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text:
|Publication number
|Publication type
|Application number
|Publication date
|Sep 28, 2004
|Filing date
|Nov 24, 1997
|Priority date
|Oct 16, 1991
|Fee status
|Publication number
|Andrew A. Potter, Mark J. Redmond, Huw P. A. Hughes
|Original Assignee
|University Of Saskatchewan
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (20), Non-Patent Citations (25), Referenced by (5), Classifications (44), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of U.S. patent application Ser. No. 08/455,970 filed on May 31, 1995 now U.S. Pat. No. 5,708,155, which is a divisional of application Ser. No. 07/960,932 filed Oct. 14, 1992 (now U.S. Pat. No. 5,422,110), which is a continuation-in-part of application Ser. No. 07/779,171 filed Oct. 16, 1991 now abandoned.
The present invention relates generally to immunological carrier systems. More particularly, the invention pertains to leukotoxin-antigen chimeras which demonstrate enhanced immunogenicity as compared to the immunogenicity of the antigen alone.
Subunit vaccines are vaccines which are devoid of intact pathogen cells. These vaccines are usually composed of substantially purified antigens. Such vaccines are generally preferable to compositions which use attenuated or inactivated pathogens. However, many subunit vaccines which include proteins, such as peptide hormones and bacterial and viral antigens, require the help of a carrier protein in order to elicit a strong immune response. This is especially true for small proteins or endogenous substances, such as hormones, which are poorly immunogenic.
The carrier serves to non-specifically stimulate T helper cell activity and to direct the antigen to the antigen presenting cell, where the antigen is processed and presented at the cell surface in the context of molecules of the major histocompatibility complex (MHC).
Several carrier systems have been developed for this purpose. For example, small peptide antigens are often coupled to protein carriers such as keyhole limpet haemocyanin (Bittle, J. L., et al., Nature (1982) 298:30-33), tetanus toxoid (Muller, G., et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79:569-573), ovalbumin, and sperm whale myoglobin, to produce an immune response. However, carriers may elicit strong immunity not relevant to the peptide antigen and this may inhibit the immune response to the peptide vaccine on secondary immunization (Schutze, M. P., et al, J. Immun. (1985) 135:2319-2322).
Antigen delivery systems have also been based on particulate carriers. For example, preformed particles have been used as platforms onto which antigens can be coupled and incorporated. Systems based on proteosomes (Lowell, G. H., et al., Science (1988) 240:800-802), immune stimulatory complexes (Morein, B., et al., Nature (1984) 308:457-460), and viral particles such as HBsAg (Neurath, A. R., et al., Mol. Immunol. (1989) 26:53-62) and rotavirus inner capsid protein (Redmond, M. J., et al., Mol. Immunol. (1991) 28:269-278) have been developed.
Other carrier systems have been devised using recombinantly produced chimeric proteins that self assemble into particles. For example, the yeast retrotransposon, Ty, encodes a series of proteins that assemble into virus like particles (Ty-VLPs; Kingsman, S. M., and A. J. Kingsman Vacc. (1988) 6:304-306). Foreign genes have been inserted into the TyA gene and expressed in yeast as a fusion protein. The fusion protein retains the capacity to self assemble into particles of uniform size.
Other chimeric protein particles have been examined such as HBsAg, (Valenzuela, P., et al., Bio/Technol. (1985) 3:323-326; U.S. Pat. No. 4,722,840; Delpeyroux, F. N., et al., Science (1986) 233:472-475), Hepatitis B core antigen (Clarke, B. E., et al., Vaccines 88 (Ed. H. Ginsberg, et al., 1988) pp. 127-131), Poliovirus (Burke, K. L., et al., Nature (1988) =:81-82), and Tobacco Mosaic Virus (Haynes, J. R., et al., Bio/Technol. (1986) 4:637-641). However, these carriers are restricted in their usefulness by virtue of the limited size of the active agent which may be inserted into the structural protein without interfering with particle assembly.
Gene fusions provide a convenient method for the production of chimeric proteins. The expression of chimeric proteins affords an efficient means of linking a carrier protein to a desired antigen.
Pasteurella haemolytica produces a cytotoxin which is a leukotoxin. See, e.g. Gentry et al. Vet. Immunology and Immunopathology (1985) 9:239-250; Shewen, P. E., and Wilkie, B. N. Infect. Immun. (1987) 55:3233-3236. The gene encoding this cytotoxin has been cloned and expressed in bacterial cells. Lo et al. Infect. Immun. (1985) 50:667-671; U.S. Pat. No. 5,055,400. The leukotoxin has been used as an antigen in vaccine formulations to fight shipping fever pneumonia in livestock (See, e.g. U.S. Pat. No. 4,957,739) as well as to produce chimeric molecules for use in vaccines against shipping fever (see, e.g. International Publication No. WO 92/03558, published Mar. 5, 1992; and U.S. Pat. No. 5,028,423). However, the use of leukotoxin as a carrier molecule to increase the immune response of antigens associated therewith has not heretofore been described.
The present invention is based on the construction of novel gene fusions between the P. haemolytica leukotoxin gene and a nucleotide sequence encoding a selected antigen. These constructs produce a chimeric protein that displays enhanced immunogenicity when compared to the immunologic reaction elicited by administration of the antigen alone.
In one embodiment, the present invention is directed to an immunological carrier system comprising an immunogenic chimeric protein. The chimeric protein comprises a leukotoxin polypeptide fused to a selected antigen, whereby the leukotoxin portion of the chimeric protein acts to increase the immunogenicity of the antigen. In particularly preferred embodiments, the selected antigen is somatostatin (SRIF), gonadotropin releasing hormone (GnRH) or rotavirus viral protein 4 (VP4).
Also disclosed are vaccine compositions comprising the chimeric proteins and a pharmaceutically acceptable vehicle and methods of using the same.
In another embodiment, the subject invention is directed to DNA constructs encoding the chimeric proteins. The DNA constructs comprise a first nucleotide sequence encoding a leukotoxin polypeptide operably linked to a second nucleotide sequence encoding the selected antigen.
In yet another embodiment, the subject invention is directed to expression cassettes comprised of (a) the DNA constructs above and (b) control sequences that direct the transcription of the construct whereby the constructs can be transcribed and translated in a host cell.
In another embodiment, the invention is directed to host cells transformed with these expression cassettes.
Another embodiment of the invention provides a method of producing a recombinant polypeptide. The method comprises (a) providing a population of host cells described above and (b) growing the population of cells under conditions whereby the polypeptide encoded by the expression cassette is expressed.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
FIG. 1 depicts the structure of the leukotoxin gene of P. haemolytica cloned in E. coli (Plasmid pAA114).
FIG. 2 depicts the structure of Plasmid pAA352 wherein tac is the hybrid trp::lac promoter from E. coli; bla represents the β-lactamase gene (ampicillin resistance); ori is the ColEl-based plasmid origin of replication; lktA is the P. haemolytica leukotoxin structural gene; and lacl is the E. coli lac operon repressor. The direction of transcription/translation of the leukotoxin gene is indicated by the arrow. The size of each component is not drawn to scale.
FIGS. 3A through 3I (SEQ ID NOS:1 and 2) shows the nucleotide sequence and predicted amino acid sequence of leukotoxin 352 (LKT 352) from plasmid pAA352. Both the structural gene for LKT 352 and the sequences of the flanking vector regions are shown.
FIG. 4 (SEQ ID NOS:3-8) shows the nucleotide sequences of SRIF, GnRH and bovine rotavirus VP4, used in the construction of the leukotoxin-antigen gene fusions.
FIG. 5 shows the structure of Plasmid pAA496 carrying a leukotoxin-SRIF (LKT-SRIF) gene fusion wherein tac is the hybrid trp::lac promoter from E. coli; bla represents the β-lactamase gene (ampicillin resistance); lktA is the P. haemolytica leukotoxin structural gene; SRIF is the somatostatin structural gene; and lacl is the E. coli lac operon repressor. The direction of transcription/translation of the leukotoxin gene is indicated by the arrow. The size of each component is not drawn to scale.
FIGS. 6A through 6J (SEQ ID NOS:9 and 10) the nucleotide sequence and predicted amino acid sequence of the LKT-SRIF chimeric protein from pAA496.
FIG. 7 shows the structure of Plasmid pAA502 carrying a leukotoxin-GnRH (LKT-GnRH) gene fusion wherein tac is the hybrid trp::lac promoter from E. coli; bla represents the β-lactamase gene (ampicillin resistance); lktA is the P. haemolytica leukotoxin structural gene; GnRH is the gonadotropin releasing hormone structural gene; and lac1 is the E. coli lac operon repressor. The direction of transcription/translation of the leukotoxin gene is indicated by the arrow. The size of each component is not drawn to scale.
FIGS. 8A through 8J (SEQ ID NOS:11 and 12) the nucleotide sequence and predicted amino acid sequence of the LKT-GnRH chimeric protein from pAA502.
