognate transport systems, which permits the bacteria to continue to replicate and overcome the non-specific defense mechanisms of the host. Continued replication, and thus each step in the infectious process, is ultimately dependent on the ability of the organism to obtain iron from its host.
With so many basic functions relying on the availability of iron, bacteria have evolved a complex regulatory network for acquiring iron under varying physiological conditions. Under anaerobic conditions, iron is present in the soluble ferrous form (Fe II) and can freely diffuse through outer membrane porins into the periplasm. For instance, in E. coli the FeoAB transport system present in the cytoplasmic membrane will transport the ferrous iron molecules into the cell cytoplasm. Under aerobic conditions and neutral pH, iron is primarily present in the insoluble ferric form (Fe III) and cannot pass through the outer membrane porins by passive diffusion. Instead, molecules called siderophores are secreted by bacteria, which have a high affinity for ferric iron. The ferric-siderophore complexes are recognized by receptors in the outer membrane collectively referred to as the TonB-dependent receptors. These receptors, once bound to loaded siderophores, are believed to interact with TonB and its associated proteins localized in the periplasm and cytoplasmic membrane. These protein-protein interactions, though poorly understood, serve to provide the energy necessary to transport the ferri-siderophore complexes across the outer membrane and through the periplasmic space. ABC transport systems present in the cytoplasmic membrane serve to transport the iron-siderophore complexes across the cytoplasmic membrane. Reductase enzymes reduce the ferric iron to its ferrous form, which dissociates it from the siderophore and releases iron into the cell.
Several species of pathogenic bacteria use additional mechanisms to obtain iron from mammalian hosts, including the direct binding of transferrin, heme, and other heme-containing compounds. The receptor proteins that bind these iron-containing molecules most likely rely on the TonB complex for the energy required to transport heme across the outer membrane, similar to the iron-siderophore complexes. Specialized ABC transporters are then used to transport the heme across the cytoplasmic membrane. In addition, some bacteria secrete hemophores, small molecules that can bind heme and present it to receptors on the bacterial cell surface. Several pathogenic species also produce hemolysins, which are toxins that lyse red blood cells, releasing heme and hemoglobin for uptake by the bacteria.
The outer membrane proteins of gram-negative bacteria control the selective permeability of many essential nutrients critical to the survival of bacteria, including all pathogenic bacteria that cause disease in animals and man. This selective permeability of nutrients is controlled by a class of membrane proteins called porins. It now appears that the majority of the outer membrane proteins on the surface of gram-negative bacteria are porins, identified as the general porins (e.g., OmpF), monomeric porins (e.g., OmpA), the specific porins (e.g., the maltose-specific porin LamB) and the TonB-dependent, gated porins (e.g., the siderophore receptor FepA). The porin class of proteins generally share structural features, including the presence of beta-barrels that span the outer membrane.
Little is known regarding the iron-acquisition by Fusobacterium spp, and genomic comparisons are difficult since the genome of only five strains of Fusobacterium nucleatum have been completely sequenced and made publicly available: F. nucleatum subspecies nucleatum, strain ATCC 25586 (Kapatral et al., J. Bacteriol., 184, 2005-2018 (2002)); Fusobacterium nucleatum subsp. vincentii 3_1_36A2; Fusobacterium nucleatum subsp. vincentii 3_1_36A2; Fusobacterium nucleatum subsp. animalis 7_1; and Fusobacterium nucleatum subsp. animalis 4_8 (Tatusova T, et al. Nucleic Acids Res 42, D553-D559 (2014). No complete sequence of Fusobacterium necrophorum strains have been published, although there are a number of partial sequences in the NCBI database The genomic sequence of ATCC 25586 was used in a comparison with a partially sequenced genome of F. nucleatum subspp. vincentii (Kapatral et al., Genome Res., 13, 1180-1189 (2003)) to investigate differences among these two subspecies. The results suggested that there were differences between the two genomes with respect to the iron uptake systems. Although iron transport systems were discovered in both genomes, the genome of strain ATCC 25586 contains three additional iron-specific ABC transport systems. In addition, hemin receptor proteins appear to be encoded by both genomes, but while the subspp. vincentii isolate encodes three receptors, the genome of strain ATCC 25586 apparently encodes five such proteins. Furthermore, the feoAB genes, encoding a putative ferrous iron transport system, are only found in the genome of the subspp. vincentii isolate. Since both organisms are obligate anaerobes and ferrous iron is the predominant form of the metal under anaerobic conditions, strain ATCC 25586 may have a second mechanism for uptake of ferrous iron. Given the differences among these two subspecies of F. nucleatum, it is likely that there will be many differences among the iron uptake systems between other Fusobacterium species. Therefore, the F. nucleatum genomic data may not be useful for predicting the presence or absence of iron acquisition systems in other species of Fusobacterium.
Fusobacterium necrophorum is ubiquitous in the environment of cattle and is considered a normal inhabitant of the intestine and rumen, and is present in feces. The organism is the causative agent of both liver abscesses and footrot. The disease is rarely fatal but can result in substantial economic losses to both the producer and packer due to cost in treatment and performance losses. It is thought that liver abscessation follows a condition of ruminal acidosis which impairs the integrity of the rumen wall allowing Fusobacterium to transverse to the blood stream to the liver and cause abscessation.
Beyond the role of iron as an essential nutrient for microbial survival, there are now many other well-defined transitional metals that play critical roles in bacterial survival, homeostasis and pathogenesis such as iron, manganese, copper, zinc, magnesium, cobalt, and nickel (Waldron and Robinson, 2009, Nature Reviews Microbiology, 7:25-35; Porcheron, 2013, Frontiers in Cellular and Infection Microbiology 3:172-194). Iron, zinc and copper are the three most abundant divalent metal ions in mammals in descending order of concentration. The ability of a bacterium to use these transitional metals by finely regulated uptake or acquisition systems significantly contributes to the virulence of pathogenic bacteria. It is well known that bacteria within the same genus/species do not have the same uptake systems for the acquisition of transitional metals owing to the difference in pathogenicity from one strain of bacteria to another. These differences in the ability of bacteria to use different transitional metals based on expressed uptake systems may specifically direct what organ or tissue an organism can invade.
Copper is the third most prevalent transitional metal behind iron and zinc and plays a major role in many enzymatic pathways. Copper is present in every tissue of the body but is stored in its highest concentration in the liver. What role copper plays in the virulence of Fusobacterium is unknown. With the concentration of copper being the highest in the liver it would make sense that Fusobacterium has adapted some mechanism to utilize this divalent metal ion once in the liver.
In the following examples we show the expression of unique proteins that are expressed in Fusobacterium when grown under iron, zinc and copper chelation.
Provided herein are compositions. In one embodiment, a composition includes at least one isolated polypeptide having a molecular weight of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, or 57 kDa to 47 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media including an iron chelator and not isolatable when grown in the media without the iron chelator; at least one isolated polypeptide having a molecular weight of 108 kDa to 98 kDa or 79 kDa to 69 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media including an iron chelator, is expressed by the Fusobacterium necrophorum when incubated in media without the iron chelator and expressed at an enhanced level during growth in media including an iron chelator; and at least one protein selected from the group consisting of a polypeptide having at least 85% similarity to SEQ ID NO:4 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:2 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:34 or a fragment thereof, and a polypeptide having at least 85% similarity to SEQ ID NO:53 or a fragment thereof.
In one embodiment, a composition includes isolated polypeptides having molecular weights of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media including an iron chelator and not isolatable when grown in the media without the iron chelator; isolated polypeptides having molecular weights of 155 kDa to 145 kDa or 89 kDa to 79 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media including an iron chelator and an iron-containing porphyrin and not isolatable when grown in the media without the iron chelator and iron-containing porphyrin, and not isolatable when grown in the media with the iron chelator and in the absence of the iron-containing porphyrin; and isolated polypeptides having molecular weights of 108 kDa to 98 kDa and 79 kDa to 69 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media including an iron chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the iron chelator and expressed at an enhanced level during growth in media including an iron chelator.
In one embodiment, a composition includes at least one isolated polypeptide having a molecular weight of 131 kDa to 121 kDa, 79 kDa to 69 kDa, or 33 kDa to 23 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media including a copper chelator and not isolatable when grown in the media without the copper chelator; and at least one isolated polypeptide having a molecular weight of 93 kDa to 83 kDa, 65 kDa to 55 kDa, or 52 kDa to 42 kDa, wherein the at least one polypeptide is isolatable from the Fusobacterium necrophorum when incubated in media including a copper chelator, is expressed by the Fusobacterium necrophorum when incubated in media without the copper chelator and expressed at an enhanced level during growth in media including an copper chelator.
In one embodiment, a composition includes at least one isolated polypeptide having a molecular weight of 131 kDa to 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, or 33 kDa to 19 kDa, wherein the polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media including a zinc chelator and not isolatable when grown in the media without the zinc chelator; and at least one isolated polypeptide having a molecular weight of 79 kDa to 69 kDa or 65 kDa to 55 kDa, wherein the at least one polypeptide is isolatable from the Fusobacterium necrophorum when incubated in media including a zinc chelator, is expressed by the Fusobacterium necrophorum when incubated in media without the zinc chelator and expressed at an enhanced level during growth in media including the zinc chelator.
In one embodiment, a composition includes isolated polypeptides having molecular weights of 131 kDa to 121 kDa, 79 kDa to 69 kDa, and 33 kDa to 23 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media including a copper chelator and not isolatable when grown in the media without the copper chelator; and isolated polypeptides having molecular weights of 93 kDa to 83 kDa, 65 kDa to 55 kDa, and 52 kDa to 42 kDa, wherein the polypeptides are isolatable from the Fusobacterium necrophorum when incubated in media including a copper chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the copper chelator and expressed at an enhanced level during growth in media including an copper chelator.
In one embodiment, a composition includes isolated polypeptides having molecular weights of 131 kDa to 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, and 33 kDa to 19 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media including a zinc chelator and not isolatable when grown in the media without the zinc chelator; and isolated polypeptides having molecular weights of 79 kDa to 69 kDa and 65 kDa to 55 kDa, wherein the polypeptides are isolatable from the Fusobacterium necrophorum when incubated in media including a zinc chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the zinc chelator and expressed at an enhanced level during growth in media including the zinc chelator.
In one embodiment, a composition can further include a polypeptide having at least 85% similarity to SEQ ID NO:4 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:2 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:34 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:53 or a fragment thereof, or a combination thereof.
In one embodiment, a composition includes an isolated polypeptide having at least 85% similarity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or a fragment thereof.
In one embodiment, a composition can further include isolated polypeptides having molecular weights of 340 kDa to 330 kDa, 247 kDa to 237 kDa, 247 kDa to 237 kDa, 235 kDa to 215 kDa, 120 kDa to 110 kDa, 51 kDa to 25 kDa, and 21 kDa to 11 kDa.
A composition can further include a pharmaceutically acceptable carrier, such as an adjuvant. In one embodiment, a composition protects an animal against challenge with Fusobacterium necrophorum.
Also provided herein are methods. In one embodiment, a method includes administering to a subject an amount of a composition described herein effective to induce the subject to produce antibody that specifically binds to at least one polypeptide of the composition.
In one embodiment, a method is for treating an infection in a subject, and the method includes administering an effective amount of a composition described herein to a subject having or at risk of having an infection caused by a Fusobacterium spp.
In one embodiment, a method is for treating a symptom in a subject, and the method includes administering an effective amount of a composition described herein to a subject having or at risk of having an infection caused by a Fusobacterium spp.
In one embodiment, a method is for decreasing colonization in a subject, and the method includes administering an effective amount of a composition described herein to a subject colonized by a Fusobacterium spp.
In one embodiment, a method is for treating an infection in a subject, and the method includes administering an effective amount of a composition to a subject having or at risk of having an infection caused by a Fusobacterium spp., wherein the composition includes antibody that specifically binds to a polypeptide described herein. In one embodiment, a method is for treating a symptom in a subject, and the method includes administering an effective amount of a composition to a subject having or at risk of having an infection caused by a Fusobacterium spp., wherein the composition includes antibody that specifically binds to a polypeptide described herein. In one embodiment, an infection causes a condition selected from metritis, hepatic abscesses, and foot rot.
In one embodiment, a method is for decreasing colonization in a subject, and the method includes administering an effective amount of a composition to a subject colonized by a Fusobacterium spp., wherein the composition includes antibody that specifically binds to a polypeptide described herein.
Also provided are kits. In one embodiment, a kit is for detecting antibody that specifically binds a polypeptide, including in separate containers an isolated polypeptide described herein, and a reagent that detects an antibody that specifically binds the polypeptide. In one embodiment, a kit is for detecting a polypeptide, including in separate containers an antibody that specifically binds an isolated polypeptide described herein, and a second reagent that specifically binds the polypeptide.
