WO2009144462A2

WO2009144462A2 - Laminin receptor binding proteins

Info

Publication number: WO2009144462A2
Application number: PCT/GB2009/001336
Authority: WO
Inventors: Dlawer Ala'aldeen; Kark Wooldridge; Jafar Mahdavi
Original assignee: The University Of Nottingham
Priority date: 2008-05-28
Filing date: 2009-05-28
Publication date: 2009-12-03
Also published as: WO2009144462A3; GB0809659D0

Abstract

The present invention relates to antigenic polypeptides expressed by the bacteria Neisseria meningitides and Haemophilus influenzae, vaccines comprising the antigenic polypeptides and therapeutic antibodies directed to the antigenic polypeptides.

Description

Laminin Receptor Binding Proteins

Field of the Invention

The invention relates to antigenic polypeptides expressed by bacteria, vaccines comprising the antigenic polypeptides and therapeutic antibodies directed to the antigenic polypeptides.

Background to the Invention

Historically, Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae and Neisseria meningitidis (meningococcus) have been the predominant causes of bacterial meningitis. Many bacteria can replicate in the bloodstream, yet this trio is unique in also binding to and crossing the endothelium of the blood-brain barrier (BBB). Penetration of the BBB allows the bacteria access to the cerebrospinal fluid of the subarachnoid space, where immune defences are initially very limited. As a consequence, bacterial multiplication in this privileged site is efficient leading to meningitis and devastating neuronal damage. While effective vaccines have been developed against a subset of these pathogens, most of the 90-plus serotypes of pneumococcus, all non-type b H. influenzae, and the highly prevalent serogroup-B meningococcus are not covered by these vaccines. A broader vaccination strategy based on common elements of pathogenesis rather than serotype could decrease global morbidity and mortality and prevent the emergence of replacement serotypes.

Bacterial seeding of a body site, such as the subarachnoid space, from the bloodstream requires that bacteria adhere to and then penetrate the vascular endothelium. It is now known that pneumococcus, H. influenzae and meningococcus share a common strategy for traversing endothelial cells: they carry surface-exposed phosphorylcholine that mediates binding to the human platelet-activating factor receptor (PAFr) (1-3). Phosphorylcholine is a crucial structural component of the chemokine platelet activating factor (PAF), and these bacterial analogs are believed to be molecular mimics. Unlike binding of PAF to the PAF receptor (PAFr), binding of bacterial phosphorylcholine does not induce activation of G-protein-mediated signaling; instead, it results in activation of β-arrestin-mediated uptake of the bacteria into a vacuole (4). Such uptake is crucial for development of pneumococcal meningitis, which does not occur in mice deficient in PAFr, even in the presence of high-titer bacteremia (4).

COrøBMAΗON COFf It has been demonstrated that that a protein from S. pneumoniae, choline binding protein A (CbpA), binds to the endothelium of the BBB via the 37/67-kDa laminin receptor (LR) and that this interaction in mediated by the R2 domain (WO 2008/039838). It is postulated that vaccines based on CbpA may be useful in the treatment of pneumococcal infections.

There remains, however, a need for further agents that are useful in the prevention and treatment of other bacterial infections associated with meningitis, in particular meningococcal infections. .

Statements of the Invention

According to a first aspect of the invention there is provided an antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii) for use as a medicament.

In a preferred aspect of the invention the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 3; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In a further preferred aspect of the invention the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 1 or 3; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In a further preferred aspect the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 4; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In a further preferred aspect the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 5; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In an alternative preferred aspect the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 6; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In a further alternative preferred aspect the antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

In a preferred aspect of the invention the medicament is a vaccine. The nucleic acid encoding the antigenic polypeptide of the first aspect of the invention may anneal under stringent hybridisation conditions to the nucleic acid sequence shown in Figures 1 to 7 or to its complementary strand.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65⁰C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55⁰C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55⁰C for 20-30 minutes each.

The nucleic acid encoding the antigenic polypeptide of the first aspect of the invention may comprise the sequence set out in Figures 1 to 7 or a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, for example 98%, or 99%, identical to the nucleic acid sequence set out in Figures 1 to 7 at the nucleic acid residue level. "Identity", as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. . Identity can be readily calculated {Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., AND Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of . methods to. measure identity between two polynucleotide or two polypeptide sequences, the term is well-known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleid Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403 (1990)).

The nucleic acid encoding the antigenic polypeptide of the first aspect of the invention may comprise of a fragment of a sequence according to the first aspect which is at least 30 bases long, for example, 40, 50, 60, 70, 80 or 90 bases in length.

The nucleic acid sequence encoding the antigenic polypeptide of the first aspect of the invention may be genomic DNA, complementary DNA (cDNA) or RNA, for example messenger RNA (mRNA).

Preferably, the antigenic polypeptide of the first aspect of the invention is expressed by a pathogenic organism, for example, a bacterium. Preferably the pathogenic organism is a bacterium. The bacterium may be a Gram-positive or Gram-negative bacterium. Preferably the bacterium is a Gram-negative bacterium for example a bacterium selected from the group consisting of: Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae and Neisseria meningitidis ^meningococcus).

In a preferred embodiment of the invention, the antigenic polypeptide of the first aspect of the invention is associated with infective pathogenicity of an organism as defined herein.

In a further preferred aspect of the invention the antigenic polypeptide comprises the amino acid sequence shown in Figures 1 to 7, or a variant sequence thereof.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by non- amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component.

The term "variant" as used herein includes polypeptides that may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative (similar) replacements: a) alanine, serine, and threonine; b) glutamic acid and asparatic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan.

Amino acid substitutions can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Amino acid substitutions are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide. Groups of functionally related amino acids within which conservative substitutions may be made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; asvariantic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tryosine/tryptophan. Polypeptides of this invention may be in glycosylated or unglycosylated form, may be modified post-translationally (e.g., acetylation, and phosphorylation) or may be modified synthetically (e.g., the attachment of a labeling group).

As used herein, the term "polypeptide" means, in general terms, a plurality of amino acid residues joined together by peptide bonds. It is used interchangeably and means the same as peptide, protein, oligopeptide, or oligomer. The term "polypeptide" is also intended to include fragments, analogues and derivatives of a polypeptide wherein the fragment, analogue or derivative retains essentially the same biological activity or function as a reference protein. The term "polypeptide" also includes peptidomimetics and structural analogues of the described sequences, and those modified either naturally (e.g. post-translational modification) or chemically, including, but not exclusively, phosphorylation, glycosylation, sulfonylation and/or hydroxylation.