FIG. 9 depicts the structure of Plasmid pAA501 carrying a leukotoxin-VP4 (LKT-VP4) gene fusion wherein tac is the hybrid trp::lac promoter froze E. coli; bla represents the β-lactamase gene (ampicillin resistance); lktA is the P. haemolytica leukotoxin structural gene; VP4 is the bovine rotavirus viral protein 4 (232-255) structural gene; and lac1 is the E. coli lac operon repressor. The direction of transcription/translation of the leukotoxin gene is indicated by the arrow. The size of each component is not drawn to scale.
FIGS. 10A through 10J (SEQ ID NOS:13 and 14) the nucleotide sequence and predicted amino acid sequence of the LXT-VP4 chimeric protein from pAA501.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, an Fritsch & Maniatis, Molecular Clonina: A Laboratory Manual, Second Edition (1989); Maniatis, Fritsch & Sambrook, Molecular Clonina: A Laboratory Manual (1982); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).
All patents, patent applications, and publications mentioned herein, whether supra or infra, are hereby incorporated by reference in their entirety.
A. Definitions
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
An "antigen" refers to a molecule containing one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is also used interchangeably with "immunogen." An antigen will include one or more epitopes from a protein molecule, such as but not limited to, bacterial and viral proteins, as well as peptide hormones which elicit an immune response. Additionally, an antigen can comprise one or more identical or different immunogenic repeating sequences of a protein. Specifically excluded from the definition for purposes of this application are cytokines such as interleukin-1 (IL1 ), interleukin-2 (IL2), interleukin-3 (IL3), interleukin-4 (IL4), and gamma-interferon (γIFN).
The term "leukotoxin polypeptide" intends a polypeptide derived from a protein belonging to the family of molecules characterized by the carboxy-terminus consensus amino acid sequence Gly-Gly-X-Gly-X-Asp (Highlander et al., DNA (1989) 8:15-28), where X is Lys, Asp(SEQ ID NOS:15), val or Asn. Such proteins include., among others, leukotoxins derived from P. haemolytica and Actinobacillus pleuropneumoniae, as well as E. coli alpha hemolysin (Strathdee, C. A., and Lo, R. Y. C. Infect. Immun. (1987) 55:3233-3236; Lo, R. Y. C., Can. J. Vet. Res. (1990) 54:S33-35; Welch, R. A., Mol. Microbiol. (1991) 5:521-528). This family of toxins is known as the "RTX" family of toxins (Lo, R. Y. C., Can. J. Vet. Res. (1990) 54:533-535). In addition, the term "leukotoxin polypeptide" refers to a leukotoxin polypeptide which is chemically synthesized, isolated from an organism expressing the same, or recombinantly produced. Furthermore, the term intends an immunogenic protein having an amino acid sequence substantially homologous to a contiguous amino acid sequence found in the particular native leukotoxin molecule. Thus, the term includes both full-length and partial sequences, as well as analogs. Although native full-length leukotoxins display leukotoxic activity, the term "leukotoxin" also intends molecules which remain immunogenic yet lack the cytotoxic character of native leukotoxins. The nucleotide sequences and corresponding amino acid sequences for several leukotoxins are known.
See, e.g., U.S. Pat. Nos. 4,957,739 and 5,055,400; Lo et al., Infect. Immun. (1985) 50:667-67; Lo et al., Infect. Immun. (1987) 55:1987-1996; Strathdee, C. A., and Lo, R. Y. C., Infect. Immun. (1987) 55:3233-3236; Highlander et al., DNA (1989) 8:15-28; Welch, R. A., Mol. Microbial. (1991) 5:521-528.
By "LKT 352" is meant a protein which is derived from the lktA gene present in plasmid pAA352 (FIG. 2, ATCC Accession No. 68283). The nucleotide sequence and corresponding amino acid sequence of this gene are described in International Publication No. W091/15237 and shown in FIG. 3A through 3I (SEQ ID NOS:1 and 2). The gene encodes a truncated leukotoxin, having 931 amino acids, which lacks the cytotoxic portion of the molecule. The derived LKT 352 is not necessarily physically derived from the sequence present in plasmid pAA352. Rather, it may be generated in any manner, including for example, by chemical synthesis or recombinant production. In addition, the amino acid sequence of the protein need only be substantially homologous to the depicted sequence. Thus, sequence variations may be present so long as the protein functions to enhance the immunogenicity of the antigen with which it is associated.
A "hapten" is a molecule containing one or more epitopes that does not stimulate a host's immune system to make a humoral or cellular response unless linked to a carrier.
The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site."
An "immunological response" to an antigen or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, such a 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 and/or γδ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
An "immunogenic protein" or "immunogenic amino acid sequence" is a protein or amino acid sequence, respectively, which elicits an immunological response in a subject to which it is administered.
A leukotoxin-antigen chimera displays "increased immunogenicity" when it possesses a greater capacity to elicit an immune response than the corresponding antigen alone. Such increased immunogenicity can be determined by administering the particular leukotoxin-antigen and antigen controls to animals and comparing antibody titers against the two using standard assays such as radioimmunoassays and ELISAs, well known in the art.
By "carrier system" is meant a system which includes a molecule that serves to increase the immunogenicity of an antigen administered therewith, as defined above. Without being bound by any particular theory, the molecule may function to increase the immunogenicity of the antigen by presenting the same to cells of the immune system, such as antigen presenting cells, macrophages, follicular dendritic cells, B cells and T cells; or by stimulating the immune system to respond at a level greater than that observed when the antigen is administered alone.
By "subunit antigen composition" is meant a composition containing at least one immunogenic polypeptide, but not all antigens, derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of intact pathogen cells or particles. Generally, a "subunit antigen composition" is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof.
The term "protein" is used herein to designate a naturally occurring polypeptide. The term "polypeptide" is used in its broadest sense, i.e., any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the term "polypeptide" includes proteins, oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.
"Native" proteins or polypeptides refer to proteins or polypeptides recovered from a source occurring in nature. Thus, the term "native leukotoxin" would include naturally occurring leukotoxin and fragments thereof.
"Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides are those prepared by chemical synthesis.
A "rotavirus VP6 protein" refers to the art-recognized major viral protein of the inner capsid from any species or strain within the family Reoviridae. See, e.g., Kapikian et al., 1985. Examples of rotavirus strains from which the VP6 protein can be isolated and employed in the present invention include, but are not limited to., Simian SA-11, human D rotavirus, bovine UK rotavirus, human Wa or W rotavirus, human DS-1 rotavirus, rhesus rotavirus, the "O" agent, bovine NCDV rotavirus, human S2 rotavirus, human KUN rotavirus, human 390 rotavirus, human P rotavirus, human N rotavirus, human Walk 57/14 rotavirus, human Mo rotavirus, human Ito rotavirus, human Nemoto rotavirus, human YO rotavirus, human McM2 rotavirus, rhesus monkey MMU18006 rotavirus, canine CU-1 rotavirus, feline Taka rotavirus, equine H-2 rotavirus, human St. Thomas No. 3 and No. 4 rotaviruses, human Hosokawa rotavirus, human Hochi rotavirus, porcine SB-2 rotavirus, porcine Gottfried rotavirus, porcine SB-1A rotavirus, porcine OSU rotavirus, equine H-1 rotavirus, chicken Ch.2 rotavirus, turkey Ty.1 rotavirus, bovine C486 rotavirus, and strains derived from them. Thus the present invention encompasses the use of VP6 from any rotavirus strain, whether from subgroup I, subgroup II, or any as yet unidentified subgroup, as well as from any of the serotypes 1-7, as well as any as yet unidentified serotypes. Such VP6 proteins can be used as immunologic carriers of polypeptides. These carrier molecules comprise amino acid sequences of rotavirus VP6 amino acid sequences which are unique to the class, or any member of the class, of VP6 polypeptides. Such unique sequences of VP6 proteins are referred to as a "rotavirus VP6 inner capsid protein amino acid sequence." VP6 carriers are further disclosed in U.S. Pat. No. 5,071,651, incorporated herein by reference in its entirety.
A carrier that is "substantially homologous to a rotavirus VP6 inner capsid protein or a functional fragment thereof" is one in which at least about 85%, preferably at least about 90%, and most preferably at least about 95%, of the amino acids match over a defined length of the molecule. A "functional fragment" of a rotavirus VP6 inner capsid protein is a fragment with the capability of acting as a carrier molecule for the novel chimeric proteins of the instant invention.
A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo or in vitro; i.e., capable of replication under its own control.
A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
A "double-stranded DNA molecule" refers to the polymeric form of deoxyribonucleotides in a double-stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA).
A DNA "coding sequence" or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic IRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence.
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bound at the 3′ terminus by the translation start codon (ATG) of a coding sequence and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Procaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
DNA "control sequences" refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell.
A coding sequence is "operably linked to" another coding sequence when RNA polymeras will transcribe the two coding sequences into mMA, which is then translated into a chimeric polypeptide encoded by the two coding sequences. The coding sequences need not be contiguous to one another so long as the transcribed sequence is ultimately processed to produce the desired chimeric protein.
A control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.
A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.
A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
Two DNA or polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, vols I & II, supra; Nucleic Acid Hybridization, supra.
The term "functionally equivalent" intends that the amino acid sequence of the subject fusion protein is one that will elicit an immunological response, as defined above, equivalent to an unmodified immunogenic leukotoxin-antigen chimeric protein.