The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
It is understood that wherever embodiments are described herein with the language “include,” “includes,” or “including,” and the like, otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
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.
Generally, a polypeptide may be characterized by molecular weight, 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, reference to 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. A molecular weight of a protein determined by SDS-PAGE is also referred to herein as an apparent molecular weight. In one embodiment, the molecular weight of a protein identified by SDS-PAGE includes molecular weights of 1, 2, 3, 4, or 5 kDa above and below the stated value.
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 Fusobacterium 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. In one embodiment, certain metal-regulated polypeptides produced by Fusobacterium 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 that also include hemin as a supplement. Table 1 summarizes the expression of proteins in the absence of different metals.
Examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low iron conditions include metal-regulated polypeptides having molecular weights of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa. Specific examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low iron conditions include polypeptides of 88 kDa, 68 kDa, 60 kDa, and 52 kDa. In one embodiment, the low iron condition is growth in the presence of 2,2′-dipyridyl.
Examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low iron conditions supplemented with an iron-containing porphyrin, such as hemin, include metal-regulated polypeptides having molecular weights of 155 kDa to 145 kDa and 89 kDa to 79 kDa. Specific examples of this type of metal-regulated polypeptide isolatable from a Fusobacterium spp. after growth in low iron conditions in the presence of an iron-containing porphyrin include polypeptides of 150 kDa and 84 kDa. In one embodiment, the low iron condition is growth in the presence of 2,2′-dipyridyl and hemin.
Examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low copper conditions include metal-regulated polypeptides having molecular weights of 131 kDa to 121 kDa, 79 kDa to 69 kDa, and 33 kDa to 23 kDa. Specific examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low copper conditions include polypeptides of 126 kDa, 74 kDa, and 28 kDa. In one embodiment, the low copper condition is growth in the presence of catechin. In one embodiment, the low copper condition is growth in the presence of quercetin.
Examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low zinc conditions include metal-regulated polypeptides having molecular weights of 131 kDa to 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, and 33 kDa to 19 kDa. Specific examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low zinc conditions include polypeptides of 126 kDa, 103 kDa, 88 kDa, 81 kDa, 68 kDa, 48 kDa, 28 kDa, and 24 kDa. In one embodiment, the low zinc condition is growth in the presence of TPEN.
In one embodiment, polypeptides described herein are expressed at detectable levels during growth of the microbe in high metal conditions but more of the polypeptide is expressed during growth in low metal conditions. The expression of such polypeptides is referred to herein as “enhanced” during growth in low metal conditions. Typically, the increase in expression of a polypeptide during growth in low metal conditions is between 20% and 500% compared to the expression of the polypeptide during growth in high metal conditions.
Examples of metal-regulated polypeptides having enhanced expression and isolatable from F. necrophorum after growth in low iron conditions include metal-regulated polypeptides having molecular weights of 108 kDa to 98 kDa and 79 kDa to 69 kDa. Specific examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low iron conditions include polypeptides of 103 kDa and 74 kDa.
Examples of metal-regulated polypeptides having enhanced expression and isolatable from F. necrophorum after growth in low copper conditions include metal-regulated polypeptides having molecular weights of 93 kDa to 83 kDa, 65 kDa to 55 kDa, and 52 kDa to 42 kDa. Specific examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low copper conditions include polypeptides of 88 kDa, 60 kDa, and 48 kDa.
Examples of metal-regulated polypeptides having enhanced expression and isolatable from F. necrophorum after growth in low zinc conditions include metal-regulated polypeptides having molecular weights of 79 kDa to 69 kDa, and 65 kDa to 55 kDa. Specific examples of metal-regulated polypeptides isolatable from a Fusobacterium spp., such as F. necrophorum, after growth in low zinc conditions include polypeptides of 73 kDa and 60 kDa.
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 this type of polypeptide isolatable from Fusobacterium spp., such as F. necrophorum, have molecular weights 340 kDa to 330 kDa, 247 kDa to 237 kDa, 235 kDa to 215 kDa, 120 kDa to 110 kDa, 51 kDa to 25 kDa, and 21 kDa to 11 kDa. Examples of molecular weights of this type of polypeptide include 335 kDa, 243 kDa, 230 kDa, 220 kDa, 115 kDa, 45 kDa, 42 kDa, 38 kDa, 35 kDa, 30 kDa, and 16 kDa.
Other proteins provided herein include a protein at SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 (
In one embodiment, a polypeptide disclosed herein, for instance at SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 lacks one or more amino acids from the amino terminus, e.g., the polypeptide lacks a signal sequence. Thus, a fragment can lack at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, or at least 63 amino acids from the amino terminus of the polypeptide.
For instance, in one embodiment a polypeptide includes the following amino acids of SEQ ID NO:2; amino acids 2 through 423, amino acids 3 through 423, amino acids 4 through 423, amino acids 5 through 423, amino acids 6 through 423, amino acids 7 through 423, amino acids 8 through 423, amino acids 9 through 423, amino acids 10 through 423, amino acids 11 through 423, amino acids 12 through 423, amino acids 13 through 423, amino acids 14 through 423, amino acids 15 through 423, amino acids 16 through 423, amino acids 17 through 423, amino acids 18 through 423, amino acids 19 through 423, amino acids 20 through 423, amino acids 21 through 423, amino acids 22 through 423, amino acids 23 through 423, amino acids 24 through 423, amino acids 25 through 423, amino acids 26 through 423, amino acids 27 through 423, amino acids 28 through 423, amino acids 29 through 423, amino acids 30 through 423, amino acids 31 through 423, amino acids 32 through 423, amino acids 33 through 423, amino acids 34 through 423, amino acids 35 through 423, amino acids 36 through 423, amino acids 37 through 423, amino acids 38 through 423, amino acids 39 through 423, amino acids 40 through 423, amino acids 41 through 423, amino acids 42 through 423, amino acids 43 through 423, amino acids 44 through 423, amino acids 45 through 423, amino acids 46 through 423, amino acids 47 through 423, amino acids 48 through 423, amino acids 49 through 423, amino acids 50 through 423, amino acids 51 through 423, amino acids 52 through 423, amino acids 53 through 423, amino acids 54 through 423, amino acids 55 through 423, amino acids 56 through 423, amino acids 57 through 423, amino acids 58 through 423, amino acids 59 through 423, amino acids 60 through 423, amino acids 61 through 423, amino acids 62 through 423, or amino acids 63 through 423.
In one embodiment, a polypeptide includes the following amino acids of SEQ ID NO:4; amino acids 2 through 714, amino acids 3 through 714, amino acids 4 through 714, amino acids 5 through 714, amino acids 6 through 714, amino acids 7 through 714, amino acids 8 through 714, amino acids 9 through 714, amino acids 10 through 714, amino acids 11 through 714, amino acids 12 through 714, amino acids 13 through 714, amino acids 14 through 714, amino acids 15 through 714, amino acids 16 through 714, amino acids 17 through 714, amino acids 18 through 714, amino acids 19 through 714, amino acids 20 through 714, amino acids 21 through 714, amino acids 22 through 714, amino acids 23 through 714, amino acids 24 through 714, amino acids 25 through 714, amino acids 26 through 714, amino acids 27 through 714, amino acids 28 through 714, amino acids 29 through 714, amino acids 30 through 714, amino acids 31 through 714, amino acids 32 through 714, amino acids 33 through 714, amino acids 34 through 714, amino acids 35 through 714, amino acids 36 through 714, amino acids 37 through 714, amino acids 38 through 714, amino acids 39 through 714, amino acids 40 through 714, amino acids 41 through 714, amino acids 42 through 714, amino acids 43 through 714, amino acids 44 through 714, amino acids 45 through 714, amino acids 46 through 714, amino acids 47 through 714, amino acids 48 through 714, amino acids 49 through 714, amino acids 50 through 714, amino acids 51 through 714, amino acids 52 through 714, amino acids 53 through 714, amino acids 54 through 714, amino acids 55 through 714, amino acids 56 through 714, amino acids 57 through 714, amino acids 58 through 714, amino acids 59 through 714, amino acids 60 through 714, amino acids 61 through 714, amino acids 62 through 714, or amino acids 63 through 714.
In one embodiment, a polypeptide includes the following amino acids of SEQ ID NO:6; amino acids 2 through 736, amino acids 3 through 736, amino acids 4 through 736, amino acids 5 through 736, amino acids 6 through 736, amino acids 7 through 736, amino acids 8 through 736, amino acids 9 through 736, amino acids 10 through 736, amino acids 11 through 736, amino acids 12 through 736, amino acids 13 through 736, amino acids 14 through 736, amino acids 15 through 736, amino acids 16 through 736, amino acids 17 through 736, amino acids 18 through 736, amino acids 19 through 736, amino acids 20 through 736, amino acids 21 through 736, amino acids 22 through 736, amino acids 23 through 736, amino acids 24 through 736, amino acids 25 through 736, amino acids 26 through 736, amino acids 27 through 736, amino acids 28 through 736, amino acids 29 through 736, amino acids 30 through 736, amino acids 31 through 736, amino acids 32 through 736, amino acids 33 through 736, amino acids 34 through 736, amino acids 35 through 736, amino acids 36 through 736, amino acids 37 through 736, amino acids 38 through 736, amino acids 39 through 736, amino acids 40 through 736, amino acids 41 through 736, amino acids 42 through 736, amino acids 43 through 736, amino acids 44 through 736, amino acids 45 through 736, amino acids 46 through 736, amino acids 47 through 736, amino acids 48 through 736, amino acids 49 through 736, amino acids 50 through 736, amino acids 51 through 736, amino acids 52 through 736, amino acids 53 through 736, amino acids 54 through 736, amino acids 55 through 736, amino acids 56 through 736, amino acids 57 through 736, amino acids 58 through 736, amino acids 59 through 736, amino acids 60 through 736, amino acids 61 through 736, amino acids 62 through 736, or amino acids 63 through 736.
In one embodiment, a polypeptide includes the following amino acids of SEQ ID NO:34; amino acids 2 through 638, amino acids 3 through 638, amino acids 4 through 638, amino acids 5 through 638, amino acids 6 through 638, amino acids 7 through 638, amino acids 8 through 638, amino acids 9 through 638, amino acids 10 through 638, amino acids 11 through 638, amino acids 12 through 638, amino acids 13 through 638, amino acids 14 through 638, amino acids 15 through 638, amino acids 16 through 638, amino acids 17 through 638, amino acids 18 through 638, amino acids 19 through 638, amino acids 20 through 638, amino acids 21 through 638, amino acids 22 through 638, amino acids 23 through 638, amino acids 24 through 638, amino acids 25 through 638, amino acids 26 through 638, amino acids 27 through 638, amino acids 28 through 638, amino acids 29 through 638, amino acids 30 through 638, amino acids 31 through 638, amino acids 32 through 638, amino acids 33 through 638, amino acids 34 through 638, amino acids 35 through 638, amino acids 36 through 638, amino acids 37 through 638, amino acids 38 through 638, amino acids 39 through 638, amino acids 40 through 638, amino acids 41 through 638, amino acids 42 through 638, amino acids 43 through 638, amino acids 44 through 638, amino acids 45 through 638, amino acids 46 through 638, amino acids 47 through 638, amino acids 48 through 638, amino acids 49 through 638, amino acids 50 through 638, amino acids 51 through 638, amino acids 52 through 638, amino acids 53 through 638, amino acids 54 through 638, amino acids 55 through 638, amino acids 56 through 638, amino acids 57 through 638, amino acids 58 through 638, amino acids 59 through 638, amino acids 60 through 638, amino acids 61 through 638, amino acids 62 through 638, or amino acids 63 through 638.
