WO2013124473A1 - Pilus proteins and compositions - Google Patents

Pilus proteins and compositions Download PDF

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Publication number
WO2013124473A1
WO2013124473A1 PCT/EP2013/053644 EP2013053644W WO2013124473A1 WO 2013124473 A1 WO2013124473 A1 WO 2013124473A1 EP 2013053644 W EP2013053644 W EP 2013053644W WO 2013124473 A1 WO2013124473 A1 WO 2013124473A1
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WIPO (PCT)
Prior art keywords
sortase
enzyme
pilus
amino acid
moiety
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PCT/EP2013/053644
Other languages
French (fr)
Inventor
Domenico Maione
Immaculada Margarit Y Ros
Roberta COZZI
Cira Daniela Rinaudo
Maddalena LAZZARIN
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Novartis Ag
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Publication date
Application filed by Novartis Ag filed Critical Novartis Ag
Priority to US14/380,260 priority Critical patent/US20150273042A1/en
Priority to CA2865028A priority patent/CA2865028A1/en
Priority to RU2014138418A priority patent/RU2014138418A/en
Priority to EP13705791.5A priority patent/EP2817320A1/en
Priority to CN201380010739.4A priority patent/CN104321335A/en
Priority to MX2014010011A priority patent/MX2014010011A/en
Priority to JP2014558136A priority patent/JP2015509713A/en
Priority to AU2013224026A priority patent/AU2013224026A1/en
Publication of WO2013124473A1 publication Critical patent/WO2013124473A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention provides methods of forming pili in vitro and mutant sortase enzymes and proteins suitable for use in these methods.
  • the invention also provides pili produced by these methods and compositions comprising these pili for the treatment and prevention of bacterial disease, in particular of conditions caused by Streptococcus.
  • the invention also provides general methods of ligating proteins and sortase enzymes for use in same.
  • Most bacterial pathogens comprise pili (also known as fimbrae), long filamentous structures extending from their surface, that are often responsible for initial adhesion of bacteria to tissues during host colonization.
  • pili also known as fimbrae
  • Gram-negative bacteria have been known for many years to have pili, typically formed by non-covalent interactions between pilin subunits.
  • Gram-positive bacteria including Streptococcus bacteria, have also been shown to have pili typically formed through covalent association of subunits by sortases that are encoded by pilus-specific pathogenicity islands.
  • the Gram-positive bacterium Streptococcus agalactiae (or "group B streptococcus", abbreviated to "GBS"), for example, has three pilus variants, each encoded by a distinct pathogenicity island, PI-1, PI-2a or PI-2b [1, 2].
  • Each pathogenicity island consists of: i) genes encoding the three structural components of the pilus (the pilus backbone protein (BP) and 2 ancillary proteins (API and AP2)); and ii) genes encoding 2 sortase proteins (SrtCl and SrtC2) that are involved in the assembly of the pilus. All GBS strains carry at least one of these 3 pathogenicity islands.
  • Streptococcus pyogenes or "group A streptococcus", abbreviated to "GAS"
  • Streptococcus pneumoniae also known as pneumococcus
  • the pathogenicity island in pneumococcus encodes the 3 structural components of the pilus (RrgA, RrgB and RrgC) and three sortases (SrtCl, SrtC2 and SrtC3) which catalyse pilus formation.
  • the FCT regions encode the backbone and accessory proteins and polymerisation of these proteins is also mediated by a sortase (SrtCl).
  • Pilus structures in these Gram-positive bacteria are considered to be interesting vaccine candidates and work has been done on assessing the immunogenicity of purified recombinant proteins from pilus structures. It is also desirable to study these proteins in their native form within assembled pili but currently, the only way to do this is by the laborious process of purifying wild-type pili from the bacteria.
  • One object of the invention is therefore to provide a process for producing recombinant pili in vitro without the need to purify wild-type pili.
  • GBS causes bacteremia and meningitis in immunocompromised individuals and in neonates.
  • GAS is a frequent human pathogen, estimated to be present in between 5-15% of normal individuals without signs of disease.
  • host defences are compromised or when GAS is introduced to vulnerable tissues or hosts, however, an acute infection occurs.
  • Diseases caused by GAS include puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotising fasciitis, myositis and streptococcal toxic shock syndrome.
  • Pneumococcus is the most common cause of acute bacterial meningitis in adults and in children over 5 years of age
  • the invention provides a method of ligating at least two moieties comprising contacting the at least two moieties with a pilus-related sortase C enzyme in vitro under conditions suitable for a sortase mediated transpeptidation reaction to occur, wherein the pilus-related sortase C enzyme comprises an exposed active site.
  • the pilus-related sortase C enzyme is from Streptococcus, more particularly from Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) and Streptococcus pyogenes (GAS).
  • the pilus-related sortase C enzyme is a sortase CI enzyme (srtCl), sortase C2 enzyme (SrtC2) or a sortase C3 enzyme (SrtC3).
  • the pilus-related sortase C enzyme mutation comprises a deletion of part or all of the lid.
  • the mutation comprises a deletion of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO: 3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another pilus-related sortase C enzyme.
  • the mutation comprises substitution of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO: 3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme.
  • the pilus-related sortase C enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and 71.
  • the method is a method of forming a recombinant or artificial pilus in vitro.
  • the at least two moieties comprise an LPxTG motif and a pilin motif.
  • the pilin motif may comprise the amino acids YPAN.
  • 'X' in any sortase recognition motif disclosed herein may be any standard or non-standard amino acid and every variation is disclosed.
  • X is selected from the 20 standard amino acids found most commonly in proteins found in living organisms.
  • the recognition motif is LPXTG or LPXT
  • X may be D, E, A, N, Q, K, or R.
  • X is selected from K, S, E, L, A, N in an LPXTG or LPXT motif.
  • the at least two moieties are from Gram-positive bacteria.
  • the at least two moieties may be from the same strain or type of Gram-positive bacteria or from different strains or types of Gram positive bacteria.
  • the at least two moieties are Streptococcal polypeptides.
  • the at least two moieties are Streptococcal backbone proteins and/or ancillary proteins.
  • the at least two moieties comprise or consist of an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97; or (b) that is a fragment of at least ' ⁇ ' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50
  • an artificial or recombinant pilus obtained or obtainable from the aforementioned method.
  • an artificial or recombinant pilus which comprises at least two variants of backbone protein GBS59. Particularly the at least two variants are selected from Group B Streptococcus strains 2603, H36B, 515, CJB111, CJB110 and DK21.
  • the artificial or recombinant pilus is a chimeric pilus comprising at least one variant of GBS backbone protein GBS59 selected from Streptococcus strains 2603, H36B, 515, CJB111, CJB110 and DK21 and at least one backbone protein from Streptococcus pneumonia selected from the group consisting of RrgA, RrgB and RrgC.
  • artificial or recombinant pili further comprise GBS80 and/or GBS 1523.
  • the artificial or recombinant pilus is for use in medicine, yet more particularly for use in preventing or treating Streptococcal infection.
  • a method of treating or preventing Streptococcal infection in a patient in need thereof comprising administering an effective amount of an artificial or recombinant pilus formed by the methods of the invention to a patient.
  • the at least two moieties comprise a first moiety comprising the amino acid motif LPXTG, wherein X is any amino acid, and a second moiety comprising at least one amino acid.
  • the first moiety is a first polypeptide and the second moiety is a second polypeptide.
  • the first polypeptide and the second polypeptide are from Gram-positive bacteria.
  • the first polypeptide and the second polypeptide may be from the same type or strain of Gram-positive bacteria or from different types or strains of Gram positive bacteria.
  • the first polypeptide and the second polypeptide are Streptococcal polypeptides.
  • the first polypeptide and the second polypeptide may be Streptococcal backbone proteins and/or ancillary proteins.
  • either the first moiety or the second moiety comprises a detectable label.
  • the detectable label may be a fluorescent label, a radio label, a chemiluminescent label, a phosphorescent label, a biotin label, or a streptavidin label.
  • the first moiety or the second moiety may be a polypeptide and the other moiety may be a protein or glycoprotein on the surface of a cell.
  • either the first moiety or the second moiety is a polypeptide and the other moiety comprises amino acids conjugated to a solid support.
  • either the first moiety or the second moiety is a polypeptide and the other moiety comprises at least one amino acid conjugated to a polynucleotide.
  • the method of the invention may be used to ligate the N-terminus of a first moiety to the N-terminus of a second moiety.
  • the method of the invention may be used to ligate the C- terminus of a first moiety to the C-terminus of a second moiety.
  • the first moiety and the second moiety are the N-terminus and C-terminus of a moiety such as a polypeptide chain, and ligation results in the formation of a circular polypeptide.
  • kits comprising a sortase CI or a sortase C2 enzyme from Streptococcus agalactiae and a moiety comprising the amino acid motif LPXTG, wherein X is any amino acid.
  • a sortase C enzyme from Streptococcus comprising a mutation in its lid region, particularly a sortase C enzyme from Streptococcus which is from Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) or Streptococcus pyogenes (GAS).
  • a sortase C enzyme from Streptococcus wherein the sortase C enzyme from Streptococcus is a sortase CI enzyme, sortase C2 enzyme or a sortase C3 enzyme.
  • a sortase C enzyme from Streptococcus wherein the mutation comprises deletion of part or all of the lid region of the sortase C enzyme.
  • the mutation comprises deletion of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme.
  • a sortase C enzyme from Streptococcus wherein the mutation comprises substitution of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme
  • a sortase C enzyme from Streptococcus which comprises a mutation in its lid region and wherein the sortase C enzyme comprises or consists of an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67,
  • Figure 1 Alignment of GBS sortase C sequences showing location of the lid region in bold and underlined.
  • Figure 2 Alignment of Streptococcus pneumoniae and Streptococcus pyogenes (GAS) sortase C sequences showing location of the lid region in bold and underlined.
  • GAS Streptococcus pneumoniae and Streptococcus pyogenes
  • Figure 3 A: conserved amino acid motifs identified in the backbone protein of GBS pilus 2a (BP-2a), GBS59 (strain 515, TIGR annotation SAL 1486).
  • Pilin motif containing a highly conserved lysine residue (Lysl89);
  • E-box containing a highly conserved glutamic acid residue (Glu589);
  • Sorting signal containing residues IPQTGG located at positions 641-646.
  • B Immunoblot performed with an antibody recognising the backbone protein of GBS pilus 2a (a-BP), showing that Lysl89 of the pilin motif of BP-2a is required for pilus polymerization by wild type sortase C.
  • a plasmid was generated encoding a mutant BP-2a carrying a substitution at Lysl89 with Ala (BP KI 89A ).
  • a GBS mutant strain lacking backbone proteins (GBS ABP ) was transformed with this plasmid (lane 2), or a control plasmid encoding wild-type BP-2a (BP WT ) (lane 1).
  • the star indicates the location of the protein bands corresponding to the monomeric, unpolymerised BP-2a protein. High molecular weight protein bands, corresponding to polymerised BP-2a, are detectable only in cell extracts of GBS transformed with the plasmid encoding wild-type BP-2a (lane 1).
  • BP WT BP WT
  • ⁇ no plasmid
  • the star indicates the location of the protein bands corresponding to the monomeric, unpolymerised BP-2a protein.
  • the triangle indicates the protein band corresponding to monomeric API protein.
  • the box indicates the protein band corresponding to BP-2a - API conjugates.
  • High molecular weight protein bands, corresponding to polymerised BP-2a are detectable only in cell extracts of GBS transformed with the plasmid encoding wild-type BP-2a.
  • Figure 4 A: Protein gel showing that wild-type GBS sortase fails to catalyse in vitro polymerization of wild-type backbone protein.
  • BP backbone protein
  • SrtC lwi wild-type sortase CI of PI-2a
  • SDS-PAGE sodium-dodecylsulfate polyacrylamide gel electrophoresis
  • the hash indicates SrtClwT- Lane 1 : BP 25 ⁇ +8 ⁇ 1 ⁇ ⁇ t0, Lane 2: BP 25 ⁇ +8 ⁇ 1 ⁇ ⁇ t24h, Lane 3 : BP 25 ⁇ +8 ⁇ 1 ⁇ ⁇ t48h; Lane 4: BP ⁇ +SrtClwT t0, Lane 5 : BP ⁇ +SrtClwT t24, Lane 6: BP ⁇ +SrtClwT t48h; Lane 7: BP 20( ⁇ M+SrtC lwT t0, Lane 8: BP 20( ⁇ M+SrtC lwT t24.
  • Figure 5 A: Protein gel showing that a mutant GBS sortase carrying a mutation in the lid region is able to catalyse in vitro polymerization of wild-type backbone protein (BP).
  • BP wild-type backbone protein
  • mutant sortase CI of PI-2a carrying a tyrosine to alanine substitution at position 86 (SrtCly86 A ) for 0, 24 or 48 hours and the proteins contained in the reaction mixture visualised by SDS- PAGE.
  • the star indicates monomeric BP.
  • High molecular weight bands >260 kDa), corresponding to polymerized BP, were detectable after 24 or 48 hours of incubation.
  • Lane 1 BP tOh
  • Lane 2 BP 100 ⁇ +8 ⁇ ⁇ 8 6 ⁇ t24h
  • Lane 3 BP 100 ⁇ +8 ⁇ 86 ⁇ t48h
  • Lane 4 BP 200 ⁇ +8 ⁇ 86 ⁇ tOh
  • Lane 5 BP
  • t24h B Immunoblot performed with an antibody recognising the backbone protein of GBS pilus 2a (BP), showing that the pattern of polymerized BP is similar to BP polymers contained in pili from wild-type bacteria (here GBS strain 515). The star indicates monomeric BP. Lane 1 : BP, Lane 2: SrtCl Y8 6A, Lane 3: BP+SrtCl Y8 6A, Lane 4: GBS515 Wild Type Pili. C: Protein gel showing the effect of different concentrations of SrtCly86 A on the efficiency of BP polymerisation.
