Classifications

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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
  • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

• CEACAMs Carcinoembryonic antigen related cell adhesion molecules
• CEACAMs are receptors for pathogens of mucosal membranes, especially for respiratory pathogens such as Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis.
• CEACAMs belong to the CarcinoEmbryonic Antigen (CEA) family, a member of the Immunoglobulin superfamily.
• CEA gene family comprises surface expressed (CEA) and secreted (pregnancy-specific glycoprotein, PSG) subfamilies.
• the membrane-associated sub-family redefined as CEACAM (CEA- related cell adhesion molecule) 20 comprises several related glycoproteins of which CEACAM1 is the most widely expressed in distinct human tissues 12 .
• the studies reported by the inventors primarily used Chinese Hamster Ovary (CHO) cells transfected with CEACAM 1 (previously termed CD66a and BGPc) that contains four extracellular domains, a TM region and a short (S) or a long (L) cytoplasmic tail (molecular formula: NA1BA2-TM-S or L).
• soluble truncated constructs containing one or more of the extracellular domains were used.
• bacteria may bind to CEACAMs on phagocytic cells and T and B lymphocytes. Such interactions may lead to bacterial cell death 8 , target cell death or inhibition of immune function, e.g. of T and B lymphcytes when N. gonorrhoeae (closely related to N. meningitidis) binds to CEACAMs of these lymphocytes 21,22 .
• CEACAM-binding ligands in Moraxella catarrhalis and Haemophilus influenzae has been a surprising find to the present inventors since CEACAMs have long been associated with outer membrane opacity- associated Opa proteins of Neisseriae and neither Haemophilus influenzae nor Moraxella catarrhalis produce Opa proteins.
• the present invention will be described with particular reference to infection of the mucosal membranes, especially of the respiratory membranes, or to infection of the ear (especially otitis media) but it is to be understood that the invention finds equal utility in other areas such as the genital mucosa or the urethrae where CEACAM receptors are implicated in infection or other receptor-binding processes or elsewhere in human infection where bacteria may become disseminated from mucosal surfaces.
• the mucosa! pathogens Neisseria meningitidis (Nm), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mx) are human specific organisms and reside in the upper respiratory tract from where they may disseminate to cause serious infections.
• Meningococcal strains of distinct serogroups may be carried within the nasopharynx of up to 25% of healthy individuals 1 .
• the organism invades the mucosal barrier to cause one of the most rapidly advancing and extremely serious diseases.
• the precise factors that increase host susceptibility to meningococcal infection are not fully understood.
• the limited protection afforded by group-specific vaccines and the non-immunogenicity of the group B polysaccharide underscore the need for fundamental studies to understand host susceptibility and identify salient sub-capsular features that could serve as common targets to combat meningococci.
• NTHi non-typable Hi
• Otitis media is one of the major problems in paediatric medicine and NTHi are responsible for over 20% of episodes in children during the first year of life 2,s .
• NTHi are also associated with acute recurrent and persistent infections in patients with chronic obstructive pulmonary disease (COPD) and cystic fibrbsis.
• COPD chronic obstructive pulmonary disease
• Moraxella catarrhalis another resident of the human respiratory tract, is often isolated from cases of localised infections together with Hi. Both organisms are associated with sinusitis and exacerbations of asthmatic conditions 5 ' 6 . Mx is the third most common cause of otitis media in children (estimated to be responsible for 3-4 million cases annually). It also causes lower respiratory tract infections in adults especially in patients with COPD 5 . On rare occasions, it has been associated with disseminated infections 5 . Both Hi and Mx cause persistent infections and are believed to escape host immune mechanisms and antibiotics by tissue penetration .
• Nm pili are long filamentous protein structures composed of multiple pilin subunits. They are generally regarded as the most important adhesins in capsulate bacteria 13,14,16 due the fact that capsule partly or totally masks outer membrane ligands resulting in their reduced functional efficacy, whilst pili traverse the capsule and remain functional in fully capsulate bacteria.
• Opa are antigenically variable family of proteins and occur in N. meningitidis as well as N. gonorrhoeae. In meningococci, 3 - 4 opa gene loci code for related transmembrane proteins with four surface exposed loops, three of which undergo sequence- variation 16,17 . Ope, another trans-membrane protein, is largely invariant 15,16 . Over the last 12 years, the present inventors have investigated structure/function relationships of Nm pili, the virulence potential of Opa and Ope proteins and identified two human receptors for the neisserial opacity proteins.
• the present invention results from the identification by the inventors of a ligand of high molecular weight isolated from Moraxella outer membrane protein which binds to CEACAM receptors.
• the ligand can be characterised by its SDS-PAGE migration pattern which is indicative of the USP-family of proteins in that it is broken down to monomers having a molecular weight of between approximately 60 and 150 kD when boiled for a prolonged period.
• the ligand has been characterised in Mx strain ATCC 25238 (MX2) as UspA1 and its amino acid sequence determined.
• the ligand has been further characterised to determine the receptor binding region or domain, i.e. peptide or peptide-associated features that bind to the receptor.
• the present invention provides a ligand isolated from Moraxella catarrhalis outer membrane protein which binds to CEACAM receptors, wherein said ligand is a polypeptide comprising or consisting of a receptor binding domain comprising or consisting of an amino acid sequence selected from the group consisting of residues 463 to 863, 527 to 623, 527 to 668, 527 to 863, 427 to 623, 427 to 668, and 427 to 863 of the sequence shown in Figure 6, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof.
• the ligand is a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of residues 527 to 623, 527 to 668, and 427 to 623 of the sequence shown in Figure 6, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof.