A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a bacterial gene, the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria. Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
A composition containing A is "substantially free of" B when at least about 85% by weight of the total of A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95%, or even 99% by weight.
The term "treatment" as used herein refers to either (i) the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms or the disease of interest (therapy).
B. General Methods
Central to the instant invention is the discovery that leukotoxin polypeptides, when coupled to selected antigens, are able to increase the immunogenicity of the antigen as compared to the immunogenicity of the antigen when presented alone. Thus, leukotoxin polypeptides can act as carrier proteins for the presentation of a desired antigen to the immune system. Accordingly, the chimeric proteins can be formulated into vaccine compositions which provide enhanced immunogenicity to the antigen presented therewith. The fusion of the leukotoxin gene to the selected antigen further functions to facilitate purification of the chimeric protein from cells expressing the same.
The leukotoxin carrier is especially useful for the presentation of small or endogenous peptide antigens, including peptide hormones, and bacterial and viral antigens, which typically elicit poor immune responses when presented without the aid of a carrier. Exemplified herein are leukotoxin chimeras which include leukotoxin fused to small peptide hormones—somatostatin (SRIF) and gonadatropin releasing hormone (GnRH). SRIF-14 has 14 amino acids and GnRH possesses 10 amino acids. The nucleotide sequences of SRIF and GnRH are depicted in FIG. 4(SEQ ID NOS:3-8). Because the sequences are relatively short, they can easily be generated using synthetic techniques, as described further below. Because these hormones are small in size and are endogenous to several mammals such as humans, bovines etc., these substances require the use of carrier proteins in order to elicit an adequate immune response in such mammals. Immunization with these hormones can regulate growth rate, lactation and reproductive efficiency. A detailed discussion of SRIF can be found in U.S. Pat. No. 5,212,156, filed Jun. 18, 1990, which is incorporated herein by reference in its entirety. GnRH is further discussed in U.S. Pat. No. 4,975,420, incorporated herein by reference in its entirety.
Also exemplified herein is a chimera comprised of leukotoxin and bovine rotavirus viral protein 4 (VP4). VP4 (molecular weight 86,719), functions as the viral hamagglutinin and forms the spike-like projections protruding from the surface of the virus. Antibodies capable of neutralizing the virus bind to the tip of the spike. VP4 appears to play a major role in viral attachment during infection. The nucleotide sequence of VP4 is depicted in FIG. 4. For a further discussion of rotavirus infection and VP4, see, Redmond, M. J. et al. in Viral Diseases (Ed. E. Kurstak, Marcel Dekker, New York, 1991, pp. 387-404); and International Publication No. WO/9207941, published May 14, 1992, both incorporated herein by reference in their entirety. Although the invention is described with respect to these particular proteins, leukotoxin polypeptides, or proteins functionally equivalent and substantially homologous thereto, can be easily fused to other antigens, based on the disclosure herein, in order to increase the immunogenicity thereof.
The leukotoxin-antigen complex can be conveniently produced recombinantly as a chimeric protein. The antigen portion of the chimera can be fused either 5′ or 3′ to the leukotoxin portion of the molecule.
Actively growing cells of P. haemolytica have been shown to secrete leukotoxin which can be cloned, the gene encoding the same isolated, and fused with a gene encoding a desired antigen, using techniques well known in the art. The resulting chimeric proteins can be expressed and used to immunize subjects against the particular antigen fused to leukotoxin.
The nucleotide sequence coding for full-length P. haemolytica Al leukotoxin has been determined. See, e.g., Lo, R. Y. C. Infect. Immun. (1987) 55:1987-1996; U.S. Pat. No. 5,055,400, incorporated herein by reference in its entirety. P. haemolytica leukotoxin can be produced using recombinant techniques and purified from, for example, bacterial cells. The leukotoxin can also be purified from native bacteria using immunoadsorbent chromatography.
Similarly, the coding sequences for numerous antigens are known or can be determined. Again, these antigens can be purified using standard techniques.
Purification of the above proteins, using standard techniques including those described herein, permits the sequencing of the same by any of the various methods known to those skilled in the art. For example, the amino acid sequences can be determined by repetitive cycles of Edman degradation, followed by amino acid analysis by HPLC. Other methods of amino acid sequencing are also known in the art. Furthermore, fragments of the proteins can be tested for biological activity and active epitopes used in compositions in lieu of the entire protein.
Once the amino acid sequences are determined, oligonucleotide probes which contain the codons for a portion of the determined amino acid sequences can be prepared and used to screen DNA libraries for genes encoding the subject proteins. The basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloninga: Vol. I, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; T. Maniatis et al., supra.
First, a DNA library is prepared. The library can consist of genomic DNA from P. haemolytica (for the isolation of the leukotoxin gene) or from appropriate cells or viruses (for the isolation of the desired antigen gene). Once the library is constructed, oligonucleotides to probe the library are prepared and used to isolate the gene encoding the desired protein. The oligonucleotides are synthesized by any appropriate method. The particular nucleotide sequences selected are chosen so as to correspond to the codons encoding a known amino acid sequence from the desired protein. Since the genetic code is degenerate, it will often be necessary to synthesize several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences which encode a particular region of the protein. Thus, it is generally preferred in selecting a region upon which to base the probes, that the region not contain amino acids whose codons are highly degenerate. In certain circumstances, one of skill in the art may find it desirable to prepare probes that are fairly long, and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in corresponding nucleic acid sequences, particularly if this lengthy and/or redundant region is highly characteristic of the protein of interest. It may also be desirable to use two probes (or sets of probes), each to different regions of the gene, in a single hybridization experiment. Automated oligonucleotide synthesis has made the preparation of large families of probes relatively straight-forward. While the exact length of the probe employed is not critical, generally it is recognized in the art that probes from about 14 to about 20 base pairs are usually effective. Longer probes of about 25 to about 60 base pairs are also used.
The selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin, using standard procedures. The labeled set of probes is then used in the screening step, which consists of allowing the single-stranded probe to hybridize to isolated ssDNA from the library, according to standard techniques. Either stringent or permissive hybridization conditions could be appropriate, depending upon several factors, such as the length of the probe and whether the probe is derived from the same species as the library, or an evolutionarily close or distant species. The selection of the appropriate conditions is within the skill of the art. See, generally, Nucleic Acid hybridization, supra. The basic requirement is that hybridization conditions be of sufficient stringency so that selective hybridization occurs; i.e., hybridization is due to a sufficient degree of nucleic acid homology (e.g., at least about 75%), as opposed to nonspecific binding. Once a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains a gene for the desired protein.
Alternatively, DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.
Once coding sequences for the desired proteins have been prepared or isolated, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra; T. Maniatis et al., supra; B. Perbal, supra.
Suitable restriction enzymes can then be employed to isolate the appropriate antigen gene or leukotoxin gene and these sequences can be ligated together and cloned to form a leukotoxin-antigen fusion gene.
The fusion gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the chimeric protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. The chimeric proteins of the present invention can be expressed using, for example, native P. haemolytica promoter, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.
In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
An expression vector is constructed so that the particular fusion coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the sequences encoding the particular chimeric protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
In some cases, it may be desirable to add sequences which cause the secretion of the polypeptide from the host organism, with subsequent cleavage of the secretory signal. It may also be desirable to produce mutants or analogs of the chimeric proteins of interest. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., T. Maniatis et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.
A number of procaryotic expression vectors are known in the art. See, e.g., U.S. Pat. Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. See, e.g., U.S. Pat. Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409; 100,561; 96,491.
Depending on the expression system and host selected, the proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The chimeric protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
An alternative method to identify proteins of the present invention is by constructing gene libraries, using the resulting clones to transform E. coli and pooling and screening individual colonies using polyclonal serum or monoclonal antibodies to the desired antigen.
The chimeric proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.
The proteins of the present invention or their fragments can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.
Monoclonal antibodies to the proteins of the present invention, and to the fragments thereof, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monotonal Antibodies (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the antigen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against.
Animals can be immunized with the compositions of the present invention by administration of the chimeric protein, or an active fragment thereof, or an analog thereof. The chimeric protein can consist of leukotoxin fused to an epitope of the desired antigen, as defined above. Thus, if the fragment or analog of the fusion protein is used, it will include the amino acid sequence of leukotoxin, or a fragment of the same which interacts with the immune system to increase the immunogenicity of the antigen or epitope thereof, linked to the antigen of interest.
Prior to immunization, it may be desirable to further increase the immunogenicity of the particular chimeric protein, or an analog of the protein, or particularly fragments of the protein. This can be accomplished in any one of several ways known those of skill in the art. For example, the antigenic peptide may be administered linked to a carrier, in addition to the leukotoxin carrier. For example, a fragment may be conjugated with a macromolecular carrier. Suitable carriers are typically large, slowly metabolized macro-molecules such as: proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
The protein substrates may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.
Other suitable carriers for the chimeric proteins of the present invention include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S. Pat. No. 5,071,651, and incorporated herein by reference. Also useful is a fusion product of a viral protein and the subject leukotoxin-antigen immunogen made by methods disclosed in U.S. Pat. No. 4,722,840. Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state Alternatively, the fusion proteins of the present invention may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.