In one embodiment, a polypeptide includes the following amino acids of SEQ ID NO:53; amino acids 2 through 1420, amino acids 3 through 1420, amino acids 4 through 1420, amino acids 5 through 1420, amino acids 6 through 1420, amino acids 7 through 1420, amino acids 8 through 1420, amino acids 9 through 1420, amino acids 10 through 1420, amino acids 11 through 1420, amino acids 12 through 1420, amino acids 13 through 1420, amino acids 14 through 1420, amino acids 15 through 1420, amino acids 16 through 1420, amino acids 17 through 1420, amino acids 18 through 1420, amino acids 19 through 1420, amino acids 20 through 1420, amino acids 21 through 1420, amino acids 22 through 1420, amino acids 23 through 1420, amino acids 24 through 1420, amino acids 25 through 1420, amino acids 26 through 1420, amino acids 27 through 1420, amino acids 28 through 1420, amino acids 29 through 1420, amino acids 30 through 1420, amino acids 31 through 1420, amino acids 32 through 1420, amino acids 33 through 1420, amino acids 34 through 1420, amino acids 35 through 1420, amino acids 36 through 1420, amino acids 37 through 1420, amino acids 38 through 1420, amino acids 39 through 1420, amino acids 40 through 1420, amino acids 41 through 1420, amino acids 42 through 1420, amino acids 43 through 1420, amino acids 44 through 1420, amino acids 45 through 1420, amino acids 46 through 1420, amino acids 47 through 1420, amino acids 48 through 1420, amino acids 49 through 1420, amino acids 50 through 1420, amino acids 51 through 1420, amino acids 52 through 1420, amino acids 53 through 1420, amino acids 54 through 1420, amino acids 55 through 1420, amino acids 56 through 1420, amino acids 57 through 1420, amino acids 58 through 1420, amino acids 59 through 1420, amino acids 60 through 1420, amino acids 61 through 1420, amino acids 62 through 1420, or amino acids 63 through 1420.
Additional examples of 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 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 terminus of the polypeptide.
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 Fusobacterium spp. genome. Such microarrays are commercially available and evaluating 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 Fusobacterium spp. Whether a polypeptide has protective immunological activity can be determined by methods known in the art such as, for example, methods described in Examples 2 and 7-11. 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 Fusobacterium spp.
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 Bacteroidaceae, such as the genus Fusobacterium. A member of the genus Fusobacterium is also referred to herein as Fusobacterium spp. Examples of Fusobacterium spp. include F. necrophorum (including F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme), F. nucleatum, F. ulcercans, F. russi, F. varium, F. mortiferum, F. gonidiaformans, F. canifelinum; F. necrogenes; and F. naviforme. An example of a representative strain is F. necrophorum 1694.
In one embodiment, 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 a reference amino acid sequence disclosed herein, for instance, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or a fragment thereof, such as a fragment that lacks one or more amino acids from the amino terminus. In one embodiment, such a polypeptide is metal-regulated when expressed by a Fusobacterium spp., such as F. necrophorum 1694.
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, or a 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.
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.
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 Bacteroidaceae, such as the genus Fusobacterium., including F. necrophorum (e.g., F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme), F. nucleatum, F. ulcercans, F. russi, F. varium, F. mortiferum, F. gonidiaformans, F. canifelinum; F. necrogenes; and F. naviforme.
Polynucleotide sequence similarity and polynucleotide sequence identity 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, a nucleotide sequence disclosed herein, such as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 78, or a fragment thereof) 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 a nucleotide sequence described herein, for example, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 78, or a fragment thereof. For example, two polynucleotide sequences can be compared using the Blastn program of the BLAST 2 search algorithm, as described by Tatusova 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.
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. In one embodiment, the whole cell is a member of the family Bacteroidaceae, such as the genus Fusobacterium, including F. necrophorum (e.g., F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme), F. nucleatum, F. ulcercans, F. russi, F. varium, F. mortiferum, F. gonidiaformans, F. canifelinum; F. necrogenes; and F. naviforme.
In one embodiment, a fusobacteria is engineered to express a recombinantly produced protein that has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:2 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:4 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:6 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:34 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:53 or a fragment thereof, or a combination thereof.
In one embodiment, a microbe, such as fusobacteria or E. coli, is engineered to express one or more recombinantly produced proteins that have structural similarity (sequence similarity or sequence identity) with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or a fragment thereof.
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, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and so on), in any combination. Unless a specific level of sequence similarity and/or identity is expressly indicated herein (e.g., at least 80% sequence similarity, at least 85% 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 or more vectors that include at least one polynucleotide encoding a polypeptide 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. Examples of host cells include, but are not limited to, E. coli and Fusobacteria. Methods for the genetic manipulation of Fusobacteria are known and routine in to art (see, for instance, Attarian et al., U.S. Pat. No. 6,962,990).
Certain compositions such as, for example, those including recombinantly-produced polypeptides, can include a maximum number of different types of polypeptides. In some embodiments, the maximum number of different types 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 17 polypeptides, no more than 16 polypeptides, no more than 15 polypeptides, no more than 14 polypeptides, no more than 13 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. A non-limiting example of a composition having no more than two polypeptides is one having the polypeptide SEQ ID NO:2 and the polypeptide SEQ ID NO:4. 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 Fusobacterium spp., or from a Fusobacterium spp. and a different microbe that is not a member of the genus Fusobacterium. 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 Fusobacterium spp. In some embodiments, a composition can include whole cell preparations from two, three, four, five, or six strains.
In one embodiment, a composition includes at least one, at least two, at least three, at least four, or at least five recombinantly produced proteins, for instance SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:34, and SEQ ID NO:53, or a fragment thereof. In one embodiment, a composition includes polypeptides expressed by a Fusobacterium spp. during growth in low iron and SEQ ID NO:2 (which is not expressed at a detectable level in low iron), SEQ ID NO:4 (which is not expressed at a detectable level in low iron), SEQ ID NO:6 (which is not expressed at a detectable level in low iron when a chelator such as 2,2-dipyridyl is used to reduce the amount of available iron and is expressed at a detectable level when 2,2-dipyridyl and hemin are present), SEQ ID NO:34, SEQ ID NO:53 (which is not expressed at a detectable level in low iron when a chelator such as 2,2-dipyridyl is used to reduce the amount of available iron and is expressed at a detectable level when 2,2-dipyridyl and hemin are present), or a combination thereof. Such compositions are not naturally occurring. A specific example of such a composition is one including proteins that are not detectable during growth of a Fusobacterium spp., such as F. necrophorum, after growth in low iron conditions (proteins having molecular weights of 88 kDa, 68 kDa, 60 kDa, and 52 kDa), proteins having enhanced expression by a Fusobacterium spp., such as F. necrophorum, after growth in low iron conditions (proteins having molecular weights of 103 kDa and 74 kDa), non-metal-regulated proteins expressed by a Fusobacterium spp., such as F. necrophorum, (335 kDa, 243 kDa, 230 kDa, 220 kDa, 115 kDa, 45 kDa, 42 kDa, 38 kDa, 35 kDa, 30 kDa, and 16 kDa), and one or more recombinantly produced proteins selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:34, SEQ ID NO:53, or a fragment thereof. Optionally, such a composition also includes metal-regulated proteins that are expressed after growth in low metal conditions supplemented with hemin (150 kDa and 84 kDa).
In one embodiment, a composition includes one or more polypeptides, for instance SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or a fragment thereof.
Optionally, a polypeptide of the present invention can be covalently bound to a carrier polypeptide to improve the immunological properties of the polypeptide. Useful carrier polypeptides are known in the art, and include, for instance, leukotoxin derived from Fusobacterium spp. The chemical coupling of a polypeptide of the present invention 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 (Harlow and Lane, Antibodies, A Laboratory Manual, generally and Chapter 5, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., N.Y. (1988)). In one embodiment, a protein described herein covalently bound to a carrier protein (such as a leukotoxin) has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:2 or a fragment thereof, has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:4 or a fragment thereof, has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:6 or a fragment thereof, has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:34 or a fragment thereof, or has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:53 or a fragment thereof.
Preferably, such compositions of the present invention include low concentrations of lipopolysaccharide (LPS). LPS is a component of the outer membrane of most gram negative microbes (see, for instance, Nikaido and Vaara, Outer Membrane, In: Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, Neidhardt et al., (eds.) American Society for Microbiology, Washington, D.C., pp. 7-22 (1987), and typically includes polysaccharides (0-specific chain, the outer and inner core) and the lipid A region. The lipid A component of LPS is the most biologically active component of the LPS structure and together induces a wide spectrum of pathophysiological effects in mammals. The most dramatic effects are fever, disseminated intravascular coagulation, complement activation, hypotensive shock, and death. The non-specific immunostimulatory activity of LPS can enhance the formation of a granuloma at the site of administration of compositions that include LPS. Such reactions can result in undue stress on the animal by which the animal may back off feed or water for a period of time, and exasperate infectious conditions in the animal. In addition, the formation of a granuloma at the site of injection can increase the likelihood of possible down grading of the carcass due to scarring or blemishes of the tissue at the injection site (see, for instance, Rae, Injection Site Reactions, available at www.animal.ufl.edu/extension/beef/documents/SHORT94/RAE.HTM, which is available through the website maintained by the Department of Animal Sciences of the University of Florida, Gainesville, Fla.).
The concentration of LPS can be determined using routine methods known in the art. Such methods typically include measurement of dye binding by LPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189 (1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, for instance, Endotoxins and Their Detection With the Limulus Amebocyte Lystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. (1982)). There are four basic commercially available methods that are typically used with an LAL test: the gel-clot test; the turbidimetric (spectrophotometric) test; the colorimetric test; and the chromogenic test. An example of a gel-clot assay is available under the tradename E-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma Technical Bulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., East Falmouth, Mass.). Typically, assay conditions include contacting the composition with a preparation containing a lysate of the circulating amebocytes of the horseshoe crab, Limulus polyphemus. When exposed to LPS, the lysate increases in opacity as well as viscosity and may gel. About 0.1 milliliter of the composition is added to lysate. Typically, the pH of the composition is between 6 and 8, preferably, between 6.8 and 7.5. The mixture of composition and lysate is incubated for 1 hour undisturbed at 37° C. After incubation, the mixture is observed to determine if there was gelation of the mixture. Gelation indicates the presence of endotoxin. To determine the amount of endotoxin present in the composition, dilutions of a standardized solution of endotoxin are made and tested at the same time that the composition is tested. Standardized solutions of endotoxin are commercially available from, for instance, Sigma Chemical (Catalog No. 210-SE), U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503), and Associates of Cape Cod, Inc., (Catalog No. E0005). In general, when a composition of the present invention is prepared by isolating polypeptides from a Fusobacterium spp. by a method as described herein (e.g., a method that includes disrupting and solubilizing the cells, and collecting the insoluble polypeptides), the amount of LPS in a composition of the present invention is less than the amount of LPS present in a mixture of same amount of the Fusobacterium spp. that has been disrupted under the same conditions but not solubilized. Typically, the level of LPS in a composition of the present invention is decreased by, in increasing order of preference, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the level of LPS in a composition prepared by disrupting, but not solubilizing, the same Fusobacterium spp.
In some aspects, a composition of the present invention does not include a leukotoxin isolatable from a Fusobacterium spp. Leukotoxins that are optionally not present in a composition of the present invention include polypeptides having a molecular weight of 335 kDa based on analysis with an 10% SDS-PAGE gel under reducing and denaturing conditions, and having an activity that is toxic to bovine leukocytes (Narayanan et al., Infect. Imun., 69, 5447-5455 (2001), and Narayanan et al., Infect. Immun., 70, 4609-4620 (2002)). Whether a polypeptide has leukotoxin activity can be determined using the monoclonal antibody F7B10 which is reactive against a F. necrophorum leukotoxin (Tan et al., Vet. Microbiol., 42, 121-133 (1994), or by determining whether the polypeptide is toxic to ruminant leukocytes. Methods for measuring the toxicity of a polypeptide for ruminant leukocytes are known in the art (Narayanan et al., Infect. Imun., 69, 5447-5455 (2001), and Narayanan et al., Infect. Immun., 70, 4609-4620 (2002).
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 of the present invention is administered in an amount sufficient to provide an immunological response to polypeptides or whole cells described herein. The amount of polypeptide present in a composition can vary. For instance, the dosage of polypeptide can be between 0.01 micrograms (μg) and 3000 milligrams (mg), typically between 10 μg and 2000 ug. When the composition is a whole cell preparation, the cells can be present at a concentration of 106 bacteria/ml, 107 bacteria/ml, 108 bacteria/ml, or 109 bacteria/ml. For an injectable composition (e.g. subcutaneous, intramuscular, etc.) the polypeptide is preferably 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 1.0-3.0 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 1.0-2.0 ml. The amount administered will vary depending on various factors including, but not limited to, the specific polypeptides or cells chosen, the weight, physical condition and age of the animal, and the route of administration. Thus, the absolute weight of the polypeptide or number of cells included in a given unit dosage form can vary, 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 skilled in the art. Other examples of dosages suitable for the invention are disclosed in Emery et al. (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. All methods of preparing a composition including a pharmaceutically acceptable carrier include the step of bringing the active compound (e.g., a polypeptide or whole cell of the present invention) 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 can also 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, dimethyldiocradecylammonium bromide (DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins (available from under the tradename EMULSIGEN from MVP Laboratories, Ralston, Nebr.), ISA-70, RIBI and other substances known in the art.
