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Title: Polynucleotides and polypeptides BASB033 from neisseria meningitidis and their uses
United States Patent: 6,627,204
Issued: September 30, 2003
Inventors: Ruelle; Jean-Louis (Limal, BE)
Assignee: SmithKline Beecham Biologicals s.a. ()
Appl. No.: 787084
Filed: July 18, 2001
PCT Filed: September 9, 1999
PCT PUB. Date: March 23, 2000
The invention provides BASB033 polypeptides and polynucleotides encoding BASB033 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.
The present invention relates to BASB033, in particular BASB033 polypeptides and BASB033 polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including prevention and treatment of microbial diseases, amongst others. In a further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting expression or activity of BASB033 polynucleotides or polypeptides.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
The invention relates to BASB033 polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of BASB033 of Neisseria meningitidis, which is related by amino acid sequence homology to Klebsiella pneumoniae outer membrane phospholipase A protein. The invention relates especially to BASB033 having the nucleotide and amino acid sequences set out in SEQ ID NO:1,3 and SEQ ID NO:2,4 respectively. It is understood that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of one embodiment of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
In one aspect of the invention there are provided polypeptides of Neisseria meningitidis referred to herein as "BASB033" and "BASB033 polypeptides" as well as biologically. diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
The present invention further provides for:
(a) an isolated polypeptide which comprises an amino acid sequence which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO:2, 4;
(b) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:1, 3 over the entire length of SEQ ID NO:1, 3 respectively; or
(c) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:2, 4;
The BASB033 polypeptides provided in SEQ ID NO:2,4 are the BASB033 polypeptides from Neisseria meningitidis strains ATCC13090 and H44/76.
The invention also provides an immunogenic fragment of a BASB033 polypeptide, that is, a contiguous portion of the BASB033 polypeptide which has the same or substantially the same immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO:2,4. That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises the BASB033 polypeptide. Such an immunogenic fragment may include, for example, the BASB033 polypeptide lacking an N-terminal leader sequence, and/or a transmembrane domain and/or a C-terminal anchor domain. In a preferred aspect the immunogenic fragment of BASB033 according to the invention comprises substantially all of the extracellular domain of a polypeptide which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2,4 over the entire length of SEQ ID NO:2
A fragment is a polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with BASB033 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of SEQ ID NO:2,4 or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, colt and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2,4, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.4.
Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis: therefore, these fragments may be employed as intermediates for producing the full-length polypeptides of the invention.
Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
The polypeptides, or immunogenic fragments, of the invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
In one aspect, the invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG1, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non-fused protein. The fusion partner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein. Preferably the fusion partner will be both an immunological fusion partner and expression enhancing partner.
Fusion partners include protein D from Haemophilus influenzae and the non-structural protein from influenzae virus, NS1 (hemaigglutinin). Another fusion partner is the protein known as LytA. Preferably the C terminal portion of the molecule is used. LytA is derived from Streptococcuts pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LytA, (coded by the lytA gene {Gene, 43 (1986) pare 265-272}) an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LytA protein is responsible for the affinity to the choline or to some choline analolgues such as DEAE. This property has been exploited for the development of E.coli C-LytA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LytA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798}. It is possible to use the repeat portion of the LytA molecule found in the C terminal end starting at residue 178, for example residues 188-305.
The present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions. whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
It is most preferred that a polypeptide of the invention is derived from Neisseria meningitidis, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
It is an object of the invention to provide polynucleotides that encode BASB033 polypeptides, particularly polynucleotides that encode the polypeptide herein designated BASB033.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding BASB033 polypeptides comprising a sequence set out in SEQ ID NO:1,3 which includes a full length gene, or a variant thereof.
The BASB033 polynucleotides provided in SEQ ID NO:1,3 are the BASB033 polynucleotides from Neisseria meningitidis strains ATCC 13090 and H44/76.
As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing BASB033 polypeptides and polynucleotides, particularly Neisseria meningitidis BASB033 polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B-and Z-DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a BASB033 polypeptide having a deduced amino acid sequence of SEQ ID NO:2,4 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a BASB033 polypeptide from Neisseria meningitidis comprising or consisting of an amino acid sequence of SEQ ID NO:2,4 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set out in SEQ ID NO:1, 3 a polynucleotide of the invention encoding BASB033 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning, and sequencing chromosomal DNA fragments from bacteria using Neisseria meningitidis cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in SEQ ID NO:1,3, typically a library of clones of chromosomal DNA of Neisseria meningitidis in E. coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence. Illustrative of the invention, each polynucleotide set out in SEQ ID NO:1,3 was discovered in a DNA library derived from Neisseria meningitidis.