The novel chimeric proteins of the instant invention can also be administered via a carrier virus which expresses the same. Carrier viruses which will find use with the instant invention include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus. By way of example, vaccinia virus recombinants expressing the novel chimeric proteins can be constructed as follows. The DNA encoding the particular leukotoxin-antigen chimeric protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant chimeric protein into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
It is also possible to immunize a subject with a protein of the present invention, or an immunogenic fragment thereof, or an analog thereof, which is administered alone, or mixed with a pharmaceutically acceptable vehicle or excipient. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The active immunogenic ingredient is often mixed with vehicles containing excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine. Adjuvants may include for example, muranyl dipeptides, avridine, aluminum hydroxide, oils, saponins and other substances known in the art. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the protein adequate to achieve the desired immunized state in the subject being treated.
Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations. For suppositories, the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about10% to about 95% of the active ingredient, preferably about 25% 35 to about 70%.
Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
Controlled or sustained release formulations are made by incorporating the chimeric protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel� copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. The chimeric proteins can also be presented using implanted mini-pumps, well known in the art.
Furthermore, the chimeric proteins (or complexes thereof) may be formulated into vaccine compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
To immunize a subject, th polypeptide of interest, or an immunologically active fragment thereof, is administered parenterally, usually by intramuscular injection in an appropriate vehicle. Other modes of administration, however, such as subcutaneous, intravenous injection and intranasal delivery, are also acceptable. Injectable vaccine formulations will contain an effective amount of the active ingredient in a vehicle, the exact amount being readily determined by one skilled in the art. The active ingredient may typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. With the present vaccine formulations, approximately 1 μg to 1 μg, more generally 5 μg to 100 μg of antigen per ml of injected solution, should be adequate to raise an immunological response when administered. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. The subject is immunized by administration of the particular antigen or fragment thereof, or analog thereof, in at least one dose, and preferably two doses. Moreover, the animal may be administered as many doses as is required to maintain a state of immunity.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
A deposit of biologically pure cultures of th following strains was made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Access to said cultures will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application. Moreover, the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.
These deposits are provided merely as a convenience to those of skill in the art, and are not an admission that a deposit is required under 35 USC �112. The nucleic acid sequences of these plasmids, as well as the amino sequences of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description herein. A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted.
Deposit Date
ATCC No.
P. haemolytica serotype 1 B122
Feb. 1, 1989
pAA352 in E. coli W1485
Mar. 30, 1990
C. Experimental
Enzymes were purchased from commercial sources, and used according to the manufacturers' directions. Radionucleotides and nitrocellulose filters were also purchased from commercial sources.
In the cloning of DNA fragments, except where noted, all DNA manipulations were done according to standard procedures. See Sambrook et al., supra. Restriction enzymes, T4 DNA ligase, E. coli, DNA polymerase I, Klenow fragment, and other biological reagents were purchased from commercial suppliers and used according to the manufacturers' directions. Double-stranded DNA fragments were separated on agarose gels.
cDNA and genomic libraries were prepared by standard techniques in pUC13 and the bacteriophage lambda gt11, respectively. See DNA CLONING: Vols I and II, supra.
P. haemolytica biotype A, serotype 1 ("A1") strain B122 was isolated from the lung of a calf which died of pneumonic pasteurellosis and was stored at −70� C. in defibrinated blood. Routine propagation was carried out on blood agar plates or in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) supplemented with 5% (v/v) horse serum (Gibco Canada Ltd., Burlington, Canada). All cultures were incubated at 37� C.
To isolate the leukotoxin gene, gene libraries of P. haemolytica A1 (strain B122) were constructed using standard techniques. See, Lo et al., Infect. Immun., supra; DNA CLONING: Vols. I and II, supra; and T. MANIATIS et al., supra. A genomic library was constructed in the plasmid vector pUC13 and a DNA library constructed in the bacteriophage lambda gt11. The resulting clones were used to transform E. coli and individual colonies were pooled and screened for reaction with serum from a calf which had survived a P. haemolytica infection and that had been boosted with a concentrated culture supernatant of P. haemolytica to increase anti-leukotoxin antibody levels. Positive colonies were screened for their ability to produce leukotoxin by incubating cell lysates with bovine neutrophils and subsequently measuring release of lactate dehydrogenase from the latter.
Several positive colonies were identified and these recombinants were analyzed by restriction endonuclease mapping. One clone appeared to be identical to a leukotoxin gene cloned previously. See, Lo et al., Infect. Immun., supra. To confirm this, smaller fragments were recloned and the restriction maps compared. It was determined that approximately 4 kilobase pairs of DNA had been cloned. Progressively larger clones were isolated by carrying out a chromosome walk (5′ to 3′ direction) in order to isolate full-length recombinants which were approximately 8 kb in length. The final construct was termed pAA114. This construct contained the entire leukotoxin gene sequence. The structure of this plasmid is shown in FIG. 1.
lktA, a MaeI restriction endonuclease fragment from pAA114 which contained the entire leukotoxin gene, was treated with the Klenow fragment of DNA polymerase I plus nucleotide triphosphates and ligated into the Smar site of the cloning vector pUC13. This plasmid was named pAA179. From this, two expression constructs where made in the ptac-based vector pGH432: lac1 digested with SmaI. One, pAA342, consisted of the 5′-AhaIII fragment of the lktA gene while the other, pAA345, contained the entire MaeI fragment described above. The clone pAA342 expressed a truncated leukotoxin peptide at high levels while pAA345 expressed full length leukotoxin at very low levels. Therefore, the 3′ end of the lktA gene (StyI BamHI fragment from pAA345) was ligated to Styl BanHI-digested pAA342, yielding the plasmid pAA352. The structure of pAA352 is shown in FIG. 2 and the nucleotide sequence and predicted amino acid sequence of P. haemloytica leukotoxin shown in FIGS. 3A through 3J(SEQ ID NOS:1and 2).
Three representative LXT-antigen fusions were constructed as follows. oligonucleotides containing sequences from the bovine rotavirus VP4, GnRH and SRIF genes were constructed on a Pharmacia Gene Assembler using standard phosphoramidite chemistry. The sequences of these oligonucleotides are shown in FIG. 4 SEQ ID NOS:3-8. The oligonucleotides were annealed and ligated into the vector pAA352 (ATCC No. 68283, and described above), which had been digested with the restriction endonuclease BamH1. This vector contains the P. haemloytica leukotoxin gene. The ligated DNA was used to transform E. coli strain JM105 (in the case of SRIF) or MH3000 (for VP4 and GnRH). Transformants containing the oligonucleotide inserts were identified by restriction endonuclease mapping. Plasmid DNA from the E. coli MH3000 strains was then isolated and used to transform the strain JM105. The recombinant plasmids were designated pAA496 (LKT-SRIF, FIG. 5), pAA502 (LKT-GnRH, FIG. 7), and pAA501 (LKT-VP4, FIG. 9). The nucleotide sequences of these three fusions are shown in FIGS. 6, 8 and 10, respectively.
The recombinant LKT-antigen fusions from Example 2 were purified using the following procedure. For each fusion, five to ten colonies of the transformed E. coli strains were inoculated into 10 ml of TB broth supplemented with 100 micrograms/ml of ampicillin and incubated at 37� C. for 6 hours on a G10 shaker, 220 rpm. Pour ml of this culture was diluted into each of two baffled Fernbach flasks containing 400 ml of TB broth+ampicillin and incubated overnight as described above. Cells were harvested by centrifugation for 10 minutes at 4,000 rpm in polypropylene bottles, 500 ml volume, using a Sorvall GS3 rotor. The pellet was resuspended in an equal volume of TB broth containing ampicillin which had been prewarmed to 37� C. (i.e., 2�400 ml), and the cells were incubated for 2 hours as described above.
3.2 ml of isopropyl-B,D-thiogalactopyranoside (IPTG, Gibco/BRL), 500 mM in water (final concentration=4 mM), was added to each culture in order to induce synthesis of the recombinant fusion proteins. Cultures were incubated for two hours. Cells were harvested by centrifugation as described above, resuspended in 30 ml of 50 mM Tris-hydrochloride, 25% (w/v) sucrose, pH 8.0, and frozen at −70� C. The frozen cells were thawed at room temperature after 60 minutes at −70� C., and 5 ml of lysozyme (Sigma, 20 mg/ml in 250 mM Tris-HCl, pH 8.0) was added. The mixture was vortexed at high speed for 10 seconds and then placed on ice for 15 minutes. The cells were then added to 500 ml of lysis buffer in a 1000 ml beaker and mixed by stirring with a 2 ml pipette. The beaker containing the lysed cell suspension was placed on ice and sonicated for a total of 2.5 minutes (5-30 second bursts with 1 minute cooling between each) with a Braun sonicator, large probe, set at 100 watts power. Equal volumes of the solution were placed in Teflon SS34 centrifuge tubes and centrifuged for 20 minutes. at 10,000 rpm in a Sorvall SS34 rotor. The pellets were resuspended in a total of 100 ml of sterile double distilled water by vortexing at high speed, and the centrifugation step repeated. Supernatants were discarded and the pellets combined in 20 ml of 10 mM Tris-HCl, 150 MN NaCl, pH 8.0 (Tris-buffered saline) and the suspension frozen overnight at −20� C.