  • Figure 6 Protein gel showing that in vitro polymerised pili structures can be successfully purified. 25 ⁇ of SrtCly86 A were incubated with 100 ⁇ of BP-2a at 37°C for 7 days. The proteins contained within the mixture were separated into fractions by size exclusion chromatography and visualised by SDS-PAGE. The high-molecular weight fractions containing purified polymerised BP elute first (white box), followed by monomeric BP (star) and SrtCly86A (cross).
  • Figure 7 Protein gel showing that mutant sortase enzymes polymerize pilus proteins from a variety of gram positive bacteria.
  • A 25 ⁇ of SrtCly86 A (GBS sortase CI of PI-2a) were incubated with 100 ⁇ of backbone protein PI-1 of GBS (also referred to as GBS 80) at 37°C for 7 days and the proteins contained in the reaction mixtures were visualised by SDS-PAGE.
  • GBS 80 also referred to as GBS 80
  • SrtCly86 A or GBS 80 alone were incubated under the same conditions.
  • the star indicates monomeric BP.
  • Lane 1 SrtCly86A
  • Lane 2 BP PI-1
  • Lane 3 SrtCl Y 86A+BP PI-1.
  • SrtCl Y8 6A GSS sortase CI of PI-2a
  • SrgB pilus protein from Streptococcus pneumoniae
  • the star indicates monomeric RrgB.
  • Lane 1 SrtCly86 A
  • Lane 2 RrgB
  • Lane 3 SrtCl Y86 A+RrgB (50 ⁇ )
  • Lane 4 SrtCl Y86A +RrgB ( ⁇ ).
  • Figure 8 Pairwise sequence alignment of homologous SrtCl sortases from PI-2a of GBS strain 515 and PI-2b of GBS strain A909.
  • the catalytic triad single underline
  • the canonical lid motif double underline
  • Figure 9 Pairwise alignment of SrtC2 sortase from PI-2b (SAK 1437) and SrtCl sortase from PI-2a (SAL 1484).
  • SrtC2 lacks the lid sequence (highlighted in box), and the C terminal trans-membrane domain. Three cysteine residues are present in PI-2b SrtC2 sequence (marked with crosses).
  • Figure 10 Western blot of total protein extracts from culture of a mutant strain derived from GBS 515 in which the PI-2a island has been deleted (515A2a) and from the wild type A909 strain complemented by a plasmid containing SrtCl and BP genes or BP gene alone. Antibodies against BP were used. High-molecular weight signals indicate pili polymerization in the complemented strains.
  • Lane 1 515A2a; Lane 2: 515A2a+BP; Lane 3: 515A2a+BP+SrtCl; Lane 4: 515A2a+BP+SrtCl; Lane 5: A909+BP; Lane 6: A909+BP+SrtCl .
  • Figure 11 SDS-PAGE of polymerization reactions.
  • Lane 1 SrtClY86A + BP-2a -515;
  • Lane 2 SrtClY86A + BP-2a -H36B;
  • Lane 3 SrtClY86A + BP-2a -CJB111;
  • Lane 4 Marker;
  • Lane 5 SrtCl Y86A + BP-2a -515-H36B-CJB111.
  • Figure 12A Western blot with polyclonal antibody against BP-1.
  • Lane 1 SrtCl Y86A; Lane 2: BP-2a - 515 variant; Lane 3: BP-2a - H36B variant; Lane 4: BP-1; Lane 5: RrgB; Lane 6: SrtClY86A + BP-1; Lane 7: SrtClY86A + BP-2a -515+ BP-1; Lane 8: SrtClY86A + BP-2a -H36B+ BP-1; Lane 9: SrtClY86A + RrgB; Lane 10: SrtClY86A + BP-2a -515+ RrgB; Lane 11 : SrtCl Y86A + BP-2a -H36B+ RrgB.
  • Figure 12B Western blot with polyclonal antibody against RrgB. .
  • Lane 1 SrtCl Y86A; Lane 2: BP-2a - 515 variant; Lane 3: BP-2a - H36B variant; Lane 4: BP-1; Lane 5: RrgB; Lane 6: SrtClY86A + BP-1; Lane 7: SrtClY86A + BP-2a -515+ BP-1; Lane 8: SrtClY86A + BP-2a -H36B+ BP-1; Lane 9: SrtClY86A + RrgB; Lane 10: SrtClY86A + BP-2a -515+ RrgB; Lane 11 : SrtCl Y86A + BP-2a -H36B+ RrgB.
  • FIG. 13 Mutant SrtC can polymerize Green Fluorescent Protein (GFP) tagged with an IPQTG sequence.
  • GFP Green Fluorescent Protein
  • Figure 14A The LPXTG motif is essential for in vitro pilus polymerization. Progression of the reaction between the SrtClY86A and recombinant BP-2a AIPQTG at TO, 48 and 72 hours of incubation at 37°C . The concentrations of both SrtClY86A and BP-2a AIPQTG were fixed at 25 ⁇ and ⁇ respectively. No formation of high molecular weight pattern could be identify, showing that the LPXTG like-motif is necessary for the BP polymerization. As controls the SrtClY86A (on the left) and BP-2a AIPQTG (on the right) were incubated alone.
  • Figure 14B The lysine of pilin motif is not essential for in vitro pilus polymerization.
  • the SrtClY86A (25 ⁇ ) and the recombinant BP-2a K189A ( ⁇ ) were mixed at 37°C and at different time points (0, 48h and 72h) the reactions were analysed by SDS-PEGE. A patter of high molecular weight could be identified, showing that the SrtClY86A used another nucleophile different from the lysinel89.
  • FIG. 14C When SrtClY86A was mixed with recombinant forms of the ancillary proteins (API -2a and AP2-2a), that in vivo can be polymerized only in the presence of the BP-2a protein (data not shown), some HMW structures were formed. These data demonstrate that SrtClY86A can use different nucleophile/s to resolve the acyl- intermediate between the enzyme and the LPXTG-like sorting signal.
  • pilus-related C-sortases in gram positive bacteria have demonstrated that the active site of many of these enzymes contains a catalytic triad of amino acids that are covered by a mobile "lid" region in the absence of substrate.
  • a feature of pilus- related sortases is the presence of a lid that not only blocks active site access, i.e. it encapsulates the active site, but also carries two key residues, generally an Asp and a hydrophobic amino acid, that interact within the catalytic cleft itself, serving as 'anchors'.
  • sequences corresponding to lid regions can be identified in all pilus-related sortases characterized to date.
  • this lid structure has been demonstrated to be present in the sortase CI enzymes from GBS PI-1, PI-2a and PI-2b [3], in the sortase CI, sortase C2 and sortase C3 enzymes from Streptococcus pneumoniae [4, 5], and in the sortase CI enzyme from GAS.
  • Mutation of the lid region in the sortase CI enzyme from GBS PI-2a has been shown not to have an adverse impact on pilus production in complementation studies [3] but until now, no studies have been conducted into the ability of mutant sortases to polymerise proteins in vitro.
  • sortase C enzymes are capable of polymerising proteins in vitro more effectively than wild-type sortase C enzyme, for example, resulting in the production of recombinant pili.
  • Wild type sortase C enzyme comprise a "mobile lid" region encapsulating the active site in a closed conformation in the absence of substrate.
  • the lid of SrtCl harbors 3 residues, Asp84, Pro85, and Tyr86 which make interactions with residues of the active site and surroundings.
  • sortase C enzymes are inactive in vitro and unable to ligate or polymerise moieties such as pilin backbone and ancillary proteins.
  • mutated enzymes of the invention possess or comprise an exposed catalytic site which is not encapsulated by a "lid” and is available to catalyze a transpeptidation reaction to form an acyl enzyme intermediate in vitro.
  • the methods of the invention can thus be used to produce artificial or recombinant pili without the need for the labourious purification procedures currently used.
  • these mutant sortase C enzymes can also be used to polymerise proteins from a variety of sources such as gram positive bacteria, not just proteins derived from the same bacteria as the mutant sortase C enzyme itself.
  • the pili resulting from these methods are immunogenic and may be used in the development of vaccines to treat or prevent diseases caused by the gram positive bacteria from which the component proteins of the pili are derived.
  • pilin subunits within the pilus contain intra-protein isopeptide bonds that form spontaneously, presumably stabilizing the structure of the pilus.
  • immunisation of a subject with proteins in the form of an artificial or recombinant pilus structure mimicking those encountered by the immune system during invasion/infection may also have advantages in terms of the presence of additional epitopes, such as structural or conformational epitopes based on three-dimensional structure.
  • additional epitopes such as structural or conformational epitopes based on three-dimensional structure.
  • Such structural or conformational epitopes may be absent from subunit vaccines when the pilus proteins are provided in compositions comprising the isolated, purified forms or as conjugates, such as glycoconjugates.
  • the polymerised pili proteins may comprise three-dimensional epitopes not predictable from the structure of the proteins alone.
  • the mutant sortase C enzyme used in the methods of the invention is derived from a wild- type sortase C enzyme from Streptococcus.
  • the mutant sortase C enzyme may, for example, be derived from a wild-type sortase C enzyme from Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) or Streptococcus pyogenes (GAS).
  • the mutant sortase C enzyme may be derived from a sortase CI enzyme, a sortase C2 enzyme or a sortase C3 enzyme.
  • the mutant sortase C enzyme is derived from a wild-type streptococcal sortase C enzyme that comprises a lid region.
  • the lid region is the structural loop of about 15-18 amino acids that covers the catalytic triad of amino acids found in the active site of a sortase C enzyme in the absence of a substrate.
  • the lid region is located within the soluble core domain of the sortase C enzyme, between the signal peptide and transmembrane (TM) region located at the N-terminal of the enzyme and the positively charged domain located at the C-terminal of the enzyme.
  • TM transmembrane
  • the location of the lid region in a variety of wild-type Streptococcal sortase C enzymes is summarised in the table below. These sequences are all wild-type sequences which include the N-terminal signal peptide.
  • Table 1 Location of lid region in Streptococcal sortases
  • the location of the lid region in other Streptococcal sortase C enzymes can readily be determined by the skilled person by structural analysis or more simply, by alignment of the sequences of these enzyme with the sequences of the Streptococcal proteins having lid regions at known locations shown in Table 1.
  • Figure 1 provides an alignment of GBS sortase C enzymes highlighting the location of the lid regions.
  • Figure 2 provides a similar alignment for sortase C enzymes from GAS and pneumococcus. Any of the sortase C enzymes shown in these Figures having a lid region may be used in the methods of the invention.
  • the sortase C enzyme from Streptococcus used in the methods of the invention comprises a mutation in its lid region.
  • the mutation may be a substitution, deletion or insertion in the amino acid sequence of the lid region of the mutant sortase C-enzyme relative to the amino acid sequence of the wild-type sortase C enzyme.
  • the mutation may comprise deletion of part or all of the lid region of the wild-type sortase C enzyme.
  • the lid region is typically around 15-18 amino acids long and the mutation may comprise deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 or more amino acids from the lid region, or deletion of all of the amino acids in the lid region.
  • the mutation may comprise deletion of amino acids at positions predicted to interact with the catalytic triad in the active site of the sortase C enzyme.
  • the mutation may comprise the deletion of amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of other sortase C enzymes.
  • the mutation may thus comprise the deletion of: i) an amino acid at position 84; ii) an amino acid at position 85; iii) an amino acid at position 86; iv) two amino acids at positions 84 and 85; v) two amino acids at positions 84 and 86; vi) two amino acids at positions 85 and 86; or vii) three amino acids at positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme.
  • Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a can readily be determined by alignment. Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) are found at:
  • the mutation may comprise the deletion of all of amino acids in the lid region.
  • the deletion may comprise further changes at positions within the remaining sortase sequence.
  • the sortase may comprise substitutions, deletions or insertions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acid positions.
  • the sortase may comprise substitutions, deletions or insertions at fewer than, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 additional amino acid positions or any range therebetween.
  • the mutation may additionally comprise deletion of part or all of the signal peptide and/or transmembrane domain of the wild-type sortase C enzyme which is N- terminal of the lid region in the wild-type enzyme.
  • the transmembrane domain comprises two alpha- helices.
  • the mutation may comprise deletion of one or both of these two alpha- helices and, optionally, may also comprise deletion of the signal peptide N-terminal of the transmembrane domain.
  • the mutation may comprise deletion of part or all of the lid region and the deletion of 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids N- terminal of the lid region.
  • the mutation may comprise deletion of part or all of the lid region and the deletion of less than 10, 20, 30, 40, 50, 60, 70, 80, 90 amino acids N-terminal of the lid region or any range therebetween.
  • the mutation comprises the deletion of all of amino acids in the lid region and all of amino acids N-terminal of the lid region.
  • the sortase C enzyme in this embodiment of the invention thus consists of the C-terminal/positively charged domain of the wild-type sortase C enzyme.
  • the mutation may consist of the deletions described above in the absence of any further mutations.
  • the mutation may consist of deletion of part or all of the lid region, deletion of part or all of the lid region and the signal peptide and/or transmembrane domain, or deletion of part or all of the lid region and the entre N-terminal region in the absence of any further mutations.
  • sequences of sortase C enzymes where the mutation consists of a) deletion of all of the lid region and the signal peptide/transmembrane domain, b) deletion of all of the lid region and the entire N- terminal regions, and c) deletion of the signal peptide/transmembrane domain and amino acids in the catalytic triad which are suitable for use in the methods of the invention are provided in Table 2 below.