• the term ligand is used herein to denote both the whole molecule which binds to the receptor and any part thereof which includes the receptor binding domain such that it retains the receptor binding property.
• ligand encompasses molecules which consist only of the receptor binding domain i.e. the peptide region or regions required for receptor binding.
• the polypeptide comprises or consists of at least one of the conserved sequences from within the region 427 to 623 of the sequence shown in Figure 6 which are identified in the alignment shown in Figure 27.
• the polypeptide comprises or consists of at least one of: QHSSDIKTLK, NVEEGLLDLSGRLIDQKADLTKDIK, NVEEGLLDLSGRLIDQKADIAKNQA, DIAQNQT, DIQDLAAYNELQD, QTEAIDALNKASS, TAELGIAENKKDAQIAKAQANENKDGIAK, NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK, NQADIANNIKNIYELA, NQADIANNI, NIYELA.
• polypeptide ligands of the invention can comprise a receptor binding domain of sequence recited herein which is modified by the addition or deletion of amino acid residues to or from the sequences recited herein at either or both the N or C termini, which modified peptides retain the ability to bind CEACAM receptors.
• the invention further provides a ligand comprising or consisting of a polypeptide in which 50, 40, 30, 20, 10, 5, 3 or 1 amino acid residues have been added to or deleted from an amino acid sequence recited herein at either or both the N or C termini, wherein said modified polypeptide retains the ability to bind CEACAM receptors and/or elicit an immune response against the non- modified peptide.
• the amino acid at position 560 is retained in the modified peptide.
• any size fragment may be used in the invention provided that the fragment retains the ability to bind CEACAM receptors. It may be desirable to isolate a minimal peptide which contains only those regions required for receptor binding.
• Polypeptide ligands according to the invention may be derived from known Moraxella catarrhalis UspA1 proteins by truncation at either or both of the N- and C- termini.
• the invention further provides a wild-type UspA1 sequence lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160 etc to 520 amino acids from the N-terminus, and/or lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180 or 200 amino acids from the C-terminus.
• the truncate retains CEACAM binding function. Possible truncates may be selected from those shown in the following table, all of which are within the scope of the invention.
• Known wild-type UspA1 sequences that may be truncated in this way are those of strains ATCC25238 (MX2; GenBank accession no. AAD43465), P44 (AAN84895), O35E (AAB96359), TTA37 (AAF40122), O12E (AAF40118), O46E (AAF36416), V1171 (AAD43469), TTA24 (AAD43467) (see Example 10, Table II).
• the UspA1 truncate of this embodiment comprises or consists of an amino acid sequence selected from the group consisting of residues 463 to 863, 527 to 623, 527 to 668, 527 to 863, 427 to 623, 427 to 668, and 427 to 863 of the sequence shown in Figure 6, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof; or comprises or consists of at least one of the conserved sequences from within the region 427 to 623 of the sequence shown in Figure 6 which are identified in the alignment shown in Figure 27, for example: QHSSDIKTLK, NVEEGLLDLSGRLIDQKADLTKDIK, NVEEGLLDLSGRLIDQKADIAKNQA, DIAQNQT, DIQDLAAYNELQD, QTEAIDALNKASS, TAELGIAENKKDAQIAKAQANENKDGIAK, NQADIQLHDKKITNL
• the invention provides fusion proteins comprising polypeptide ligands according to the invention.
• a fusion protein according to this embodiment is less than 50%) identical to any known full length sequence over its entire length.
• Homologous peptides may be identified by sequence comparison.
• Homologous peptides are preferably at least 60% identical, more preferably at least 70%, 80%, 90%, 95% or 99% identical in ascending order of preference to the peptide sequences disclosed herein or fragments thereof over their entire length.
• the homologous peptide retains the ability to bind CEACAM receptors and/or elicit an immune response against the peptide sequences disclosed herein or fragment thereof.
• the amino acid at position 560 or homologous position is lysine.
• Figure 20 shows an alignment of peptide sequences of different origin which indicates regions of sequence that are capable of being modified whilst retaining function (i.e. CEACAM binding ability).
• the inventors have determined that the CEACAM binding ability of the peptide ligand is associated with an -helical based conformation as determined by circular dichroism (CD) spectroscopy, as opposed to a random coil structure.
• CD circular dichroism
• the peptide ligand or receptor binding domain of the invention or fragment or homologue or other derivative thereof adopts an ⁇ -helical structure.
• the structure is a coiled coil structure.
• CD spectroscopy is performed as described in the accompanying examples.
• the invention provides peptides which are structurally homologous to the peptides disclosed herein or fragments thereof.
• a structurally homologous peptide is a homologous peptide as described above which gives a circular dichroism (CD) spectroscopy trace indicative of an ⁇ -helical based conformation as shown in Figure 18.
• CD circular dichroism
• Mimotopes may comprise D-amino acids or non-natural amino acid substitutions but still retain the functional characteristics of the peptides disclosed herein, including a CD trace indicative of an ⁇ -helical based conformation.
• the ⁇ -helical based conformation revealed by the inventors indicates that the peptides disclosed herein possess a globular sub-unit structure that does not rely on an associated membrane to achieve the appropriate conformation for CEACAM binding.
• the invention provides a globular sub-unit molecule comprising the peptide ligand or receptor binding domain of the invention which is not a full-length UspA1 protein and which is capable of binding CEACAM receptors without the need for an outer membrane to be present.
• the globular sub-unit molecule comprises less than 200 amino acids, more preferably less than 100 amino acids.