Moreover, each DNA sequence set out in SEQ ID NO:1,3 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in SEQ ID NO:2, 4 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID NO:1, between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 1126 of SEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.
The polynucleotide of SEQ ID NO:3, between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 1123 of SEQ ID NO:3, encodes the polypeptide of SEQ ID NO:4.
In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of:
(a) a polynucleotide sequence which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:1,3 over the entire length of SEQ ID NO:1,3 respectively; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO:2, 4 over the entire length of SEQ ID NO:2, 4 respectively.
A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than Neisseria meningitidis, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45-65oC. and an SDS concentration from 0.1-1%) with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO:1,3 or a fragment thereof; and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in SEQ ID NO: 1, 3. Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites. Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals.
The polynucleotide sequence may also comprise additional coding sequence encoding, additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
The nucleotide sequence encoding BASB033 polypeptide of SEQ ID NO:2, 4 may be identical to the polypeptide encoding sequence contained in nucleotides 1 to 1125 of SEQ ID NO:1, or the polypeptide encoding sequence contained in nucleotides 1 to 1122 of SEQ ID NO:3, respectively. Alternatively it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2, 4.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Neisseria meningitidis BASB033 having an amino acid sequence set out in SEQ ID NO:2, 4. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA recognization) together with additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of SEQ ID NO:2, 4.
Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding BASB033 variants, that have the amino acid sequence of BASB033 polypeptide of SEQ ID NO:2, 4 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of BASB033 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding BASB033 polypeptide having an amino acid sequence set out in SEQ ID NO:2, 4, and polynucleotides that are complementary to such polynucleotides. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of SEQ ID NO:1, 3.
In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to BASB033 polynucleotide sequences, such as those polynucleotides in SEQ ID NO:1, 3.
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is overnight incubation at 42oC. in a solution comprising: 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6). 5xDenhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1xSSC at about 65oC. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO:1, 3 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:1, 3, or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding BASB033 and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the BASB033 gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs. Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs. Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have less than 30 nucleotide residues or base pairs.
A coding region of a BASB033 gene may be isolated by screening using a DNA sequence provided in SEQ ID NO:1, 3 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al. PNAS USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon.TM. technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon.TM. technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an `adaptor` sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oliconucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying, out a separate full-length PCR using the new sequence information for the design of the 5' primer.
The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a sequence of SEQ ID NOS:1-4 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
The invention also provides polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
For each and every polynucleotide of the invention there is provided a polynucleotide complementary to it. It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in describing certain polynucleotides of the invention. "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (Wu et al., J Biol Chem. (1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment (Tan, et al. Nature (1992) 356:152, Eisenbraun et at., DNA Cell Biol (1993) 12: 791) and in vivo infection using cloned retroviral vectors (Seedier et al. PNAS USA (1984) 81: 849).
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis, Haemophilus influenzae and Neisseria menigitidis; fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albictans and Aspergillus; insect cells such as cells of Drosophila S2 and Spodoptera St9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells: and plant cells, such as cells of a gymnosperm or angiosperm.
A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses, retroviruses, and alphaviruses and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterolocgous signals.
Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, ion metal affinity chromatography (IMAC) is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and or purification.
The expression system may also be a recombinant live microorganism, such as a virus or bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses. Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox). alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus). herpesviruses (varicella zoster virus, etc), Listeria. Salmonella, Shigella, Neisserin BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of BASB033 polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of BASB033 polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the BASB033 gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled BASB033 polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al., Science, 230: 1242 (1985). Sequence chances at specific locations also may be revealed by nuclease protection assays, such as RNase, V1 and S1 protection assay or a chemical cleavage method. See, for example, Cotton et al., Proc. Natl. Acad Sci. USA, 85: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASB33 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al., Science, 274: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO:1, 3, or a fragment thereof;
(b) a nucleotide sequence complementary to that of (a),
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2, 4 or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2, 4.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, among others.