  • GAS sortase CI SEQ ID NO: 18 SEQ ID NO:27 SEQ ID NO:36
  • Mutant sortase enzymes used in the methods of the invention may thus comprise or consist of an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.
  • Mutant sortase enzymes used in the methods of the invention may also comprise or consist of an amino acid sequence selected from SEQ ID NO: 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 except for the substitution, deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the mutation may comprise one or more amino acid substitutions in the lid region compared to the wild-type sortase C enzyme sequence.
  • the substitution(s) may be at positions in the lid region predicted to interact with amino acids in the catalytic site such that the substitutions abolish normal lid function.
  • the mutation may comprise the substitution of amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution deletion of amino acids at corresponding positions in the amino acid sequence of other sortase C enzymes.
  • the mutation may thus comprise the substitution of: i) an amino acid at position 84; ii) an amino acid at position 85; iii) an amino acid at position 86; iv) two amino acids at positions 84 and 85; v) two amino acids at positions 84 and 86; vi) two amino acids at positions 85 and 86; or vii) three amino acids at positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme.
  • Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a can readily be determined by alignment. Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) are found at:
  • substitutions at positions corresponding to position 84 and/or position 85 and/or position 86 may comprise replacement of the wild-type residue at these positions with an alanine residue.
  • the mutation may comprise replacement of the aspartate residue at position 90 with an alanine residue (D90A) and/or replacement of the proline residue at position 91 with an alanine residue (P91A), and/or replacement of the tyrosine residue at position 92 with an alanine residue (Y92A).
  • the mutation may comprise replacement of the aspartate residue at position 84 with an alanine residue (D84A) and/or replacement of the proline residue at position 85 with an alanine residue (P85A), and/or replacement of the phenylalanine residue at position 86 with an alanine residue (F86A).
  • the mutation may comprise replacement of the aspartate residue at position 84 with an alanine residue (D84A) and/or replacement of the proline residue at position 85 with an alanine residue (P85A), and/or replacement of the tyrosine residue at position 86 with an alanine residue (Y86A).
  • the mutation may comprise replacement of the aspartate residue at position 88 with an alanine residue (D88A) and/or replacement of the proline residue at position 89 with an alanine residue (P89A), and/or replacement of the tyrosine residue at position 90 with an alanine residue (Y90A).
  • the mutation may comprise replacement of the methionine residue at position 53 with an alanine residue (M53A) and/or replacement of the lysine residue at position 54 with an alanine residue (K54A), and/or replacement of the tryptophan residue at position 55 with an alanine residue (W55A).
  • the mutation may comprise replacement of the aspartate residue at position 58 with an alanine residue (D58A) and/or replacement of the proline residue at position 59 with an alanine residue (P59A), and/or replacement of the tryptophan residue at position 60 with an alanine residue (W55A).
  • the mutation may comprise replacement of the aspartate residue at position 50 with an alanine residue (D50A) and/or replacement of the proline residue at position 51 with an alanine residue (P51A), and/or replacement of the phenylalanine residue at position 52 with an alanine residue (F52A).
  • the mutation may comprise replacement of the aspartate residue at position 74 with an alanine residue (D74A) and/or replacement of the proline residue at position 75 with an alanine residue (P75A), and/or replacement of the phenylalanine residue at position 76 with an alanine residue (F76A).
  • the mutation may comprise replacement of the aspartate residue at position 46 with an alanine residue (D46A) and/or and/or replacement of the phenylalanine residue at position 48 with an alanine residue (F48A).
  • the GAS sortase CI enzyme already comprises an alanine residue at position 47.
  • the mutation may comprise further amino acid changes at positions other than at positions corresponding to positions 84 and/or 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3).
  • the mutation may comprise substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acid positions.
  • the mutation may comprise deletions and/or insertions.
  • the mutation may comprise substitutions at positions corresponding to positions 84 and/or 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) and deletion of a) the signal peptide and/or transmembrane domain, or b) deletion of the entire N- terminal region of the wild-type sortase enzyme.
  • the sortase may consist of substitutions at positions 84 and/or 85 and/or 86 in the absence of any further mutations.
  • sequences of sortase C enzymes consisting of substitutions at positions that are equivalent to positions 84 and/or 86 of the lid region of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) and also consisting of deletion of the signal peptide/transmembrane region which are suitable for use in the methods of the invention are provided in Table 3 below.
  • Mutant sortase enzymes used in the methods of the invention may thus comprise or consist of an amino acid sequence selected from SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71.
  • Mutant sortase enzymes used in the methods of the invention may also comprise or consist of an amino acid sequence selected from SEQ ID NO:37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71 except for the substitution, deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • mutant sortase C enzymes suitable for use in the methods of the invention described above are also embodiments of the invention in their own right.
  • Particularly sortase mutants are the SrtCly92A and SrtC2 F 86A because the stability of these enzymes is higher, they are better expressed and more soluble in comparison with, for example SrtCl- ⁇ and SrtC2-ANT deletion mutants. This is surprising since the Vmax of the cleavage reaction for the Y92A and F86A mutants was lower than that of the SrtCl- ⁇ and SrtC2- ⁇ mutants which are also more difficult to purify.
  • Sortases cleave the LPXTG motif of, for example, pilin proteins and covalently join the C terminus of one moiety, such as a pilin subunit, to a Lys side-chain NH2 group on the next moiety or subunit.
  • Two recognition events are involved in this sortase action. Firstly, the sortase recognition motif (LPXTG or a variant) of the substrate protein must be recognised and bound. Secondly, the acceptor substrate, to which the substrate protein will be transferred, must be recognised and bound, and a specific amino group brought into position to attack the thioacyl intermediate.
  • mutant sortase C enzymes Bacterial polypeptides polymerised by the mutant sortase C enzymes
  • the mutant sortase C enzymes described above may be used to polymerise one or more polypeptides.
  • the mutant sortase C enzymes are brought into contact with the one or more polypeptides in vitro and following a period of incubation, polymerised polypeptides are detected, for example by identifying a pattern of high molecular weight bands on SDS gels.
  • Incubation may be carried out at 37°C. Incubation may be carried out for 1 , 2, 3, 4, 5, 6, 7 days or more.
  • the polypeptides and the mutant sortase C enzymes may be incubated in the presence of a reducing agent, for example DTT lmM, to keep the catalytic cysteine of the mutant sortase C enzyme active. Incubation may be carried out at around pH 7-8.
  • the wild-type sortase C enzymes fail to polymerise polypeptides in vitro.
  • in vitro refers to the use of isolated and/or purified components of a cell, such as an enzyme, to effect pilus polymerisation without requiring the presence of the cell itself.
  • the polypeptides polymerised by the mutant sortase C enzymes of the invention typically comprise the LPxTG motif. They may further comprise a pilin motif (consensus WxxxVxVyPK) and/or an E-Box motif (consensus YxLxETxAPxGY) shown to be important for pilus assembly [6].
  • the polypeptides may comprise a conserved lysine (K) residues, for example, found in the pilin motif.
  • the polypeptides do not comprise a conserved lysine (K) residue in the pilin motif, i.e. wherein the presence of the conserved lysine residue is excluded.
  • the polypeptides polymerised by the mutant sortase C enzymes of the invention may comprise an N-terminal glycine residue.
  • Other sequence motifs will be apparent to one skilled in the art and may include, by way of non-limiting example: LPETGG, LPXT, LPXTG, LPKTG, LPATG, LPNTG, IPQTG, IQTGGIGT.
  • the mutant sortase C enzyme may be brought into contact with a backbone protein found in a pilus from GBS, GAS or Streptoccoccus pneumoniae.
  • the mutant sortase C enzyme may be brought into contact with the backbone protein from GBS PI-1 (GBS80/SAG0645), the backbone protein from GBS PI-2a (GBS59/SAG1407), the backbone protein from GBS PI-2b (Spbl/SAN1518), the backbone protein from Streptococcus pneumoniae (RrgB), or the backbone protein from GAS (fee6, spyl28, orf80, eftLSLA).
  • the mutant sortase C enzyme may be brought into contact with an ancillary protein found in a pilus from GBS, GAS or Streptococcus pneumoniae.
  • the mutant sortase C enzyme may be brought into contact with the ancillary protein 1 (AP-1) from GBS PI-1 (GBS104), the AP-1 from GBS PI-2a (GBS67/SAG1408), the AP-1 from GBS PI-2b (SAN1519), the AP-1 from Streptococcus pneumoniae (RrgA) or the AP-1 from GAS (cpa), the ancillary protein 2 (AP-2) from GBS PI-1 (GBS52), the AP-2 from GBS PI-2a (GBS150/SAG1404), the AP-2 from GBS PI-2b (SAN1516), the AP-2 from Streptococcus pneumoniae (RrgC) or the AP-2 from GAS spyl30, orf82, orf2).
  • AP-1 ancill
  • mutant sortase C enzymes of the invention may be used to polymerise homologues, fragments or variants of the wild-type backbone protein and ancillary protein sequences, provided that these homologues, fragments and variants retain the sequences described above necessary for polymerisation by mutant sortase C enzymes.
  • variants of these polypeptides that may be used in the methods of the invention include backbone proteins and/or ancillary protein sequences from which the transmembrane domain has been deleted compared to the wild-type sequence.
  • variants may comprise the additional of a glycine residue at the N-terminal to promote polymerisation.
  • sequences some of these polypeptides which may be polymerised by the mutant sortase enzymes of the invention are provided below for reference.
  • sequences of additional polypeptides which may be polymerised by the mutant sortases of the invention can be readily determined by the skilled person. Further details of these polypeptides are provided in reference [7].
  • GBS80 The amino acid sequence of full length GBS80 as found in the 2603 strain is given as SEQ ID NO: 72 herein.
  • Wild-type GBS80 contains a N-terminal leader or signal sequence region at amino acids 1-37 of SEQ ID NO:72.
  • One or more amino acids from the leader or signal sequence region of GBS80 can be removed, e.g. SEQ ID NO:73.
  • GBS 1523 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% or more identity (e.g.
  • SEQ ID NO: 110 SEQ ID NO: 110; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 110, wherein ' ⁇ ' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • ' ⁇ ' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • the wild-type sequence contains an amino acid motif indicative of a cell wall anchor (LPSTG) at amino acids 468-472 of SEQ ID NO: 110.
  • LSTG cell wall anchor
  • An E box containing a conserved glutamic residue has also been identified at amino acids 419-429 of SEQ ID NO: l 10, with a conserved glutamic acid at residue 423.
  • the E box motif may be important for the formation of oligomeric pilus-like structures, and so useful fragments of GBS1523 may include the conserved glutamic acid residue.
  • GBS1523 has been identified in which the glutamine (Q) at position 41 of SEQ ID NO: l 10 is substituted for a lysine (K), as a result of a mutation of a codon in the encoding nucleotide sequence from CAA to AAA. This substitution may be present in the GBS1523 sequences and GBS1523 fragments (e.g. SEQ ID NO: 112).
  • SEQ ID NO: 112 A further variant of GBS1523 COH1 without signal sequence region is provided as SEQ ID NO: 111.
  • the amino acid sequence of full length GBS59 as found in the 2603 strain is given as SEQ ID NO: 74 herein.
  • Variants of GBS59 exist in strains H36B, 515, CJBl l l, DK21 and CJB110.
  • the amino acid sequence of full length GBS59 as found in the H36B, 515, CJB111, CJB110 and DK21 strains are given as SEQ ID NOs: 75, 76, 77, 78, and 79.
  • Spbl GBS PI-2b
  • SEQ ID NO: 80 The amino acid sequence of full length Sbpl as found in the COH1 strain is given as SEQ ID NO: 80 herein.
  • Wild-type Spbl contains a N-terminal leader or signal sequence region.
  • One or more amino acids from the leader or signal sequence region of Spbl can be removed, e.g. SEQ ID NO:81.
  • the RrgB pilus subunit has at least three clades.
  • Reference amino acid sequences for the three clades are SEQ ID NOs: 82, 83 and 84 herein.
  • GBS PI-1 GBS104/SAG0649
  • GBS67 The amino acid sequence of full length GBS 104 as found in the 2603 strain is given as SEQ ID NO: 85 herein.
  • AP-1 from GBS PI-2a GBS67
  • SEQ ID NO: 86 The amino acid sequence of full length GBS67 as found in the 2603 strain is given as SEQ ID NO: 86 herein.
  • a variant of GBS67 (SAI1512) exists in strain H36B.
  • the amino acid sequence of full length GBS67 as found in the H36B strain is given as SEQ ID NO: 87.
  • Variants of GBS67 also exists in strains CJBl l l, 515, NEM316, DK21 and CJBl lO.
  • amino acid sequences of full length GBS67 as found in the C JB 111 , 515, NEM316, DK21 and CJB110 strains are given as SEQ ID NOS: 88, 89, 90, 91, and 92 herein.
  • RrgA The amino acid sequence of full length RrgA is given as SEQ ID NO: 94 herein.
  • GBS052/SAG0646 The amino acid sequence of full length GBS052/SAG0646 as found in the 2603 strain is given as SEQ ID NO: 95 herein.
  • AP-2 from GBS PI-2a (GBS150/SAG1404
  • amino acid sequence of full length GBS150/SAG1404 as found in the 2603 strain is given as SEQ ID NO: 96 herein.
  • polypeptides for use with the invention may thus comprise or consist of an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97 or to any other backbone or ancillary protein sequences described above; or (b) that is a fragment of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.
  • 'n' is less than 20 or less than 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or less than 150.