In another embodiment, a composition of the invention 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-alpha, IFN-gamma, and other cytokines that effect immune cells. A composition can also include an antibiotic, preservative, anti-oxidant, chelating agent, etc. Such components are known in the art.
This disclosure also provides methods for obtaining the polypeptides and whole cells described herein. Polypeptides and whole cell preparations described herein may be obtained by incubating a member of the genus Fusobacterium under conditions that promote expression of one or more of the polypeptides described herein. The polypeptides and whole cells as described herein may be isolatable from a member of the family Bacteroidaceae, such as the genus Fusobacterium, including F. necrophorum (such as F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme), F. nucleatum, F. ulcercans, F. russi, F. varium, F. mortiferum, F. gonidiaformans, F. canifelinum; F. necrogenes; and F. naviforme. An example of a representative strain is F. necrophorum 1694. Microbes useful for obtaining polypeptides 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 in 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.
The present invention also includes compositions prepared by the processes disclosed herein. Typically, such conditions are low metal conditions. As used herein, the phrase “low metal conditions” refers to an environment, typically bacteriological media that contains amounts of a free metal that cause a microbe to express a metal regulated polypeptide 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, 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, copper, or zinc.
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 a 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 a combination thereof. Examples of metal chelators include natural and synthetic compounds. Examples of natural compounds include plant phenolic compounds, such as flavonoids. Examples of flavonoids include the copper chelators catechin, naringenin, and quercetin, and the iron chelator myricetin. Examples of synthetic copper chelators include, for instance, ammonium tetrathiomolybdate, and examples of synthetic zinc chelators include, for instance, N,N,N′,N′-Tetrakis (2-pyridylmethyl)-ethylene diamine (also referred to as TPEN). Examples of synthetic iron chelators include 2,2′-dipyridyl (also referred to in the art as α,α′-bipyridyl), 8-hydroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxamine methanesulfonate (desferol), transferrin, lactoferrin, ovotransferrin, biological siderophores, such as the catecholates and hydroxamates, and citrate.
In one embodiment, 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 0.0025 micrograms/milliliter (μg/ml), at least 0.025 μg/ml, or at least 0.25 μg/ml. High levels of 2,2′-dipyridyl can be 10 μg/ml, 20 μg/ml, or 30 μg/ml.
In one embodiment, a medium is supplemented with an iron-containing porphyrin, such as hemin. Typically, hemin is added to the medium at a concentration of 20 ug/ml, and other concentrations can be used.
In one embodiment, quercetin is used for the chelation of copper. Typically, quercetin is added to the media at a concentration of 50 uM, and concentrations between 25 μM and 100 μM can be used.
In one embodiment, TPEN is used for the chelation of zinc. Typically, TPEN is added to the media at a concentration of 50 μM is used, and it is expected that higher concentrations can be used.
It is expected that a Fusobacterium spp. with a mutation in a fur gene will result in the constitutive expression of many, if not all, of the metal regulated polypeptides of the present invention. A potential fur gene has been identified in a F. nucleatum (Kapatral et al., J. Bacteriol. 184 (7), 2005-2018 (2002)). The production of a fur mutation in a Fusobacterium spp. can be produced using routine methods including, for instance, electroporation and genetic constructs useful for gene knock-out in gram negative bacteria.
In one embodiment, the fusobacteria used to make a composition described herein, e.g., a composition including isolated polypeptides or a composition including whole cells, may be produced using a fusobacteria that has been engineered to recombinantly express a protein that has structural similarity (sequence similarity or sequence identity) with SEQ ID NO:2 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:4 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:6 or a fragment thereof, a portion thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:34 or a fragment thereof, structural similarity (sequence similarity or sequence identity) with SEQ ID NO:53 or a fragment thereof, or a combination thereof. In one embodiment, such a fusobacteria is incubated in the presence of low iron conditions, and the one of more recombinant polypeptides are expressed during the incubation in the low iron conditions. The result is a fusobacteria that expresses iron-regulated proteins and the one of more recombinant polypeptides.
Many Fusobacterium spp. are able to grow in low metal conditions in vitro in artificial media only after adaptation. For instance, a Fusobacterium spp., such as the isolate given the identification number MS 040525 and F. necrophorum 1694 can be adapted to low iron conditions in vitro by growth in the presence of low concentrations of an iron chelator after growth in a medium containing the chelator, gradually increasing the concentration of the chelator. For instance, a Fusobacterium spp. can be adapted to growth in low iron conditions by adding 0.0025 μg/ml of 2,2′-dipyridyl to a medium, and exposing the culture to gradually increasing concentrations of the chelator to a greater concentration, for instance 20 μg/ml as previously reported Straub et al. (U.S. Pat. No. 8,329,192). Adaptation of Fusobacterium spp. to reduced zinc and copper is also possible. Repeat passage of at least five consecutive passes in 50 μM TPEN adapted Fusobacterium spp. to reduced zinc. Repeat passage of at least five consecutive passes in 50 or in 100 uM quercetin, repeat passage of at least five consecutive passes in 100 uM catechin, or repeat passage of at least five consecutive passes in 100 μM Naringenin adapted Fusobacterium spp. to reduced copper. Culture of adapted Fusobacterium spp. in the presence of any of these chelators resulted in increased expression of unique proteins. Adaptation of other Fusobacterium spp. strains to low metal conditions can be accomplished in this way.
The medium used to incubate the microbe is not critical, and conditions useful for the culture of fusobacteria are known to the skilled person. In one embodiment, supplements may be added to a culture medium, such as, but not limited to, hemin. The volume of medium used to incubate the microbe can vary. When a Fusobacterium spp. microbe is being evaluated for the ability to produce 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 in the art. The conditions used for growing a microbe preferably include a metal chelator, more preferably an iron chelator, for instance 2,2′-dipyridyl, TPEN, or quercetin, a pH of between 6.5 and 7.5, preferably between 6.9 and 7.1, and a temperature of 37° C. When a fermenter is used, the culture may be purged with an appropriate gas, for instance, nitrogen, to maintain anaerobic conditions. Members of the genus Fusobacterium are obligate anaerobes, thus growth conditions do not include levels of oxygen that will prevent growth.
In some aspects of the invention, a Fusobacterium spp. 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 and/or centrifugation. Typically, the concentrated microbe is suspended in decreasing amounts of buffer. Preferably, the final buffer includes a metal chelator, preferably, ethylenediaminetetraacetic acid (EDTA). An example of a buffer that can be used contains Tris-base (7.3 grams/liter) and EDTA (0.9 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 and preferably, the concentrated microbe is frozen at −20° C. or below until disrupted. In one embodiment, bacterial cells may be concentrated into a pellet by, for instance, centrifugation, and the concentrated cells suspended in osmotic shock buffer (OMS; 7.26 grams/liter Tris-base and 0.93 grams/liter EDTA adjusted to a pH of 8.5). The ratio of cells to OMS may be 50 grams cell pellet, 60 grams cell pellet, or 70 grams cell pellet to 1 liter of OMS. The suspension of cells in OMS can be incubated at 2-8° C. for at least 24 hours, at least 48 hours, or at least 60 hours to remove excess endotoxin from the cells. In one embodiment, the incubation is for no greater than 72 hours. After the incubation the suspension is centrifuged again and the supernatant discarded to remove free endotoxin and any extracellular material, e.g., secreted proteins.
When the Fusobacterium spp. 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 Fusobacterium spp. is to be used to prepare polypeptides of the present invention, the Fusobacterium spp. may be disrupted using chemical, physical, or mechanical methods routine and known in the art, including, for example, french press, sonication, or homogenization. Preferably, homogenization is used. 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 in 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. The temperature during disruption is typically kept at 4° C., to further minimize proteolytic degradation.
The disrupted microbe is solubilized in a detergent, for instance, an anionic, zwitterionic, nonionic, or cationic detergent. Preferably, the detergent is sarcosine, more preferably, sodium lauroyl sarcosinate. 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. The conditions for solubilization preferably result in the aggregation of polypeptides of the present invention into insoluble aggregates that are large enough to allow easy isolation by, for instance, centrifugation.
Preferably, the sarcosine is added such that the final ratio of sarcosine to gram weight of disrupted microbe is between 1.0 gram sarcosine per 4.5 grams pellet mass and 6.0 grams sarcosine per 4.5 grams pellet mass, preferably, 4.5 gram sarcosine per 4.5 grams pellet mass. The solubilization of the microbe may be measured by methods that are routine and known in the art, including, for instance, changes in optical density. Typically, the solubilization is allowed to occur for at least 24 hours, more preferably, at least 48 hours, most preferably, at least 60 hours. The temperature during disruption is typically kept low, preferably at 4° C.
The insoluble aggregates that include the polypeptides described herein may be isolated by methods that are routine and known in the art, such as centrifugation, filtration, or a combination thereof. In one embodiment, the insoluble aggregates are isolated by filtration, such as tangential or crossflow filtration. Examples of a molecular weight cutoff to use with tangential filtration are 40 kDa, 50 kDa, or 60 kDa. In one embodiment, a tangential filtration system has a molecular weight cutoff of 50 kDa. Tangential filtration may aid in removal of residual sarcosine from the protein suspension. Tangential filtration results in concentration of the protein suspension. Thus, the insoluble aggregates can be isolated at a significantly lower cost.
In one embodiment, the sarcosine is removed from the isolated polypeptides. Methods for removing sarcosine from the isolated polypeptides are known in the art, and include, for instance, diafiltration, precipitation, hydrophobic chromatography, ion-exchange chromatography, and/or affinity chromatography, and ultrafiltration and washing the polypeptides in alcohol, such as isopropyl alcohol, by diafiltration. After isolation, the polypeptides suspended in buffer and stored at low temperature, for instance, −20° C. or below.
Polypeptides of the present invention may also be isolated from Fusobacterium spp. using methods that are known in the art. The isolation of the polypeptides may be accomplished as described in, for instance, Hussain, et al. Infect. Immun., 67, 6688-6690 (1999); Trivier, et al., FEMS Microbiol. Lett., 127, 195-199 (1995); Heinrichs, et al., J. Bacteriol., 181, 1436-1443 (1999).
In those aspects of the present invention where a whole cell preparation is to be made, after growth of a Fusobacterium spp. the 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 of the invention may be prepared recombinantly. When prepared recombinantly, a polynucleotide encoding the polypeptide may be identified and cloned into an appropriate expression host. The recombinant expression host may be grown in an appropriate medium, disrupted, and the polypeptides isolated as described above.
Also provided are methods of using the polypeptides described herein. The methods include administering to an animal an effective amount of a composition that includes at least one polypeptide described herein. The composition may further include a pharmaceutically acceptable carrier. As used herein, an “effective amount” of a composition of the present invention is the amount able to elicit the desired response in the recipient. The composition can be administered at a time that maternal antibody may be present, for instance, as early as one day of age, or at a later time during the life of the animal. The animal can be, for instance, an ungulate, a companion animal, or a human. Examples of ungulates include animals that are bovine (including, for instance, cattle), caprine (including, for instance, goats), ovine (including, for instance, sheep), porcine (including, for instance, swine), equine (including, for instance, horses), avian (including, for instance, turkeys and chickens), members of the family Cervidae (including, for instance, deer, elk, moose, caribou and reindeer), and Bison (including, for instance, buffalo). Examples of companion animals include dogs and cats. In one embodiment, an animal is a mouse. In one embodiment, an animal is a hooved animal. 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, 1 to 8 weeks, preferably 2 to 4 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 annual boosters will not be necessary, as an animal will be challenged in the field by exposure to members of the genus Fusobacterium expressing polypeptides having epitopes that are identical to or structurally related to epitopes present on the polypeptides present in the composition administered to the animal.
In one aspect, the invention is directed to methods for making antibody to a polypeptide described herein, for instance, by inducing the production of antibody in an animal, or by recombinant techniques. The antibody produced includes antibody that specifically binds at least one polypeptide present in the composition. In this aspect of the invention, 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 antibodies that specifically bind polypeptides present in a composition of the present invention can be determined as described herein.
As used herein, an antibody that can “specifically bind” a polypeptide is an antibody that interacts only with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. An antibody that “specifically binds” to an epitope will, under the appropriate conditions, interact with the epitope even in the presence of a diversity of potential binding targets.
In one aspect the invention is also directed to treating an infection in an animal caused by a member of the genus Fusobacterium. The infection may be caused exclusively by Fusobacterium spp., or may be a mixed infection of Fusobacterium spp. and, for instance, Bacteroides nodosus. The method includes administering an effective amount of the composition to an animal having an infection caused by a member of the genus Fusobacterium, and determining whether the Fusobacterium spp. causing the infection has decreased. Methods for determining whether an infection is caused by a member of the genus Fusobacterium are routine and known in the art. It is expected that compositions made with polypeptides isolatable from one species of Fusobacterium will be useful in the methods described herein against other species of Fusobacterium.