A "variant thereof of an antigenic polypeptide according to the invention may thus include a fragment or subunit of the antigenic polypeptide wherein the fragment or subunit is sufficient to induce an antigenic response in a recipient. Thus the present invention encompasses an antigenic polypeptide comprising an amino acid sequence as represented in Figure 4, 5, 6 or 7, or a fragment thereof or a variant polypeptide wherein said variant is modified by addition, deletion or substitution of at least one amino acid residue of the amino acid sequence presented in Figure 4, 5, 6 or 7 and wherein said variant polypeptide is sufficient to induce an antigenic response in a recipient and is capable of interacting with the laminin receptor. As used herein "a fragment of a polypeptide comprising the amino acid sequence as shown in Figure 4, 5, 6 or 7" includes fragments that contain between 1 and 50 amino acids, for example between 1 and 30 amino acids such as between 10 and 30 amino acids.

According to a second aspect of the invention there is provided a vector comprising a nucleic acid sequence encoding a polypeptide according to the first aspect of the invention.

The vector of the second aspect of the invention may be a plasmid, cosmid, phage or virus based vector. The vector may include a transcription control sequence (promoter sequence) which mediates ceil specific expression, for example, a cell specific, inducible or constitutive promoter sequence. The vector may be an expression vector adapted for prokaryotic or eukaryotic gene expression, for example, the vector may include one or more selectable markers and/or autonomous replication sequences which facilitate the maintenance of the vector in either a eukaryotic cell or prokaryotic host (Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach VoI III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, lnc.(1994). Vectors which are maintained autonomously are referred to as episomal vectors.

Promoter is an art-recognised term and may include enhancer elements which are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3¹ to a gene sequence or even located in intronic sequences and is therefore position independent). Enhancer activity is responsive to trans acting transcription factors (polypeptides e.g. phosphorylated polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues which include intermediary metabolites (eg glucose, lipids), environmental effectors ( eg light, heat,).

Promoter elements also include so called TATA box and RNA polymerase initiation selection (RIS) sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

The vector of the second aspect of the invention may include a transcription termination or polyadenylation sequences. This may also include an internal ribosome entry sites (IRES). The vector may include a nucleic acid sequence that is arranged in a bicistronic or multi-cistronic expression cassette.

According to a third aspect of the invention there is provided a method for the production of a recombinant antigenic polypeptide according to any previous aspect of the invention comprising: (i) providing a cell transformed/transfected with a vector according to the second aspect of the invention; (ii) growing said cell in conditions suitable for the production of said polypeptides; and (iii) purifying said polypeptide from said cell, or its growth environment.

In a preferred aspect of the method of the third aspect, the vector encodes, and thus said recombinant polypeptide is provided with, a secretion signal to facilitate purification of said polypeptide.

According to a fourth aspect of the invention there is provided a cell or cell-line transformed or transfected with the vector according to the second aspect of the invention.

In a preferred embodiment of the invention said cell is a prokaryotic cell, for example, r a bacterium such as E. coli. Alternatively said cell is a eukaryotic cell, for example a yeast or other fungal cell, insect, amphibian, or mammalian cell, for example, COS, CHO cells, Bowes Melanoma and other suitable human cells, or plant cell.

According to a fifth aspect of the invention there is provided a vaccine comprising at least one antigenic polypeptide, or variant thereof, according to the first aspect of the invention. Preferably said vaccine further comprises an adjuvant/carrier.

The vaccine may comprise an antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

The vaccine may comprise an antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence is selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 3; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii). The vaccine may comprise an antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 4; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

The vaccine may comprise an antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 5; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

The vaccine may comprise an antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 6; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

The vaccine may comprise an antigenic polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

The vaccine may comprise the antigenic polypeptides, or variant thereof, encoded by the isolated nucleic acid sequences selected from the group consisting of: i) (a) a nucleic acid sequence selected from Figures 1 to 6; and (b) the nucleic acid sequence shown in Figure 7; ii) nucleic acid sequences which hybridise to the sequences identified in (i) above; and iii) nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequences defined in (i) or (ii).

The vaccine may comprise the antigenic polypeptides, or variant thereof, encoded by the isolated nucleic acid sequences selected from the group consisting of: i) (a) a nucleic acid sequence as shown in Figure 5 and/or 6; and (b) a nucleic acid sequence as shown in Figure 7; ii) nucleic acid sequences which hybridise to the sequences identified in (i) above; and iii) nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequences defined in (i) or (ii).

In one embodiment of the invention the vaccine further comprises the antigenic polypeptide, or variant thereof, encoded by the isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figure 8; ii) a nucleic acid sequence which hybridises to the sequences identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences defined in (i) or (ii).

The vaccine according to the fifth aspect may be a subunit vaccine in which the immunogenic part of the vaccine is a fragment or subunit of the antigenic polypeptide according to the first aspect of the invention.

An adjuvant is a substance or procedure that augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, Freunds adjuvant, squalene, phosphate adjuvants and aluminium salts

(e.g. aluminium hydroxide or aluminium phosphate). Others may include muramyl dipeptides, liposomes. A carrier is an immunogenic molecule which, when bound to a second molecule, augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the "carrier" protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen. Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.

In yet a further aspect of the invention there is provided a method to immunise an animal against a pathogenic microbe comprising administering to said animal at least one polypeptide, or variant thereof, according to the first aspect of the invention. Preferably, the polypeptide is in the form of a vaccine according to the fifth aspect of the invention.

In a preferred method of the invention the animal is human.

Preferably the antigenic polypeptide of the first aspect, or the vaccine of the fifth aspect, of the invention can be delivered by direct injection either intravenously, intramuscularly, subcutaneously. Further still, the vaccine or antigenic polypeptide, may be taken orally. The polypeptide or vaccine may be administered in a pharmaceutically acceptable carrier, such as the various aqueous and lipid media, such as sterile saline, utilized for preparing injectables to be administered intramuscularly and subcutaneously. Conventional suspending and dispersing agents can be employed. Other means of administration, such as implants, for example a sustained low dose releasing bio- observable pellet, will be apparent to the skilled artisan.

The vaccine may be against a bacterial species selected from the group consisting of Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae and Neisseria meningitidis (meningococcus).

The vaccine may be against the bacterial species Neisseria meningitidis.

The vaccine may be against the bacterial species Haemophilus influenzae.

The vaccine may be against the bacterial species Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae and Neisseria meningitidis (meningococcus). It will also be apparent that vaccines or antigenic polypeptides are effective at preventing or alleviating conditions in animals other than humans, for example and not by way of limitation, companion animals (e.g. domestic animals such as cats and dogs), livestock (e.g. cattle, sheep, pigs) and horses.

According to a further aspect of the invention there is provided an agent that binds to at least one antigenic polypeptide, or variant thereof, according to the invention. Preferably the agent is an antagonist. Preferably the agent inhibits the activity of said antigenic polypeptide. As used herein the term "inhibits" refers to a species which retards, blocks or prevents an interaction, for example binding between an antigenic polypeptide according to the invention and the laminin receptor. Typically, inhibition does not result in 100% blockage but rather reduces the amount and/or speed of interaction.