  • the methods of the invention may involve polymerisation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 polypeptides having 50% identity to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, or of or of fragments of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more).
  • 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150
  • the methods of the invention may involve polymerisation of 1, 2, 3, 4, 5 or 6 polypeptides having 50% identity e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 74, 75, 76, 77, 78 and 79, or of fragments of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g.
  • the methods of the invention may involve polymerisation of 1, 2, or 3 polypeptides having 50% identity e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%o, 99%), 99.5%> or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 82, 83 and 84, or of fragments of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more).
  • Amino acid fragments of these backbone and ancillary proteins may comprise an amino acid sequence of e.g up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 125, up to 150, up to 175, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, up to 550, up to 600, up to 650, up to 700, up to 750, up to 800, up to 850, up to 900, up to 950, up to 1000, up to 1 100, up to 1200, up to 1300, up to 1400, up to 1500, consecutive amino acid residues of the sequences provided above.
  • Other fragments omit one or more polypeptide domains, for example the transmembrane domain.
  • the mutant sortase C enzymes of the invention polymerise these polypeptides in a manner that is analogous to the polymerisation of backbone proteins and accessory proteins by wild-type Streptococcal sortase C enzymes in vivo to form a pilus.
  • the polymerised polypeptides produced according to these methods are thus structurally similar to a pilus produced by a Streptococcal bacterium in vivo.
  • Pili in Gram-positive bacteria are constructed from either two or three types of pilin subunits.
  • the shaft of the pilus is formed by multiple copies of a major pilin subunit, while the tip of the pilus contains a single copy of a minor 'tip' pilin subunit that typically functions as an adhesin.
  • Three-component pili are similar, but they also contain a minor 'basal' pilin subunit that is covalently attached to the cell wall.
  • EM transmission electron microscopy
  • immuno-gold labelling studies have led to the conclusion that the minor 'basal' pilin subunits are also interspersed throughout the shaft of the pilus, presumably because the sortase enzymes are promiscuous in the substrates they recognize.
  • the mutant sortase C enzymes may be brought into contact with 1 polypeptide, leading to the formation of a monomeric pilus.
  • the mutant sortase enzyme may be brought into contact with GBS80, GBS59 or RrgB, leading to the formation of a monomeric pilus comprising subunits of GBS80, GBS59 or RrgB respectively.
  • the polypeptide is from a Gram positive bacterium
  • the mutant sortase enzyme that is used to polymerise that polypeptide need not be from the same Gram positive bacterium.
  • a mutant sortase C enzyme derived from GBS can be used to polymerise proteins not just from GBS but also from Streptococcus pneumoniae and/or GAS.
  • pili polymerised in vitro may include a combination of GBS59 variants from GBS strains 515, CJB111, H36B, 2603, DK21 and 090, more particularly a combination of GBS59 variants from GBS strains 515, CJB111, H36B and 2603.
  • Such pili comprising two or more variants of GBS59 are not found in nature because strains of wild type bacteria express only one variant of back-bone protein (BP-2a/GBS59).
  • the mutant sortase C enzymes may be brought into contact with 2, 3, 4, 5 or more different polypeptides which may be from 1, 2, 3, 4, 5 or more Gram positive bacteria, leading to the formation of a chimeric pilus.
  • the mutant sortase C enzymes may be brought into contact with the backbone and accessory proteins from a single Gram positive bacterium which are found in combination in a natural Streptococcal pilus from that bacterium, resulting in a chimeric pilus that is equivalent in structure to a naturally- occurring pilus.
  • Such chimeric pili are a useful tool to enable the study of pilus properties without the laborious purification process currently used to isolate pili from Gram positive bacteria.
  • the three-dimensional structures of the monomeric and chimeric pili produced by the methods of the invention make them particularly convenient and effective for immunisation purposes compared to the administration of individual recombinant proteins.
  • protection assays have shown that these pili are more effective at inducing protection against the Streptococcus bacteria from which they are derived than monomeric recombinant proteins. It is postulated that this may be because the pili contain epitopes present in pili in vivo that are not replicated in monomeric recombinant proteins, particularly such epitopes are structural epitopes.
  • the invention includes pili obtained or obtainable using the methods of the invention.
  • the combinations of polypeptides found in these pili differ from the combination of polypeptides found in naturally-occurring pili in Streptococcal bacteria.
  • Examples of pili that may be produced according to the methods of the invention include pili comprising or consisting of the backbone proteins and/or the ancillary proteins from Streptococcus described above. In some embodiments, these pili do not contain the combinations of polypeptides found in naturally-occurring pili found in GBS, GAS or Streptococcal pneumoniae.
  • pili polymerised in vitro differ from naturally- occuring pili in terms of their composition, for example, because the acyl enzyme intermediate is not attached to a wild type sortase but is attached to a mutant sortase of the invention.
  • pili polymerised in vitro do not comprise cell wall/membrane components such as lipid II or precursors of peptidoglycan such as MurNAc-N-acetyl- muramic acid.
  • pili polymerised in vitro comprise combinations of pilus proteins not found in nature.
  • pili polymerised in vitro can be differentiated from those occurring naturally.
  • the term "artificial" refers to a synthetic, or non-cell derived composition, particularly a structure which is synthesized in vitro and which is not identical to structures found in native bacteria such as Streptococcus.
  • Immunogenic compositions comprising pili
  • the invention provides immunogenic compositions comprising the pili described above, which may be obtained or obtainable by the methods of the invention.
  • immunogenic is used to mean that the pilus is capable of eliciting an immune response, such as a cell-mediated and/or an antibody response, against the polypeptide or polypeptides making up the pilus when used to immunise a subject (preferably a mammal, more preferably a human or a mouse). Particularly, the immune response is a protective immune response which provides protective immunity.
  • Immunogenic compositions of the invention may be useful as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.
  • prophylactic vaccines do not guarantee complete protection from disease because even if the patient develops antibodies, there may be a lag or delay before the immune system is able to fight off the infection. Therefore, and for the avoidance of doubt, the term prophylactic vaccine may also refer to vaccines that ameliorate the effects of a future infection, for example by reducing the severity or duration of such an infection.
  • protection against infection and/or “provide protective immunity” means that the immune system of a subject has been primed (e.g by vaccination) to trigger an immune response and repel infection.
  • the immune response triggered is capable of repelling infection against a number of different strains of bacteria. A vaccinated subject may thus get infected, but is better able to repel the infection than a control subject.
  • compositions may thus be pharmaceutically acceptable. They will usually include components in addition to the antigens e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in reference [8].
  • compositions will generally be administered to a mammal in aqueous form. Prior to administration, however, the composition may have been in a non-aqueous form. For instance, although some vaccines are manufactured in aqueous form, then filled and distributed and administered also in aqueous form, other vaccines are lyophilised during manufacture and are reconstituted into an aqueous form at the time of use. Thus a composition of the invention may be dried, such as a lyophilised formulation.
  • the composition may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the vaccine should be substantially free from (i.e. less than 5 ⁇ g/ml) mercurial material e.g. thiomersal-free. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly preferred.
  • a composition may include a temperature protective agent. Further details of such agents are provided below.
  • a physiological salt such as a sodium salt.
  • Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml e.g. about 10+2mg/ml NaCl.
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
  • Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290-310 mOsm/kg.
  • Compositions may include one or more buffers.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer.
  • Buffers will typically be included in the 5-20mM range.
  • the pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.
  • the composition is preferably sterile.
  • the composition is preferably non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • the composition is preferably gluten free.
  • the composition may include material for a single immunisation, or may include material for multiple immunisations ⁇ i.e. a 'multidose' kit).
  • the inclusion of a preservative is preferred in multidose arrangements.
  • the compositions may be contained in a container having an aseptic adaptor for removal of material.
  • Human vaccines are typically administered in a dosage volume of about 0.5ml, although a half dose ⁇ i.e. about 0.25ml) may be administered to children.
  • Immunogenic compositions of the invention may also comprise one or more immunoregulatory agents.
  • one or more of the immunoregulatory agents include one or more adjuvants.
  • the adjuvants may include a TH1 adjuvant and/or a TH2 adjuvant, further discussed below.
  • Adjuvants which may be used in compositions of the invention include, but are not limited to:
  • mineral salts such as aluminium salts and calcium salts, including hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates) and sulphates, etc. [e.g. see chapters 8 & 9 of ref. 9];
  • oil-in-water emulsions such as squalene-water emulsions, including MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer)
  • squalene-water emulsions including MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer)
  • CFA complete Freund's adjuvant
  • IF A incomplete Freund's adjuvant
  • VLPs virus-like particles
  • bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives [24, 25], immunostimulatory oligonucleotides [26-31], such as IC-31TM [32] (deoxynucleotide comprising 26- mer sequence 5'-(IC)i3-3' (SEQ ID NO: 46) and polycationic polymer peptide comprising 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 47)) and ADP-ribosylating toxins and detoxified derivatives thereof [33 - 42];
  • LPS enterobacterial lipopolysaccharide
  • LPS Lipid A derivatives
  • immunostimulatory oligonucleotides such as IC-31TM [32] (deoxynucleotide comprising 26- mer sequence 5'-(IC)i3-3' (SEQ ID NO: 46) and polycationic polymer peptide comprising 11-
  • human immunomodulators including cytokines, such as interleukins (e.g. IL-1 , IL- 2, IL-4, IL-5, IL-6, IL-7, IL-12 [43, 44], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor;
  • cytokines such as interleukins (e.g. IL-1 , IL- 2, IL-4, IL-5, IL-6, IL-7, IL-12 [43, 44], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor;
  • bioadhesives and mucoadhesives such as chitosan and derivatives thereof, esterified hyaluronic acid microspheres [45] or mucoadhesives, such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulos [46];
  • microparticles i.e. a particle of -lOOnm to ⁇ 150um in diameter, more preferably ⁇ 200nm to ⁇ 30um in diameter, and most preferably ⁇ 500nm to ⁇ 10 ⁇ in diameter
  • materials that are biodegradable and non-toxic e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycapro lactone, etc.
  • muramyl peptides including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-normuramyl-l-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2-( -2'-dipalmitoyl-5/7- glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE); and
  • imidazoquinolone compounds including Imiquamod and its homologues ⁇ e.g.
  • Immunogenic compositions and vaccines of the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
  • the following adjuvant compositions may be used in the invention: (1) a saponin and an oil- in- water emulsion [55]; (2) a saponin ⁇ e.g. QS21) + a non-toxic LPS derivative ⁇ e.g. 3dMPL) [56]; (3) a saponin ⁇ e.g. QS21) + a non-toxic LPS derivative ⁇ e.g. 3dMPL) + a cholesterol; (4) a saponin ⁇ e.g.
  • RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • LPS such as 3dMPL
  • aluminium hydroxide and/or aluminium phosphate adjuvant are particularly preferred, and antigens are generally adsorbed to these salts.
  • Calcium phosphate is another preferred adjuvant.
  • Other preferred adjuvant combinations include combinations of Thl and Th2 adjuvants such as CpG & alum or resiquimod & alum.
  • a combination of aluminium phosphate and 3dMPL may be used (this has been reported as effective in pneumococcal immunisation [59]).
  • compositions of the invention may elicit both a cell mediated immune response as well as a humoral immune response.
  • This immune response will preferably induce long lasting (e.g. neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to infection.
  • CD8 T cells Two types of T cells, CD4 and CD8 cells, are generally thought necessary to initiate and/or enhance cell mediated immunity and humoral immunity.
  • CD8 T cells can express a CD8 co-receptor and are commonly referred to as Cytotoxic T lymphocytes (CTLs).
  • CTLs Cytotoxic T lymphocytes
  • CD8 T cells are able to recognized or interact with antigens displayed on MHC Class I molecules.
  • CD4 T cells can express a CD4 co-receptor and are commonly referred to as T helper cells.
  • CD4 T cells are able to recognize antigenic peptides bound to MHC class II molecules.
  • the CD4 cells Upon interaction with a MHC class II molecule, the CD4 cells can secrete factors such as cytokines. These secreted cytokines can activate B cells, cytotoxic T cells, macrophages, and other cells that participate in an immune response.
  • Helper T cells or CD4+ cells can be further divided into two functionally distinct subsets: THl phenotype and TH2 pheno types which differ in their cytokine and effector function.
  • Activated THl cells enhance cellular immunity (including an increase in antigen- specific CTL production) and are therefore of particular value in responding to intracellular infections.
  • Activated THl cells may secrete one or more of IL-2, IFN- ⁇ , and TNF- ⁇ .
  • a THl immune response may result in local inflammatory reactions by activating macrophages, NK (natural killer) cells, and CD8 cytotoxic T cells (CTLs).
  • a THl immune response may also act to expand the immune response by stimulating growth of B and T cells with IL-12.
  • THl stimulated B cells may secrete IgG2a.
  • Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections.
  • Activated TH2 cells may secrete one or more of IL-4, IL-5, IL- 6, and IL-10.
  • a TH2 immune response may result in the production of IgGl , IgE, IgA and memory B cells for future protection.
  • An enhanced immune response may include one or more of an enhanced THl immune response and a TH2 immune response.
  • a TH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFN- ⁇ , and TNF- ⁇ ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a.
  • the enhanced TH1 immune response will include an increase in IgG2a production.
  • a TH1 immune response may be elicited using a TH1 adjuvant.
  • a TH1 adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant.
  • TH1 adjuvants suitable for use in the invention may include for example saponin formulations, virosomes and virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides.
  • LPS enterobacterial lipopolysaccharide
  • Immunostimulatory oligonucleotides such as oligonucleotides containing a CpG motif, are preferred TH1 adjuvants for use in the invention.