This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferable, SEQ ID NO:1, 3, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan, RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers complementary to a polynucleotide encoding BASB033 polypeptide can be used to identify and analyze mutations.
The invention further provides primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. These primers may be used for, among other things, amplifying BASB033 DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing disease, preferably bacterial infections, more preferably infections caused by Neisseria meningitidis, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of SEQ ID NO:1, 3. Increased or decreased expression of a BASB033 polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over-expression of BASB033 polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a BASB033 polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
The polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or rids. These high density arrays are particularly useful for diagnostic and prognostic purposes. For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as using hybridization or nucleic acid amplification, using a probe obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual. Such a presence may indicate the presence of a pathogen, particularly Neisseria meningitidis, and may be useful in diagnosing and/or prognosing disease or a course of disease. A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO:1, 3 are preferred. Also preferred is a grid comprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2, 4.
The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.
In certain preferred embodiments of the invention there are provided antibodies against BASB033 polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975): Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms or animals, such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.
Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-BASB033 or from naive libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks, et al., (199) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al., (1991) Nature 352: 628).
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against BASB033-polypeptide or BASB033-polynucleotide may be employed to treat infections, particularly bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized," where the complimentary determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology 9, 266-273.
Antagonists and Agonists--Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonist or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be. Further, the screenings methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measurings BASB033 polypeptide and/or polynucleotide activity in the mixture, and comparing the BASB033 polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and BASB033 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).
The polynucleolides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of BASB033 polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal. The method of screening may involve high-throughput techniques. For example, to screen for arsonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising BASB033 polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a BASB033 agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the BASB033 polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously. i.e., without inducing the effects of BASB033 polypeptide are most likely to be good antagonists. Molecules that bind sell and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to calorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in BASB033 polynucleotide or polypeptide activity, and binding assays known in the art.
Another example of an assay for BASB033 agonists is a competitive assay that combines BASB033 and a potential agonist with BASB033-binding molecules, recombinant BASB033 binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. BASB033 can be labeled, such as by radioactivity or a colorimetric compound, such that the number of BASB033 molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing BASB033-induced activities, thereby preventing the action or expression of BASB033 polypeptides and/or polynucleotides by excluding BASB033 polypeptides and/or polynucleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular bindings molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neurochem, 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of BASB033.
In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoalobulin is the constant part of the heavy chain of human IgG, particularly IgG1, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
Each of the polynucleotide sequences provided herein may be used in the discovery and development of anitibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide, arsonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial BASB033 proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
In accordance with yet another aspect of the invention, there are provided BASB033 agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
In a further aspect, the present invention relates to mimotopes of the polypeptide of the invention. A mimotope is a peptide sequence, sufficiently similar to the native peptide (sequentially or structurally), which is capable of being recognised by antibodies which recognise the native peptide; or is capable of raising antibodies which recognise the native peptide when coupled to a suitable carrier.
Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides may be modified for the purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine. In addition it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic terminus distal from the conjugated terminus of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier protein. Thereby presenting the peptide in a conformation which most closely resembles that of the peptide as found in the context of the whole native molecule. For example, the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Alternatively, the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide.
Alternatively, peptide mimotopes may be identified using antibodies which are capable themselves of binding to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267 B1). This technique, generates a large number, of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native polypeptide.
Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, preferably humans, which comprises inoculating the individual with BASB033 polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Neisseria meningitidis infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of BASB033 polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressing BASB033 polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cvtotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not. One example or administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.
A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a BASB033 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant BASB033 polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said BASB033 polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+T cells.
A BASB033 polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Haemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protein which solubilizes the protein and facilitates production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein. The co-protein may be attached to either the amino- or carboxy-terminus of the first protein.
Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Neisseria meningitidis. Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Neisseria meningitidis infection, in mammals, particularly humans.
The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteristatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition o the sterile liquid carrier immediately prior to use.
The vaccine formulation of the invention may also include adjuvant systems for enhancing the immunogenicity of the formulation. Preferably the adjuvant system raises preferentially a TH1 type of response.
An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed TH1-type responses (cell-mediated response), and TH2 -type immune responses (humoral response).