• Structurally and/or functionally equivalent receptor binding domains may also occur in other UspA-like proteins since hybrid proteins occur in Mx that may contain mosaic epitopes derived from both UspA1 and UspA2 proteins 23 .
• Ligands comprising such equivalent receptor binding domains are also within the scope of the invention.
• the CEACAM binding property of the peptide ligand means that it has utility as both an antigen (i.e. in a vaccine) and as an "antibiotic" whereby it is administered in order to block CEACAM binding and so prevent binding and entry of the pathogen.
• the ligand or receptor binding domain is preferably suitable for use in the prevention or treatment of infection.
• the present invention also provides a nucleic acid sequence encoding the ligand protein of the present invention together with homologues, fragments, polymorphisms, degenerates and splice variants thereof.
• the ligand of the invention, or combinations thereof may be used in a vaccine or other prophylactic treatment of infection.
• the vaccine or other prophylactic treatment may comprise any known adjuvant, vehicle, excipient, binder, carrier, preservatives and the like, to provide a pharmaceutically acceptable preparation of the ligand for use in the treatment of a patient.
• the invention also provides a pharmaceutically acceptable preparation of the ligand for use in medicine.
• the pharmaceutically acceptable preparation of the ligand may be used in the treatment or prevention of any disease where CEACAM receptors are implicated, for example in the treatment or prevention of infection, respiratory disease, neoplastic diseases and associated conditions of neoplastic diseases, and angiogenesis.
• the infection is of, or has occurred via, the mucosal membrane, especially a respiratory infection.
• the ligand is used as a vaccine for or in other prophylaxis or treatment of Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis.
• the ligand is used as a vaccine for or in other prophylaxis or treatment of otitis media.
• the ligand of the present invention may also be used to identify novel blocking reagents for use as therapeutic agents to protect vulnerable groups and the public in general against several mucosal pathogens.
• the ligand may be used to identify receptor analogs which are useful for this purpose.
• the present invention also provides a screening assay for the identification of novel blocking reagents for use as therapeutic agents, the assay comprising the steps of screening potential therapeutic agents for their ability to mimic or for their homology to the ligand of the present invention.
• the invention further provides therapeutic agents identified by the aforementioned screening assay.
• Effective vaccine components may be produced by using the information of the receptor targeting mechanisms identified by the present invention such as biologically active peptide mimics.
• Bacterially derived biologically active peptide sequences identified by the ligand of the present invention could be used to study the roles of CEACAMs in cancer and development as the molecules are associated with these processes. These also have potential as anti-cancer agents and to control or otherwise in the treatment of angiogenesis.
• the information generated by the inventors regarding the conformation of the peptide ligand of the invention could be used to design a synthetic nano- structure e.g. from plastic. Such a structure would have the advantages of being resistant to biological degradation and non-immunogenic.
• the invention provides the use of a CEACAM receptor-binding ligand in the manufacture of a medicament for the treatment or prophylaxis of a disease in which CEACAM receptors are involved in cellular targeting of the pathogen which causes the disease, wherein the ligand comprises or consists of an amino acid sequence selected from the group consisting of residues 463 to 863, 527 to 623, 527 to 668, 527 to 863, 427 to 623, 427 to 668, and 427 to 863 of the sequence shown in Figure 6, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof.
• ligands suitable for use in this aspect of the invention are polypeptides comprising or consisting of at least one of the conserved sequences from within the region 427 to 623 of the sequence shown in Figure 6 which are identified in the alignment shown in Figure 27, for example: QHSSDIKTLK, NVEEGLLDLSGRLIDQKADLTKDIK, NVEEGLLDLSGRLIDQKADIAKNQA, DIAQNQT, DIQDLAAYNELQD, QTEAIDALNKASS, TAELGIAENKKDAQIAKAQANENKDGIAK, NQADIQLHDKKITNLGILHSMVARAVGNNTQGVATNKADIAK, NQADIANNIKNIYELA, NQADIANNI, NIYELA.
• the disease is selected from the group consisting of infection, respiratory disease, neoplastic disease and associated conditions of neoplastic disease, and angiogenesis.
• Medicaments as described above are of particular utility where the pathogen infects, or enters via, a mucosal membrane.
• Medicaments as described herein are particularly useful in the treatment or prevention of infections (disease) caused by Moraxella catarrhalis.
• ligands as described herein are useful in the manufacture of medicaments for the treatment of any disease where CEACAM receptors are implicated such as diseases caused by Neisseria meningitidis and Haemophilus influenzae.
• the disease is otitis media.
• Ligands of the invention may also be used in the treatment of diseases caused by other oral bacteria, such as dental caries.
• the oral bacterium Fusobacterium nucleatum is associated with gum disease but has also been linked with otitis media, still births and in rare cases with bacteraemia.
• Recent work by the inventors has shown that several isolates of Fusobacterium nucleatum bind to CEACAMs and that binding of CEACAM 1 to F. nucleatum can be inhibited by a polypeptide ligand as disclosed herein suggesting that ligands or receptor binding domains of the invention have utility in the treatment or prevention of diseases caused by this pathogen.
• D-7 a preferred polypeptide ligand according to the invention, to block interactions of non-capsulate (not shown) or capsulate bacteria with HeLa-CC1 H (Example 7), to block binding to multiple CEACAMs and its efficacy against a number of mucosal opportunist species (Example 7), makes it an anti-microbial agent with significant potential.
• its ability to evoke antibody response that block Mx adhesion suggests its potential as a vaccine candidate, alone or as a part of a multicomponent vaccine (together with other Mx antigens such as: UspA2, Hag/MID, OMPCD, Mcap) to prevent otitis media or lung infections in which Mx is often implicated 5 .