In another aspect, the present invention is directed to methods for treating one or more symptoms of certain conditions in animals that may be caused by infection by a member of the genus Fusobacterium. Examples of conditions caused by Fusobacterium spp. infections include hepatic abscesses, foot rot, laminitis, purulent dermatitis, interdigital dermatitis, contagious ecthyma, necrotic rhinitis, skin ulcers, peritonsillar abscesses, septic arthritis, Lemierre's syndrome, endocarditis, metritis, and shipping fever. Treatment of these conditions can be prophylactic or, alternatively, can be initiated after the development of a condition described herein. Treatment that is prophylactic, for instance, initiated before a subject manifests symptoms of a condition caused by Fusobacterium spp., is referred to herein as treatment of a subject that is “at risk” of developing the condition. Typically, an animal “at risk” of developing a condition is an animal likely to be exposed to a Fusobacterium spp. causing the condition. For instance, the animal is present in an area where the condition has been diagnosed in at least one other animal, or is being transported to an area where a Fusobacterium spp. is endemic, and/or where conditions caused by Fusobacterium spp. are prevalent. 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, including 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 condition, decrease the severity of the symptoms of a condition, and/or completely remove the symptoms.
The potency of a composition described herein can be tested according to standard methods. For instance, the use of mice as an experimental model for Fusobacterium spp. infection in humans and large animals such as cattle is well established (Conion et al, Infect. Immun, 15, 510-517 (1977), Garcia and McKay, Can. J. Comp. Med, 42, 121-127 (1978), Abe et al, Infect. Immun, 13, 1473-1478 (1976), Emery and Vaughan, Vet. Microbiol, 12, 255-268 (1986), Smith et al, Epidemiol. Infect, 110, 499-506 (1993), and Narayanan et al., Vet. Micro. 93, 335-347 (2003)). A mouse model of Fusobacterium infection is available, and is recognized as correlating to with abscess formation and useful for evaluating the in vivo efficacy of antimicrobial agents (Nagaoka et al., 2013, J. Med. Micriobiol., 62(11):1755-1759). This model has proven to be a valuable model to evaluate the immunogenicity and identification of various target antigens provided by various fusobacteria species. Alternatively, when the condition is present in an animal such as, for instance, a sheep or cow, a controlled experimental trial can be run by vaccinating animals with varying levels of the composition and challenging vaccinated and unvaccinated animals with a Fusobacterium spp. Methods for determining whether an animal has the conditions disclosed herein and symptoms associated with the conditions are routine and known in the art. Symptoms often associated with hepatic abscesses can be a range of pathologies, from small foci of lymphocyte inflammation surrounded by low numbers of degenerating hepatcytes, to pronounced foci with necrosis and hemorrhage, loss of hepatocytes, fibrin and mixed inflammatory cells at the margin of the necrotic area.
A composition of the invention can be used to provide for passive immunization against infection by Fusobacterium spp. For instance, the composition can be administered to an animal to induce the production of immune products, such as antibodies, which can be collected from the producing animal and administered to another animal to provide passive immunity. Immune components, such as antibodies, can be collected to prepare antibody compositions from serum, plasma, blood, colostrum, etc. for passive immunization therapies. Antibody compositions including monoclonal antibodies, anti-idiotypes, and/or recombinant antibodies can also be prepared using known methods. 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. However, the antibodies may also be isolated from serum, plasma, blood, colostrum, and the like, using known methods and spray dried or lyophilized for later use in a concentrated or reconstituted form. Passive immunizing preparations may be particularly advantageous for treatment of acute systemic illness, or passive immunization of young animals that failed to receive adequate levels of passive immunity through maternal colostrum.
Another aspect of the present invention provides methods for detecting antibody that specifically binds polypeptides of the present invention. These methods are useful in, for instance, detecting whether an animal has antibody that specifically binds polypeptides of the present invention, and diagnosing whether an animal may have an infection caused by Fusobacterium spp. Preferably, such diagnostic systems are in kit form. The methods include contacting an antibody with a preparation that includes at least one polypeptide of the present invention to result in a mixture. Preferably, the antibody is present in a biological sample, more preferably blood, milk, or colostrum. The method further includes incubating the mixture under conditions to allow the antibody to specifically bind a 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 present in a composition of the present invention 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 and peroxidase.
The methods for detecting the presence of antibodies that specifically bind to polypeptides of the present invention can be used in various formats that have been used to detect antibody, including radioimmunoassay and enzyme-linked immunosorbent assay.
The present invention also provides a kit for detecting antibody that specifically binds polypeptides of the present invention. The kit includes at least one polypeptide of the present invention 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. 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 known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the polypeptides can be used for detecting antibodies induced by infection with Fusobacterium spp. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to detect such antibodies. As used herein, the term “package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the polypeptides. Thus, for example, a package can be a microtiter plate well to which microgram quantities of polypeptides have been affixed. “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.
Embodiment 1. A composition comprising:
at least one isolated polypeptide having a molecular weight of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, or 57 kDa to 47 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator,
at least one isolated polypeptide having a molecular weight of 108 kDa to 98 kDa or 79 kDa to 69 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator, is expressed by the Fusobacterium necrophorum when incubated in media without the iron chelator and expressed at an enhanced level during growth in media comprising an iron chelator, and
at least one protein selected from the group consisting of a polypeptide having at least 85% similarity to SEQ ID NO:4 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:2 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:34 or a fragment thereof, and a polypeptide having at least 85% similarity to SEQ ID NO:53 or a fragment thereof,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 2. A composition comprising:
isolated polypeptides having molecular weights of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator,
isolated polypeptides having molecular weights of 108 kDa to 98 kDa and 79 kDa to 69 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the iron chelator and expressed at an enhanced level during growth in media comprising an iron chelator, and
at least one protein selected from the group consisting of a polypeptide having at least 85% similarity to SEQ ID NO:4 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:2 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:34 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:53 or a fragment thereof,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 3. A composition comprising:
isolated polypeptides having molecular weights of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator,
isolated polypeptide having molecular weights of 155 kDa to 145 kDa and 89 kDa to 79 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator and an iron-containing porphyrin and not isolatable when grown in the media without the iron chelator and iron-containing porphyrin, and not isolatable when grown in the media with the iron chelator and in the absence of the iron-containing porphyrin, and
isolated polypeptides having molecular weights of 108 kDa to 98 kDa and 79 kDa to 69 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media comprising an iron chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the iron chelator and expressed at an enhanced level during growth in media comprising an iron chelator, and
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 4. A composition comprising:
at least one isolated polypeptide having a molecular weight of 131 kDa to 121 kDa, 79 kDa to 69 kDa, or 33 kDa to 23 kDa, wherein the at least one polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media comprising a copper chelator and not isolatable when grown in the media without the copper chelator, and
at least one isolated polypeptide having a molecular weight of 93 kDa to 83 kDa, 65 kDa to 55 kDa, or 52 kDa to 42 kDa, wherein the at least one polypeptide is isolatable from the Fusobacterium necrophorum when incubated in media comprising a copper chelator, is expressed by the Fusobacterium necrophorum when incubated in media without the copper chelator and expressed at an enhanced level during growth in media comprising an copper chelator,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 5. A composition comprising:
at least one isolated polypeptide having a molecular weight of 131 kDa to 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, or 33 kDa to 19 kDa, wherein the polypeptide is isolatable from a Fusobacterium necrophorum when incubated in media comprising a zinc chelator and not isolatable when grown in the media without the zinc chelator,
at least one isolated polypeptide having a molecular weight of 79 kDa to 69 kDa or 65 kDa to 55 kDa, wherein the at least one polypeptide is isolatable from the Fusobacterium necrophorum when incubated in media comprising a zinc chelator, is expressed by the Fusobacterium necrophorum when incubated in media without the zinc chelator and expressed at an enhanced level during growth in media comprising the zinc chelator,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 6. A composition comprising:
isolated polypeptides having molecular weights of 131 kDa to 121 kDa, 79 kDa to 69 kDa, and 33 kDa to 23 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media comprising a copper chelator and not isolatable when grown in the media without the copper chelator, and
isolated polypeptides having molecular weights of 93 kDa to 83 kDa, 65 kDa to 55 kDa, and 52 kDa to 42 kDa, wherein the polypeptides are isolatable from the Fusobacterium necrophorum when incubated in media comprising a copper chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the copper chelator and expressed at an enhanced level during growth in media comprising an copper chelator,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 7. A composition comprising:
isolated polypeptides having molecular weights of 131 kDa to 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, and 33 kDa to 19 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum when incubated in media comprising a zinc chelator and not isolatable when grown in the media without the zinc chelator, and
isolated polypeptides having molecular weights of 79 kDa to 69 kDa and 65 kDa to 55 kDa, wherein the polypeptides are isolatable from the Fusobacterium necrophorum when incubated in media comprising a zinc chelator, are expressed by the Fusobacterium necrophorum when incubated in media without the zinc chelator and expressed at an enhanced level during growth in media comprising the zinc chelator,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 8. The composition of any one of embodiments 1-7 further comprising:
a polypeptide having at least 85% similarity to SEQ ID NO:4 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:2 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:34 or a fragment thereof, a polypeptide having at least 85% similarity to SEQ ID NO:53 or a fragment thereof, or a combination thereof,
wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 9. A composition comprising:
an isolated polypeptide having at least 85% similarity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or a fragment thereof, wherein the composition protects an animal against challenge with Fusobacterium necrophorum.
Embodiment 10. The composition of any one of embodiments 1-9 further comprising:
isolated polypeptides having molecular weights of 340 kDa to 330 kDa, 247 kDa to 237 kDa, 247 kDa to 237 kDa, 235 kDa to 215 kDa, 120 kDa to 110 kDa, 51 kDa to 25 kDa, and 21 kDa to 11 kDa, wherein the polypeptides are isolatable from a Fusobacterium necrophorum.
Embodiment 11. The composition of any one of embodiments 1-10 further comprising a pharmaceutically acceptable carrier.
Embodiment 12. The composition of any one of embodiments 1-11 further comprising an adjuvant.
Embodiment 13. A method comprising:
administering to a subject an amount of the composition of any one of embodiments 1-12 effective to induce the subject to produce antibody that specifically binds to at least one polypeptide of the composition.
Embodiment 14. A method for treating an infection in a subject, the method comprising:
administering an effective amount of the composition of any one of embodiments 1-12 to a subject having or at risk of having an infection caused by a Fusobacterium spp.
Embodiment 15. A method for treating a symptom in a subject, the method comprising:
administering an effective amount of the composition of any one of embodiments 1-12 to a subject having or at risk of having an infection caused by a Fusobacterium spp.
Embodiment 16. A method for decreasing colonization in a subject, the method comprising:
administering an effective amount of the composition of any one of embodiments 1-12 to a subject colonized by or at risk of being colonized by a Fusobacterium spp.
Embodiment 17. A method for treating an infection in a subject, the method comprising:
administering an effective amount of a composition to a subject having or at risk of having an infection caused by a Fusobacterium spp., wherein the composition comprises antibody that specifically binds to a polypeptide of the composition of any one of embodiments 1-12.
Embodiment 18. A method for treating a symptom in a subject comprising:
administering an effective amount of a composition to a subject having or at risk of having an infection caused by a Fusobacterium spp., wherein the composition comprises antibody that specifically binds to a polypeptide of the composition of any one of embodiments 1-12.
Embodiment 19. A method for decreasing colonization in a subject, the method comprising:
administering an effective amount of a composition to a subject colonized by a Fusobacterium spp., wherein the composition comprises antibody that specifically binds to a polypeptide of the composition of embodiment any one of embodiments 1-12.
Embodiment 20. The method of any one of embodiments 13-19 wherein the subject is a mammal.
Embodiment 21. The method of any one of embodiments 13-20 wherein the mammal is a human, a bovine, or an ovine.
Embodiment 22. The method of any one of embodiments 13-21 wherein the Fusobacterium spp. is F. necrophorum.
Embodiment 23. The method of any one of embodiments 13-22 wherein at least 10 micrograms (μg) and no greater than 2000 μg of polypeptide is administered.
Embodiment 24. The method of any one of embodiments 13-23 wherein the infection causes a condition selected from metritis, hepatic abscesses, and foot rot.
Embodiment 25. A kit for detecting antibody that specifically binds a polypeptide, comprising in separate containers:
an isolated polypeptide of the composition of any one of embodiments 1-12; and
a reagent that detects an antibody that specifically binds the polypeptide.