Extreme TH1-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice TH1-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgG1 type antibodies. TH2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgG1, IgA, and IgM.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines. High levels of TH1-type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
The distinction of TH1 and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly TH1 or predominantly TH2. However, it is often convenient to consider the families of cytokinies in terms of that described in murine CD4+ve T cell clones by Mosmann and Coffman (Mosmnn, T. R. and Coffman R. L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properities, Annual Review of Immunology, 7, p145-173). Traditionally, TH1-type responses are associated with the production of the INF-.gamma. and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of TH1-type immune responses are not produced by T-cells, such as IL-12. In contrast, TH2-type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suited to the stimulation of either TH1 or TH2-type cytokine responses. Traditionally the best indicators of the TH1:TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of TH1 or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the IgG1:IgG2a ratio of antigen specific antibody responses.
Thus, a TH1-type adjuvant is one which preferentially stimulates isolated T-cell populations to produce high levels of TH1-type cytokines when re-stimulated with antigen in vitro, and promotes development of both CD8+cytotoxic T lymphocytes and antigen specific immunoglobulin responses associated with TH1-type isotype.
Adjuvants which are capable of preferential stimulation of the TH1 cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).
Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22 micron membrane (European Patent number 0 689 454). 3D-MPL will be present in the range of 10 .mu.g-100 .mu.g preferably 25-50 .mu.g per dose wherein the antigen will typically be present in a range 2-50 .mu.g per dose.
Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina. Optionally this may be admixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with a carrier.
The method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful TH1 stimulating adjuvants when formulated together with an antigen.
Further adjuvants which are preferential stimulators of TH1 cell response include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
Combinations of different TH1 stimulating adjuvants, such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of TH1 cell response. For example, QS21 can be formulated together with 3D-MPL. The ratio of QS21: 3D-MPL will typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1 and often substantially 1:1. The preferred range for optimal synergy is 2.5:1 to 1:1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to the invention. The carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tween 80. In a particularly preferred aspect the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion. Additionally the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of 1 .mu.g-200 .mu.g, such as 10-100 .mu.g, preferably 10 .mu.g-50 .mu.g per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210.
The present invention also provides a polyvalent vaccine composition comprising a vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating, cancers, autoimmune diseases and related conditions. Such a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.
While the invention has been described with reference to certain BASB033 polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides.
The antigen can also be delivered in the form of whole bacteria (dead or alive) or as subcellular fractions, these possibilities do include N.meningitidis itself.
Compositions, Kits and Administration
In a farther aspect of the invention there are provided compositions comprising a BASB033 polynucleotide and/or a BASB033 polypeptide for administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/or a polypeptide discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other deterrents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, solutions, powders and the like.
For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 .mu.g/kg of subject.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3-dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as the GCG program package.
Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded protein sequences. Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, DNA, RNA and protein structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, codon usage analysis, nucleic acid base trimming, and sequencing chromatogram peak analysis.
A computer based method is provided for performing homology identification. This method comprises the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
A computer based method is also provided for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.
Claim 1 of 8 Claims
What is claimed is:
1. An isolated polypeptide comprising a member selected from the group consisting of
(a) an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4;
(b) an immunogenic polypeptide comprising a fragment sequence of at least 15 amino acids that corresponds to an aligned contiguous segment of SEQ ID NO:2 or SEQ ID NO:4;
wherein the isolated polypeptide, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell immune response to a polypeptide having the sequence of SEQ ID NO:2 or SEQ ID NO:4.


text: We had the pleasure of photographing family portraits for this beautiful family, the Wong's. Their baby daughter Sabrina was full of energy and super cute in front of the camera. They are a really sweet family, and we are so honored that they used the photographs for their Christmas cards!

subset: commoncrawl


text:
No bail for man charged with molesting 13-year-old girl with autism
BY JON SEIDEL Staff Reporter email@example.com June 23, 2013 3:42PM
Edwin Hernandez / photo provided by Chicago Police
Updated: July 25, 2013 6:37AM
An Albany Park man who prosecutors said admitted to sexually assaulting a 13-year-old autistic girl he later described as "sick in the head" was held without bail Sunday.
Edwin Hernandez, 30, of the 4600 block of North Lawndale, has been charged with aggravated criminal sexual assault and home invasion for allegedly molesting the girl who Assistant State's Attorney Bridget O'Brien said has the mental capacity of a 2- or 3-year-old.