• Vaccines based on adhesins have been successfully used, for example against uropathogenic Escherichia coli in a mouse cystitis model by systemic vaccination 29 .
• Inclusion of a ligand according to the invention as a prophylactic drug may be considered in a variety of situations where the risk of acquiring particularly virulent or antibiotic resistant strains is high and may be delivered by direct topical application or via probiotics.
• soluble carbohydrates have successfully prevented infections in in vitro and animal models 30, 31 ' 32 .
• Topical application of a synthetic peptide corresponding to a region of Streptococcus mutans protein SAI/II was shown to inhibit binding by S.
• mutans in human subjects The study used peptide at 1mg. ml "1 in a mouthwash daily for 2 weeks and this was sufficient to prevent colonization 33 .
• Probiotics in the shape of lactobacilli have been used to prevent numerous infections 34, 35 .
• a recombinant E. coli strain has been used as a probiotic in which LPS genes were modified to encode a structural mimic of the Shiga toxin receptor. Oral administration of this strain was shown to prevent death in mice from lethal challenge with shiga-toxin producing E. coli 36 .
• Topically applied interfering peptides have a further advantage in that they can be delivered when and where required by the use of transitory probiotics or by expression vectors that can be controlled for the timing or the levels of expression.
• the length of exposure to the anti-microbial agent can be controlled 37, 38 .
• Interference by targeting the binding domain of the receptor mimics bacterial adherence and is unlikely to have deleterious effect over and above that of binding of native commensal bacterial ligands.
• such specific strategy ensures tolerance towards other commensal microflora, very few of which bind to CEACAMs 7
• the monomeric and monovalent nature of the predominating form of the peptide is less likely to trigger undesirable signaling which, for CEACAMs, appears to be induced on receptor clustering 11 ' 39 .
• Scope may exist for improvement of D-7 by further identifying critical amino acids involved in CEACAM interaction and modifications to reduce its size whilst ensuring binding as well as its longevity in vivo. Such modifications could include incorporation of unnatural or D-amino acids 33 . Resistance to such anti-adhesive / anti-invasive treatments is unlikely to occur since any changes in the bacterial ligand are likely themselves to lead to a loss of function and in this case colonisation/infection.
• CD33-Fc binding in each case was negligible (black, right hand bars).
• Strains 1 , 2, 3 MX2 (ATCC 25238), MX3, MX4 (clinical isolates) respectively. Binding was determined in a dot blot overlay and the intensity of reactions quantified by densitometric analysis using NIH Scion Image program.
• Figure 2 shows a Western blot of the strain MX2 proteins separated under undissociating (unheated, lane 1 ) or after boiling for 10 min. (lane 2). Blot was overlaid with CEACAM1-Fc (1 ⁇ g.ml "1 ) and the receptor binding detected with anti-human Fc antibody conjugated to horseradish peroxidase and its substrate.
• Figure 3 shows Western blots of denatured whole cell lysates of strains MX2, -3, -4 (lanes 1-3 respectively) were overlaid with CEACAM1-Fc (1 ⁇ g. ml "1 ; a), anti-UspA1 peptide antibody (10 ⁇ g.ml " ; b) and anti-UspA2 peptide antibody (10 ⁇ g.ml "1 ; c). Note the similar migration profile of CEACAM 1-Fc binding proteins and anti-UspA1 binding proteins in the three strains. Remnants of undissociated proteins at c.
• FIG. 5 shows Western blot analysis of tryptic fragments of UspA1 of MX2.
• UspA1 -specific peptide used to raise antisera in rabbits is shown in bold.
• the CEACAM-binding region is contained in the underlined C-terminal fragment of UspA1 of MX2.
• Figure 7 shows the separation of tryptic peptides reacting with CEACAMs.
• M. catarrhalis strain MX2 was treated with 1 mg/ml trypsin at 37°C for 10 min. Trypsinised sample was subjected to SDS-PAGE. After staining, 50 kDa region was electroeluted overnight. Electroeluted protein was freeze dried and resuspended in buffer and applied to a second gel.
• Figure 8 shows the amino acid sequence of a tryptic peptide of MX2 UspAI protein (SEQ ID NO:3). The 50 kDa tryptic peptide shown (amino acids 463 to 863) binds CEACAM and antiserum against UspAI peptide (amino acids 753-780 underlined). The N-terminal sequence of the c.
• CEACAM binding peptide is "ALESNVEEGL" (SEQ ID NO:4) that occurs after the trypsin cleavage site at amino acid 462.
• Figure 9 shows a diagrammatic representation of the positions of primers used to generate uspAI gene fragments for the expression of recombinant peptides.
• the CEACAM 1 binding site was encoded by DNA amplified by primers P4 and P7, additional primers throughout this region (letters A-l) were designed and employed.
• Figure 10 shows the sequence of the recombinant fragment 4-7 (SEQ ID NO:5).
• the underlined region is the N-terminal region of the CEACAM 1- reactive tryptic peptide.
• the predicted molecular weight of the fragment 4-7 is c.
• FIG. 11 is a diagram showing the general cloning, expression and purification strategy for recombinant UspAI peptides, as exemplified by the pQE30 system.
• Figure 12 is a map of the vector pQE30.
• Figure 13 shows binding of CEACAM 1-Fc in blot overlay to recombinants 4-8 (lane 3), 4-T (lane 4) and 4-7 (lane 5) polypeptides.
• Lane 1 contained a Treponemal control recombinant peptide and lane 2 contained lysates of non- induced M15 containing 4-8 construct.