Preferably the agent is an antibody or active binding fragment thereof. The antibody, or active binding fragment, may be a polyclonal antibody or a monoclonal antibody. Preferably the antibody, or active binding fragment, is a monoclonal antibody.

Antibodies or immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant. The L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region. The variable region contains complementarity determining regions or CDR's which form an antigen binding pocket. The binding pockets comprise H and L variable regions which contribute to antigen recognition. It is possible to create single variable regions, so called single chain antibody variable region fragments (scFv's). If a hybridoma exists for a specific monoclonal antibody it is well within the knowledge of the skilled person to isolate scFv's from mRNA extracted from said hybridoma via RT PCR. Alternatively, phage display screening can be undertaken to identify clones expressing scFv's. Alternatively said fragments are "domain antibody fragments". Domain antibodies are the smallest binding part of an antibody (approximately 13 kDa). Examples of this technology is disclosed in US6, 248, 516, US6, 291, 158, US6.127, 197 and EP0368684 which are all incorporated by reference in their entirety.

In a preferred embodiment of the invention said antibody fragment is a single chain antibody variable region fragment.

In a further preferred embodiment of the invention said antibody is a humanised or chimeric antibody.

A chimeric antibody is produced by recombinant methods to contain the variable region of an antibody with an invariant or constant region of a human antibody. A humanised antibody is produced by recombinant methods to combine the complementarity determining regions (CDRs) of an antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody. Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used. The complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

In a further preferred aspect of the invention said antibody is a chimeric antibody produced by recombinant methods to contain the variable region of said antibody with an invariant or constant region of a human antibody.

In a further preferred aspect of the invention, said antibody is humanised by recombinant methods to combine the complimentarity determining regions of said antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.

Preferably said antibody is provided with a marker including a conventional label or tag, for example a radioactive and/or fluorescent and/or epitope label or tag. T/GB2009/001336

15

Preferably said humanised monoclonal antibody to said polypeptide is produced as a fusion polypeptide in an expression vector suitably adapted for transfection or transformation of prokaryotic or eukaryotic cells.

Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not illicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases. Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.

In another aspect of the invention there is provided a vector comprising a nucleic acid sequence encoding the humanised or chimeric antibodies according to the invention.

In a yet further aspect of the invention, there is provided a cell or cell line which comprises the vector encoding the humanised or chimeric antibody according to the invention. The cell or cell line may be transformed or transfected with the vector encoding the humanised or chimeric antibody according to the invention.

In a yet further aspect of the invention there is provided a hybridoma cell line which produces a monoclonal antibody as hereinbefore described.

In a further aspect of the invention there is provided a method of producing monoclonal antibodies according to the invention using hybridoma cell lines according to the invention.

In a yet further aspect of the invention there is provided a method for the production of the humanised or chimeric antibody according to the invention comprising:

(i) providing a cell transformed or transfected with a vector which comprises a nucleic acid molecule encoding the humanised or chimeric antibody according to the invention; (ii) growing said cell in conditions suitable for the production of said antibody; and purifying said antibody from said cell, or its growth environment.

In a further aspect of the invention there is provided a method for preparing a hybridoma cell-line according to the invention comprising the steps of: i) immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide having an amino acid sequence as represented in Figures 1 to 7, or fragments thereof; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step

(ii) for binding activity to the amino acid sequences of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and v) recovering the monoclonal antibody from the culture supernatant.

The immunocompetent mammal may be a mouse, rat or rabbit.

The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, "Basic Facts about Hybridomas" in Compendium of Immunology V.ll ed. by Schwartz, 1981 , which are incorporated by reference.

A further aspect of the invention provides a pharmaceutical composition comprising an effective amount of at least one antigenic polypeptide, vaccine or agent according to the invention. When administered, the pharmaceutical compositions and formulations of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents. The compositions and formulations of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. When antibodies are used therapeutically, one particular route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation.

The compositions and formulations of the invention are typically administered in effective amounts. An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response.

When administered, the pharmaceutical preparations and formulations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically- acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

Pharmaceutical compositions and formulations may be combined if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term

"carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

In a further aspect of the invention there is provided the use of an antigenic polypeptide according to the first aspect of the invention in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection or a bacteria related disorder.

Preferably, the bacterial infection is caused by a bacterial pathogen derived from a bacterial species selected from the group consisting of: Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae and Neisseria meningitidis (meningococcus).

In a preferred embodiment the bacterial infection is a meningococcal infection.

The bacteria related disorder may be a meningococcal-associated disorder. A meningococcal-associated disorder may include, for example, meningitis (meningococcal meningitis), septicaemia and septic shock.

The bacteria related disorder may be a Haemophilus influenzae-associated disorder. A Haemophilus influenzae-associated disorder may include, for example, , influenza, bacteremia and meningitis. Other disorders may include septicaemia and septic shock, cellulitis, osteomyelitis, epiglottitis, joint infections, respiratory tract infection, otitis media, conjunctivitis, sinusitis and pneumonia.

Preferably the bacteria related disorder is meningitis.

A further aspect of the invention there is provided the use of antibodies according to the invention in the manufacture of a medicament for the treatment of a bacterial infection.

In a further aspect of the invention there is provided a method of treating a patient comprising administering to the patient an antigenic polypeptide according to the first aspect of the invention, or a vaccine according to the fifth aspect of the invention, or an antibody according to the invention. Preferably the method is for the treatment of a meningococcal infection for example meningitis.

The present invention also provides the use of an antigenic polypeptide, or variant thereof, in the identification of agents which modulate the interaction of laminin receptor with said polypeptide wherein the polypeptide, or variant thereof, is encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii).

The present invention also provides a use of laminin receptor in the identification of agents which modulate the interaction of laminin receptor with an antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii)

According to a further aspect of the invention there is provided a kit comprising an agent specifically reactive with a polypeptide encoded by a nucleic acid sequence as represented in any of Figures 1 to 7, or a fragment or variant thereof as defined herein, or an agent specifically reactive with a polypeptide comprising an amino acid sequence as represented in any of Figures 1 to 7, or a fragment or variant thereof as defined herein.

In a preferred embodiment of the invention said kit further comprises an oligonucleotide or antibody specifically reactive with said nucleic acid molecule or said polypeptide.

Preferably said kit comprises a thermostable DNA polymerase and components required for conducting the amplification of nucleic acid. Preferably said kit includes a set of instructions for conducting said polymerase chain reaction and control nucleic acid.

In an alternative preferred embodiment of the invention said kit comprises an antibody specifically reactive with a polypeptide comprising an amino acid sequence as represented in Figures 1 to 7, or a fragment or variant thereof as defined herein. According to a further aspect of the invention there is provided a method to screen for an agent that modulates the activity of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii); wherein the method comprises a) forming a preparation comprising a polypeptide, or sequence variant thereof, and at least one agent to be tested; b) determining the activity of said agent with respect to the activity of said polypeptide.