Embodiment 26. A kit for detecting a polypeptide, comprising in separate containers:
an antibody that specifically binds an isolated polypeptide of the composition of any one of embodiments 1-12; and
a second reagent that specifically binds the polypeptide.
Embodiment 27. A composition comprising:
isolated antibody that specifically binds to a polypeptide of the composition of any one of embodiments 1-12.
Embodiment 28. A composition comprising:
an isolated whole cell that comprises a polypeptide of the composition of any one of embodiments 1-12.
Embodiment 29. The composition of embodiment 28 wherein the isolated whole cell comprises the polypeptides of the composition of any one of embodiments 1-12.
Embodiment 30. A composition comprising:
isolated antibody that specifically binds to a whole cell of any one of embodiments 28-29.
In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
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.
In the following studies we examined the expression of proteins of Fusobacterium necrophorum subsp. necrophorum under various conditions of metal ion restriction in order to mimic the expression of novel proteins that may be expressed during systemic invasion and colonization of the liver.
More than a dozen clinical isolates of Fusobacterium necrophorum were isolated from infected livers of beef cattle obtained from multiple processing plants. To preserve the original isolates a master seed stock of each isolate was prepared under anaerobic conditions. Briefly, single colonies were selected off blood agar plates grown under anaerobic conditions at 37° C. for approximately 48 hours. Colonies were transferred to 10 mL modified Trypticase Soy Broth (mTSB) (Becton Dickenson and Company, Franklin Lakes, N.J.) containing 0.05% cysteine HCL and 5 g/L yeast extract. A frozen master seed of each isolate was prepared by inoculating the isolate into 10 ml mTSB. The cultures were incubated anaerobically for 16 hours at 37° C. Five ml of each culture was transferred into 100 ml bottles of mTSB with either 15 μg/ml 2,2′-dipyridyl (DP) (Sigma Chemicals, St Louis, Mo.) or 20 μg/mL FeCl3 (Sigma) and allowed to grow anaerobically for 6 hours at 37° C. Fifty ml of the resulting cultures was combined with 50 ml of fresh mTSB containing 15 ug/ml DP and 20% glycerol, and the bacterial suspensions were sterilely dispensed into 2 ml cryogenic vials (1.5 ml per vial) and stored at −80° C. until use. Master seed stocks of each isolate were expanded into a working seeds. Briefly, one vial of the previously prepared master seed was grown as previously described, sterilely dispensed into 1.5 ml per vials and stored at −80° C. until use.
One isolate was selected based on its ability to grow well in metal ion chelators and showed good outer membrane protein expression. The isolate selected as the Master for the production of the Fuso-SRP Extract was designated as Master Seed 1694.
To test for novel protein expression under conditions of iron, zinc and copper chelation, frozen working seeds of Fusobacterium necrophorum as described above were transferred into 100 ml deplete modified Tryptic Soy Broth (TSB) containing 0.05% cysteine HCL and 5 g/L yeast extract and one of four metal ion chelators, 2,2′-dipyridyl (Dp), 2-pyridylmethyl-ethylene diamine (TPEN), catechin, and naringenin (all obtained from Sigma, St. Louis, Mo.). The metal ion chelators were used at the following concentration; 50 uM N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN); 50 and 100 uM quercetin; 100 uM catechin; or 100 uM Naringenin respectively. Cultures were grown with each chelator for 12 hours, at which point the culture was subcultured a second time for an additional 12 hours. Each culture was subcultured for five consecutive passes at 12-hour intervals. At the end of the fifth pass, each culture was harvested by centrifugation at 10,000×g for 20 minutes. Each culture was washed twice by centrifugation at 10,000×g and resuspended in 20 ml Tris-buffered saline, pH 7.2 at 4° C.
The bacterial cell suspensions were disrupted by sonication for 1.5 minutes at 4° C. using a Branson 450 equipped with a half inch disruption horn (Branson, Danbury Conn.). The disrupted bacterial suspensions were clarified by centrifugation at 32,000×g for 12 minutes. The supernatants were collected and solubilized by the addition of sodium lauroyl sarcosinate (4% vol/vol) at 4° C. for 24 hours. The detergent-insoluble protein-enriched fractions were collected by centrifugation at 32,000×g for 2.5 hours at 4° C. The protein pellets were resuspended in 200 μl Tris-buffer (pH 7.2) and stored at −90° C.
Cell extracts, derived from each metal chelation, were size-fractionated on SDS-PAGE gels using a 4% stacking gel and 10% resolving gel. Samples for electrophoresis were prepared by combining 30 μg of sample with 30 μl of SDS reducing sample buffer (62.5 mM Tris-HCL pH 6.8, 20% glycerol, 2% SDS, 5% beta-mercaptoethanol) boiled for 4 minutes. A sample of each extract was resolved on a 10% SDS-PAGE gel per standard methods and visualized by Coomassie Blue staining.
The SDS-PAGE patterns of Fusobacterium necrophorum 1694 grown under iron, zinc, and/or copper chelation showed unique banding patterns that were different when compared to the same isolate when grown under iron-restriction in the presence of 2,2′-dyipyridyl. For example, when the Fusobacterium necrophorum 1694 isolate was grown under iron-restriction or in the presence of the chelator 2,2′-dyipyridyl, unique iron-regulated proteins were expressed at the 88, 84, 68, 60, and 52 kDa regions (Table 1). These proteins were not detected when the isolate was grown in the presence of ferric chloride. Growth of Fusobacterium necrophorum 1694 in iron restriction also resulted in the increased expression of proteins having molecular weights of 103 and 74 kDa (Table 1). These proteins were detected when the isolate was grown in the presence of ferric chloride, but expressed at higher levels during growth under iron restriction.
When Fusobacterium necrophorum 1694 isolate was grown under copper-restriction, unique copper-regulated proteins were expressed at the 126, 74, and 28 kDa regions (Table 1). These proteins were not detected when the isolate was grown in the presence of free copper. Growth of Fusobacterium necrophorum 1694 in copper restriction also resulted in the increased expression of proteins having molecular weights of 88, 60, and 48 kDa (Table 1). These proteins were detected when the isolate was grown in the presence of free copper, but expressed at higher levels during growth under copper restriction.
When Fusobacterium necrophorum 1694 isolate was grown under zinc-restriction, unique zinc-regulated proteins were expressed at the 126, 103, 88, 81, 68, 48, 28, and 24 kDa regions (Table 1). These proteins were not detected when the isolate was grown in the presence of free zinc. Growth of Fusobacterium necrophorum 1694 in zinc restriction also resulted in the increased expression of proteins having molecular weights of 73 and 60 kDa (Table 1). These proteins were detected when the isolate was grown in the presence of free zinc, but expressed at higher levels during growth under zinc restriction.
We show for the first time a novel subset of proteins expressed by Fusobacterium necrophorum when the organism is grown under Iron, copper and zinc-restriction that are not expressed when the same isolate is grown under non-restricted conditions. Since transitional metals are used by organisms to build enzymes that catalyze various biochemical reactions, the metal ions may play a vital role in microbial survival during a systemic infection and/or the tissues they infect. It is perhaps for this reason that during sepsis there is a transient decrease in the availability of these transitional metals, making them unavailable for growth of the organism. These novel proteins could very well enhance the protective efficacy of the existing composition grown under iron-restriction because they may also be expressed by the bacteria under the metal ion restriction.
The electrophoretic banding profiles of the proteins isolated from the Fusobacterium necrophorum isolate grown in the following five conditions as described above were compared: iron-deplete media (400 ml mTSB containing 15 μg 2,2-dipyridyl), and controlled fermentation conditions (the iron-deplete fermentation conditions of Example 4); mTSB containing 100 uM naringenin; mTSB containing 100 uM catechin; mTSB containing 50 or 100 uM Quercetin; and mTSB containing 100 μM of N,N,N,N Tetrakis. The results revealed different banding profiles between each sample grown under different metal-depleting conditions.
In the presence of catechin (Quinde-Axtell et al., 2006, J. Agric. Food Chem., 54(26):9978-9984), a unique protein of ˜60 kDa by SDS-PAGE was visibly upregulated that was not enhanced in the presence of the other flavonoids and chelators (
In the presence of Quercetin (copper restriction) a protein of ˜48 kDa by SDS-PAGE, was shown to be preferentially upregulated, as compared to the other flavonoids and chelators (
A protein of ˜81 kDa by SDS-PAGE was shown to be upregulated in the presence of N,N,N,N tetrakis, a mostly zinc chelator (
A cryogenic vial of the working seed of Fusobacterium necrophorum 1694 (1 ml at 109 CFU/ml) was used to inoculate 250 ml of 37° C. modified TSB (mTSB) containing 5 g/L yeast extract and 0.05% cysteine (Sigma) and incubated in an anaerobic chamber. The culture was incubated at 37° C. for 20 hours at which point was sterilely transferred into 1.25 liters of the above media plus 25 micrograms (μg) 2,2-dipyridyl. This second culture was allowed to grow for an additional 3 hours at 37° C. This culture was used to inoculate a 15-liter Bioflo IV bench-top fermentor, (New Brunswick Scientific Co, Edison N.J.) charged with 9.5 liters of the above-described media. The pH was held constant between 6.9 and 7.1 by automatic titration with 50% NaOH and 20% H3PO4. The stirring speed was adjusted to 250 revolutions per minute (rpm), and the culture purged with pure nitrogen to maintain an anaerobic condition. The culture was allowed to grow continuously at these conditions for 24 hours at which point the fermentation was terminated by raising the pH to 8.5.
The bacterial cells were concentrated by centrifugation (Beckman Coultier, Brea, Calif.) at 7,000 rpm for 20 minutes. The bacterial pellet was then resuspended at a ratio of 60 g cell pellet to 1 liter sterile Osmotic Shock Buffer (OMS) containing 7.26 grams/liter Tris-base and 0.93 grams/liter EDTA adjusted to a pH of 8.5. The cell suspension was then incubated at 2-8° C. for 24 hours to remove excess endotoxin from the cells. The resulting suspension was then centrifuged again and the supernate discarded to remove free endotoxin and any extracellular material, e.g., secreted proteins. The cell pellet was resuspended in 3 liters of OMS. The cell suspension was mixed thoroughly and dispensed into a sterile four liter Nalgene containers and placed into a −20° C. freezer for storage. The pellet mass was calculated by centrifuging 30 ml samples of the fermented culture and final harvest. Briefly, pre-weighted 50 ml Nalgene conical tubes were centrifuged at 39,000×g for 90 minutes in a Beckman J2-21 centrifuge using a JA-21 rotor (Beckman Coulter, Brea, Calif.). At the end of the run, the supernate was poured off and the tubes were weighed again. The pellet mass was calculated for each stage.
One liter of the harvested three liter frozen bacterial cell slurry in OMS was thawed at 4° C. (60 gram pellet mass). The liquid culture suspension was disrupted by homogenization. Briefly, the tank containing the bacterial suspension was connected to a model Emulsiflex C500B Homogenizer, (Avisten Inc, Ottowa, Canada). A second process tank (empty) was connected to the homogenizer such that the fluid in the process tank could be passed through the homogenizer, into the empty tank and back again, allowing for multiple homogenizing passes while still maintaining a closed system. The temperature during homogenization was kept at 4° C. At the start of each pass, fluid was circulated at 40-65 psi through the homogenizer and back to the tank of origin, while the homogenizer pressure was adjusted to ≥20,000 psi. Prior to the first pass, two pre-homogenizing samples were withdrawn from the homogenizer to establish a baseline for determining the degree of disruption and monitoring of pH. The degree of disruption was monitored by transmittance (% T at 540 nanometers (nm) at 1:100 dilution) compared to the non-homogenized sample. The bacterial suspension was passed three times through the homogenizer to give a final percent transmittance >80% T at a 1:100 dilution.
After homogenization, Sodium Lauroyl Sarcosinate (Hamptosyl L-30, Chem/Serv, Minneapolis, Minn.) was aseptically added to the homogenized bacterial suspension for solubilization. The amount of Sarcosine (30%) added equaled 0.5% of the solubilizing volume, in liters. The process tank was removed from the homogenizer and kept at 4° C. while shaking at 120 rpm for 16-24 hours.