• Figure 14 shows Western blots showing recombinant peptide reactivity with anti-His tag antibody (top) and CEACAM 1-Fc (bottom).
• Lanes 2-4 contained 6-8 peptide, lane 1 contained 4-8 as a control. Predicted migration positions of the peptides are shown on the right. Both peptides bind anti-His. antibody. However, whilst 4-8 binds to CEACAM 1-Fc, 6-8 does not.
• Figure 15 shows that D-8 polypeptide binds CEACAM 1-Fc (lane 1) but not CD33-Fc used as a control (lane 2). The origin of this peptide was confirmed by reactivity with the anti-His tag antibody (lane 3).
• Figure 16 is a schematic diagram showing the relative sizes and positions of rUspAI fragments. Recombinant 4-7 has been used for bacterial blocking - see Figure 17.
• Figure 17 shows CHO-CEACAM1 transfectants were incubated in the absence of peptides (A) or with recombinant control peptide (a treponemal peptide, B) or UspAI r4-7 peptide (sequence corresponding to the strain MX2, C and D) at the concentrations shown and bacteria added for a period of 2 hours. At the end of this incubation, unbound bacteria were washed off and bound bacteria detected with anti- M. catarrhalis polyclonal antiserum and TRITC conjugated secondary antibody. At 1 ⁇ g/ml significant inhibition of a heterologous M. catarrhalis strain (MX1 ) was obtained and at 10 ⁇ g per ml, H.
• MX1 heterologous M. catarrhalis strain
• FIG 18 is a graph showing the circular dichroism spectra of recombinant D-7 peptide and D-7 with K 560 I mutation. The spectra indicate that D-7 has an ⁇ -helical structure whereas D-7 (K 560 I) adopts a random coil formation.
• Figure 19 shows a series of linear peptides spanning the D-T region which do not bind CEACAM1. The K residue corresponding to K560 of D-7 is underlined.
• Figure 20 is an alignment of the D-7 region of the amino acid sequences of the UspAI proteins of ten strains of Mx: TTA24, TTA37, p44, O12E, O35E, O46E, MX2, V1171 , MX3 and MX4.
• the top line shows the majority sequence.
• Figure 21 is a manual alignment of O35E D-7 versus MX2 D-7.
• the top line shows the majority sequence.
• Figure 22 is a table showing sequence identity of D-7 regions of Mx isolates as determined by MegAlignTM (DNASTAR Inc.).
• Figure 23 shows recombinant peptide D-7 (but not recombinant 6-8 i.e.
• Figure 24 is a series of charts showing inhibition of bacterial-CC1-Fc interactions by D-7.
• Recombinant D-7 black columns but not D-8 ⁇ (grey columns) inhibits the binding of multiple strains of both homologous and heterologous species (as indicated above each graph) to CCI-Fc.
• Strains used in a and b were: Mx (MX1 ), Nm (C751 D), Hi (THi, Rd) and Hi-aeg (A3), (c) Inhibition of Nm (C751 D) binding to distinct CEACAMs by D-7.
• HeLa cells expressing CEACAM1 (CC1), CEA or CEACAM6 (CC6) were preincubated with either no peptide, control peptide (2 ⁇ g.ml "1 ; grey columns), or D-7 (2 ⁇ g.ml "1 ; black columns).
• FIG. 27 shows adhesion of N. meningitidis to HMEC1 cells alone (A) or in the presence of control peptide (B) or the blocking peptide D-7 (C). Note the virtual complete inhibition in the presence of D-7.
• Figure 28 is a multiple alignment of fragments of known UspAI protein sequences corresponding to fragment 4-T of MX2 sequence.
• Residues that are conserved in all of the sequences analysed are shaded black. Residues that are conserved in all sequences where they are present, but there is a deletion of the corresponding position in one or more sequences are shaded dark grey. Where there is variation in the residue present at that position, the consensus residues are shaded light grey.
• Moraxella catarrhalis strains bind to human CEACAM 1-Fc via the UspAI proteins
• the strains of Moraxella catarrhalis (Mx) used in this study include clinical isolates (MX3 and MX4) as well as reference strain purchased from American Type Culture Collection (ATCC 25238, a clonal culture of this was designated MX2).
• Receptor overlay assays show that Mx bind to CEACAM 1-Fc receptor constructs.
• bacteria c. 4 - 8 x 10 6
• Nitrocellulose strips were overlaid with either CEACAM 1-Fc (1-2 ⁇ g ml "1 ) alone or in the presence of the CEACAM1 N-domain antibody YTH71.3.
• CD33-Fc 1-2 ⁇ g ml "1 ) was used as a negative control.
• Chimeric protein constructs were prepared as previously described 11 .
• ETNNRQDQKIDQLGYALKEQGQHFNNR SEQ ID NO:1 ; UspAI -peptide
• KDEHDKLITANKTAIDANKAS SEQ ID NO:6; UspA2-peptide
• Peptides were coupled to KLH via an N-terminal cysteine residue that was incorporated and were used to immunise rabbits (200 ⁇ g peptide per rabbit) at 14 day intervals, initially with complete Freund's adjuvant and thereafter with incomplete Freund's adjuvant. Rabbits were bled at day 0 and at 14 day- intervals post immunisation. Polyclonal antibodies were purified using the appropriate peptide coupled to AminoLink Plus columns (Pierce).