The amino acid sequences represented in Figures 1 to 7, can be used for the structure- based design of molecules which modulate e.g. inhibit) the interaction of the polypeptide with the laminin receptor Such "structure based design" is also known as "rational drug design". The proteins can be three-dimensionally analysed by, for example, X-ray crystallography, nuclear magnetic resonance or homology modelling, all of which are well-known methods. The use of structural information in molecular modelling software systems is also encompassed by the invention. Such computer-assisted modelling and drug design may utilise information such as chemical conformational analysis, electrostatic potential of the molecules, protein folding etc. One particular method of the invention may comprise analysing the three-dimensional structure of the protein of Figures 1 to 7 for likely binding sites of targets, synthesising a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described above.

In a method of the invention said agent may be an antagonist. Agents identified by the screening method of the invention may include, antibodies, small organic molecules, (for example peptides, cyclic peptides), and dominant negative variants of the polypeptides herein disclosed.

As mentioned above, the invention also provides, in certain embodiments, "dominant negative" polypeptides derived from the polypeptides hereindisclosed. A dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect of the active protein. For example, a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the ligand. Likewise, a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphorylate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal. Similarly, a dominant negative transcription factor which binds to another transcription factor or to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.

It will be apparent to one skilled in the art that modification to the amino acid sequence of polypeptides or agents according to the present invention could enhance the binding and/or stability of the peptide with respect to its target sequence. Modifications include, by example and not by way of limitation, acetylation and amidation. Alternatively or preferably, said modification includes the use of modified amino acids in the production of recombinant or synthetic forms of peptides. It will be apparent to one skilled in the art that modified amino acids include, for example, 4-hydroxyproline, 5-hydroxylysine, N⁶- acetyllysine, N⁶-methyllysine, N⁶,N⁶-dimethyllysine, N⁶,N⁶,N⁶-trimethyllysine, cyclohexyalanine, D-amino acids, ornithine. Other modifications include amino acids with a C₂, C₃ or C₄ alkyl R group optionally substituted by 1 , 2 or 3 substituents selected from halo ( eg F, Br, I), hydroxy or C₁-C₄ alkoxy. It will also be apparent to one skilled in the art that polypeptides could be modified by cyclisation. Cyclisation is known in the art, (see Scott et al Chem Biol (2001), 8:801-815; Gellerman et al J. Peptide Res (2001), 57: 277-291; Dutta et al J. Peptide Res (2000), 8: 398-412; Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following materials, methods and figures:

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BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 to 3 show the DNA and amino acid sequence of overlapping peptides PP18, PP19 and PP21 of loop 4 of PorA from Neisseria meningitidis;

Figure 4 shows the DNA corresponding to the end of loop 3, all of loop 4 and the beginning of loop 5 of PorA. The amino acid sequence corresponds to loop 4 of PorA

Figure 5 shows the DNA and amino acid sequence of PorA from MC58;

Figure 6 shows the DNA and amino acid sequence of PiIQ from MC58;

Figure 7 shows the DNA and amino acid sequence of the outer membrane protein OmpP2 from Haemophilus influenzae Rd KW20; Figure 8 shows the DNA and amino acid sequence of choline binding protein A (CbpA) from Streptococcus pneumoniae D39;

Figure 9 Identification of LR-binding proteins of meningococcus and H. influenzae, a) Adhesion (OD₄₀₅nm) of digoxigenin-labeled N. meningitidis MC58 (Nm), S. pneumoniae T4 (Sp) and H. influenzae Rd (Hi) to rLR (solid phase antigen) after pre-incubation with soluble rLR (light bars) or BSA (dark bars). ***p<0.001 compared to adhesion without pre-incubation. b) Specific binding of digoxigenin-labeled wild-type (black bars) or isogenic mutant (white bars) bacteria to LR-coated ELISA plates (mean±SD, n=4) was determined by subtracting the absorbance in BSA-coated wells from that in LR-coated wells. * p<0.005 compared to wild-type value. c,d) Meningococcal (c) PorA- and PiIQ- dependent and H. influenzae (d) OmpP2-dependent binding of LR. MC58 (wild-type) and isogenic pilQ^~, porA^~, and pilQ^~porA^~ double-mutant meningococcus or H. influenzae Rd (wild-type) and its isogenic oxx\pP2^~ mutant were labeled with CSFC and incubated with Cy5-conjugated LR. After washing, co-localization (white) was detected by confocal microscopy, e) Cranial window images showing representative adhesion of beads bearing PorA or PiIQ protein to cerebral vasculature (n=3 each).

Figure 10 Common LR-binding characteristics of pathogens, a) Schematic representation of LR ligand binding domains (αLR :anti-LR antibody; VEE: Venezuelan equine encephalitis virus; E. coli toxin: cytotoxic necrotizing factor-1). Numbers indicate amino acid residues, b) LR siRNA (grey bars) reduces adherence of wild-type (wt) pneumococcus (Sp), H. influenzae (Hi) and N. meningitidis (Nm) to rBCECβ cells but does not affect mutants lacking CbpA, OmpP2, PorA or PiIQ, respectively. MAP kinase siRNA (white bars) was used as a negative control. * p<0.05 compared to untransfected control (black bars); 100% values: Sp 10⁴, Hi 10⁵, Nm 4.3x10⁵ cfu/ml. c) Inhibition of adherence of FITC-labeled pneumococcus (black), H. influenzae (white) and meningococcus (grey) to human cerebral endothelial cells by LR peptides 161-182, 263- 282 or scrambled 263-282 (Scr). (mean±SD, n=3); * p<0.05 compared to no-peptide control, d) Colony blots of pneumococcus, H. influenzae and meningococcus treated with anti-CbpA antibody. cbpA- = negative control, (pre-immune rabbit serum was nonreactive with all), e) Inhibition of adherence to rBCEC₆ cells by anti-CbpA antibody. 100%: Sp 1.28 x 10⁵; Hi 7.6 x 10⁴; Nm 2.7 x 10⁴ cfu/ml. Pre-immune serum did not decrease adherence of any of the bacteria below 95% of control. Data representative of n=3. Figure 11 Calcium signalling response induced by bacterial LR ligands in human BBB cells. FLUO-4 loaded human brain microvascular endothelial cells were exposed to bacterial LR-binding proteins and visualized for quantitative fluorescence, a) Representative view of positive Ca²⁺ signal, b) Background response with no bacterial proteins added, c) CbpA LR binding peptide 379-408 alone, d) CbpA non-LR binding peptide 414-443 alone, e) rPilQ alone, f) rPilQ signaling inhibited by CbpA peptide 379- 408, g) rPorA alone, h) rPorA signaling inhibited by CbpA peptide 379-408, i) rOmpP2 alone, j) rOmpP2 signalling inhibited by CbpA peptide 379-408. Data plotted is fluorescent intensity quantification of >5000 frames for each data set. Scale bar = 100 μm. Data representative of n=3. Upward trend = positive. Flat or downward trend = negative.