Hernandez is friends with the girl's baby sitter, O'Brien said, and met the victim at the baby sitter's home. He learned where the girl lives, O'Brien said, and watched her through her bedroom window at least three times.
Once, the prosecutor said, Hernandez entered the victim's home but was startled and fled when he heard someone.
But on June 16, on the way home from the Puerto Rican Festival, O'Brien said Hernandez again went to the girl's window around 2 a.m., pushed it open, climbed into the room where she was sleeping and began to molest her.
O'Brien said someone heard him and alerted the girl's parents after he left. When her parents entered the victim's room, O'Brien said, they found muddy footprints on the window sill and called police.
The prosecutor said police matched Hernandez to DNA found on the victim. His fingerprints were also found outside her bedroom window.
Hernandez was arrested Friday night in the 5000 block of North Pulaski, according to police reports.
O'Brien said Hernandez admitted to the assault and said he thought the girl was 10 or 11. He allegedly told police he knew the girl was "sick in the head, and her mind was not right."


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Discover Chocolate and Live La Vida Cocoa!
I am going to be participating in a 1000 person event where I have to give out sample bars...I am looking for a 2 or 3 oz. bar mold that doesn't look wimpy. Any suggestions? And also, I normally package my bars in cello bags for sale out of my store but think I need something a slight step up from this...any suggestions?
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1460 University Dr
Winchester, VA 22601-5195
(540) 665-4500
Winchester, VA 22601-5195
(540) 665-4500
Website: www.su.edu
Location: Southeast
Location: Southeast
Calendar: Semester
Get a snapshot of Shenandoah University with this general overview, including student enrollment and graduation rates, average test scores, student demographics, and more.
Mission StatementShenandoah University prepares individuals to be critical, reflective thinkers; lifelong learners; and ethical, compassionate citizens who are committed to making responsible contributions within a community, a nation and the world. Shenandoah distinguishes itself by providing opportunities to gain knowledge and develop skills in a collaborative, personalized environment that intertwines professional and liberal learning. A Shenandoah education incorporates scholarship, experiential learning and sophisticated technologies, as well as practical wisdom. To this end, the university has identified six core values that provide a foundation for the decisions it makes and the programs it offers: - Development of an enduring passion for learning - Commitment to self-reflection and personal development - Respect for diverse cultures, experiences, and perspectives - Celebration of creative performance, expression, teaching, and discovery - Cultivation of leadership to advance positive change and growth - Dedication to citizenship, professional service, and global outreach As an institution affiliated with the United Methodist Church, Shenandoah University practices the highest ethical standards in its interactions with the community and with students of all faiths. Within a community of scholars, Shenandoah promotes the welfare of the whole person by fostering a nurturing environment in which students learn, grow, and flourish.
School Characteristics
Private not-for-profit
$22,340 per year
Length of Study: Four or more years
Religious Affiliation: United Methodist
Test Scores & GPAAverage GPA: 3.1
Average ACT: 20
Average SAT: 1000
School ProfileTotal Enrollment: 3,393
Retention Rate: 65%
Graduation Rate: 45%
School AccreditationNational: No
Regional: Not Available
Federal: No
Male / Female Enrollment
Total Enrollment
School data was obtained from the US Dept. of Education's Integrated Postsecondary Education Data System (IPEDS). Data may vary depending on school and academic year.


text:
Does anybody know if you are billed for a 911 call and EMT visit? I know you are billed for ambulance transport, but if parents are just overly cautious and there is no transport are you also billed?
The back story to my question is that we called 911 on Saturday and had a visit from our local fire dept. DD woke from a nap and started hyper-ventilating and could not catch her breath. Luckily, by the time they arrived she was breathing normally and seemed to be thrilled to have so many guests at our house. She was getting over a bad cold and fever and they suspect that maybe she choked on some phglem.
I believe it is dependent on where you live. I call 911 once (2004) because Logan got his leg wedged in his crib and was not charged. We lived in Seattle and it was the city EMS. Things could have changed. I know up here in Edmonds they are talking about charging for 911 calls because they don't have the $$ in the budget for EMS...so sad.?
Logan was probably a little under 1 when it happened and boy there was like a storm of EMTs in my house. Very exciting for a 1 y/o. I felt like a dumbarse.?