• UspA- specific antibodies were used at a concentration of 1-10 ⁇ g ml "1 for Western blot overlays and detected with goat anti-rabbit secondary antibody coupled to alkaline phosphatase. Blots were developed as described earlier. The migration of the CEACAM1-Fc binding proteins on SDS-PAGE of the three Mx strains was identical to that of the UspAI -antibody binding proteins but not UspA2-antibody binding proteins (Fig. 3) M.
• catarrhalis ligand co-precipitating with CEACAM1-Fc is identified as UspAI Overnight cultures of bacteria were suspended in 100 mM octyl ⁇ D glucopyranoside in PBSB containing a protease inhibitor cocktail (pic; PMSF 1 mM, E-64 1 ⁇ M, pepstatin A 1 ⁇ M, bestatin 6 nM and EDTA 100 ⁇ M). Samples were mixed end over end overnight at 4°C.
• the co-precipitated proteins were further identified as UspAI proteins since they bound to anti- UspA1 peptide antibody.
• the MX4 protein was subjected to MALDl-TOF mass spectrometry (see below).
• CEACAM 1 ligand identification by MALDI-TOF mass spectrometrv (a) Western overlay samples. Whole cell lysates of MX2 and MX3 were subjected to SDS-PAGE in trench gels and the protein band corresponding to CEACAMI-Fc binding ligand was electroeluted from the gel. The sample was concentrated and reapplied in a single lane of a second gel, subjected to electrophoresis prior to in-gel trypsin digestion of the appropriate protein. The resulting peptides were analysed by Matrix-assisted Laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry.
• MALDI-TOF Matrix-assisted Laser desorption/ionisation-time-of-flight
• 150 kDa peptides were subjected to N-terminal sequencing.
• the N-terminal sequences were ALESNVEEGL (SEQ ID NO:4) (c. 50 kDa peptide) and ALESNV (SEQ ID NO:7) (c. 150 kDa peptide).
• the 150 kDa protein is apparently a trimer of the 50 kDa protein as they have the same N-terminal sequence.
• the N-terminal sequence of this peptide of MX2 UspAI is shown in Figure 8.
• N. meningitidis and H. influenzae target human CEACAM molecules via ligands that bind to overlapping sites on CEACAM.
• the Mx ligand/s of the present invention also target the N domain.
• HV1 and HV2 hyper-variable loops of Opa
• phage display which can be used to identify mimotopes (random sequences to mimic the binding domains) 19 as well as aptamers (sequences more closely related to the original structure that inhibit ligand binding) 20 .
• the current invention also makes it highly feasible that the Mx ligand domain that binds CEACAMs will act as a mimic for other CEACAM-binding mucosal pathogens and the structural features of this ligand will help identify the salient features required in Nm and Hi ligands for CEACAM N-domain targeting.
• Antibodies to the Mx domain could have the potential of identifying other ligands that have the capacity to target the same, similar or closely positioned region of the receptor. Identification of the minimal CEACAM1 binding domain of MX2 UspAI is being undertaken using known methods of protein engineering and recombinant DNA technology.
• Recombinant peptides can be screened in vitro by receptor overlay assays described above to detect the domain of MX2 that binds to the receptor. His-Tagged peptides can be separated on a nickel column, His-Tag cleaved as required and used for immunising rabbits and mice to obtain antibodies for further investigations. A peptide of suitable length for biological applications may be determined by examining immunological stimulatory properties as well other functions such as blocking of receptor binding.
• Example 3 Salient Features of the Receptor Reguired for Ligand Interactions Since the N-domain of CEACAM is sufficient for the interactions of Opa proteins, the present inventors are using phage display technique also to investigate adhesive epitopes of the CEACAM N-domain. The knowledge of the ligand-binding region/s on the receptor, which has been studied by the present inventors by alanine scanning mutagenesis of the receptor 9 has facilitated this study. In this case also, a ligand overlay assay is available for biopanning (affinity concentration) of chimeric phages that bear receptor sequences.
• Receptor analogues have the potential to block multiple strains independently of the Opa type produced since our studies have already shown that despite their antigenic variation, distinct Opa proteins require common features on the receptor for primary adhesion 9 . It is also of interest to note that CEA antigens are shed from the gut mucosa and may block adhesion of E. coli strains also known to target CEACAMs. This has been proposed as a mechanism of innate immunity vs. enteric pathogens. 12 Thus these receptor analogues act as therapeutic agents.
• the UspAI fragment- 1-5 was produced using the pBAD system.
• the required PCR products were TA cloned into pBAD vector and TOP10 E. coli strain was used for amplification.
• the rest of the procedure was as shown in Fig. 11 with the exception that pBAD was induced using arabinose.
• the fragment 4-7 was produced using both the pBAD and pQE30 (Fig. 11) systems with similar CEACAM-binding results. The rest of the fragments were produced using the strategy in Fig. 11.
• the vector pQE30 (Fig. 12) was used in conjunction with E. coli strain M15.
• M15 contains the plasmid pREP4 encoding a repressor, which restricts transcription of DNA cloned into pQE30.
• IPTG a concentration of 1 mM prevents coding of this repressor and thus transcription of the cloned fragments in pQE30.
• PCR amplimers were first ligated into pCR2.1. This provided a stable host from which the amplimer could be recovered by restriction digest ( ⁇ a/r.HI/Ps-1), ensuring that each end of the gene fragment was cut.