  • a TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgGl, IgE, IgA and memory B cells.
  • the enhanced TH2 immune resonse will include an increase in IgGl production.
  • a TH2 immune response may be elicited using a TH2 adjuvant.
  • a TH2 adjuvant will generally elicit increased levels of IgGl production relative to immunization of the antigen without adjuvant.
  • TH2 adjuvants suitable for use in the invention include, for example, mineral containing compositions, oil-emulsions, and ADP-ribosylating toxins and detoxified derivatives thereof. Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use in the invention.
  • the invention includes a composition comprising a combination of a TH1 adjuvant and a TH2 adjuvant.
  • a composition elicits an enhanced TH1 and an enhanced TH2 response, i.e., an increase in the production of both IgGl and IgG2a production relative to immunization without an adjuvant.
  • the composition comprising a combination of a TH1 and a TH2 adjuvant elicits an increased TH1 and/or an increased TH2 immune response relative to immunization with a single adjuvant (i.e., relative to immunization with a TH1 adjuvant alone or immunization with a TH2 adjuvant alone).
  • the immune response may be one or both of a TH1 immune response and a TH2 response.
  • immune response provides for one or both of an enhanced TH1 response and an enhanced TH2 response.
  • the enhanced immune response may be one or both of a systemic and a mucosal immune response.
  • the immune response provides for one or both of an enhanced systemic and an enhanced mucosal immune response.
  • the mucosal immune response is a TH2 immune response.
  • the mucosal immune response includes an increase in the production of IgA.
  • compositions of the invention may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition).
  • the composition may be prepared for topical administration e.g. as an ointment, cream or powder.
  • the composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured).
  • the composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared as a solid dosage form for parenteral or needleless administration, for example intra-dermal administration.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as drops.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient.
  • kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
  • kits may comprise two vials, or it may comprise one ready- filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of the pilus, as well as any other components, as needed.
  • 'immunologically effective amount' it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Examples of an immunologically effective amount are around 0. ⁇ g-l( ⁇ g pilus, for example 0 ⁇ g-l( ⁇ g pilus.
  • a composition may include a temperature protective agent, and this component may be particularly useful in adjuvanted compositions (particularly those containing a mineral adjuvant, such as an aluminium salt).
  • a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point e.g. to reduce the freezing point to below 0°C.
  • the temperature protective agent also permits freezing of the composition while protecting mineral salt adjuvants against agglomeration or sedimentation after freezing and thawing, and may also protect the composition at elevated temperatures e.g. above 40°C.
  • a starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1-80% by volume of the final mixture.
  • Suitable temperature protective agents should be safe for human administration, readily miscible/soluble in water, and should not damage other components ⁇ e.g. antigen and adjuvant) in the composition.
  • Examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG).
  • PEGs may have an average molecular weight ranging from 200-20,000 Da.
  • the polyethylene glycol can have an average molecular weight of about 300 Da ('PEG-300').
  • the invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention, or a pilus of the invention.
  • the immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity.
  • the method may raise a booster response.
  • the invention also provides immunogenic combinations or compositions for use as a medicament e.g. for use in raising an immune response in a subject, such as a mammal.
  • the invention also provides the use of the pilus of the invention in the manufacture of a medicament for raising an immune response in a mammal.
  • the mammal By raising an immune response in the mammal by these uses and methods, the mammal can be protected against diseases caused by the bacteria from which the polypeptides in the pilus are derived.
  • the mammal can be protected against disease caused by Streptococcal bacteria, including GAS, GBS and Streptococcus pneumoniae.
  • the invention also provides a delivery device pre-filled with an immunogenic composition of the invention.
  • the mammal is preferably a human, a large veterinary mammal (e.g. horses, cattle, deer, goats, pigs) and/or a domestic pet (e.g. dogs, cats, gerbils, hamsters, guinea pigs, chinchillas).
  • the mammal is a human, e.g. human patient.
  • the vaccine is for prophylactic use, the human may be a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human may be a teenager or an adult.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • a mammal e.g.
  • Maternal passive immunity is a type of naturally acquired passive immunity, and refers to antibody- mediated immunity conveyed to a fetus by its mother during pregnancy.
  • Maternal antibodies are passed through the placenta to the fetus by an FcRn receptor on placental cells. This occurs around the third month of gestation. Particularly the antibodies are Immunoglobulin G (IgG) or Immunoglobulin A (IgA).
  • IgGy antibody isotypes can pass through the placenta during pregancy. Passive immunity may also provided through the transfer of IgA antibodies found in breast milk that are transferred to the gut of the infant, protecting against bacterial infections, until the newborn can synthesize its own antibodies.
  • One way of checking efficacy of therapeutic treatment involves monitoring infection after administration of the compositions of the invention.
  • One way of checking efficacy of prophylactic treatment involves monitoring immune responses, systemically (such as monitoring the level of IgGl and IgG2a production) and/or mucosally (such as monitoring the level of IgA production), against the antigen(s) in the pilus of the invention after administration of the composition.
  • antigen-specific serum antibody responses are determined post-immunisation but pre-challenge whereas antigen-specific mucosal antibody responses are determined post-immunisation and post-challenge.
  • compositions of the present invention Another way of assessing the immunogenicity of the compositions of the present invention is to express the proteins recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays. A positive reaction between the protein and the patient sample indicates that the patient has mounted an immune response to the protein in question. This method may also be used to identify immunodominant antigens and/or epitopes within antigens.
  • the efficacy of compositions of the invention can also be determined in vivo by challenging animal models of infection, e.g., guinea pigs or mice, with the vaccine compositions.
  • compositions of the invention will generally be administered directly to a patient.
  • Direct delivery may be accomplished by parenteral injection ⁇ e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or mucosally, such as by rectal, oral ⁇ e.g. tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • the invention may be used to elicit systemic and/or mucosal immunity, preferably to elicit an enhanced systemic and/or mucosal immunity.
  • the enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response.
  • the enhanced immune response includes an increase in the production of IgGl and/or IgG2a and/or IgA.
  • Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart ⁇ e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). Vaccines prepared according to the invention may be used to treat both children and adults.
  • a human patient may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old.
  • Preferred patients for receiving the vaccines are the elderly (e.g. >50 years old, >60 years old, and preferably >65 years), the young (e.g. ⁇ 5 years old), hospitalised patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients.
  • the vaccines are not suitable solely for these groups, however, and may be used more generally in a population.
  • Vaccines produced by the invention may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre) other vaccines e.g. at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H.
  • other vaccines e.g. at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H.
  • influenzae type b vaccine an inactivated polio virus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Y vaccine), a respiratory syncytial virus vaccine, etc.
  • compositions of the invention are antigenic components of compositions of the invention.
  • compositions further comprising at least one further antigen.
  • the invention also provides a composition comprising a polypeptide of the invention and one or more of the following further antigens:
  • - a saccharide antigen from N. meningitidis serogroup A, C, W135 and/or Y (preferably all four).
  • - a saccharide or polypeptide antigen from Streptococcus pneumoniae [e.g. 61, 62, 63].
  • an antigen from hepatitis A virus such as inactivated virus [e.g. 64, 65].
  • an antigen from hepatitis B virus such as the surface and/or core antigens [e.g. 65, 66].
  • a diphtheria antigen such as a diphtheria toxoid [e.g. chapter 3 of ref. 67] or the CRMi97 mutant [e.g. 68].
  • tetanus antigen such as a tetanus toxoid [e.g. chapter 4 of ref. 67].
  • Bordetella pertussis such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 69 & 70].
  • PT pertussis holotoxin
  • FHA filamentous haemagglutinin
  • agglutinogens 2 and 3 optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 69 & 70].
  • a saccharide antigen from Haemophilus influenzae B [e.g. 71].
  • - polio antigen(s) e.g. 72, 73] such as IPV.
  • influenza antigen(s) e.g. chapter 19 of ref. 67
  • haemagglutinin and/or neuraminidase surface proteins such as the haemagglutinin and/or neuraminidase surface proteins.
  • composition may comprise one or more of these further antigens.
  • Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [70]).
  • diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
  • Saccharide antigens are preferably in the form of conjugates.
  • Carrier proteins for the conjugates include diphtheria toxin, tetanus toxin, the N. meningitidis outer membrane protein [80], synthetic peptides [81,82], heat shock proteins [83,84], pertussis proteins [85,86], protein D from H. influenzae [87], cytokines [88], lymphokines [88], streptococcal proteins, hormones [88], growth factors [88], toxin A or B from C. difficile [89], iron- uptake proteins [90], etc.
  • a preferred carrier protein is the CRM 197 mutant of diphtheria toxin [91].
  • Antigens in the composition will typically be present at a concentration of at least ⁇ g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
  • nucleic acid preferably DNA e.g. in the form of a plasmid
  • encoding the antigen may be used.
  • Antigens are preferably adsorbed to an aluminium salt.
  • mutant sortases of the invention can use different nucleophile/s to resolve the acyl-intermediate between the enzyme and the LPXTG-like sorting signal.
  • wild type sortases from which the mutant sortases are derived require the presence of a lysine residue.
  • Mutant sortases of the invention are effective in vitro at catalysing transpeptidation reactions and forming polymers of GBS pilus proteins. Mutant sortases of the invention are further useful in a variety of protein engineering applications. The structural differences between the sortases of the present invention and other pilus-related sortases in gram positive bacteria may provide new functionality and enable new in vitro methods to be performed, or may allow polymerisation and ligation reactions to be performed more efficiently.
  • mutant sortase enzymes of the invention are useful for performing ligation reactions between any moiety that comprises the LPXTG recognition motif (or those listed above) and any moiety that comprises an amino acid residue that can provide the nucleophile to complete the transpeptidation reaction.
  • mutant sortases of the invention are able to cleave and polymerise backbone proteins and ancillary proteins comprising the LPXTG motif.
  • Previous work has demonstrated that bacterial sortases require only a single amino acid to provide the nucleophile to complete the transpeptidation reaction (Proft., Biotechnology Letters, 2010, 32: 1-10; Popp et al, Current Protocols in Protein Science, 2009, 15, WO2010/087994).
  • either the first moiety or the second moiety in the ligation is a polypeptide and the other moiety is a protein or glycoprotein on the surface of a cell.
  • the sortases of the invention can be used to attach polypeptides to proteins on the cell surface. This can be particularly useful for, for example, labelling specific proteins on the cell surface.
  • the cell has been transfected to express the surface protein of interest with a LPXTG motif. This motif can then be targeted for ligation using a sortase of the invention.
  • the protein label may comprise the motif.
  • Sortases for ligation of substrates other than pilus proteins
  • mutant sortases of the invention are used to ligate proteins to a solid support and either the first moiety or the second moiety is a polypeptide and the other moiety comprises amino acids conjugated to a solid support.
  • the protein comprises the LPXTG motif and the solid support has amino acids, such as lysine, conjugated to it.
  • the solid support is a bead, such as a polystyrene bead or gold bead or particle such as a nanoparticle.
  • the methods of the invention allow circularisation of polypeptide chains.
  • the first moiety and the second moiety are the N-terminus and C-terminus of a polypeptide chain, and ligation results in the formation of a circular polypeptide.
  • Sortases are also of significant interest for protein modification and engineering applications. Sortases promote pilin formation in vivo by catalysing a transpepditation reaction between backbone and ancillary proteins. Sortases recognise and cleave a recognition motif (for example, LPXTG) and form an amide linkage with a target protein. By utilising the recognition motif, a variety of protein engineering functions can be performed. Ligation reactions performed using sortases are flexible, efficient and require fewer steps than comparable chemical ligation techniques. Therefore, another object of the invention is to provide improved sortases for protein engineering applications. The techniques of Sortagging are known in the art.
  • sortases SrtCl and SrtC2 from GBS pathogenicity island PI-2b may be used for ligation.
  • sortases SrtCl and SrtC2 from GBS pathogenicity island PI-2b may be used for ligation.
  • SrtCl as used in the methods of the invention does not comprise a signal peptide or N-terminal transmembrane domain (as in SEQ ID NO:98, SEQ ID NO:99 or SEQ ID NO: 100).
  • SrtCl as used in the methods of the invention comprises SEQ ID NO: 101, which corresponds to the cloned soluble domain.
  • SrtCl may have a W55F mutation (as in SEQ ID NO: 102).
  • W55 may be important in regulating the activity of SrtCl , because it is located in the region that the canonical sortases lid motif is normally found in Streptococcal sortases. W55 may mimic the function of the lid found in other sortases.
  • the SrtCl as used in the methods of the invention may have a C188A mutation (as in SEQ ID NO: 103).
  • CI 88 may be a catalytic cysteine.
  • the amino acid sequence of wild type SrtC2 from PI-2b is presented in SEQ ID NO: 105.
  • the SrtC2 as used in the methods of the invention may have its cysteines substituted with alanines (as in SEQ ID NO: 106).
  • SrtC2 as used in the methods of the invention does not comprise a signal peptide or N-terminal transmembrane domain (as in SEQ ID NO: 108 or SEQ ID NO: 109).
  • the skilled person is capable of identifying any signal peptide or N-terminal transmembrane domain.
  • PI-2b sortase CI and sortase C2 enzymes for use with the invention may thus comprise or consist of an amino acid sequence: (a) having 70% or more identity (e.g. 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs:5, 98,100, 101 , 102, 103, 105, 106, 108 and 109; or (b) that is a fragment of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 100 or more (e.g.
  • PI-2b sortase CI and sortase C2 enzymes for use with the invention retain the ability to perform ligation and polymerisation reactions.