The protein suspension (1 Liter) was adjusted to 5 liters using sterile Tris-buffer, pH 8.5. The suspension was washed and dialyzed using a Optisep 1000 SmartFlow Tangential Flow Filter device (NCSRT Inc, Apex, N.C.), equipped with a 0.8 ft2 screen-channel series Alpha 50 kDa Centrasette filter (Pall Filtron) to remove residual sarcosine. The protein solution was concentrated by filtration to a target volume of 1 liter at which point 10 liters of Tris-buffer pH 7.2 containing 10% isopropyl alcohol was slowly added to the concentrate from a second process tank. Isopropyl alcohol is thought to cause a slight unfolding of the protein structure allowing for the removal of bound sarcosine without compromising the immunogenicity of the proteins. Diafiltration continued until the pH stabilized to 7.2 at which point 5 liters Tris-buffer pH 7.2 was slowly added by diafiltration to remove residual alcohol. The Fuso-SRP Extract suspension was then concentrated to approximately 325 ml. The protein concentrate was stored at −20° C. until use.
Alternative methods for bacterial harvest can be used. Bacterial harvest may be performed by the use of hollow fiber filter methods. Bacterial culture is harvested using filter cartridges ranging in size from 0.2 μM to 5 kDa; preferably with a 750 kDa cartridge. Culture is reduced in volume from 2-20× and subsequently washed 1-5× by diafiltration with buffer prior to storage at 4° C. or freezing at −20° C. In this manner, undesired media proteins, bacterial proteins and LPS are removed from the culture. In another alternative, bacterial harvest may be performed by the use of industrial scale centrifugation, for example, by use of a disc-stack centrifuge.
The full nucleotide sequence of the rZinc protein, including signal peptide, was submitted to GenScript USA Inc. (Piscataway, N.J.) for gene synthesis. The amino acid sequence was optimized for expression in Escherichia coli. Synthesized DNA was cloned in to plasmid pET-20b+(Novagen) by GenScript using the NdeI-XhoI cloning sites allowing for a C-terminal 6× Histidine tag. The resulting plasmid is named rZinc_pET-20b+.
Plasmid pTHV (Epitopix, LLC) was amplified using primers 3 and 4 (Table 2) to exclude the existing gene insert and only amplify the plasmid backbone. Fragment rZinc, excluding the signal peptide, was amplified from plasmid FT_pET-20b+ using oligonucleotide primers 1 and 2 (Table 2). The primers include nucleotides that overlap with the destination plasmid, pTHV. The vector and fragment PCR products were assembled using the NEBuilder® HiFi DNA assembly protocol (New England Biolabs) and transformed into NEB 10-beta competent E. coli for expression.
The full nucleotide sequence of the rHemin protein, including signal peptide, was submitted to GenScript USA Inc. (Piscataway, N.J.) for gene synthesis. The amino acid sequence was optimized for expression in Escherichia coli. Synthesized DNA was cloned in to plasmid pET-20b+(Novagen) by GenScript using the NdeI-XhoI cloning sites allowing for a C-terminal 6× Histidine tag. The resulting plasmid is named rHemin_pET-20b+.
Plasmid pTHV (Epitopix, LLC) was amplified using primers 7 and 8 (Table 3) to exclude the existing gene insert and only amplify the plasmid backbone. Fragment rHemin, excluding the signal peptide, was amplified from plasmid rHemin_pET-20b+ using oligonucleotide primers 5 and 6 (Table 3). The primers include nucleotides that overlap with the destination plasmid, pTHV. The vector and fragment PCR products were assembled using the NEBuilder® HiFi DNA assembly protocol (New England Biolabs) and transformed into NEB 10-beta competent E. coli for expression.
Convalescent serum was collected as part of a vaccination and challenge study in which steers with an average weight of approximately 350 pounds were used for generation of sera. Calf number 72 was an unvaccinated control animal challenged via the portal vein according to the method of K. Lechtenberg et al (Am J Vet Res. 1991 June; 52(6)803-9) with approximately 6×108 cfu of a virulent Fusobacterium necrophorum strain.
Blood samples were collected from all steers on day 66 (10 days post-challenge). All blood was collected in sterile 13×75 millimeter vacutainer collection tubes (SST No. 369783, Becton Dickinson, Franklin Lakes, N.J.). After clotting the blood tubes were centrifuged at 800×g for thirty minutes and frozen at −20° C.
The proteins in the vaccine composition as described in Example 2 were subjected to electrophoresis followed by western blot analysis with convalescent serum as described in Example 7. Briefly, the membrane proteins derived from Fusobacterium necrophorum grown under iron-limiting conditions were size-fractionated on an SDS-PAGE gel using a 4% stacking gel and 7.5% resolving gel. A 10 μl sample was combined with 10 μl of SDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4 minutes. Samples were electrophoresed at 18 mA constant current for 5 hour at 4° C. using a Protein II xi cell and model 1000/500 power supply (BioRad Laboratories, Richmond, Calif.). Band migration was visualized using broad range kaleidoscope standards (BioRad) to aid in the electro-blot transfer while biotinylated broad range standards were used as molecular weight references on the blot, see
The purpose of this analysis was to determine which of the proteins present in the immunizing composition induced antibody responses following challenge of steers. The results revealed unique immunological reactivity with proteins at 48 kDa in the presence of the copper chelator Quercetin, catechin, or narangenin (
These results demonstrated that the membrane proteins of the composition described in Example 2 reacted strongly with the convalescent sera described in Example 7, suggesting that these components of the vaccine may provide protection against disease. However, the sensitivity limits of the assay may have prevented the detection of weaker interactions, that, although less evident, may still contribute to the vaccine's effectiveness by augmenting the immune response to the composition. In addition, the proteins that were not sero-reactive in this assay may elicit responses other than antibody production, such as stimulation of cytokines, intereferon, interleukins, T-cells, or colony-stimulating factors. Such responses could enhance, direct, or restore the ability of the host's immune system to fight disease.
The composition made from Fusobacterium necrophorum strain 1694 of Example 4 was used as the vaccine in this experimental study. The vaccine was prepared from the composition by diluting the antigen into phosphate buffered saline (PBS) containing 8.0 NaCl, 0.2 KCl, 1.44 g/l Na2HPO4 and 0.24 g/l KH2PO4 pH 7.4 The suspension (500 μg total protein/ml) was then emulsified into the commercial adjuvant, EMULSIGEN, (MVP Laboratories, Ralston, Nebr.) using the syringe method of emulsification. The process can be summarized as follows: (1) force an amount of adjuvant from syringe B by pushing it into syringe A filled with antigen solution to mingle with the latter; (2) push the same volume of the mix from syringe A back to syringe B slowly; (3) repeat the above mixing process until the mixed portion becomes milky white. A mouse dose was administered to give a final dose of 100 μg total protein in a 0.1 ml injectable volume with an adjuvant concentration of 22.5% vol/vol. A placebo was prepared by replacing the antigen with physiological saline in the above formulation and emulsifying the suspension into EMULSIGEN to give an adjuvant concentration of 22.5%.
The efficacy of the Fuso-SRP Extract derived from Fusobacterium necrophorum 1694 was carried out against a live virulent challenge in mice. Eighty (N=80) female CF-1 mice obtained from Charles River Laboratories (Wilmington Del.) weighing 16-22 grams were equally distributed into two groups (40 mice/group). Mice were housed in polycarbonate cages in a self-contained HEPA filtered Mobile Housing System (Thoren Caging systems; Hazleton; PA). Treatment groups were designated as Group-A (Placebo) and Group-B (Fuso-SRP Extract Vaccinated). Food and water was supplied ad libitum to all mice. Mice were vaccinated subcutaneously twice at 21 day intervals. The volume administered was 0.1 ml/mouse see Table 4.
Twenty eight days after the second vaccination, mice in groups A and B were intravenously challenged. The Fusobacterium necrophorum isolate 1694 as previously described in Example 1 was used as the challenge strain. Briefly, a cryogenic vial of the frozen working seed of Fusobacterium necrophorum 1694 of Example 1 was used for challenge. Briefly, the frozen stock was thawed at 4° C. then diluted 1:10 in cold mTSB and the resulting dilution was used for challenge. All mice in groups A and B were intravenously challenged via the caudal vein with 0.1 ml of Fusobacterium necrophorum (˜1×108 colony forming units per ml) as previously enumerated as described in Example 7 Just prior to challenge, 100 μl of the above bacterial suspension was serially diluted tenfold to enumerate the number of CFU/dose. Mortality was recorded daily for 10 days post challenge at which point the experimental trial was terminated. All surviving mice from Groups A- and B were euthanized by carbon dioxide. The liver from all dead and surviving mice was aseptically removed and gross examination was performed to determine differences in liver abscessation.
The results showed a strong protective index against a caudal vein challenge as seen in Table 5. Ten out of 40 (25%) of the placebo-vaccinated mice (Group A) died within 10 days after challenge. In contrast, no mortality (0 out of 40) was seen in the vaccinated mice of Group B (degree of significance of P=0.001).
bLiver
cPercent
aMortality (%)
aThe mortality of mice that died within 10 days after IV challenge with 3.0 × 108 CFU of Fusobacterium necrophorum.
bThe percent of mice that had visible liver abscess upon death or at 10 days post challenge (two-sided P value) was P = 0.0143.
cPercent Survivability; 100 percent of the vaccinated mice survived challenge compared to the non-vaccinated controls where only 75 percent survived (two-sided P value) was P = 0.0010.
Gross examination of each liver revealed a dramatic difference in the number of abscesses between the Placebo and Vaccinated mice. It was clearly evident that mice given the vaccine rapidly reduced the number of bacteria able to proliferate successfully in the liver as indicated by the reduction in visible abscesses as compared to the placebo vaccinated mice (Table 5). The difference in the number of abscessed livers of Placebo vaccinated controls and the vaccinated group was statistically significant (degree of significance of P=0.0143), indicating a direct correlation in the reduction of lesions through vaccination by preventing the proliferation and colonization of Fusobacterium necrophorum in the liver The number of mice with abscesses was 9 out of 40 (22.5%) in the placebo vaccinated group as compared to only 1 out of 40 (2.5%) in the vaccinated group (Table 5).
The Fuso-SRP Extract vaccine of Group B showed a high degree of systemic protection as compared to non-vaccinated mice of Group A; (Placebo vaccinated). The vaccine prepared from Fusobacterium necrophorum was highly efficacious in preventing mortality associated with an intravenous challenge with Fusobacterium necrophorum in a standardized mouse model as well as reducing the formation of liver abscesses.
The purpose of the following experimental study was to evaluate the vaccine efficacy of two recombinant proteins, rZinc and rHemin of Fusobacterium necrophorum. In addition, a vaccine formulation consisting of the rZinc protein in combination with the Fuso-SRP extract and the Fuso-SRP extract as a stand-alone vaccine formulation was evaluated as illustrated in Table 6. The bovine strain of Fusobacterium necrophorum 1694 was used as the challenge strain as previously described in Example 1. The outcome parameters used to evaluate vaccine efficacy in this experiment were 1) serological response to vaccination 1) the reduction in the incidence of lesions between vaccinates and placebo control mice 2) the difference in the size of lesions based on a lesion score, where a lesion ≤0.5 cm=1 and a lesion ≥0.5=2) the difference in the Prevented Fraction which is defined as the percentage of animals in each treatment group that is protected against liver lesions calculated as:
1−p2/p1
p2=affected fraction in vaccine group
p1=affected fraction in control group
where, the prevented fraction is the complement of the risk ratio 1−p2/p1; where p2 is the affected fraction in the experimental product and p1 is the affected fraction in the placebo group. The precision of the estimate is evaluated by determining the 95% confidence interval.
Briefly, three hundred twenty (N=320) female Harlan CF-1 mice obtained from Charles River Laboratory (Wilmington, Mass.) weighing 16-22 grams were equally divided into 8 treatment groups (40 mice/group) designated as groups A-H (Table 6). Mice were housed in polycarbonate cages in a self-contained HEPA filtered Mobile Housing System (Thoren Caging systems; Hazleton; PA) at 5 mice per cage with food and water supplied ad libitum. All mice were allowed to acclimate one week prior to the first vaccination.
Vaccines of the recombinant Zinc and Hemin proteins as well as the Fusobacterium necrophorum SRP extract was prepared at their appropriate dosage levels in phosphate buffered saline (PBS) containing 8.0 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na.sub.2HPO.sub.4 and 0.24 g/l KH.sub.2PO.sub.4 pH 7.4 formulated with 10 percent Rehydragel HPA; (General Chemical; Berkeley Heights; New Jersey). The antigen/aluminum hydroxide suspensions was stirred for 24 hours at 4° C. to allow maximum adsorption of the protein to the adjuvant. The antigen/aluminum hydroxide suspension was then emulsified into the commercial adjuvant, EMULSIGEN, (MVP Laboratories, Ralston, Nebr.) to give and adjuvant concentration of 22.5% vol/vol. The rZinc vaccine of groups B and C was formulated at 100 μg and 250 μg respectively. The combination vaccine of Group D was formulated containing 10 μg of the Fuso-SRP extract as previously described in Example 4 and 50 μg of the rZinc protein to give a mouse dose of 60 μg total protein. The rHemin protein of Groups E and F was formulated at 25 μg and 100 μg dose levels respectively, while the Fuso-SRP extract of Groups G and H was formulated at 10 and 100 μg total protein respectively. All vaccines of Groups A-H were formulated to be delivered at 0.1 ml injectable volume. A placebo vaccine was prepared by substituting physiological saline for the aqueous protein suspension as described above. Mice were vaccinated subcutaneously two times at 21 day intervals and then challenged 14 days following the last vaccination. Blood was taken randomly from five mice from each group three times during the course of the study 1) first vaccination (pre-immune); 2) second vaccination and 3) 24 hours pre-challenge. Individual blood samples were equally pooled and stored at −80° C. until analyzed by western blot and ELISA to determine the serological response to vaccination.