• pQE30 was similarly digested and recovered by gel purification, which also ensured that both restriction sites had been cut. Digested pQE30 and UspAI amplimer were ligated overnight at 16°C with T4 DNA ligase and transformed into CaCI 2 competent E. coli M15. Transformants were selected for on LB agar supplemented with ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
• buffer B 8M urea, 50mM Tris, 10% ethanol, 2% Tween, 5mM imidazole, pH 7
• Supematants were incubated with nickel resin for 1-2 hrs on a rotary mixer, and passed through a polypropylene column. The retained resin was washed with 5-10 ml buffer B and bound protein eluted with 0.5 ml elutions of buffer B supplemented with 100 mM imidazole. Eluted proteins were checked by SDS- PAGE before dialysis to remove urea and other salts.
• Recombinant fragments A Fragments 1-5 and 4-8 These fragments produced by pBAD system showed that 1-5 did not bind CEACAM1 but 4-8 did.
• Fragments 4-8, 4-8T, 4-7 and 6-8 produced using pQE30 system.
• Fragment 4-8 was the first rUspAI fragment produced and was found to bind CEACAM1 with a high affinity in blot-overlay assays.
• This peptide designated 4-T
• This peptide also bound CEACAM 1 and appeared to be expressed at lower levels than 4-8 (Fig. 13).
• Sequence analysis of pQE30/4-T found that a mismatch (CAA to TAA) led to a termination codon at residue Q624 (see Fig. 10).
• Biological activity of recombinant peptide 4-7 M. catarrhalis strain MX1 and H. influenzae strain Rd bind to Chinese Hamster Ovary (CHO) cells transfected with CEACAM1. Their binding can be blocked with M. catarrhalis UspAI recombinant peptide 4-7 but not a control peptide (Fig. 17).
• recombinant peptide D-7 but not recombinant 6-8 i.e. residues E659-K863 of UspAI of MX2
• recombinant peptide D-7 but not recombinant 6-8 i.e. residues E659-K863 of UspAI of MX2 inhibits binding of both homologous and heterologous strains to transfected CHO cells expressing CEACAM 1 (Fig. 23).
• Example 5 Characterisation of CEACAM1 -binding peptides Peptide D-7 was found to be the strongest binding recombinant peptide. Therefore, region D-7 (142 amino acids; see Fig. 16) of MX2 contains the CEACAM1 binding information. Truncated peptide 4-T (197 amino acids) retains weak binding (Fig. 13). Therefore region D-T may contain a region with CEACAM 1 binding ability. A single amino acid substitution, K 560 I, in D-7 was found to nearly abolish CEACAM1 binding. Deletion of the region 571-632 in the peptide D-8 (D-8 ⁇ ) resulted in a loss of CEACAM1 binding. Linear overlapping peptides spanning the D-T region (Fig. 19) were made and tested for their ability to bind CEACAM1. No binding to CEACAM1 was observed.
• Circular dichroism spectroscopy The peptide D-7 and the mutant D-7 (K 560 I) were analysed by circular dichroism (CD) spectroscopy. Circular dichroism spectra were obtained at room temperature using a Jobin-Yvon CD6 spectropolarimeter. Spectra of recombinant D-7 and D-7 (K 560 I) at concentrations 0.1 mg/ml were measured in quartz cuvettes. Al! spectra are averages of 8 scans with relevant protein-free buffer spectra subtracted and were plotted using Excel (Microsoft Inc.). The spectra obtained show that D-7 has an ⁇ -helical structure whereas D-7 (K 560 I) adopts a random coil formation (Fig. 18). Without wishing to be bound by any particular theory, it is proposed that CEACAM1 binding of Mx UspAI requires an ⁇ -helical based conformation, perhaps a coiled coil structure.
• D-7 peptide means that the peptide, and derivatives, homologues or fragments thereof, could have particular utility as a sub-unit vaccine or therapeutic.
• Derivatives of the D-7 peptide having the required ⁇ -helical based conformation may be identified by the unique "fingerprint" circular dichroism spectrum.
• Example 6 Sequence analysis An alignment of the D-7 region of the amino acid sequences of the UspAI proteins of ten strains of Mx is shown in Figure 20. Six out of ten strains are identical over the sequenced region. Strains MX3 and MX4 are 100% identical over the available sequence of the D-7 region and have overall identities 90.85% and 88.03%> respectively, taking into account the N-terminal 13 and 17 amino acid residues for MX3 and MX4 respectively that have not been determined. The remaining two strains TTA37 and O35E have deletions as shown (Fig. 20) which occur within the region D-T. Overall identity including the gaps in D-7 is 70.4% and 50% respectively. TTA37 is identical in the remaining region of 100 amino acids whereas O35E is identical in 71 out of 72 amino acids when aligned manually (Fig. 21). It is known that O35E does not bind to CEACAM 1. TTA37 is not available for testing.
• Example 7 Adhesion blocking properties of D-7 Results
• the potential of D-7 as an anti-adhesive agent effective against homologous and heterologous strains of Mx, Nm, Ng and Hi was first examined using the soluble receptor.
• CEACAM 1-Fc CC1-Fc
• Recombinant D-7 was shown to inhibit binding of CCI-Fc significantly to a heterologous strain MX1 and to the homologous strain MX2 in a dose dependent manner (Fig. 24 a). Inhibition was significant and appeared to reach a plateau above 0.01 ⁇ g. ml "1 .
• meningitidis has been shown to target other members of the human CEACAM family, including epithelial CEA and CEACAM6 (NCA) 9 , this is also the case with some Mx strains, Hi strains tend to target CEACAM 1 as a preferred receptor (data not shown).
• D-7 (2 ⁇ g. ml "1 )
• inhibition of Nm binding was observed to transfected HeLa cells expressing CEACAM 1 , 6 and CEA (Fig. 25 c).