Figure 12 Identification of meningococcal and H. influenzae ligands for LR. Whole meningococcus MC58 (a) or H. influenzae Rd (b) were biotin-labeled with cross-linked LR₁ and labeled bacterial proteins were identified by MALDI-TOF after separation by SDS-PAGE. Meningococcal proteins of 37-kDa, 40-kDa, and 60-kDa and a large protein that did not enter the gel (not visible in this figure) were identified as PorA, EF-Tu, GroEL and PiIQ, respectively (a, lane 1). Similar profiles were observed for isogenic pilQ^' (a, lane 2), porA^' (a, lane 3) and porA^'-pilQ^' double mutant meningococcus (a, lane 4), except for the absence of the PorA and PiIQ bands from the respective deletion mutants. In wild-type H. influenzae extracts (b), the proteins GroEL, EF-Tu and outer membrane protein (Omp) P2 were identified. For both bacteria, LR binding to EF-Tu and GroEL was deemed an artifact, as binding persisted after deletion of the adhesins (a, lane 4) or when wild-type cells were probed with labelled KLH (not shown), c) Inhibition of binding of decreasing concentrations of digoxigenin-labeled rLR to ELISA plates coated with N. meningitidis by preincubation with PorA (white bar) or PiIQ (grey bar) (black bar = no inhibitor) (mean ± SD, n=2). Absorbance in BSA-coated wells was subtracted from that in bacteria-coated wells to obtain specific binding. Binding was significantly reduced in all dilutions. ** p<0.01 and *** p<0.001 compared to binding without inhibitor.

EXAMPLE

Materials and Methods

Bacterial strains and growth conditions. E. coli strains, XLIO-GoId, BL21 (DE3), TOP10P (both Invitrogen) and JM109 (Promega) and their derivatives containing plasmids were grown at 37°C in Luria-Bertani broth with agitation or on Luria-Bertani agar supplemented, where appropriate, with ampicillin (100 μg/ml), spectinomycin and streptomycin (100 μg/ml each) or kanamycin (50 μg/ml). S. pneumoniae serotype 2 strain D39X; serotype 4 strain TIGR4; the unencapsulated TIGR4 derivative, T4R; and 20 clinical isolates (collection of ET) were grown on tryptic soy agar (Difco, Detroit, Ml) plates supplemented with 3% defibrinated sheep blood or in defined semisynthetic casein liquid media supplemented with 0.5% yeast extract (1, 2). The choline-binding protein A mutant (CbpA^~) forms of unencapsulated T4R and wild-type T4 were created as described previously (3), and chloramphenicol (50 μg/ml) and erythromycin (1 μg/ml) (Sigma, St. Louis, MO) were added to the growth media as appropriate. N. meningitidis strain MC58, its mutant derivatives and 70 clinical isolates (collection of DAA) and H. influenzae strains Rd and ATCC 10211 , their derivatives and 38 clinical isolates (collection of DAA) were cultured on chocolate agar (Oxoid) at 37⁰C in 5% CO₂. For selection of mutants, meningococcal cells were cultured on Mueller-Hinton agar plates supplemented with Vitox (Oxoid) and, where appropriate, streptomycin and spectinomycin (each 100 μg/ml) or kanamycin (100 μg/ml). All mutants retained wild-type growth rates (data not shown). The ompP2 mutant of H. influenzae strain Rd was a gift from Joachim Reid (4). Liquid cultures of N. meningitidis were grown in Mueller-Hinton broth supplemented with Vitox at 37°C with agitation.

DNA manipulation. Chromosomal DNA was prepared by using a DNeasy tissue kit

(Qiagen) and the protocol for bacterial cells recommended by the manufacturer. Plasmid DNA was prepared by using a QIAprep spin kit (Qiagen) according to the manufacturer's recommendations. Restriction enzymes were purchased from New England Biolabs or Fermentas and used according to the directions of the manufacturer. T4 DNA ligase was purchased from Boehringer Mannheim. Expand Taq DNA polymerase (Roche Diagnostics GmbH) was used in all PCR reactions. Unless otherwise stated, PCR reactions contained 100 ng of chromosomal DNA or 1 ng of plasmid DNA. All reactions contained each primer at 200 nM; 200 μM concentrations each of dATP, dCTP, dGTP, and dTTP; Expand polymerase buffer (including MgCI₂ to a final concentration of 1.5 mM); and 0.6 U of Expand polymerase in a total volume of 25 μl. After initial incubation at 95⁰C for 3 min, reactions comprised 30 cycles of incubation at 50-55⁰C for 1 min, 65- 68⁰C for 1 to 6 min, and 94⁰C for 1 min, with final incubations at 50-55⁰C for 1 min and 65-68°C for 10 min. Products of restriction enzyme digestion and PCR were analyzed by agarose gel electrophoresis. Mutagenesis of N. meningitidis. The porA gene (NMB1429) and flanking DNA was amplified from the chromosomal DNA of strain MC58 using primers PorA-M1 (ATCAGAAACCTAAAATCCCGTCAT) and PorA-M2

(TCCTCTGTTTTGAAACCCTGAC). The amplicon was cloned into pGEM-T Easy and subjected to inverse PCR mutagenesis with the primers PorA-M3 (GCGGGATCCTCGGGCAAACACCCGATAC) and PorA-M4 (GCGGGATCCATCGGGGCGGTGAAGC). This procedure resulted in removal of the porA coding sequence and introduction of a unique BamHl site, which was used to introduce a Ω cassette (encoding resistance to spectinomycin and streptomycin) (5). The resulting plasmid was used to mutate the meningococcal strain MC58 by natural transformation and allelic exchange as described previously (6). The deletion in the resulting mutant (MC58por/\) was confirmed by PCR analysis. The pilQ gene (NMB1218) and its flanking sequence were amplified from chromosomal DNA of strain MC58 by PCR using the primers PiIQFI (GCCGTCTGAAACAGCTGCCGACAGATGC) and PiIQRI (AAACCAGTACGGCGTTGCCTCGC). The amplicon was cloned into the plasmid pGEM-T Easy (Promega) and subjected to inverse PCR mutagenesis with the primers PΪIQF2 (CGCGGATCCCTTTCACCGTAACCTCAATCGC) and PHQR2 (CGCGGATCCCTGTAATGTTTCCTGCCGATGC). This procedure resulted in deletion of the ORF and introduction of a unique BamHl site that was used to introduce the kanamycin resistance cassette digested with BamHl from plasmid pJMK30 (7). The resulting plasmid was used to mutate both the wild-type strain MC58 and the porA mutant, yielding strains MC58p//Q and MC58p//Q, porA, respectively. Successful mutagenesis of the pilQ gene in both mutants was confirmed by PCR and immunoblot analysis. All mutants showed growth equivalent to wild type (data not shown).