  • the nucleotide sequences encoding SrtCl and SrtC2 are provided in SEQ ID NO: 104 and SEQ ID NO: 107.
  • Particular recognition motifs may include LPETGG, LPXTG, LPXT, LPKTG, LPATG, LPNTG, LPET, VPDT, IPQT, YPR , LPMT, LAFT, LPQT, NSKT, NPQT, NAKT, NPQS, LPKT, LPIT, LPDT, SPKT, LAET, LAAT, LAET, LAST, LPLT, LSRT.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do no materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • Consisting of is generally taken to mean that the invention as claimed is limited to those elements specifically recited in the claim (and may include their equivalents, insofar as the doctrine of equivalents is applicable).
  • the term "about” in relation to a numerical value x means, for example, x+10%.
  • references to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 100.
  • a preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith- Waterman homology search algorithm is disclosed in ref. 101.
  • the percent identity of a first polypeptide and a second polypeptide is generally determined by counting the number of matched positions between the first and second polypeptides and dividing that number by the total length of the shortest polypeptide followed by multiplying the resulting value by 100. For fragments of polypeptides this value is usually around 100% and therefore has little meaning. Therefore, in the context of fragments of the present invention, the term "proportion of reference polypeptide" (expressed as a percentage) is used. Proportion of reference polypeptide is calculated by counting the number of matched positions between the fragment and reference polypeptides and dividing that number by the total length of the reference polypeptide followed by multiplying the resulting value by 100.
  • fragments will comprise less than 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25 or less than 20% of the sequence of the reference polypeptide.
  • MODES FOR CARRYING OUT THE INVENTION Example 1: Functional regulation of GBS SrtCl: a single mutation in the lid region enhances BP polymerization in vitro.
  • Cell-surface pili are important virulence factors and promising vaccine candidates.
  • Gram- positive bacteria elaborate pili via a sortase C-catalyzed transpeptidation mechanism from backbone and ancillary pilin substrates.
  • specific residues and/or motifs such as the pilin motif and the conserved LPxTG sorting signal are absolutely necessary.
  • Site-directed mutagenesis of GBS sortase CI of Pi- a (SrtCl) reveals the specific involvement of Tyr86 in the lid-regulatory site in the activation of recombinant SrtCl .
  • This example shows that recombinant BP high molecular weight pili structures can be obtained in vitro using catalytic enzyme concentrations. This provides direct evidence of self-inhibition of sortase C enzymes by the presence of the lid and opens a field for studying pili assembly by using recombinant pili polymerized by a sortase-active mutant, reducing the necessity to purify high amount of wild type pili from pathogenic bacteria.
  • GBS Group B Streptococcus
  • Streptococcus agalactiae is the leading cause of life- threatening diseases in newborn and is also becoming a common cause of invasive disease in nonpregnant, elderly or immune-compromised adults [102].
  • Pili long filamentous fibers protruding from bacterial surface, have been discovered in Gram-Positive pathogens as important virulence factors and potential vaccine candidates. From the analysis of the eight sequenced genomes of GBS, two genomic islands, each coding for three different pili, have been identified [103; 1].
  • the srtA locus that encodes the " housekeeping' sortase A (SrtA) is present in a different genome region in all analyzed GBS strains [103].
  • Each pilus genomic island codes for three LPXTG proteins: the backbone protein (BP) representing the main pilus subunit, and two ancillary proteins (API and AP2).
  • BP backbone protein
  • API and AP2 ancillary proteins
  • each island encodes at least two class C sortases, each having specificity for one of the ancillary proteins [1; 104].
  • the crystal structure of the soluble core of GBS SrtCl-2a, containing its catalytic domain indicates that SrtCl employs a catalytic triad composed of Hisl57-Cys219-Arg228, essential for pilus fiber formation and covered by a loop, known as "lid", which is dispensable for sortase activity in vivo [3].
  • the crystal structure suggests that SrtCl is folded as an auto -inactivated enzyme, by the presence of the lid that sterically blocks the active site.
  • the function of the lid region in enzyme regulation and activity is still unclear, but is supposed to have a role in selecting the proper pilus proteins for polymerization. In this work we show, for the first time, efficient recombinant BP high molecular weight structures by using catalytic enzyme concentrations.
  • GBS 515 strain and mutants were grown in Todd Hewitt Broth (THB) or in Trypticase soy agar (TSA) supplemented with 5% sheep blood at 37°C.
  • the proteins SrtC I 43- 292 (SEQ ID NO:3 without signal and transmembrane domains), SrtCl Y8 6A (SEQ ID NO:48) and SrtC 1 ALID (SEQ ID NO: 12) were expressed as His-MBP, TEV cleavable, fusion proteins and purified as previously described [3].
  • Recombinant BP 30 - 649 containing both the pilin motif and the sorting signal, was cloned in speedET vector and expressed as previously described [107], and BP KI 89 A was generated by PIPE site-directed mutagenesis using wild type BP 30 -649.
  • Recombinant BP 30 -64o, lacking the C- terminal LPxTG motif was cloned in speedET vector and expressed and purified as N terminal His-tag, TEV cleavable, fusion protein using the same protocol used for wild type BP.
  • Antisera specific for the BP-2a and API -2a proteins were produced by immunizing CD1 mice with the purified recombinant proteins [107, 108].
  • complementation vectors and site-specific mutagenesis GBS knock-out (KO) mutant strain for BP was generated as previously reported [1].
  • DNA fragments corresponding to wild type BP SAL 1486
  • gene was PCR amplified from GBS 515 genome and the product was cloned into the E. co/z ' -streptococcal shuttle vector pAM401/gbs80P+T, previously described [11, 27] and containing the promoter and terminator regions of the gbs80 gene (TIGR annotation SAG 0645).
  • Site-directed mutagenesis of pAM BP was performed using the PIPE (Polymerase Incomplete Primer Extension) method [19].
  • the complementation vectors PAM BPALPXTG and pAM_BP K i 89A were electroporated into the KO strain ⁇ . Complementation was confirmed by checking BP expression by Western Blotting.
  • Mid-exponential phase bacterial cells were resuspended in 50mM Tris-HCl containing 400U of mutanolysin (Sigma-Aldrich) and COMPLETE protease inhibitors (Roche). The mixtures were then incubated at 37°C for lh and cells lysed by three cycles of freeze- thawing. Cellular debris were removed by centrifugation and protein concentration was determined using BCA protein assay (Pierce, Rockford, IL). Total protein extracts (20 ⁇ g) or recombinant pili were resolved on 3-8% or 4-12% NuPAGE precast gels (Invitrogen) by SDS-PAGE and transferred to nitrocellulose.
  • Membranes were probed with mouse antiserum directed against BP and API proteins (1 :1,000 dilution) followed by a rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). Bands were then visualized using an Opti-4CN substrate kit (Bio-Rad).
  • Lysine 189 in the putative pilin motif and IPQTG sorting signal of BP-2a are essential for pilus formation by wild-type sortase C.
  • BP-2a (strain 515, TIGR annotation SAL 1486) we identified a putative pilin motif containing a highly conserved lysine residue (Lysl89) and the IPQTGG motif at residue 641-646 as the C terminus sorting motif (Fig. 3 A).
  • PIPE Polymerase Incomplete Primer Extension
  • the LPXTG-like sorting signal is essential for the transpeptidation reaction mediated in vitro by the SrtCly86A mutant but the pilin motif is NOT.
  • Wild-type SrtCl-2a is not able to induce recombinant BP polymerization in vitro.
  • Recombinant GBS major pilin subunit BP carrying the pilin motif Kl 89 and the C-terminal LPxTG recognition site was mixed with WT SrtCl, at various ratios and incubated at 37°C for different times reaching also the high enzyme amounts used for S. pneumoniae SrtCl [4].
  • SDS-page analysis of these samples showed no formation of high molecular weight bands that could represent pilus polymers (Fig. 4A), but only the formation of a complex compatible with the formation of a hetero-dimer formed by rSrtCl and rBP, as previously described for S. pneumoniae [4] and a dimer BP-BP that is formed also in absence of SrtCl (Fig. 4B).
  • BP high molecular weight structures can be assembled in vitro by recombinant SrtCl lid mutant.
  • the polymerization in vitro was tested by incubating ⁇ -BP ALPXT G and r-BP K i 89A with SrtCly86A confirming that the polymerization occurs through the cleavage of the LPXTG sorting signal and the subsequent linking to the pilin motif of the next subunit.
  • Fig. 7 shows that mutant sortase enzymes polymerize pilus proteins from a variety of gram positive bacteria.
  • SrtCly86 A GBS sortase CI of PI-2a was incubated with backbone protein PI-1 of GBS (also referred to as GBS 80) (Fig. 7A) or with pilus protein from Streptococcus pneumoniae (also referred to as RrgB) (Fig. 7B).
  • the in vitro polymerized pili structures may be used in immunisation studies in mice. For example, 10 ⁇ g of purified recombinant pili may be mixed with an adjuvant (e.g. alum) and injected into mice in a final volume of 200 ⁇ . This may be followed by one or more booster immunisations. The mice may then be analysed for an immune response to the pili structures. This immune response may be protective against the bacteria from which the monomeric pilus proteins were originally derived.
  • An immunisation study has been conducted in which mice were immunised with monomeric pili comprising GBS59 generated according to the methods of the invention in combination with alum, and the protective immune response was assessed following subsequent challenge with GBS.
  • the GBS strains used in this work were 2603 V/R (serotype V), 515 (la), CJBl l l (V), H36B (serotype lb), 5401 (II) and 3050 (II). Bacteria were grown at 37°C in Todd Hewitt Broth (THB; Difco Laboratories) or in trypticase soy agar supplemented with 5% sheep blood.
  • TTB Todd Hewitt Broth
  • trypticase soy agar supplemented with 5% sheep blood.
  • Genomic DNA was isolated by a standard protocol for gram-positive bacteria using a NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer's instructions.
  • the full length recombinant BP-2a proteins corresponding to 515, CJB111 and 2603 allelic variants (TIGR annotation SAL1486, SAM1372 and SAG1407, respectively), were produced as reported in Margarit et al, Journal of Infectious Diseases, 2009, 199: 108-115, whilst the full length H36B variant (TIGR annotation SAI 1511) was cloned in pET24b+ (Novagen) using strain H36B as source of DNA. Primers were designed to amplify the coding regions without the signal peptide and the 3' terminal sequence starting from the LPXTG motif.
  • the cultures were maintained at 25°C for 5h after induction with ImM IPTG for the pET clones or with 0.2% arabinose for the SpeedET clones. All recombinant proteins were purified by affinity chromatography and gel filtration. Briefly, cells were harvested by centrifugation and lysed in "lysis buffer", containing lOmM imidazole, lmg ⁇ ml lysozyme, 0.5 mg ⁇ ml DNAse and COMPLETE inhibitors cocktail (Roche) in PBS.
  • lysis buffer containing lOmM imidazole, lmg ⁇ ml lysozyme, 0.5 mg ⁇ ml DNAse and COMPLETE inhibitors cocktail (Roche) in PBS.
  • the lysate was clarified by centrifugation and applied onto His-Trap HP column (Armesham Biosciences) pre-equilibrated in PBS containing lOmM imidazole. Protein elution was performed using the same buffer containing 250mM imidazole, after two wash steps using 20mM and 50mM imidazole buffers. The eluted proteins were then concentrated and loaded onto HiLoad 16/60 Superdex 75 (Amersham Biosciences) pre-equilibrated in PBS.
  • Antisera specific for each protein were produced by immunizing CD1 mice with the purified recombinant proteins as previously described (WO90/07936). Protein-specific immune responses (total Ig) in pooled sera were monitored by ELISA.
  • Example 4 In vitro polymerization with two type of backbone proteins (BP-2a + pilus 1 BP (BP-1) and/or Pneumococcus RrgB) : Following the procedures outlined above, chimeric pili comprising backbone proteins from both Streptococcus agalactiae and Pneumococcus were prepared:
  • Example 4 Mutant SrtC can polymerize GFP-IPQTG
  • the "IQTGGIGT” sequence was added at the C-terminus of the GFP protein DNA sequence using mutagenesis:.
  • GFP-lpxtg_F attccacaaacaggtggtattggtacaTAACGCGACTTAATTAAACGG
  • the SrtCly 86A mutant was able to polymerise GFP-IPQTG.
  • Example 5 Recombinant PI-2b SrtCl and SrtC2 proteins are active in vitro and are able to cleave fluorescent peptides carrying the LPXTG-like motif of pilus proteins
  • Full-length SrtCl and C2 were cloned (using strain COH1 as template) in fusion with a His-MBP-tag.
  • Recombinant enzymes were then expressed in E.coli and purified with IMAC or IMAC and MBP-trap column.
  • FRET assays with purified sortases were carried out using synthetic fluorescent peptides carrying the LPXTG sorting motif of PI-1 backbone protein and of PI-1 minor ancillary protein in order to assess the catalytic activity.
  • the PI-2b SrtCl and SrtC2 enzymes are able to cleave the fluorescent peptides. These data demonstrate that thePI-2b SrtCl and SrtC2 enzymes are active in vitro and are suitable for use in ligating and polymerising proteins.
  • Tecan plate reader - 300 cycles with a measurement every 10 minutes, temperature [34- 37.5° C°] with 37° C for optimum and wavelength [400nm-600 nm] have been obtained with maximum absorption provided to 500nm.
  • Example 6 SrtCl is effective for polymerising BP
  • the activity of SrtCl was further assayed by using a mutant GBS strain that does not express any pili (515A2a).
  • This strain was transformed with complementation vectors PAMp80/t80 carrying genes coding for BP alone or BP with PI-2b SrtCl .