The recombinant suspension was thawed at room temperature and added to 100 ml of 8 M Guanidine HCl (Sigma) in Tris-buffered saline and mixed vigorously. A magnetic stir bar was placed in the bottle and the solubilized sample was mixed at room temperature for 30 minutes. The solution was transferred to a 2000 ml Ehrlenmyer flask and 1200 ml of Tris-buffered saline was quickly added. This mixture was stirred at room temperature for an additional 2 hours. 500 ml aliquots were placed in dialysis bags (Spectrum, 63.7 mm diameter, 6,000-8,000 MW cutoff, #132670, from Fisher scientific) and these were placed in 4,000 ml beakers containing 3,500 ml of Tris-buffered saline+0.5 M Guanidine HCl The beakers were placed in a 40� C. room on a magnetic stirrer overnight after which dialysis buffer was replaced with Tris-buffered saline+0.1 M Guanidine HCl and dialysis continued for 12 hours. The buffer was then replaced with Tris-buffered saline+0.05 M Guanidine HCl and dialysis continued overnight. The buffer was replaced with Tris-buffered saline (no guanidine), and dialysis continued for 12 hours. This was repeated three more times. The final solution was poured into a 2000 ml plastic roller bottle (Corning) and 13 ml of 100 mM PMSF (in ethanol) was added to inhibit protease activity. The solution was stored at −200� C. in 100 ml aliquots.
To confirm that the fusion proteins had been isolated, aliquots of each preparation were diluted 20-fold in double distilled water, mixed with an equal volume of SDS-PAGE sample buffer, placed in a boiling water bath for five minutes and run through 12% polyacrylamide gels. Recombinant leukotoxin controls were also run. Western blots of the purification products were performed by reacting the LKT-SRIF preparation with swine anti-SRIF serum at a 1:500 dilution and the LKT-GnRH and LXT-VP4 preparations with mouse anti-VP4 serum at a 1:50 dilution. The only band visible in the LKT-SRIF western blot was that associated with the LKT-SRIF protein sample. No cross-reactivity with the leukotoxin was observed. Both the LKT-GnRH and LKT-VP4 proteins had similar apparent molecular weights, however, the anti-VP4 serum reacted only with the LKT-VP4 a fusion protein.
All fusion proteins were expressed at high levels as inclusion bodies. The predicted molecular weights based on the DNA sequences of the three proteins (depicted in FIGS. 6, 8 and 10) were 101,366 (LKT-SRIF); 100,521 (LKT-GnRH); and 102,120 (LKT-VP4). The molecular weight of the recombinant leukotoxin molecule was 99,338. Both the SRIF and VP4 fusions were shown to react with monospecific antisera against the corresponding peptide.
To test for enhanced immunogenicity of the LXT-antigen fusions as compared to the antigens alone, LKT-SRIF fusion protein was purified from E. coli cultures induced with IPTG, as described in Example 2. Aggregated protein was dissolved by treating with guanidine-HCl at a final concentration of three molar. The leukotoxin concentration of this material was assayed using a standard quantitative leukotoxin specific ELISA. The assay utilized recombinant leukotoxin in 4 N guanidine-HCl (2 mg/ml) as a standard. Rabbit anti-leukotoxin antiserum was used as a detection and quantitation system.
A vaccine was formulated to a volume of 1 ml by mixing equal volumes of LKT-SRIF, diluted in Hanks Buffered Saline, and Emulsigen Plus (MVP Laboratories, Ralston, Nebr.). Four three month old lambs were immunized twice with 100 micrograms of fusion protein (containing an equivalent of approximately 1.4 micrograms of SRIF peptide). Blood samples were taken 10 days after the second injection and were analyzed for leukotoxin and SRIF specific antisera. All of the animals were found to have anti-leukotoxin titers of greater than 1 in 50,000, as determined by a leukotoxin specific ELISA. SRIF titers were assayed by a radioimunoassay as described in Laarveld, B., et al., Can. J. Anim. Sci. (1986) 66:77-83. Two animals were found to have titers greater than 1 in 100.
To further test the ability of the LKT-SRIF chimeras to induce an anti-SRIF immunological response in vivo, and to compare this response to that produced by other SRIF conjugates, the following vaccination trial was performed. Three groups of 8 female pigs, approximately 8 weeks of age (35-50 kg) were used which were Specific Pathogen Free. The animals were maintained in a minimal disease facility and were vaccinated on days 0, 21 and 35 of the trial with the following formulations:
Group 1—placebo which was saline formulated in Emulsigen Plus adjuvant containing 15 mg DDA (Kodak) (2 ml);
Group 2—LKT-SRIF (250 μg LKT, prepared as described above) formulated in th same adjuvant (2 ml);
Group 3—SRIF-avidin, biotinylated SRIF (10 μg) and 2.5 μg avidin, formulated in the same adjuvant (2 ml).
Blood samples were taken on days 0, 21 and 35, allowed to clot, centrifuged at 1500 g, and the serum removed. The serum antibody titers against SRIF were measured using the RIA procedure of Laarveld et al., Can. J. Anim. Sci. (1986) 6:77-83.
7 of the 8 animals immunized with the LKT-SRIF formulation produced significant titers against SRIF (>1:700) whereas only 2 of 8 animals immunized with the SRIF-Avidin responded with serum titers of >700.
This example demonstrates that leukotoxin chimeric molecules are highly immunogenic. It has been reported by Laarveld, et al., Can. J. Animal Sci. (1986) 66:77, that repeated immunization with greater than 100 micrograms of SRIF peptide conjugated to an ovalbumin carrier was necessary to evoke an immune reaction.
To test for the ability of LKT-GNRH to induce an anti GnRH immunological response in vivo, and to compare this response to other GnRH carrier conjugates, the following vaccination trial was performed. Three groups of 8 male pigs, approximately 8 weeks of age (35-50 kg) were used which were Specific Pathogen Free. The animals were maintained in a minimal disease facility and were vaccinated on days 0 and 21 of the trial with the following formulations:
Group 1—placebo which consisted of saline formulated in Emulsigen Plus adjuvant containing 15 mg of DDA (2 ml);
Group 2—LKT-GnRH (250 μg LXT, prepared as described in the previous examples) formulated in the same adjuvant (2 ml);
Group 3—VP6-GnRH, 0.5 μg VP6 and 5 μg GnRH, formulated in the same adjuvant (2 ml). The VP6 preparation was made as described in U.S. Pat. No. 5,071,651, using the binding peptide described therein.
Blood samples were taken on days 0, 21 and 35, allowed to clot, centrifuged at 1500 g, and the serum removed. The serum antibody titers against GnRH were measured using the RIA procedure of Silversides et al., J. Reprod. Immunol. (1985) 7:171-184.
The results of this trial indicated that only those animals immunized with the LKT-GnRH formulation produced significant titers against GnRH (titers >1:70). Neither the placebo nor the VP6-GnRH groups produced anti-GnRH titers. Previously, multiple vaccinations with doses of GnRH of more than 100 μg, conjugated to other carrier proteins, were required to induce anti-hormone titers.
Thus, chimeric proteins including leukotoxin fused to a selected antigen, have been disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.
|Cited Patent
|Filing date
|Publication date
|Sep 19, 1985
|Feb 2, 1988
|Chiron Corporation
|Hybrid particle immunogens
|Sep 13, 1984
|Sep 19, 1989
|Cytogen Corporation
|Antibody-therapeutic agent conjugates
|Aug 13, 1987
|Sep 18, 1990
|Board Of Regents, The University Of Texas System
|Pharmaceutical compositions of a 105 kD P. Haemolytica derived antigen useful for treatment of Shipping Fever
|Sep 30, 1987
|Dec 4, 1990
|University Of Saskatchewan
|Agents and procedures for provoking an immune response to GnRH and immuno sterilizing mammals
|Jun 29, 1988
|Jul 2, 1991
|Immunex Corporation
|Immunogenic conjugates comprising leukotoxin peptide fragments
|Nov 26, 1986
|Oct 8, 1991
|University Of Guelph
|Leukotoxin gene of pasteurella haemolytica
|Aug 22, 1990
|Aug 24, 1993
|Veterinary Infectious Disease Organization
|Interleukin-2-leukotoxin gene fusions and uses thereof
|Oct 16, 1991
|Dec 28, 1993
|University Of Saskatchewan
|Gamma-iterferon-leukotoxin gene fusions and uses thereof
|Oct 14, 1992
|Jun 6, 1995
|University Of Saskatchewan
|Enhanced immunogenicity using leukotoxin chimeras
|Feb 9, 1993
|Dec 19, 1995
|University Of Saskatchewan
|Pasteurella haemolytica leukotoxin compositions and uses thereof
|Dec 20, 1993
|Jan 14, 1997
|University Of Saskatchewan
|Chimeric protein comprising an RTX-family cytotoxin and interferon-2 or interferon
|May 31, 1995
|Jan 13, 1998
|University Of Saskatchewan
|Enhanced immunogenicity using leukotoxin chimeras
|Feb 10, 1995
|Mar 3, 1998
|University Of Saskatchewan
|GnRH-leukotoxin chimeras
|Aug 9, 1996
|Nov 17, 1998
|University Of Saskatchewan
|GnRH-leukotoxin chimeras
|Jun 19, 1997
|Oct 19, 1999
|University Of Saskatchewan
|GNRH-leukotoxin chimeras
|Jul 29, 1998
|Feb 8, 2000
|University Of Saskatchewan
|GnRH-leukotoxin chimeras
|Aug 26, 1999
|Feb 18, 2003
|University Of Saskatchewan
|Polynucleotides encoding LKT 111
|Mar 9, 1990
|Sep 20, 1990
|Praxis Biologics, Inc.