You do not get billed for a 911 call, you already pay for it through your taxes. Billing people would actually scare people from using the public service. I'm a former 911 dispatcher.
EMT service? I have no idea, you could just call down to your local fire department and ask.


text:
Now Hiring Medical Assemblers in Maple Grove & Plymouth
Don't miss this rewarding opportunity to enhance your career while working for a globally recognized company.
Acquire work skills desired by employers while building lifesaving medical devices
Choose from multiple shifts to fit your schedule
Earn extra money through overtime opportunities
Work in a team environment
Full time work!
Take advantage now!
High School Diploma or GED equivalent.
Criminal background check and drug screen administered.
Ability to commit to this position for 12 months.
Work experience preferred.
Basic reading/math/measuring skills, computer literacy and manual dexterity.
Please click APPLY NOW button or submit your resume to firstname.lastname@example.com.
Atterro/Pro Staff is an Equal Opportunity Employer.
© 2013 Atterro, Inc.
All Rights Reserved.


text:
Point P Wall Sconce
Price: $238.95
+ Free Shipping100% Price Match
Pin It Houzz
Related Info
Design by Arter & Citton.By Alt Lucialternativa, from Leucos.
The Point P Wall Sconce is available in halogen or compact fluorescent lamping and three finish options. Transparent, red, or blue glass disks and a chrome plated diffuser make up the face of this wall sconce. Satin white glass comprises its body. Ideal for contemporary bedrooms, hallways, or entryways. ADA Certified. Dimmable.
Shown in blue.
Material(s): Glass, Metal
Dimensions: 6"D X 3" extension
Lamp Type: HALOGEN, COMPACT FLUORESCENT
Bulbs: Halogen: 1 X 40W 120V G9 halogen lamp (not included) Compact Fluorescent: 1 X 9W 120V GX35 compact fluorescent lamp (not included)
Listing: UL, ADA
Manufacturer Specifications: Click to download specifications.
Model(s): 105061013459 105061013464 105061014859 105061014864 105061014359 105061014364
Founded in 1962, Leucos is an Italian manufacturer of contemporary-styled lighting products. Leucos lighting designs incorporate the renowned craftsmanship of Murano glassmakers with the latest, award-winning technologies. Early Leucos products were the first to marry design and the tradition of glass making production. In line with this innovative spirit, much attention was devoted to the creative process - something that has become emblematic of the brand and strengthened its recognition throughout the world.
Free shipping
on most orders
Expert advice
+ service
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of modern designs
Price guarantee
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text:
Using Gmail and Calendar
Date and Time:Wednesday, March 19, 2014 - 3:00pm - 4:00pm
Event Contact Name:Joseph Blankson, PhD
Event Contact E-mail:firstname.lastname@example.com
Event Location:Mathile 300
Learn to use Google apps; Gmail, Calendar and Google Drive. Create new documents, spreadsheets, and presentations to share and collaborate with your students or colleagues in real time. All your changes are saved automatically.


text:
San Francisco is our kind of town. It's passionate about wine, brims with great restaurants and offers a relaxed yet sophisticated way of life. You could almost say it's Wine Spectator 's hometown. Though the magazine was born in San Diego, it moved to San Francisco when only 3 years old, in 1979, and veteran editors James Suckling, James Laube, Harvey Steiman and Kim Marcus came on board during the San Francisco years.
For the full article, check out the new issue of Wine Spectator, on newsstands October 15, 2006.
Do you have a Wine Spectator magazine subscription? Save 50% on your Online Membership right now!
Sips & Tips | Wine & Healthy Living
Video Theater | Collecting & Auctions
» View samples
» Or sign up now!
» Manage my newsletter preferences
The marketplace for all your wine needs, including:
Wine Storage | Wine Clubs
Dining & Travel | Wine Auctions
Wine Shops | Wine Accessories


text:
Shark® Electronic Steam Mop
Does your hard floor need some TLC? No need to get all hot and bothered over a mop and bucket – keeping floors clean, and sanitised into the bargain, this slimline mop from Shark is just what you've been waiting for. Making mopping less of a chore, it utilises super-heated continuous steam to leave floors immaculately clean and practically dry straightaway so you can get on with more enjoyable pursuits. And if your carpets are looking a little tired, clip on the carpet glider and this little gem will revive them in no time at all. With two steam settings, one for light cleaning and dusting and the second for a more intense, deep-down clean, it comes with 2 machine washable, dual-sided microfibre floor pockets – one side normal, the other 'scrubby' – and a carpet glider.