  • the ability of the complementation vectors to restore pili polymerisation was analysed by western blot. As shown in Figure 10, transfection with BP alone did not result in any polymerisation. However, transfection with BP and SrtCl resulted in the formation of high molecular weight polymers. Strain A909, which expresses pilus 2b, was used as a positive control.
  • Figure 10 provides a western blot of the membrane preparation from the 515A2a mutant strain and from the wild type A909 strain complemented by a plasmid containing SrtCl and BP genes or BP gene alone. Antibodies against SrtCl were used. Expected signals at 30 kDa confirm the expression and correct localization of SrtC 1.
  • Vaccine Adjuvants Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.

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Abstract

The invention provides methods of forming pili in vitro and proteins suitable for use in these methods. The invention also provides pili produced by these methods and compositions comprising these pili for the treatment and prevention of bacterial disease, in particular of conditions caused by Streptococcus.

Description

PILUS PROTEINS AND COMPOSITIONS TECHNICAL FIELD
The invention provides methods of forming pili in vitro and mutant sortase enzymes and proteins suitable for use in these methods. The invention also provides pili produced by these methods and compositions comprising these pili for the treatment and prevention of bacterial disease, in particular of conditions caused by Streptococcus. The invention also provides general methods of ligating proteins and sortase enzymes for use in same.
BACKGROUND ART
Most bacterial pathogens comprise pili (also known as fimbrae), long filamentous structures extending from their surface, that are often responsible for initial adhesion of bacteria to tissues during host colonization. Gram-negative bacteria have been known for many years to have pili, typically formed by non-covalent interactions between pilin subunits. More recently, Gram-positive bacteria, including Streptococcus bacteria, have also been shown to have pili typically formed through covalent association of subunits by sortases that are encoded by pilus-specific pathogenicity islands.
The Gram-positive bacterium Streptococcus agalactiae (or "group B streptococcus", abbreviated to "GBS"), for example, has three pilus variants, each encoded by a distinct pathogenicity island, PI-1, PI-2a or PI-2b [1, 2]. Each pathogenicity island consists of: i) genes encoding the three structural components of the pilus (the pilus backbone protein (BP) and 2 ancillary proteins (API and AP2)); and ii) genes encoding 2 sortase proteins (SrtCl and SrtC2) that are involved in the assembly of the pilus. All GBS strains carry at least one of these 3 pathogenicity islands.
Similar pathogenicity islands are present in other Gram-positive bacteria including Streptococcus pyogenes or "group A streptococcus", abbreviated to "GAS"), and Streptococcus pneumoniae (also known as pneumococcus). The pathogenicity island in pneumococcus encodes the 3 structural components of the pilus (RrgA, RrgB and RrgC) and three sortases (SrtCl, SrtC2 and SrtC3) which catalyse pilus formation. In GAS, the FCT regions encode the backbone and accessory proteins and polymerisation of these proteins is also mediated by a sortase (SrtCl). Pilus structures in these Gram-positive bacteria are considered to be interesting vaccine candidates and work has been done on assessing the immunogenicity of purified recombinant proteins from pilus structures. It is also desirable to study these proteins in their native form within assembled pili but currently, the only way to do this is by the laborious process of purifying wild-type pili from the bacteria. One object of the invention is therefore to provide a process for producing recombinant pili in vitro without the need to purify wild-type pili.
The streptococcal bacteria discussed above are associated with serious disease. GBS causes bacteremia and meningitis in immunocompromised individuals and in neonates. GAS is a frequent human pathogen, estimated to be present in between 5-15% of normal individuals without signs of disease. When host defences are compromised or when GAS is introduced to vulnerable tissues or hosts, however, an acute infection occurs. Diseases caused by GAS include puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotising fasciitis, myositis and streptococcal toxic shock syndrome. Pneumococcus is the most common cause of acute bacterial meningitis in adults and in children over 5 years of age
Investigations have been conducted into the development of protein-based vaccines against these Streptococcal bacteria but currently, no protein-based vaccines are commercially available. There therefore remains a need for effective vaccines against Streptococcal infection. It is a further object of the invention to provide immunogenic compositions which can be used in the development of vaccines against streptococcal infection.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a method of ligating at least two moieties comprising contacting the at least two moieties with a pilus-related sortase C enzyme in vitro under conditions suitable for a sortase mediated transpeptidation reaction to occur, wherein the pilus-related sortase C enzyme comprises an exposed active site.
Particularly the pilus-related sortase C enzyme is from Streptococcus, more particularly from Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) and Streptococcus pyogenes (GAS). Yet more particularly the pilus-related sortase C enzyme is a sortase CI enzyme (srtCl), sortase C2 enzyme (SrtC2) or a sortase C3 enzyme (SrtC3). In certain embodiments the pilus-related sortase C enzyme mutation comprises a deletion of part or all of the lid. Particularly the mutation comprises a deletion of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO: 3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another pilus-related sortase C enzyme.
In other embodiments the mutation comprises substitution of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO: 3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme.
Particularly the pilus-related sortase C enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and 71.
In one embodiment of the invention, the method is a method of forming a recombinant or artificial pilus in vitro. This, the at least two moieties comprise an LPxTG motif and a pilin motif. For example, the pilin motif may comprise the amino acids YPAN. 'X' in any sortase recognition motif disclosed herein may be any standard or non-standard amino acid and every variation is disclosed. In some embodiments, X is selected from the 20 standard amino acids found most commonly in proteins found in living organisms. Where the recognition motif is LPXTG or LPXT, X may be D, E, A, N, Q, K, or R. In particular, X is selected from K, S, E, L, A, N in an LPXTG or LPXT motif.
Particularly the at least two moieties are from Gram-positive bacteria. The at least two moieties may be from the same strain or type of Gram-positive bacteria or from different strains or types of Gram positive bacteria. Yet more particularly, the at least two moieties are Streptococcal polypeptides. Still yet more particularly, the at least two moieties are Streptococcal backbone proteins and/or ancillary proteins.
For example, the at least two moieties comprise or consist of an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97; or (b) that is a fragment of at least 'η' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more).
In other aspects of the invention, there is provided an artificial or recombinant pilus obtained or obtainable from the aforementioned method. In one embodiment there is provided an artificial or recombinant pilus which comprises at least two variants of backbone protein GBS59. Particularly the at least two variants are selected from Group B Streptococcus strains 2603, H36B, 515, CJB111, CJB110 and DK21. Yet more particularly, the artificial or recombinant pilus is a chimeric pilus comprising at least one variant of GBS backbone protein GBS59 selected from Streptococcus strains 2603, H36B, 515, CJB111, CJB110 and DK21 and at least one backbone protein from Streptococcus pneumonia selected from the group consisting of RrgA, RrgB and RrgC. In other embodiments artificial or recombinant pili further comprise GBS80 and/or GBS 1523.
In particular aspects of the invention, the artificial or recombinant pilus is for use in medicine, yet more particularly for use in preventing or treating Streptococcal infection. Thus, in another embodiment there is provided a method of treating or preventing Streptococcal infection in a patient in need thereof comprising administering an effective amount of an artificial or recombinant pilus formed by the methods of the invention to a patient.
In a second aspect of the invention, there is provided a method wherein the at least two moieties comprise a first moiety comprising the amino acid motif LPXTG, wherein X is any amino acid, and a second moiety comprising at least one amino acid.
Particularly the first moiety is a first polypeptide and the second moiety is a second polypeptide. In certain embodiments, the first polypeptide and the second polypeptide are from Gram-positive bacteria. For example, the first polypeptide and the second polypeptide may be from the same type or strain of Gram-positive bacteria or from different types or strains of Gram positive bacteria. In some embodiments, the first polypeptide and the second polypeptide are Streptococcal polypeptides. For example, the first polypeptide and the second polypeptide may be Streptococcal backbone proteins and/or ancillary proteins. In some embodiments of the invention, either the first moiety or the second moiety comprises a detectable label. By way of non-limiting example, the detectable label may be a fluorescent label, a radio label, a chemiluminescent label, a phosphorescent label, a biotin label, or a streptavidin label. In some embodiments, the first moiety or the second moiety may be a polypeptide and the other moiety may be a protein or glycoprotein on the surface of a cell. In yet further embodiments, either the first moiety or the second moiety is a polypeptide and the other moiety comprises amino acids conjugated to a solid support. In still yet further embodiments, either the first moiety or the second moiety is a polypeptide and the other moiety comprises at least one amino acid conjugated to a polynucleotide.
The method of the invention may be used to ligate the N-terminus of a first moiety to the N-terminus of a second moiety. The method of the invention may be used to ligate the C- terminus of a first moiety to the C-terminus of a second moiety. Alternatively, the first moiety and the second moiety are the N-terminus and C-terminus of a moiety such as a polypeptide chain, and ligation results in the formation of a circular polypeptide. Thus, there is provided conjugate obtained or obtainable from the method described herein.
In other aspects of the invention, there is provided a kit comprising a sortase CI or a sortase C2 enzyme from Streptococcus agalactiae and a moiety comprising the amino acid motif LPXTG, wherein X is any amino acid.
In another aspect of the invention, there is provided a sortase C enzyme from Streptococcus comprising a mutation in its lid region, particularly a sortase C enzyme from Streptococcus which is from Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) or Streptococcus pyogenes (GAS). Yet more particularly a sortase C enzyme from Streptococcus wherein the sortase C enzyme from Streptococcus is a sortase CI enzyme, sortase C2 enzyme or a sortase C3 enzyme. In certain embodiments, there is provided a sortase C enzyme from Streptococcus wherein the mutation comprises deletion of part or all of the lid region of the sortase C enzyme. Particularly the mutation comprises deletion of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme. In other embodiments there is provided a sortase C enzyme from Streptococcus wherein the mutation comprises substitution of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme Particularly there is provided a sortase C enzyme from Streptococcus which comprises a mutation in its lid region and wherein the sortase C enzyme comprises or consists of an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Alignment of GBS sortase C sequences showing location of the lid region in bold and underlined. Figure 2: Alignment of Streptococcus pneumoniae and Streptococcus pyogenes (GAS) sortase C sequences showing location of the lid region in bold and underlined.
Figure 3: A: Conserved amino acid motifs identified in the backbone protein of GBS pilus 2a (BP-2a), GBS59 (strain 515, TIGR annotation SAL 1486). Pilin motif: containing a highly conserved lysine residue (Lysl89); E-box: containing a highly conserved glutamic acid residue (Glu589); Sorting signal: containing residues IPQTGG located at positions 641-646. B: Immunoblot performed with an antibody recognising the backbone protein of GBS pilus 2a (a-BP), showing that Lysl89 of the pilin motif of BP-2a is required for pilus polymerization by wild type sortase C. A plasmid was generated encoding a mutant BP-2a carrying a substitution at Lysl89 with Ala (BPKI 89A). A GBS mutant strain lacking backbone proteins (GBSABP) was transformed with this plasmid (lane 2), or a control plasmid encoding wild-type BP-2a (BPWT) (lane 1). The star indicates the location of the protein bands corresponding to the monomeric, unpolymerised BP-2a protein. High molecular weight protein bands, corresponding to polymerised BP-2a, are detectable only in cell extracts of GBS transformed with the plasmid encoding wild-type BP-2a (lane 1). C: Immunoblots performed with antibodies recognising the backbone protein of GBS pilus 2a (a-BP) (lanes 1, 2 and 3) or ancillary protein of GBS pilus 2a (a- API) (lanes 4 and 5), showing that the IPQTG motif of BP-2a is required for pilus polymerization. A plasmid was generated encoding a mutant BP-2a carrying a deletion of the IPQTG sorting signal (BPAIPQTG). A GBS mutant strain lacking backbone proteins (GBSABP) was transformed with this plasmid (lanes 3 and 4). As controls, a control plasmid encoding wild-type BP-2a
(BPWT) was used (lane 1), or no plasmid (ΔΒΡ) (lanes 2 and 5). The star indicates the location of the protein bands corresponding to the monomeric, unpolymerised BP-2a protein. The triangle indicates the protein band corresponding to monomeric API protein. The box indicates the protein band corresponding to BP-2a - API conjugates. High molecular weight protein bands, corresponding to polymerised BP-2a, are detectable only in cell extracts of GBS transformed with the plasmid encoding wild-type BP-2a. Figure 4: A: Protein gel showing that wild-type GBS sortase fails to catalyse in vitro polymerization of wild-type backbone protein. Various concentrations of recombinant backbone protein (BP) (25, 100 and 200 μΜ) were incubated at 37°C with wild-type sortase CI of PI-2a (SrtC lwi) for 0, 24 and 48 hours. The proteins contained in the reaction mixture were resolved by sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualised. No formation of high molecular weight bands, corresponding to polymerized BP, was detectable. The star indicates monomeric BP. The hash indicates SrtClwT- Lane 1 : BP 25μΜ+8ι·^1 ντ t0, Lane 2: BP 25μΜ+8Γ^1 ντ t24h, Lane 3 : BP 25μΜ+8ι^1 ντ t48h; Lane 4: BP ΙΟΟμΜ+SrtClwT t0, Lane 5 : BP ΙΟΟμΜ+SrtClwT t24, Lane 6: BP ΙΟΟμΜ+SrtClwT t48h; Lane 7: BP 20(^M+SrtC lwT t0, Lane 8: BP 20(^M+SrtC lwT t24. B: Protein gel showing that wild- type backbone protein (BP) can form BP-BP homodimers in the absence of catalytic sortase activity, explaining the additional bands observed in panel A. Various concentrations of recombinant BP (25 and 100 μΜ) were incubated for 0, 24, 48 and 72 hours and the proteins contained in the reaction mixture were visualised by SDS-PAGE. Lane 1 : BP 25μΜ tOh, Lane 2: BP 25μΜ t24h, Lane 3 : BP 25μΜ t48h, Lane 4: BP 25 μΜ t72; Lane 5 : BP ΙΟΟμΜ tOh, Lane 6: BP ΙΟΟμΜ t24h, Lane 7: BP ΙΟΟμΜ t48h, Lane 8: BP ΙΟΟμΜ t72h.