|Vaccines for nontypable haemophilus influenzae
|May 25, 1990
|Oct 17, 1991
|University Of Saskatchewan
|Compositions and treatments for pneumonia in animals
|Aug 22, 1991
|Mar 5, 1992
|Andrew Potter
|Interleukin-2-leukotoxin gene fusions and uses thereof
|Adams et al., J. Anim. Sci. (1990) 68:1691-1698.
|Adams et al., J. Anim. Sci. (1990) 68:2793-2802.
|Bittle et al., Nature (1982) 298:30-33.
|Bruggemann et al., Bio Techniques (1991) 10(2):202-209.
|Burke et al., Nature (1988) 332:81-82.
|Clarke et al., Vaccines 88 Ginsberg, H., et al., Eds., (1988) pp. 127-131.
|Delpeyroux et al., Science (1986) 233:472-475.
|Forestier et al., Infection and Immunity (1991) 59(11):4212-4220.
|Gentry et al., Vet. Immunology and Immunopathology (1985) 9:239-250.
|Haynes et al., Bio/Technology (1986) 4:637-641.
|Hughes et al., Infection and Immunity (1992) 10(2):545-570.
|Jeffcoate et al., Theriogenology (1982) 18:65-77.
|Kingsman et al., Vaccine (1988) 6:304-306.
|Lo et al., Infect. Immun. (1985) 50(3):667-671.
|Lowell et al., Science (1988) 240:800-802.
|Morein et al., Nature (1984) 308:457-460.
|Muller et al., Proc. Natl. Acad. Sci. (1982) 79:569-573.
|Neurath et al., Mol. Immunol. (1989) 26(1):53-62.
|Phalipon et al., Gene (1987) 55:255-263.
|Que et al., Infection and Immunity (1988) 56(10):2645-2649.
|Redmond et al., Mol. Immunol. (1991) 28(3):269-278.
|Sad et al., Immunology (1991) 74:223-227.
|Schutze et al., J. Immunol. (1985) 135(4):2319-2322.
|Strathde et al., Infect. Immun. (1987) 55(12):3233-3236.
|Valenzuela et al., Bio/Technology (1985) 3:323-326.
|Citing Patent
|Filing date
|Publication date
|Jun 19, 2001
|May 22, 2007
|The Regents Of The University Of California
|Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of Moraxella bovis infections
|Oct 6, 2006
|Sep 14, 2010
|The Board Of Regents For Oklahoma State University
|Mannheimia haemolytica chimeric outer membrane protein PlpE and leukotoxin epitopes as a vaccine or vaccine component against shipping fever
|Apr 6, 2010
|May 27, 2014
|University Of Saskatchewan
|Methods and compositions for treating and preventing Shiga toxin-producing Escherichia coli infection
|Oct 6, 2006
|Jun 14, 2007
|Sahlu Ayalew
|Mannheimia haemolytica chimeric outer membrane protein PIpE and leukotoxin epitopes as a vaccine or vaccine component against shipping fever
|Mar 16, 2007
|Nov 22, 2007
|The Regents Of The University Of California
|Moraxella bovis cytotoxin, cytotoxin gene, antibodies and vaccines for prevention and treatment of moraxella bovis infections
|U.S. Classification
|424/192.1, 424/193.1, 424/241.1, 424/255.1, 424/195.11, 424/236.1, 424/185.1, 530/351, 530/350
|International Classification
|Cooperative Classification
|European Classification
|Apr 4, 2008
|Surcharge for late payment
|Apr 4, 2008
|Fee payment
Year of fee payment: 4
|Apr 7, 2008
|Maintenance fee reminder mailed
|May 14, 2012
|Maintenance fee reminder mailed
|Sep 28, 2012
|Lapse for failure to pay maintenance fees
|Nov 20, 2012
|Expired due to failure to pay maintenance fee
Effective date: 20120928

subset: commoncrawl


text:
Movies Home / Entertainment Channel / Bullz-Eye Home
12 – 20. Jose Canseco, Mike Scioscia, Ozzie Smith, Don Mattingly, Steve Sax, Roger Clemens, Wade Boggs, Ken Griffey, Jr., and Darryl Strawberry
EPISODE: "Homer at the Bat" (Season 3)
EXCUSE FOR APPEARANCE: After Homer's inspirational performance with his homemade bat, Mr. Burns make a million-dollar bet that the nuclear power plant's softball team can beat the softball team for the Shelbyville Nuclear Power Plant…but having no faith in his employees, Burns hires a team of ringers.
|Jose Canseco: I get $50,000 to play one game?
Smithers: That's right, Mr. Canseco.
Jose Canseco: Well, it's a pay cut, but what the hey. It sounds like fun.
|Dr. Hibbert: Uh, Mike, try to lift your arm.
Mike Scioscia: Can't…lift…arm…or…speak…at…normal…rate.
Dr. Hibbert : Well, I'm afraid you have a case of acute radiation poisoning, Mr. Scioscia.
Mike Scioscia: Will…I…be…able…to…play…softball… tomorrow?
Dr. Hibbert: (Laughs) No, by tomorrow, you'll barely be able to breathe.
Mike Scioscia: Oh…man!
|Ozzie Smith: How long does it take to see this thing? I'm kind of in a hurry.
Mystery Spot Owner: Well, it's hard to say, my friend. Once you go in, you may never come out.
Ozzie Smith: Wow! One, please! (Falls down an endless hole) Ahhhhhhhhhh…!
|Smithers: Hello, are you Don Mattingly?
Don Mattingly: (wearing rubber gloves, sporting a dish towel over his shoulder, and holding a plate) Yes! Yes, I am! Come in, come in! Can I get you something? What's your name?
|Officer Lou: (during a traffic stop) I heard some guy got killed in New York City and they never solved the case. But you wouldn't know anything about that now, would you, Steve?
Steve Sax: But there are hundreds of unsolved murders in New York City.
Officer Lou: You don't know when to keep your mouth shut, do you, Saxxy Boy?
|Homer: Please, please, please, I want to make the team. Clemens, did I make the team?
Roger Clemens: You sure did!
Homer: I did? Woo-hoo! Woo-hoo! In your face, Strawberry!
Roger Clemens: Wait a minute, are you Ken Griffey, Jr.?
Homer: No.
Roger Clemens: Sorry. Didn't mean to get your hopes up.
|Barney: And I say, England's greatest Prime Minister was Lord Palmerston!
Wade Boggs: Pitt the Elder!
Barney: Lord Palmerston!
Wade Boggs: Pitt the Elder! (Pokes Barney)
Barney: Okay, you asked for it, Boggs! (Punches out Wade Boggs)
|Ken Griffey, Jr.: Wow! It's like there's a party in my mouth, and everyone's invited!
|Homer: You're Darryl Strawberry.
Darryl Strawberry: Yes?
Homer: You play right field.
Darryl Strawberry: Yes?
Homer: I play right field, too.
Darryl Strawberry: So?
Homer: Well, are you better than me?
Darryl Strawberry: Well, I never met you, but...yes.
21. Beverly D'Angelo
CHARACTER: Lurleen Lumpkin, a waitress turned country singer whose career Homer decides to manage.
EPISODE: "Colonel Homer" (Season 3)
|Homer: I can't get your song out of my mind. I haven't felt this way since "Funkytown!" Can I get a copy?
Lurleen: (Tapping her head) Sorry, Homer. All my songs are up here. "I'm Basting a Turkey with My Tears," "Don't Look Up my Dress Unless You Mean It," "I'm Sick of Your Lying Lips and False Teeth"…
22. Spinal Tap
EPISODE: "The Otto Show" (Season 3)
EXCUSE FOR APPEARANCE: The band's world tour brings them to London, Paris, Munich…and Springfield.
|David St. Hubbins: Well, when the Berlin Wall fell, our records started selling on the dismal side of the Iron Curtain, so, naturally, that gave us a boost.
Nigel Tufnel: We're very big in Bulgaria. And...what's his name? The other "Garia."
St. Hubbins: Hungaria.
Tufnel: Yeah, whatever.
Derek Smalls: I can't think of anyone who's benefited more from the fall of Communism than us.
Tufnel: Well, maybe people who actually lived in the Communist countries.
Smalls: Oh, yeah. Hadn't thought about that. I'll bet you're right.