Purification of recombinant laminin receptor (rLR). E. coli strain BL21 (DE3) containing plasmid pET28LRP was cultured in LB containing ampicillin overnight at 37⁰C with agitation. Cells were harvested by centrifugation; the pellet was washed in PBS containing 0.05% v/v Tween 20 (PBS/T) and centrifuged at 6,500 * g for 5 min. The supernatant was replaced with fresh PBS/T. This step was repeated 3 times before cells were sonicated in an ice bath for 15 cycles of 10 s with 15 s of cooling. Samples were solubilized in 1% SDS sample buffer and the suspension was incubated at 37°C for 30 min. The lysate was separated by SDS-PAGE using a ready-made 7.5% polyacrylamide preparative gel (Bio-Rad), stained with SimpleBlue™ SafeStain™ (Invitrogen) for 30 min and de-stained in dH₂O overnight. A vertical section of the gel was removed for immunoblot analysis and the band containing rLRP was excised from the remaining gel, cut into small cubes and placed in dialyzer midi D-tubes (Calbiochem) in 1 ml PBS/T. The protein was eluted in SDS-PAGE running buffer at 100 V for 2 h, the current was reversed for 2 min, and the eluate was then collected by pipetting. The gel was discarded and the eluate was put back into the D-tube for dialysis against 1 liter of PBS/T at 4°C for at least 24 h.

Colony immunoblotting. Bacterial strains T4R, H. influenzae and N. meningitidis were streaked onto agar plates and grown overnight at 37°C. CbpA-/T4R was used as a negative control. PVDF membrane was activated in methanol, rinsed with water and overlaid on the colonies. Blots were removed, blocked in 5% milk and probed overnight with polyclonal anti- CbpA (1:2000). Binding was detected with anti-rabbit IgG-horseradish peroxidase (1 :10,000; BioRad)and Supersignal chemiluminescent substrate.

Identification of H. influenzae and N. meningitidis LR ligands by retagging. Bacterial laminin receptor-binding proteins were purified as previously described for the H. pylori SabA adhesion protein (13), with some modifications. N. meningitidis, H. influenzae and S. pneumoniae were incubated with purified rl_R to which the Sulfo-SBED cross-linker (Pierce, Rockville, IL.) had been conjugated according to the manufacturer's recommendations. The photo-reactive cross-linker group was activated by 2 min of UV irradiation, and the biotin-(re)tagged proteins were purified with streptavidin-coated magnetic beads as described previously (13). Extracted biotin-tagged proteins were separated by SDS-PAGE and bands were digested with sequencing-grade trypsin (Promega) and analyzed using a Micromass Tof-Spec E (Micromass, Manchester, England). The nanoflow LC-MS/MS was done on a 7-Tesla (Linear Trap Quadrupole- Fourier Transform) LTQ-FT mass spectrometer (Thermo Electron) equipped with a nanospray source modified in-house. The spectrometer was operated in data-dependent mode, automatically switching to MS/MS mode. MS-spectra were acquired in the FTICR, while MS/MS-spectra were acquired in the LTQ-trap. For each scan of FTICR, the three most intense, doubly or triply charged, ions were sequentially fragmented in the linear trap by collision-induced dissociation. All the tandem mass spectra were searched by MASCOT (Matrix Science, London) against the databases for the N. meningitidis strain MC58, H. influenzae strain Rd KW20 and S. pneumoniae strain TIGR4 as appropriate.

ELISA rLR. Purified rLR or BSA (5-50 μg/ml) diluted in carbonate buffer (150 mM; 142 mM NaHCO₃, 8 mM Na2SO₃, pH9.0) was used to coat amino-reactive 96-well microtiter plates (Immoblizer Amino; NUNC) for 2 h at room temperature. Bacterial strains were grown in liquid culture and washed three times in PBS/T (0.05% Tween 20 in PBS) before being resuspended in carbonate buffer to an optical density of 0.1 at 600 nm. 10 μg of digoxigenin (Roche) per 1 ml bacterial suspension was added and the suspensions were incubated for 2-4 h at room temperature. Liquid was removed from the plates, plates were washed three times with PBS/T, 100 μl of bacterial suspensions were added to each well and plates were incubated for 2-4 h at room temperature. Plates were washed several times with PBS/T and incubated with polyclonal anti-digoxigenin Fab fragment (POD)-conjugated antibody (1 :5000; Roche) in 1% BSA in PBS/T (blocking buffer) at 100 μl per well. Plates were incubated at room temperature for an additional 1 h and washed several times as above. 100 μl ABTS-tablets (5 mg/ml) (Roche) were added to each well and the absorbance was measured at 405 nm after 30 min using an ELISA plate reader. Inhibition assays were performed as described above except that bacteria were pre-incubated with 20 μg/ml rLR for 2 h at room temperature and washed twice before being added to the ELISA plates. In the reverse inhibition assay, wells were coated with either N. meningitidis or H. influenzae OD₆₀₀ 0.1, and then incubated with digoxigenin-labeled rLR. In this experiment, rLP was pre-incubated with rPorA, rPilQ, rOMPP2 or nothing. Wells coated with BSA or ethanolamine were used as negative controls.

Confocal Microscopy. Bacterial suspensions were adjusted to an optical density of 0.1 at 600 nm and 1 ml of suspension was labeled with 10 μg of carboxylic fluorescein diacetate succinimidyl ester (CSFC; Molecular Probes/lnvitrogen) for 10 min at room temperature, after which the cells were harvested by centrifugation and washed in 1 ml of PBS/T. Purified rLR was labeled with Cy5 (Amersham Pharmacia Biotech) according to the manufacturer's recommendations. CSFC-labeled bacterial cells were incubated with Cy5-labeled rLRP for 2-4 h. Cells were washed 2 times in PBS/T and transferred to the confocal plates. Images were captured using a Zeiss LSM510uv META Combi confocal system on a Zeiss Axioverti 00 microscope with a Zeiss C-Apochromat 63x/1.2W objective lens. Sequential scanning with an argon 488-nm laser was used to excite the CSFC with a 505-550 nm band pass filter and a HeNe 633 nm laser with a 650 nm long pass filter for the Cy5. The image size was 1024 x 1024 pixels with 4 averages taken per line and a zoom of 3. All images were captured with the same laser, gains and zoom settings. Colocalization analysis was performed using the Zeiss LSM software and its pixel spreadgram function. Fluo-4 labeling and detection of Calcium signaling. Human brain microvascular endothelial cells (Sciencell) were grown to 70% confluence in D-MEM (Invitrogen) supplemented with 10% fetal bovine serum (Biosera) in Mattek coverslip based Petri dishes. Following activation for 1 h with TNFα (10ng/ml), cells were incubated with media containing 1 μM Fluo-4™AM (Invitrogen) and 2.5 mM Probenecid (Sigma) for 45 min at 37°C. They were washed in HEPES buffered saline containing 2.5 mM probenecid and left for 30 min before imaging. For signaling, 4 μg/ml recombinant protein or CbpA peptide (residues 379-408: RLEKIKTDRKKAEEEAKRKAAEEDKVKEK as negative control or residues 414-443: KCELELVKEEAKEPRNEEKVKQAKAECESK as the LR binding region; Hartwell Center for Biotechnology at St Jude Children's Research Hospital) was added 10 s after the start of imaging. Cells were imaged at room temperature with a Zeiss 20x/0.5 NA objective and emission on a Zeiss Axio Observer D1 inverted microscope with a Yokagowa confocal spinning disk system controlled through Improvision Volocity software. Fluo4 was excited with a 491 nm DPSS laser and emission collected with a LP 520nm filter. Capture rate was at either 30 or 12 frames/sec. To examine the inhibitory effect of CbpA peptides, endothelial monolayers were pre-incubated with 4 μg/ml peptide for 2 h before labeling with Fluo-4.