The Fusobacterium necrophorum isolate 1694 as previously described in Example 1 was used as the challenge strain. Briefly, a cryogenic vial of the frozen working seed of Fusobacterium necrophorum 1694 of Example 1 was used for challenge. The frozen stock was thawed at 4° C. then diluted 1:10 in cold mTSB and the resulting dilution was used for challenge. All mice in groups A through H were intravenously challenged via the caudal vein with 0.1 ml of Fusobacterium necrophorum (˜1×108 colony forming units per ml) as previously enumerated as described in Example 7. Just prior to challenge, 100 μl of the above bacterial suspension was serially diluted tenfold to enumerate the number of CFU/dose. Mortality was recorded daily for 7 days post challenge at which point the experimental trial was terminated. All surviving mice from Groups A-H were euthanized by carbon dioxide. The liver from all dead and surviving mice was aseptically removed and gross examination was done to determine differences in liver abscessation.
Seven days post challenge the livers of all dead and surviving mice were aseptically removed and the difference in incidence and size of lesions was determined between vaccinates and placebo controls. Gross examination of each liver revealed a dramatic difference in both the size and incidence of lesions between the Placebo and Vaccinated mice. For example; thirty percent of the Placebo control mice had well defined foci in the livers in contrast to vaccinates; (Table 7;
aLiver
bPrevented
aLiver lesions - The difference in the number of mice having lesions calculated as a percent between treatment groups compared to controls.
bPrevented fraction is the percentage of mice in each treatment group that was protected against liver lesions.
The only vaccinated group that was not significantly different than the placebo controls was the rZinc protein at the 100 μg dose level of Group B. This vaccine was inadvertently administered only one time rather than the proposed two time vaccine regimen (Table 6). These results clearly show that a single dose of the rZinc protein at a 100 μg is not sufficient to induce a proper protective response. The rZinc protein administered at the 250 μg dose level of Group C was highly effective in reducing both the incidence and the size of lesions, clearly demonstrating a dose response; as the dose increased the incidence and size of lesions decreased. It's interesting to speculate that if the dose of the rZinc protein was increased beyond the 250 μg dose level if one could have obtained a greater degree of protection that would have been equivalent to the Fuso-SRP Extract. These results clearly demonstrate that a single recombinant protein at an optimal dose can protect against a systemic challenge of Fusobacterium. The rZinc protein reduced the incidence and the size of lesions.
Not unlike the rZinc protein, the rHemin protein was also effective as a vaccine candidate in reducing both the incidence and the size of lesions compared to the non-vaccinated controls. Both the 25 μg and 100 μg dose levels of the rHemin protein reduced the incidence and overall size of lesions in the liver (
In addition, the results show the calculated Prevented Fraction as described in Example 14 for each treatment group compared to the non-vaccinated placebo controls. For example, the Fuso-SRP Extracts at both the 10 μg and 100 μg dose levels had calculated Prevented Fractions of 100 and 92 percent with p-values of 0.0002 and 0.0015 respectively (Table 8). In fact the only other group that equaled these values was the combo vaccine of Group D consisting of 50 μg of the rZinc protein plus 10 μg of the Fuso-SRP Extract having a Prevented Fraction of 92 percent. These results showed a high degree of statistical significance having a p-value of 0.0015. The rZinc at the 250 μg dose level had a Prevented Fraction of 73 percent with a degree of significance of p=0.048. The rHemin protein at the 25 μg and 100 μg dose levels had Prevented Fractions of 58 and 50 percent with degrees of significance of p=0.099 and p=0.180 respectively. The rHemin protein showed a reduction in the incidence and the size of lesions when compared to the non-vaccinated controls but was not statistically significant. Results may have been different if a more rigorous dose finding regiment would have been performed. Nevertheless, all vaccine formulations tested except for Group B showed a reduction in the incidence and the overall size of liver lesions.
First the rZinc; rHemin and Fuso-SRP Extract were subjected to electrophoresis followed by western blot analysis with the sera taken 24 hours pre-challenge as described in Example 15. Briefly, rZinc; rHemin and Fuso-SRP Extract were size-fractionated on an SDS-PAGE gel using a 4% stacking gel and 7.5% resolving gel. A 10 μl sample was combined with 10 μl of SDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4 minutes. Samples were electrophoresed at 18 mA constant current for 5 hour at 4° C. using a Protein II xi cell and model 1000/500 power supply (BioRad Laboratories, Richmond, Calif.). Band migration was visualized using broad range kaleidoscope standards (BioRad) to aid in the electro-blot transfer while biotinylated broad range standards were used as molecular weight references on the blot, see
When the rHemin protein of Lane A5 was probed with sera derived from mice given the 250 μg rZinc vaccine of Group C no reactivity was seen; as expected.
The western blot (B) of the Fuso-SRP Extract grown under iron deplete conditions was probed with sera derived from the combo vaccine of Group D (10 μg Fuso-SRP Extract plus 50 μg rZinc protein). Note; multiple bands reacted in Lane B1 probed with sera derived from mice vaccinated with the combo vaccine. In contrast; the rZinc protein of Lane B2 was probed with sera derived from the combo vaccine of Group D consisting of 10 μg Fuso-SRP Extract plus 50 μg rZinc protein. Please note; the single rZinc band at the ˜81 kDa region (Lane-B2) showing immunological reactivity and a band in Lane B1 but with a slightly lower molecular weight than the rZinc protein of Lane-B2 with an approximate molecular weight between the 76 kDa-79 kDa region. Results clearly have shown that the zinc protein is not expressed under iron-deplete conditions; please refer to Lane-A2; (Fuso-SRP Extract probed with sera derived from the 250 μg rZinc vaccine of Group C) showing no reactivity.
The Western Blot showing the serological response to the rHemin protein is illustrated in
Lane 3 shows the rZinc protein probed with sera derived from the 100 μg rHemin vaccine of Group F. Note the lack of reactivity in this lane (3) clearly showing that the rZinc protein has no homology to rHemin protein. In contrast; the rHemin protein run in lanes Lanes A4 and A5 probed with sera derived from the 25 and 100 μg rHemin vaccinated mice reacted strongly with the purified recombinant protein in lanes A4 and A5 respectively.
The immunological response to the Fuso-SRP Extract and individual recombinant proteins after vaccination was determined by measuring the IgG titers by ELISA. In brief, the two recombinant proteins were coated in 5M urea, 100 mM NaCl, 20 mM Sodium Phosphate Buffer and the Fuso-SRP Extract was coated in the Carbonate Coating Buffer (Sigma S8875 Capsules). 100 μl of each antigen was added at 250 ng/well of a 96-well Immulon 2HB plate and incubated overnight at 4° C. with gentle agitation. The plate was washed three times with PBS wash buffer (PBS containing 0.05% Tween 20) followed by the addition of 200 μl/well 1% PVA/PBS and incubated at 37 degrees Celsius, gentle agitation. After one hour, the plate was washed three times with PBS wash buffer. 100 μl of PVA/PBS was placed into columns 2-11, all rows. Serial 4 fold dilutions of the primary antisera were performed in the plate by the addition of 133 μl of a 1:100 dilution to rows 1 and 12, mixing 3-4 times, with transfer of 33 μl to the next row, towards the center of the plate for a total of 6 dilutions for each sample. The plate was incubated for 1 hour at 37 degrees Celsius followed by three washes and addition of 100 μl/well of an HRP conjugated goat anti-mouse IgG, (H+L) chain antibody (KPL #074-1806) at a 1:10,000 dilution. After 1 hour incubation, the plate was washed three times followed by the addition of 100 μl 2 component ABTS Peroxidase Substrate System (KPL 50-62-01). Color was allowed to develop for 15 minutes. The absorbance was measured at a wavelength of 405-490 nm.
The serological response to vaccination was monitored by ELISA as described in Example 19. Blood samples were taken at the time first vaccination (pre-immune); second vaccination and 24 hours pre-challenge. Individual blood samples were equally pooled and analyzed by ELISA to determine the serological response to vaccination.
The 1694 culture of example 1 was inoculated into 20 ml mTSB and incubated overnight at 37° C. in an anaerobic chamber. A 2.5 mg/ml solution of hemin was prepared by adding 0.05 g of hemin (Sigma, St Louis, Mo.) to 20 ml of 0.1 Normal Sodium Hydroxide solution and vortexed to mix. The solubilized hemin was then sterilized through a 0.2 micron filter into a sterile 50 ml conical tube. Three sets of mTSB were prepared according to Table 9.
2,2′ bipyridyl was added to medium A at 15 ug/ml and autoclaved for 30 minutes at 121° C. The sterile hemin solution was added to media A at 800 uL per 100 mL for a final concentration of 20 ug/ml. Media B contained no hemin and 20 ug/ml 2,2′ bipyridyl. Medium C contained no hemin or bipyridyl, but contained FeCl3 at 20 ug/ml.
Five mL of the overnight culture was transferred to 100 mL of each medium A, B and C and incubated anaerobically for 7 hours at 37° C. After 7 hours, 25 ml of the cultures were transferred to fresh 500 ml volumes of their respective media and allowed to incubate overnight at 37° C. The following morning, strong growth was observed as measured by visual turbidity. All cultures were then centrifuged for 20 minutes at 7,500×G. The supernatant was decanted and discarded. The cell pellet was resuspended in 35 ml sterile Tris buffered water with 0.93 g/l EDTA salt. The cell suspension was then frozen at −80° C. for a minimum of 2 hours. The bacterial cell suspensions were disrupted by sonication for 90 seconds at 4° C. using a Branson 450 equipped with a half inch disruption horn (Branson, Danbury Conn.). The disrupted bacterial suspensions were clarified by centrifugation at 39,000×g for 20 minutes. The supernatants were collected and solubilized by the addition of sodium lauroyl sarcosinate (1% vol/vol) at 4° C. for 18 hours. The detergent-insoluble protein-enriched fractions were collected by centrifugation at 39,000×g for 2.5 hours at 4° C. The protein pellets were resuspended in 200 μl Tris-buffer (pH 7.2) and stored at −90° C.
The iron-restricted hemin-supplemented SRP extract, the iron-restricted SRP extract and the iron-supplemented SRP Extract were subjected to electrophoresis followed by western blot analysis with the mouse sera taken 24 hours pre-challenge as described in Example 16, and with the calf convalescent sera described in Example 7. Briefly, the SRP Extracts were size-fractionated on a Criterion TGX stain free pre-cast SDS-PAGE gel (BioRad Laboratories, Richmond Calif.) with a 4% stacking gel and 7.5% resolving gel. A 10 μl sample was combined with 10 μl of SDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4 minutes. Samples were electrophoresed at 200 volt constant current for 44 minutes at 4° C. using a Criterion cell and model 1000/500 power supply (BioRad). Band migration was visualized using broad range kaleidoscope standards (BioRad) to aid in the electro-blot transfer while biotinylated Precision Plus standards (BioRad) were used as molecular weight references on the blot, see
The SDS-PAGE showing the upregulation of the rHemin protein and the hemagglutinin protein is illustrated in
Lane 3 shows the iron restricted formulation B of table 9. Note the lack of expression of these two proteins in the presence of iron restriction alone without hemin supplementation. Lane 4 shows the iron replete formulation C of table 9. Note the lack of expression of the rHemin and hemagglutinin proteins in the presence of ferric iron. This demonstrates that iron restriction alone is not enough to upregulate these proteins; only by limiting iron and adding back hemin as an iron source are these proteins expressed.
In addition to the upregulation of the rHemin protein, a protein of ˜150 kDa by SDS-PAGE was shown to be upregulated in the presence of hemin in an otherwise iron deplete media (
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 their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. 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 the benefit of U.S. Provisional Application Ser. No. 62/252,951, filed Nov. 9, 2015, which is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 62252951 | Nov 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15774168 | May 2018 | US |
| Child | 16921243 | US |