We recommend customers who live in a hard water area, use distilled water. Alternatively, you can use our Lakeland Water Softener which treats up to 10 litres of water
Cat Ref: SKU 23482
Free delivery
We can offer you free UK delivery on this item
Click & Collect, Free from your local store
Take advantage of our Click & Collect service available in our UK stores.
Please check availability estimates during checkout for your nearest store.
Add the Shark® Electronic Steam Mop to your basket
In Stock
Recycle your electricals
Lakeland has joined a scheme whereby customers can take their unwanted electricals to recycling points set up around the country.
Visit www.recycle-more.co.uk to find out where your nearest recycling point is.
Useful documents
Customer Reviews - Shark® Electronic Steam Mop
Showing 3 of 31 reviews See all reviews
Impressed" by Jane
I bought this to replace my old steam mop of another brand and this is streets ahead in every way. It feels sturdy, the machine and pads are very good quality and it glides easily over my vinyl floor (no pad slipping or difficulty pushing over the floor like my old one). The high power setting is very powerful and great for more stubborn marks. I washed the pad in the machine as directed and it came very clean. Very pleased.
Buy this one!" by Clare
This is the fourth floor steam cleaner I have bought or tried in the last few months (inc the Bissell steam and clean) - thank goodness its by far the best! I had nearly given up hope of finding a good one .... I use this on large open plan area of polished tiles and also on lino in the bathroom. No puddles or streaks, it does exactly what I had hoped. Its light yet sturdy, heats up in no time and is very easy to use. Comes with 4 different cleaning pads too. The flip over cleaning head is also a really clever design. If you are going to buy one, buy this!
This review is for an older version of this product
Upright Steam Cleaner" by Brenda
I am delighted with my new Shark steam floor cleaner. It is far superior to my previous cylinder cleaner. The steam is more powerful; the floors dry very quickly and the long lead enables me to clean 2 rooms using one socket. It is an excellent machine, lightweight and well designed. I am very happy that I made the change.
You may also like:
- Shark Sonic Duo Floor Cleaner
- £179.99
Can we also suggest
- Lakeland Steam Mop
- £59.99
- Lakeland Electronic Steam Mop
- Save: £40.00 Now £49.99
- Bissell® Vac and Steam
- £149.99
- Lakeland Water Softener
- £4.99
- Bissell® Steam Shot
- Save: £1.00 Now £38.99


text:
Linux Foundation Announces Open Compliance Program
Major names in open source and enterprise lend support.
The Linux Foundation began LinuxCon with a bang when it announced the formation of the Open Compliance Program, Tuesday.
The Open Compliance Program is an initiative meant to help companies abide by open source licenses and alleviate legal concerns within the marketplace. The program consists of six elements:
Training: The Linux Foundation offers additional documentation and training models that cover open source licensing fundamentals and activities geared toward enterprise. The foundation also will offer offer on-site or online training.
Tools: The Linux Foundation also developed complementary tools to improve due diligence. This is done via three tools, a dependency checker that identifies code combinations at dynamic and static levels, a Bill of Material (BoM) Difference Checker, and a code janitor that provides linguistic review capabilities. The Code Janitor ensures that developers don't leave comments in the source code by scanning source code files for a series of keywords stored in a database.
Self-Assessment Checklist: A checklist developed by The Linux Foundation that gives companies a list of compliance criteria to self-evaluate a given project's level of compliance.
The SPDX Standard and Workgroup: A labeling standard meant to easily identify and categorize open source components within a project. Visit www.linuxfoundation.org/workgroup/spdx/ for more details.
A Compliance Directory and Rapid Alert System: The Linux Foundation created a master list of compliance officers at companies implementing open source code in their commercial products. The database can be accessed and added to by visiting http://www.linuxfoundation.org/programs/legal/compliance/directory/
Community: These tools join the FOSSBazaar workgroup. The workgroup can be found at FOSSBazaar.org or http://www.linuxfoundation.org/workgroups/fossbazaar/.