Results

Interaction of meningococcus and H. influenzae with LR.

To determine whether meningococcus and H. influenzae bind LR, we measured bacterial binding to immobilized recombinant LR in the presence and absence of soluble LR by ELISA. Both bacteria bound immobilized LR; in each case, binding was drastically reduced in the presence of soluble rLR (p<0.001 ; Fig. 9A). Binding to LR was conserved among clinical isolates: 68 of 70 meningococcal strains and all 38 H. influenzae strains bound LR in ELISA (data not shown). Because murine cells are deficient in CD46, the endothelial receptor targeted by the type IV pilus major adhesion of the meningococcus, we had the opportunity to study bacterial adherence to LR in the absence of interaction with CD46. Adherence of both pathogens to rBCEC₆ cells was partially reduced by anti- LR antibody (meningococcus, 57%±7% of serum control, p=0.003; H. influenzae, 73%+20%, p=0.03) and by LR peptide 263-282 (45%±8%, p=0.001 and 64%±17%, p=0.007, respectively). We used retagging, a contact-dependent cross-linking approach (20), in conjunction with MALDI-TOF to identify surface molecules of meningococcus and H. influenzae that bind LR. This approach independently verified CbpA binding to LR (data not shown). Both the piius secretin protein PiIQ (NMB1812) and the major outer membrane porin PorA (NMB1429) were identified as meningococcal LR-binding surface proteins by this method (Fig. 12a). When the same retagging approach was applied to H. influenzae, the porin OmpP2 (P2) was identified (21) (Fig. 12b). Binding for both species was confirmed by using biotin-labeled cross-linked proteins purified with magnetic beads (data not shown). PiIQ and PorA were shown to bind LR specifically and independently, as PorA was detected in lysates of the pilCT mutant (Fig. 12a, lane 2) and PiIQ was detected in lysates of the porA^~ mutant (not shown; the protein complex was too large to enter the resolving gel).

When both the porA and pilQ genes were inactivated, LR binding was reduced to <7% of wild-type in ELISA (p=0.005) (Fig. 9b); single mutants bound LR at levels similar to the wild-type (data not shown). Wild-type H. influenzae bound well to LR-coated ELISA plates, whereas the ompPT mutant showed dramatically less adherence (p=0.005) (Fig. 9b). Conversely, binding of LR to plates coated with meningococcus was inhibited by recombinant PiIQ and PorA in a dose-dependent fashion (Fig. 12C). Cy5-LR bound strongly to wild-type meningococcus, while pilQT and porA^~ mutants were labelled to a lesser degree and the double mutant was virtually unlabeled (Fig. 9c). Wild-type H. influenzae was strongly labelled with Cy5-LR, whereas ompPT mutant was labelled to a much lesser degree (Fig. 9d).The physiologic relevance of PorA- and PilQ-mediated adherence was demonstrated by binding of beads bearing these proteins in the cranial window assay (Fig. 9e).

Shared binding to LR When LR expression was reduced by transfection of rBCEC₆ cells with LR siRNA, pneumococcal binding was reduced to 53% ± 8% of the binding of controls transfected with irrelevant RNA (Fig. 10b); the binding of CbpA^" bacteria was unaffected. Inhibition of LR expression by siRNA decreased adherence of wild-type meningococcus (67%±5%), but not that of the PorA^" and PiIQ^" mutants, to endothelial cells (Fig. 10b). LR siRNA also decreased the adherence of wild-type H. influenzae (64%±8%), but not that of the ompP2^~ mutant, to endothelial cells (Fig. 10b).

Not only did the three meningeal pathogens target LR, but they also appeared to bind the same domain of LR, as defined by peptides and antibodies developed previously to characterize LR interactions with laminin (Fig. 10a). Adherence not only to rat but also to human brain microvascular endothelial cells was inhibited by the laminin peptide 263- 282 but not by 161-182 or by a scrambled peptide 263-282 (Fig. 10c). Inhibition of the meningococcus to the host cells by peptide 263-282 was not highly significant, due possibly to the combined affinity of PorA and PiIQ which may bind to additional LR motifs. Adherence of H. influenzae and meningococcus to activated rBCEC₆ cells was reduced by pre-incubation of the cells with rCbpA (49%±12% and 46%±12%, respectively). Conversely, pre-incubation of rBCEC₆ cells with H. influenzae or meningococcus reduced pneumococcal adherence to 39%±21% and 68%±17%, respectively, of control values. Anti-CbpA antibody bound pneumococcus, H. influenzae and meningococcus in colony blot assays (Fig. 1Od) and blocked adherence of the three pathogens to cells (Fig. 1Oe). These results suggested that, although the adhesins have diverse primary sequences, a similar tertiary structure may underlie their common binding affinity for the LR carboxyl terminus.

The bacterial LR-ligands induce cell signaling.

LR causes calcium influx into the cell after interaction with its ligands (22). To determine whether the identified bacterial LR ligands could induce similar cell-signalling events, TNFα stimulated human brain microvascular endothelial cells were loaded with the intracellular calcium indicator FLUO4 and then exposed to recombinant PorA, PiIQ, OmpP2 or CbpA peptides either in soluble form (Fig. 11) or as coated beads (data not shown). Confocal microscopy of these cells confirmed that addition of recombinant PiIQ, PorA, OmpP2 or CbpA peptide 379-408 (encompassing the LR-binding domain of CbpA; Fig. 11e, g, i, c respectively) caused a transient increase in calcium in the endothelial cells. Addition of CbpA peptide 414-443 (a non-LR-binding segment of CbpA) failed to induce any increase in calcium levels (Fig 11d). Importantly, the transient Ca²⁺ wave evoked in response to recombinant PiIQ, PorA and OmpP2 proteins could be inhibited by pre-treatment of the cells with CbpA peptide 379-408 (Fig. 11f, h, j), providing additional confirmation that the binding of the bacterial ligands was specific and functionally cross-reactive.