23. Bob Hope
EPISODE: "Lisa the Beauty Queen" (Season 4)
EXCUSE FOR APPEARANCE: After winning the Little Miss Springfield pageant, Lisa ends up as part of a USO appearance at Fort Springfield, where she's introduced by Hope.
|Bob Hope: (Backstage) What's the mayor's name?
Bob's Manager: Quimby.
Bob Hope: Beautiful. (Walks onstage) Hello, this is Bob "What The Hell Am I Doing In Springfield?" Hope. Hey, how 'bout that Mayor Quimby? He's some golfer. His golf ball spends more time underwater than Greg Louganis. And, now, I wanna show you what you're fighting for… (Pauses) …if there was a war on: Little Miss Springfield! Isn't she beautiful?
24. Tom Jones
EPISODE: "Marge Gets a Job" (Season 4)
EXCUSE FOR APPEARANCE: After Marge gets a job at the nuclear power plant, Mr. Burns falls in love with her and wants to impress her, so he gets Tom Jones to serenade her…after a little coercion.
|Tom Jones: (To Marge, while manacled to a stage) Get help, love. Call INTERPOL, get me a hacksaw…anything!
25. Sara Gilbert
EPISODE: "New Kid on the Block" (Season 4)
CHARACTER: Laura Powers, the teenage girl who moves next door to the Simpsons…and steals Bart's heart.
|Laura Powers: (To Bart) You know, if you were only old enough to grow a bad teen-aged moustache, I'd go out with you in a second.
26. Adam West
EPISODE: "Mr. Plow" (Season 4)
EXCUSE FOR APPEARANCE: After wrecking his car during a snow storm, Homer and the kids attend a car show, where the original Batmobile from the '60s is on display…along with its original driver.
|Lisa: Dad, that's not the real Batman.
Adam West: Of course I'm Batman. See? (Pulls out a photo) Here's a picture of me with Robin.
Bart: Who the hell's Robin?
Adam West: (Stiffens) Oh. I guess you're only familiar with the new Batman movies…? Michelle Pfeiffer. Ha! The only true Catwoman is Julie Newmar, Lee Merriwether, or Eartha Kitt. And I didn't need a molded plastic suit to improve my physique. (Taps chest) Pure, West.
27. Linda Ronstadt
EPISODE: "Mr. Plow" (Season 4)
EXCUSE FOR APPEARANCE: After beating Homer at the snowplow game, Barney makes a TV commercial in his new identity as The Plow King and brings in a guest to sing the advertising jingle.
|Linda Ronstadt: Barney, I'd like to put a Spanish version of your jingle on my next album. (Sings) Senor Plow no es macho / Es solamente un boracho....
Barney: Yeah, you do that.
28. Leonard Nimoy
EPISODE: "Marge vs. the Monorail" (Season 4)
EXCUSE FOR APPEARANCE: When Springfield launches the inaugural (and, as it turns out, final) run of its new monorail, the guest of honor is none other than Mr. Spock himself.
|Mayor Quimby: And now, I'd like to turn things over to our Grand Marshall, Mr. Leonard Nimoy.
Leonard Nimoy: I'd say this vessel could do at least Warp Five. (Audience laughs)
Mayor Quimby: And let me say, "May the Force be with you!"
Leonard Nimoy: (Annoyed) Do you even know who I am?
Mayor Quimby: (Uncertainly) I think I do. Weren't you one of the Little Rascals?
29. Barry White
EPISODE: "Whacking Day" (Season 4)
EXCUSE FOR APPEARANCE: When the town of Springfield gathers for its annual tradition of Whacking Day, Barry White is invited as the Grand Marshall…and is horrified to discover that the event involves pummeling snakes with clubs.
|Bart: You like snakes, don't you, Barry White?
Barry White: I love the sexy slither of a lady snake. Ohhhhhhhh, baby….
30 – 35. Johnny Carson, Bette Midler, Hugh Hefner, Red Hot Chili Peppers (Anthony Kiedis, Chad Smith, Flea, Arik Marshall), Luke Perry and
Elizabeth Taylor
EPISODE: "Krusty Gets Kancelled" (Season 4)
EXCUSE FOR APPEARANCE: When Krusty the Klown's show gets cancelled, he launches a comeback, with the help of as many superstars as he can wrangle.
|Johnny Carson: Krusty, wanna stay for dinner?
Krusty: Nah, sorry.
Johnny Carson: Ah, that's too bad, 'cause tonight my guests will be Dr. Carl Sagan and, from the San Diego Zoo, Joan Embry.
|Lisa: Miss Midler, I can't believe you do this all by yourself.
Bette Midler: We're Americans. We deserve clean highways. (A truck drives by, and the driver – Snake, Springfield's favorite lowlife scum – throws a can out of the window) You pigs! (Begins running after the truck)
Snake: (Looks in rear view mirror) Oh, no! Bette Midler!
Bette Midler: (Leaps at the truck, hangs onto side mirror, throws can through window, causing Snake to crash his truck, then walks back to Lisa and Bart) Now…where were we?
|Hugh Hefner: A lot of people know about the Grotto and the Game Room…
Bart: Of course.
Hugh Hefner: …but few know about the laboratory, the biosphere the alternative energy research center.
Bart: Fascinating. (Looks out window) De-salinization plant…?
Hugh Hefner: Yes. The Grotto uses so much water, the bunnies felt we should go this way.
Bart: Smart bunnies, Hef. I can call you Hef, can't I?
Hugh Hefner: No.
|Krusty: Now boys, the network has a problem with some of your lyrics. Do you mind changing them for the show?
Anthony Kiedis: Forget you, clown!
Chad Smith: Yeah, our lyrics are like our children, man. No way!
Krusty: Well okay, but here where it says, "What I got you gotta get and put it in ya," how about just, "What I'd like is I'd like to hug and kiss ya"?
Flea: Wow! That's much better!
Arik Marshall: Everyone can enjoy that!
|Luke Perry: (after being shot out of a cannon, into the Quik-E-Mart, and through Apu's display of half-price acid) Ahhhhhhh! My face! My valuable face!
|Elizabeth Taylor: (After passing on her opportunity to be on Krusty's comeback special, then seeing Hugh Hefner playing Prokofiev on wine glasses) I've got to fire that agent.
36. George Harrison
EPISODE: "Homer's Barbershop Quartet" (Season 5)
EXCUSE FOR APPEARANCE: Homer forms a barbershop quartet – called The Be Sharps – that becomes world-famous, resulting in an encounter with the former Beatle.
|George Harrison: (after spotting The Be Sharps offering their farewell performance on a rooftop) It's been done.
37. The Ramones (Joey Ramone, Johnny Ramone, C.J. Ramone and
Marky Ramone)
EPISODE: "Rosebud" (Season 5)
EXCUSE FOR APPEARANCE: The band is inexplicably booked to play Mr. Burns' birthday party.
|Smithers: Here are several fine young men who I'm sure are gonna go far. Ladies and gentlemen, the Ramones!
Mr. Burns: Ah, these minstrels will soothe my jangled nerves.
Joey Ramone: I'd just like to say this gig sucks!
Johnny Ramone: Hey, up yours, Springfield!
Joey Ramone: One, two, three, four! (Singing) Happy Birthday to you! / Happy Birthday to you! / Happy Birthday, Burnsey / Happy Birthday to you!
C.J. Ramone: Go to hell, you old bastard. (Curtain falls)
Marky Ramone: Hey, I think they liked us!
Burns: Have the Rolling Stones killed.
Smithers: Sir, those aren't --
Burns: Do as I say!
38. Ernest Borgnine
EPISODE: "Boy Scoutz 'N The Hood" (Season 5)
EXCUSE FOR APPEARANCE: Bart accidentally joins the Junior Campers while on a sugar high, and when one of the Campers' dads can't go on the upcoming rafting trip, troupe leader Ned Flanders arranges for him to have a special celebrity dad.
|Ernest Borgnine: (Watching Homer and Flanders' raft shoot off in a different direction) Hey, where are the sissy and the bald guy going?
39. Michelle Pfieffer
CHARACTER: Mindy Simmons, the nuclear power plant's new "token" female employee with whom Homer becomes instantly smitten.
EPISODE: "The Last Temptation of Homer" (Season 5)
|Homer: Uh...so, let's have a conversation. Um...I think we'll find we have very little in common.
Mindy Simmons: Can't talk. Eating.
Homer: Hey, my favorite -- raspberry swirl with a double glaze.
Mindy Simmons: Double glaze... (Starts drooling)
Homer: D'oh! Okay, so we have one thing in common. But you know what I hate? Drinking beer and watching TV.
Mindy Simmons: Oh, not me. That's my idea of heaven.
Homer: D'oh! Me too.
Mindy Simmons: Really? I can see I'm gonna love working with you. Well, gotta go. (Whispers) I wanna sneak in a quick nap before lunch. (Walks off)
Homer: Foul temptress. I'll bet she thinks Ziggy's gotten too preachy, too!
40. Werner Klemperer
CHARACTER: Homer's guardian angel, who takes the form of someone that Homer recognizes and reveres: Colonel Klink, from "Hogan's Heroes."
EPISODE: "The Last Temptation of Homer" (Season 5)
|Homer: Colonel Klink! Did you ever get my letters?