"Our mission is to enable the expansion of free and open source software, so we created this program to give companies the information, tools and processes they need to get the most out of their investment, while governing the software," Jim Zemlin, executive director of The Linux Foundation said.
Along with those tools, The Linux Foundation also announced the founding participants in the program. HP, Intel, IBM, AMD, ARM Limited, Cisco Systems, Google , Novell, Samsung, Adobe, Nokia, NEC, Motorola, Sony Electronics, and Software Freedom Law Center are all members of the Open Compliance Program.
"By creating the Open Compliance Program, The Linux Foundation once again has stepped up to the challenge of providing the unifying force in an arena experiencing explosive growth, while decreasing the FUD around Linux and Open Source. IBM proudly supports the Open Compliance Program, which is an invaluable step in furthering the standards, tools, training and certification so needed by the industry," said Dan Frye, VP open systems development at IBM.
Currently, the foundation has released initial builds of the complimentary tools and encourages developers to contribute to them. The BoM Difference Checker will be available later this year. A finalized version of the Self-Assessment Checklist will be formally released in Q4 2010. For more information about the Open Compliance Program, visit http://www.linuxfoundation.org/programs/legal/compliance/.
Issue 170/2015
Buy this issue as a PDF
TGXf Project Warns of File Transfer through Screen Pixels
Weird data transfer technique avoids all standard security measures.
Industry Giants Announce a Fix for the Password Mess
FIDO alliance declares the beginning of the end for old-style login authentication.
Poll: Vote for the Best Open Source Software for Photographers
The Linux New Media Awards have honored the most significant products, projects, people, and organizations for open source/Linux every year since 2000.
Debian Gets Forked
Legendary Uber-distro splits over the systemd controversy.
Linux Mint Project Releases Mint 17.1
New LTS version offers many refinements for the Cinnamon and Mate desktops and significant improvement under the hood.
CeBIT Open Source Forum Call for Papers
One of CeBIT's most successful forums returns in 2015.
TOR Network Isn't So Anonymous
A new study says it is possible to unmask 81% of TOR users.
.NET Goes Open Source
Redmond joins the revolution by turning the .NET Core Runtime into a GitHub project.
Drupal Advisory Unleashes a Torrent of Attacks
Users only had 7 hours to update before the intrusions started.
HTML5 Gets Promoted to a Standard
It's official: The new web arrives


text:
A UEFA Champions League winner with FC Bayern München in 2001, Willy Sagnol thinks that his old club "need a European title very soon in order to continue their progress".
This content is streamed in such a way that it is protected and available only in a Flash format. Your device seems not to be compatible with our Flash video player.


text:
Betsy Lehman
• Zero Harm Event Materials and Research Reports
• Events
Betsy Lehman Center for Patient Safety and Medical Error Reduction
2 Boylston Street
Suite 212
Boston, MA 02116
BetsyLehmanCenter@ MassMail.state.ma.us
Betsy Lehman Center for Patient Safety and Medical Error Reduction is an independent state agency with a broad mandate to improve patient safety in Massachusetts. It is named for Betsy Lehman, a Boston Globe health care reporter and mother who died at the age of 39 in 1994 as the result of an overdose of a chemotherapy drug.
In the years since Betsy's death, improved patient safety measures have been implemented in Massachusetts and nationwide. But preventable harm through mistakes made during medical care remains a critical issue.
Up to 400,000 deaths each year are associated with preventable adverse events in American hospitals. Medical errors also occur in doctors' offices, nursing homes, clinics, pharmacies and patients' homes, and can result in additional or prolonged treatment, disability or death. This makes medical error the third-leading cause of death in the country, behind only heart disease and cancer.
The high costs – both financial and suffering by patients and families – demand even greater strides towards reducing medical error today.
Betsy Lehman Center (BLC) was originally launched in 2004 within the state's Executive Office of Health and Human Services and operated within the Department of Public Health. It was reestablished in 2013 as an independent agency within the Center for Health Information and Analysis (CHIA). BLC is now led by Executive Director Barbara Fain who was appointed by a Board comprised of Attorney General Martha Coakley, Secretary of Health and Human Services John Polanowicz, Undersecretary of Consumer Affairs Barbara Anthony, and Áron Boros, Executive Director of CHIA.Learn more about Betsy Lehman Center


text:
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