Table 1

The sequence of Overlapping peptide related to loop 4 from PorA with inhibitory effect in Ca2+ signal transduction mediate by rPorA, rPilQ (N. menigitidis) and rP2 (Haemofilus influenzae):

PP18

G F S G S V Q F V P I Q N S K S A Y T P Best inhibitory effect PP19

P I Q N S K S A Y T P A Y Y T K D T N N Moderate-low inhibitory effect

PP21

T L V P A V V G K P G S D V Y Y A G L N Best inhibitory effect

References for Methods

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13. J. Mahdavi et al., Science 297, 573 (2002).

Claims

1. An antigenic polypeptide, or variant thereof, encoded by an isolated nucleic acid sequence selected from the group consisting of: i) a nucleic acid sequence as shown in Figures 1 to 7; ii) a nucleic acid sequence which hybridises to the sequence identified in (i) above; and iii) a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) or (ii) for use as a medicament.

2. An antigenic polypeptide, or variant thereof, as claimed in claim 1 wherein the nucleic acid sequence in (i) is as shown in Figures 1 to 3.

3. An antigenic polypeptide, or variant thereof, as claimed in claim 1 wherein the nucleic acid sequence in (i) is as shown in Figure 4.

4. An antigenic polypeptide, or variant thereof, as claimed in claim 1 wherein the nucleic acid sequence in (i) is as shown in Figure 5.

5. An antigenic polypeptide, or variant thereof, as claimed in claim 1 wherein the nucleic acid sequence in (i) is as shown in Figure 6.

6. An antigenic polypeptide, or variant thereof, as claimed in claim 1 wherein the nucleic acid sequence in (i) is as shown in Figure 7.

7. An antigenic polypeptide as claimed in any one preceding claim wherein the medicament is a vaccine.

8. An antigenic polypeptide as claimed in any preceding claim wherein the nucleic acid encoding the antigenic polypeptide anneals under stringent hybridisation conditions to the nucleic acid sequence shown in Figures 1 to 7 or to its complementary strand.

9. An antigenic polypeptide as claimed in claim 1 wherein the antigenic polypeptide comprises the amino acid sequence shown in Figure 1 to 7 or a variant sequence thereof.

10. A vector comprising a nucleic acid sequence encoding an antigenic polypeptide as claimed in any one of claims 1 to 6.

11. A method for the production of a recombinant antigenic polypeptide as claimed in any one of claims 1 to 6 comprising:

(i) providing a cell transformed/transfected with a vector according to claim 10; (ii) growing said cell in conditions suitable for the production of said polypeptides; and (iii) purifying said polypeptide from said cell, or its growth environment.

12. A cell or cell-line transformed or transfected with a vector according to claim 10.

13. A vaccine comprising at least one antigenic polypeptide, or variant thereof, as claimed in any one of claims 1 to 6.

14. A vaccine as claimed in claim 13 wherein the antigenic polypeptide, or variant thereof, is encoded by isolated nucleic acid sequences selected from the group consisting of: i) (a) a nucleic acid sequence selected from Figures 1 to 6; and

(b) the nucleic acid sequence shown in Figure 7; ii) nucleic acid sequences which hybridise to the sequences identified in (i) above; and iii) nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequences defined in (i) or (ii).

15. A vaccine as claimed in claim 14 wherein the antigenic polypeptides, or variant thereof, is encoded by isolated nucleic acid sequences selected from the group consisting of: i) (a) a nucleic acid sequence as shown in Figure 5 and/or 6; and

(b) a nucleic acid sequence as shown in Figure 7; ii) nucleic acid sequences which hybridise to the sequences identified in (i) above; and iii) nucleic acid sequences that are degenerate as a result of the genetic code to the nucleic acid sequences defined in (i) or (ii). 36

16. A vaccine as claimed in any one of claims 13 to 15 wherein the vaccine further comprises a carrier and/or adjuvant.

17. A vaccine as claimed in any one of claims 13 to 16 wherein the vaccine is a subunit vaccine in which the immunogenic part of the vaccine is a fragment or subunit of the antigenic polypeptide according to any one of claims 1 to 6.

18. A method to immunise an animal against a pathogenic microbe comprising administering to said animal at least one antigenic polypeptide, or part thereof, according to any one of claims 1 to 6.

19. A method as claimed in claim 18 wherein the polypeptide is in the form of a vaccine according to any one of claims 13 to 17.

20. A pharmaceutical composition comprising an effective amount of at least one of the antigenic polypeptides as claimed in any one of claims 1 to 6, or a vaccine as claimed in any one of claims 13 to 17, in combination with a pharmaceutically acceptable carrier or diluent.

21. An antibody, or active binding fragment thereof, which binds at least one antigenic polypeptide, or variant thereof, according to any one of claimsi to 6.

22. An antibody as claimed in claim 21 wherein the antibody is a monoclonal antibody.

23. A hybridoma cell line which produces a monoclonal antibody as claimed in claim 22.

24. An antibody as claimed in claim 21 or 22 wherein the antibody is a chimeric antibody.

25. An antibody as claimed in claim 21 or 22 wherein the antibody is a humanised antibody comprising the complimentarity determining regions of said antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.

26. A vector comprising a nucleic acid sequence encoding a chimeric antibody according to claim 24 or a humanised antibody according to claim 25.

27. A cell or cell line transformed or transfected with the vector of claim 26.

28. A method for the production of a humanised or chimeric antibody comprising: i) providing a cell transformed or transfected with a vector according to claim 26; ii) growing said cell in conditions suitable for the production of said antibody; and purifying said antibody from said cell, or its growth environment.

29. A method for preparing a hybridoma cell-line comprising the steps of: i) immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide having an amino acid sequence as represented in Figures 1 to 7, or fragments thereof; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step (ii) for binding activity to the amino acid sequences of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and v) recovering the monoclonal antibody from the culture supernatant.

30. Use of an antigenic polypeptide as claimed in any one of claims 1 to 6 in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection or a bacteria-related disorder.

31 . Use as claimed in claim 30 wherein the bacterial infection is caused by a bacterial pathogen belonging to a bacterial species selected from the group consisting of: Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae and Neisseria meningitidis (meningococcus).

32. Use as claimed in claim 31 wherein the bacterial infection is caused by Neisseria meningitidis (meningococcus).

33. Use as claimed in claim 31 or 32 wherein the bacteria related disorder is a meningococcal-associated disorder.

34. Use as claimed in claim 33 wherein the meningococcal-associated disorder is meningitis (meningococcal meningitis) or septicaemia.

35. Use of an antibody as claimed in claim 21 or 22 in the manufacture of a medicament for the treatment of a bacterial infection.

36. A method of treating a patient comprising administering to the patient an antigenic polypeptide as claimed in any one of claims 1 to 6, or a vaccine as claimed in any one of claims 13 to 17, or an antibody as claimed in claim 21 or 20.