• the efficacy of D-7 was further tested using HeLa-CC1 H, a cell line generated to express high-levels of CEACAM 1 to mimic, in part, a possible in vivo inflammation state of epithelial cells.
• Bacterial isolates and culture Mx and Nm were grown on BHI agar supplemented with 10 % heated horse blood whereas, Hi were grown on brain heart infusion (BHI) agar supplemented with Levinthal base.
• Ng strains were cultured on GC agar. All bacteria were cultured at 37°C for up to 16h in a 5% CO2 incubator.
• Mx strains were clinical isolates obtained from cases of otitis media and COPD and represent isolates from several countries.
• Eagan, d and f1 are typable Hi with capsules of type b, c and f respectively, Rd is an acapsulate derivative of a type d strain.
• Strains A930065 and NT1 are NTHi.
• Nm isolates C751A, C751 B and C751 D are three distinct Opa expressing isolates of a serogroup A strain C751.
• Other isolates were of the following serogroups: PMC17(A), C311 and MC58 (B), and C114 (C) PMC2 (29E), PMC4 (W135) and PMC10 (Y).
• Ng isolates P9-13, 16 and 35 are intrastrain Opa variants of strain P9 and other clinical isolates were of worldwide origin. The majority of strains employed in the current study have been described further previously 10, 26, 28 .
• Anti-poly histidine mouse monoclonal antibody was purchased from Qiagen and used at 0.2 ⁇ g. ml "1 . Polyclonal antisera against Mx, Nm and Hi strains were raised in rabbits using .standard protocols and whole cell lysates of multiple strains as antigens. Anti-UspA1 antibody (R38) used in this study has been described in Example 1 and 26 . Polyclonal antiserum against D-7 was generated in rabbits by immunisation with peptide bound to Ni-NTA resin (Qiagen; 100-200 ⁇ g of peptide per immunization). Complement was inactivated by heating the antisera at 56°C for 30 min. Anti-D-7 antibodies were affinity purified using peptide D-7 coupled to AminoLink Plus column according to the manufacturer's protocol (Pierce).
• Soluble receptor constructs & cell lines Soluble CEACAMI-Fc and CHO cells transfected with CEACAM1 used in this study have been described previously 11, 9 .
• HeLa cells expressing a range of CEACAM molecules were a gift from Professor Wolfgang Zimmerman (University of Munich, Germany) and Dr Scott Gray-Owen (University of Toronto, Canada) and were grown in RPMI 1640 containing 10% foetal calf serum (FCS).
• FCS foetal calf serum
• A549 human lung carcinoma cells (Flow laboratories) were cultured in F12 Ham medium containing 10% (FCS).
• HeLa cells expressing high levels of CEACAM were generated by using Tet-OnTM (Clontech) gene expression system.
• the ceacaml gene was cloned into the pTRE-2hyg response plasmid (Clontech) and transformed into HeLa cells that contained the regulatory gene (Clontech) using Fugene-6 (Roche). Transfectants were selected using 400 ⁇ g. ml "1 hygromycin. Those transfectants that were positive for CEACAM expression were selected using FACS and limiting dilution. HeLa-CC1 H clone in the presence of 0.25 ⁇ g. ml "1 doxycycline produced the highest levels of the receptor.
• Numbers of bacteria adhering to HeLa-CEACAM expressing cells were obtained by direct counting using an Olympus IX70 microscope, with X400 magnification. Mean values of bacteria bound were obtained after counting adherent bacteria to 20 cells chosen at random from duplicate experiments. Bacterial adherence to HeLa-CC1 H was determined by cfu analysis as described above.
• Example 8 Anti D-7 antibody inhibits Mx-CEACAM1 interactions
• Rabbit antisera generated against D-7 contained antibodies that were cross-reactive with UspAI from several Mx strains in Western blot overlay of whole cell lysates (data not shown). No binding of anti-D-7 was observed to Nm. Opa or Hi P5 by Western blotting using affinity purified antibodies (data not shown). Incubation of whole cell lysates of a range of Mx strains with anti- D-7 (10 ⁇ g. ml "1 ) prior to CC1-Fc overlay resulted in a significant inhibition of the receptor binding with the majority of strains showing greater than 80% inhibition (Fig. 26).
• D-7 could offer protection against Mx infection whereas D-7 may serve as a more general anti-microbial peptide for a diverse range of CEACAM targeting bacteria.
• Example 9 Inhibition of bacterial adherence to CEACAM expressing human endothelial cells by peptide D-7
• HMEC-1 cells confluent monolayers of HMEC-1 cells were used.
• the human microvasular endothelial cells were preincubated with either D-7 or a recombinant control molecule (both at I ⁇ g.ml "1 ) for 60 min.
• Bacteria (OpaD-expressing isolate of N. meningitidis strain C751 ) were then added at infection ratio of 100 bacteria per cell and incubated for 1hr at 37°C. Following this time, non-adherent bacteria were removed by washing and the monolayer fixed in methanol for 10 min at room temperature. Adherent bacteria were detected by overlay with rabbit polyclonal antisera against N.
• D-7 is capable of inhibiting Opa-CEACAM mediated adhesion of N. meningitidis to endothelial cells as well as epithelial cells.
• the above results indicate that the region 4-T is highly conserved among different strains, with sequence identity of 95% or more for all strains tested except O35E (85% identity). As previously noted, O35E does not bind to CEACAM. Accordingly, peptides comprising or consisting of conserved regions of sequence 4-T (427-623), as shown in Figure 28, are preferred peptides according to the invention with utility for the treatment or prophylaxis of disease, in particular diseases where CEACAM receptors are implicated.

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