Guardian Angel: I'm not actually Colonel Klink; I'm just assuming his form.
Homer: Hee, hee, hee! Did you know that Hogan had tunnels all over your camp? Ha, ha, ha!
Guardian Angel: (In the same annoyed voice Colonel Klink used to take with Hogan) Homerrrrrrr!!! That's not why I'm here!


text:
- Description
Quick Overview
Every day your skin comes in contact with dirt, pollution, wind, sun & sweat. Inspite of these setbacks, you can still have the softest, most gentlest skin. Natural nutrients gently & effectively lightens dark spots for a more youthful & even toned radiant skin. The rich lather rinses completely leaving no residue, while orange peel powder deep cleanses to remove dirt, bacteria and oil & exfoliates dead skin cells for blemish free dear skin, Works wonders on troubled teenage & oily skin. Glycerin has humectants properties, attracting moisture to your skin making it softer & smoother, one of the reasons that our soaps are so beautiful on the skin. This soap does not use any animal produce like milk or honey.
Orange essential oil: It has antiseptic & anti-inflammatory benefits that help in acne & other skin problems. When you use honey on skin it will retain moisture needed for fresh healthy skin plus replace the moisture if needed. Provides support in stressful situations & helps promote mental concentration. Encourages dry, mature skin to produce its own oil & also treats acne for those with dry skin.
Glycerine: Glycerin is a colorless, odorless and viscous organic compound. It is a neutral substance which has a high boiling point and freezes to form a thick gummy paste. Glycerin is a good solvent as it dissolves a lot of things easier than water or alcohol. It absorbs moisture from the air. It has soothing effect on your skin when diluted with water and is also known to soften the skin. This is the reason why it is so widely used many soaps.
How to use product
Our soap is made with vegetable oils of olive, castor, coconut & palm oil. During the natural saponification of these oils, soap is produced. Glycerin is a natural byproduct of our soap making & has humectant properties, attracting moisture to your skin making it softer & smoother, one of the reasons that our soap is so beautiful on the Normal skin. this Soap does not use any animal produce like milk or honey.
Explore more from our Herbal/Ayurvedic Soap collection here
Expiry Date : Click Here January 2018
All our products are far from expiry, and procured directly from the brand/authorized resellers of the brand.
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text: I am looking at getting a full JDM STi interior for my car and assumed that the door cards would fit without issue. However someone is telling me they dont think that they will, and now I just want to double check that. Did some searching around but couldnt find anything concrete. Does anyone know one way or another if they will or wont work?


text:
The City of Perry will hold a "sneak peek" of Rosehead Park's Grand Pavilion for the community Friday, April 5, from 6-7 p.m.
The free event will feature music from Ernie Evans and the Florida State Bluegrass Band, one of the headliners from the 11th annual Florida State Bluegrass Festival.
Subscribe to our e-Edition and read the rest of the story. Already a subscriber? Click here to sign in.


text:
Physical Science
posted by Rachel .
A 0.6-g peanut is burned beneath 50 g of water, which increases in temperature from 22degreesC to 50 degreesc. What is the exact food value of the peanut? (Assuming a 40% efficiency)
heat to water = mass x specific heat x delta T.
mass of water = 50 g in the problem.
specific heat water = 1 calorie/gram*C
delta T = 50-22 = 28 degrees.
Calculate heat produced by the peanut to heat the water. Let's call that value x.
If the process was only 40% efficient, then
x/0.40 = all of the heat produced which we will call ??. That is the food value of the peanut.
Now here you can get into an argument since what I have calculated is the number of calories for the 0.6 g peanut. My calculations give a value of about 5800 calories OR about 5.8 kilocalories. (you need to do the exact math). Why the argument? Many foods, in calorie tables, show the calorie content as kilocalories BUT (and this is an important BUT) they call it calories. When your problem says to list the EXACT FOOD VALUE of the peanut, it makes me think they want the so-called BIG calorie and that is the kilocalorie so I would list 5.8; however, about 5800 calories is the correct answer. You may have a multiple guess answer sheet and that may be of some help in knowing what answer they want.


text:
About this Business
- HoursSunClosedMon-Fri9:00 AM - 5:00 PMSatClosed
From Our Editors
Designed by founder and renaissance woman Lynne Brick and her accomplished husband, Victor, Brick Bodies and Lynne Brick's health clubs share a fitness cornucopia of group classes, personal-training sessions, and women's health tips. They stock their workout facilities with premium cardio and strength-training equipment, as well as offer amenities that may include pools, childcare services, and volcano-free saunas. Each of the seven locations sports its own personality, including the Owings Mills and Belvedere facilities, which operate as all-female communities.
The Brick's also employ a team of certified instructors, who lead more than 30 types of group fitness classes, allowing students of all skill levels access to sessions that range from low-impact workouts, such as BodyVive, to more intense courses, such as the kickboxing-inspired BodyCombat classes.


text:
KUED's nationwide search for a new general manager landed on a man who's career has taken him nationwide.
Well, not quite. But James Morgese, who takes over at Channel 7 on Dec. 1, has been at a number of stations in a number of states in the past 30 years - most recently in Bowling Green, Ky., where he was the general manger of both the public television and public radio stations.
He's also worked at PBS stations in Colorado, Idaho and Florida and NBC affiliates in Colorado and New York.
"James brings a wealth of public television experience to the position and KUED's viewers will be the beneficiaries," says Fred Esplin, director of institutional development at the University of Utah. "The university is delighted that he has accepted the position and has full confidence in his ability to build on the great tradition of service for which KUED is known."<


text:
Need Plumbing help in basement!
1 Attachment(s)
thanks for the great website, I am a real estate agent, and know little bit about plumbing and the structure,
recently I have started to finish my basement which was partially finished, I have designed the basement model and drain lines on the computer.
If you get chance to look at it please and advice,
and also should I remove the old lines when installing new ones or leave it there if they don't conflict with the new ones.
*please check attached picture for diagram, thanks
Whats old and why don't you want to use it? I would direct drains on the shortest route to the main line. Use 3" from the toilet not 4" if your main is 3".
thanks for the reply,
the old lines meaning, i had washroom installed but I wanted to move everything according to my new plan so i can fit the laundry in.
sure i will use 3" if the drain is 3".. good point...
any other suggestion?
I know what old lines are. What I want to which one of the pictured lines are the old work and which are the proposed new lines.
Need Plumbing help in basement!
1 Attachment(s)
picture attached for new lines
All those are new? Way too complicated. 2" drain line for the floor drains. Direct the drains for the sink, washer and floor drain (by the washer) on the shortest path (1 to3 ft long ) directly into the main (or into a short 3" line off the main between the sink and washing machine). The rest looks ok. Might need a vent for the floor drains.
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text:
Just vote for 1911 then - w/ the options being limited (there is a max), no way that he could put EVERY manufacturer...
So far for me it's Para Ordnance but they are not on the list.
Just vote for 1911 then - w/ the options being limited (there is a max), no way that he could put EVERY manufacturer...
That's like choosing your favorite child. I can bring it down to two... SIG and H&K.
As for customer service, yeah, both have failed badly there in the not too distant past and may well again. Then again, you have to face the fact that the civilian market isn't their primary interest. Just be thankful you have as many choices as you do and act when the time comes you may not.
If you really want to know the favorite manufacturer the 1911's should be broken into separate/individual manufacturers. The 1911 style handgun is made by a huge number of companies so breaking it down would certainly be cumbersome.
Yes, U are right - there is a limit to the # of poll options...Originally Posted by bambam
I think that S&W is as good as it gets.
Anything but a Bryco. I think that covers it.
Saint John Moses Browning and Colt says it all
another 1911 fan here
I voted for the XD cause I have one and like it alot...but 1911's rule!
1911's and ruger revolvers. Savage hands down for rifles Randall
All my hand guns are S & W but my wife has some off brands.
In my experience I like Ruger and S&W about the same, but my Ruger is preferred out of what I have.
Based on the number of units I own, I would have to say Walther!!
Based on what fits my hand the best, I would have to say Kahr.


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Sara Lee is working to integrate RFID tags into its operations so that it can meet a mandate from Wal-Mart. Ray Hagedorn, vice president of business solutions for the IT department in the Sara Lee Foods division, talks about the bumps on the road - and Sara Lee's attempts to see how else they can benefit from the technology.
This story, "Interview: Sara Lee wrestles with RFID, looks for benefits" was originally published by Computerworld.


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Petits Fours Icing
Use this icing to make the decorative petits fours with butterflies by Karen Porter of Tilly's Cakes.
- Yield: Makes enough for 32 petits fours
Source: The Martha Stewart Show, April Spring 2007
- 9 cups sifted confectioners' sugar
- 1/2 cup light corn syrup
- 1 teaspoon clear vanilla extract
- 1/2 teaspoon almond extract
- Gel food coloring, in desired colors
In a heatproof bowl set over (but not touching) simmering water, mix sugar, corn syrup, 1/2 cup water, vanilla and almond extracts together until they are warm, well combined, and smooth. Stir in enough food coloring until desired color is reached. Do not overheat. Let cool about 8 minutes before pouring.