Figure 5: A: Protein gel showing that a mutant GBS sortase carrying a mutation in the lid region is able to catalyse in vitro polymerization of wild-type backbone protein (BP). Various concentrations of recombinant BP (100 and 200 μΜ) were incubated with mutant sortase CI of PI-2a carrying a tyrosine to alanine substitution at position 86 (SrtCly86A) for 0, 24 or 48 hours and the proteins contained in the reaction mixture visualised by SDS- PAGE. The star indicates monomeric BP. High molecular weight bands (>260 kDa), corresponding to polymerized BP, were detectable after 24 or 48 hours of incubation. Lane 1 : BP
Figure imgf000009_0001
tOh, Lane 2: BP 100μΜ+8ΓΐΟΥ86Α t24h, Lane 3 : BP 100μΜ+8ΓΐΟγ86Α t48h; Lane 4: BP 200μΜ+8ΓΐΟγ86Α tOh, Lane 5 : BP
Figure imgf000009_0002
t24h B: Immunoblot performed with an antibody recognising the backbone protein of GBS pilus 2a ( BP), showing that the pattern of polymerized BP is similar to BP polymers contained in pili from wild-type bacteria (here GBS strain 515). The star indicates monomeric BP. Lane 1 : BP, Lane 2: SrtClY86A, Lane 3: BP+SrtClY86A, Lane 4: GBS515 Wild Type Pili. C: Protein gel showing the effect of different concentrations of SrtCly86A on the efficiency of BP polymerisation. 10, 50 or 100 μΜ of SrtCly86A were mixed with BP and incubated for 0 hours, 48 hours, 3 and 4 days and the proteins contained in the reaction mixtures were visualised by SDS-PAGE. The star indicates monomeric BP. D: Protein gel showing the effect of different concentrations of BP on the efficiency of BP polymerisation. 25, 50 or 100 μΜ of BP were mixed with 25 μΜ of SrtCly86A and incubated for 0 hours, 3 days, 5 days and 7 days and the proteins contained in the reaction mixtures were visualised by SDS-PAGE. The star indicates monomeric BP.
Figure 6: Protein gel showing that in vitro polymerised pili structures can be successfully purified. 25 μΜ of SrtCly86A were incubated with 100 μΜ of BP-2a at 37°C for 7 days. The proteins contained within the mixture were separated into fractions by size exclusion chromatography and visualised by SDS-PAGE. The high-molecular weight fractions containing purified polymerised BP elute first (white box), followed by monomeric BP (star) and SrtCly86A (cross).
Figure 7: Protein gel showing that mutant sortase enzymes polymerize pilus proteins from a variety of gram positive bacteria. A: 25 μΜ of SrtCly86A (GBS sortase CI of PI-2a) were incubated with 100 μΜ of backbone protein PI-1 of GBS (also referred to as GBS 80) at 37°C for 7 days and the proteins contained in the reaction mixtures were visualised by SDS-PAGE. As controls, SrtCly86A or GBS 80 alone were incubated under the same conditions. The star indicates monomeric BP. Lane 1 : SrtCly86A, Lane 2: BP PI-1, Lane 3: SrtClY86A+BP PI-1. B: 25 μΜ of SrtClY86A (GBS sortase CI of PI-2a) were incubated with 50 or 100 μΜ of pilus protein from Streptococcus pneumoniae (also referred to as RrgB) at 37°C for 3 days and the proteins contained in the reaction mixtures were visualised by SDS-PAGE. As controls, SrtCly86A or RrgB alone were incubated under the same conditions. The star indicates monomeric RrgB. Lane 1 : SrtCly86A, Lane 2: RrgB, Lane 3: SrtClY86A+RrgB (50μΜ), Lane 4: SrtClY86A+RrgB (ΙΟΟμΜ). Figure 8: Pairwise sequence alignment of homologous SrtCl sortases from PI-2a of GBS strain 515 and PI-2b of GBS strain A909. The catalytic triad (single underline) is conserved, while the canonical lid motif (double underline) is not present in PI-2b SrtCl . Instead there is a tryptophan that appears to mimic the lid function.
Figure 9: Pairwise alignment of SrtC2 sortase from PI-2b (SAK 1437) and SrtCl sortase from PI-2a (SAL 1484). SrtC2 lacks the lid sequence (highlighted in box), and the C terminal trans-membrane domain. Three cysteine residues are present in PI-2b SrtC2 sequence (marked with crosses).
Figure 10: Western blot of total protein extracts from culture of a mutant strain derived from GBS 515 in which the PI-2a island has been deleted (515A2a) and from the wild type A909 strain complemented by a plasmid containing SrtCl and BP genes or BP gene alone. Antibodies against BP were used. High-molecular weight signals indicate pili polymerization in the complemented strains. M: Marker; Lane 1 : 515A2a; Lane 2: 515A2a+BP; Lane 3: 515A2a+BP+SrtCl; Lane 4: 515A2a+BP+SrtCl; Lane 5: A909+BP; Lane 6: A909+BP+SrtCl .
Figure 11: SDS-PAGE of polymerization reactions. Lane 1 : SrtClY86A + BP-2a -515; Lane 2: SrtClY86A + BP-2a -H36B; Lane 3: SrtClY86A + BP-2a -CJB111; Lane 4: Marker; Lane 5: SrtCl Y86A + BP-2a -515-H36B-CJB111.
Figure 12A: Western blot with polyclonal antibody against BP-1. Lane 1 : SrtCl Y86A; Lane 2: BP-2a - 515 variant; Lane 3: BP-2a - H36B variant; Lane 4: BP-1; Lane 5: RrgB; Lane 6: SrtClY86A + BP-1; Lane 7: SrtClY86A + BP-2a -515+ BP-1; Lane 8: SrtClY86A + BP-2a -H36B+ BP-1; Lane 9: SrtClY86A + RrgB; Lane 10: SrtClY86A + BP-2a -515+ RrgB; Lane 11 : SrtCl Y86A + BP-2a -H36B+ RrgB.
Figure 12B: Western blot with polyclonal antibody against RrgB. . Lane 1 : SrtCl Y86A; Lane 2: BP-2a - 515 variant; Lane 3: BP-2a - H36B variant; Lane 4: BP-1; Lane 5: RrgB; Lane 6: SrtClY86A + BP-1; Lane 7: SrtClY86A + BP-2a -515+ BP-1; Lane 8: SrtClY86A + BP-2a -H36B+ BP-1; Lane 9: SrtClY86A + RrgB; Lane 10: SrtClY86A + BP-2a -515+ RrgB; Lane 11 : SrtCl Y86A + BP-2a -H36B+ RrgB.
Figure 13: Mutant SrtC can polymerize Green Fluorescent Protein (GFP) tagged with an IPQTG sequence.
Figure 14A: The LPXTG motif is essential for in vitro pilus polymerization. Progression of the reaction between the SrtClY86A and recombinant BP-2a AIPQTG at TO, 48 and 72 hours of incubation at 37°C . The concentrations of both SrtClY86A and BP-2a AIPQTG were fixed at 25μΜ and ΙΟΟμΜ respectively. No formation of high molecular weight pattern could be identify, showing that the LPXTG like-motif is necessary for the BP polymerization. As controls the SrtClY86A (on the left) and BP-2a AIPQTG (on the right) were incubated alone. Figure 14B: The lysine of pilin motif is not essential for in vitro pilus polymerization. The SrtClY86A (25μΜ) and the recombinant BP-2a K189A (ΙΟΟμΜ) were mixed at 37°C and at different time points (0, 48h and 72h) the reactions were analysed by SDS-PEGE. A patter of high molecular weight could be identified, showing that the SrtClY86A used another nucleophile different from the lysinel89. Figure 14C: When SrtClY86A was mixed with recombinant forms of the ancillary proteins (API -2a and AP2-2a), that in vivo can be polymerized only in the presence of the BP-2a protein (data not shown), some HMW structures were formed. These data demonstrate that SrtClY86A can use different nucleophile/s to resolve the acyl- intermediate between the enzyme and the LPXTG-like sorting signal. DETAILED DESCRIPTION OF THE INVENTION
Structural studies of pilus-related C-sortases in gram positive bacteria have demonstrated that the active site of many of these enzymes contains a catalytic triad of amino acids that are covered by a mobile "lid" region in the absence of substrate. Thus, a feature of pilus- related sortases is the presence of a lid that not only blocks active site access, i.e. it encapsulates the active site, but also carries two key residues, generally an Asp and a hydrophobic amino acid, that interact within the catalytic cleft itself, serving as 'anchors'. Generally sequences corresponding to lid regions can be identified in all pilus-related sortases characterized to date. In particular, this lid structure has been demonstrated to be present in the sortase CI enzymes from GBS PI-1, PI-2a and PI-2b [3], in the sortase CI, sortase C2 and sortase C3 enzymes from Streptococcus pneumoniae [4, 5], and in the sortase CI enzyme from GAS. Mutation of the lid region in the sortase CI enzyme from GBS PI-2a has been shown not to have an adverse impact on pilus production in complementation studies [3] but until now, no studies have been conducted into the ability of mutant sortases to polymerise proteins in vitro. The inventors have now found that sortase C enzymes are capable of polymerising proteins in vitro more effectively than wild-type sortase C enzyme, for example, resulting in the production of recombinant pili. Wild type sortase C enzyme comprise a "mobile lid" region encapsulating the active site in a closed conformation in the absence of substrate. For example, the lid of SrtCl harbors 3 residues, Asp84, Pro85, and Tyr86 which make interactions with residues of the active site and surroundings. Thus, sortase C enzymes are inactive in vitro and unable to ligate or polymerise moieties such as pilin backbone and ancillary proteins. The inventors have now discovered that by mutating the lid region, the catalytic site can be exposed rendering these mutated enzymes active in vitro. As discussed below, and surprisingly, these mutated enzymes are more active than their wild- type counterparts and yet more surprisingly are capable of recognising a broader range of amino acids. Particularly, mutated enzymes of the invention possess or comprise an exposed catalytic site which is not encapsulated by a "lid" and is available to catalyze a transpeptidation reaction to form an acyl enzyme intermediate in vitro.
The methods of the invention can thus be used to produce artificial or recombinant pili without the need for the labourious purification procedures currently used. Surprisingly, these mutant sortase C enzymes can also be used to polymerise proteins from a variety of sources such as gram positive bacteria, not just proteins derived from the same bacteria as the mutant sortase C enzyme itself. Furthermore, the pili resulting from these methods are immunogenic and may be used in the development of vaccines to treat or prevent diseases caused by the gram positive bacteria from which the component proteins of the pili are derived.
Some pilin subunits within the pilus contain intra-protein isopeptide bonds that form spontaneously, presumably stabilizing the structure of the pilus. Thus, in the context of vaccines, immunisation of a subject with proteins in the form of an artificial or recombinant pilus structure mimicking those encountered by the immune system during invasion/infection may also have advantages in terms of the presence of additional epitopes, such as structural or conformational epitopes based on three-dimensional structure. Such structural or conformational epitopes may be absent from subunit vaccines when the pilus proteins are provided in compositions comprising the isolated, purified forms or as conjugates, such as glycoconjugates. Thus, the polymerised pili proteins may comprise three-dimensional epitopes not predictable from the structure of the proteins alone.
Mutant sortase C enzymes The mutant sortase C enzyme used in the methods of the invention is derived from a wild- type sortase C enzyme from Streptococcus. The mutant sortase C enzyme may, for example, be derived from a wild-type sortase C enzyme from Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) or Streptococcus pyogenes (GAS). The mutant sortase C enzyme may be derived from a sortase CI enzyme, a sortase C2 enzyme or a sortase C3 enzyme. The mutant sortase C enzyme is derived from a wild-type streptococcal sortase C enzyme that comprises a lid region. The lid region is the structural loop of about 15-18 amino acids that covers the catalytic triad of amino acids found in the active site of a sortase C enzyme in the absence of a substrate. The lid region is located within the soluble core domain of the sortase C enzyme, between the signal peptide and transmembrane (TM) region located at the N-terminal of the enzyme and the positively charged domain located at the C-terminal of the enzyme. The location of the lid region in a variety of wild-type Streptococcal sortase C enzymes is summarised in the table below. These sequences are all wild-type sequences which include the N-terminal signal peptide. Table 1 : Location of lid region in Streptococcal sortases
Figure imgf000014_0001
The location of the lid region in other Streptococcal sortase C enzymes can readily be determined by the skilled person by structural analysis or more simply, by alignment of the sequences of these enzyme with the sequences of the Streptococcal proteins having lid regions at known locations shown in Table 1. Figure 1 provides an alignment of GBS sortase C enzymes highlighting the location of the lid regions. Figure 2 provides a similar alignment for sortase C enzymes from GAS and pneumococcus. Any of the sortase C enzymes shown in these Figures having a lid region may be used in the methods of the invention.
The sortase C enzyme from Streptococcus used in the methods of the invention comprises a mutation in its lid region. The mutation may be a substitution, deletion or insertion in the amino acid sequence of the lid region of the mutant sortase C-enzyme relative to the amino acid sequence of the wild-type sortase C enzyme.
Deletion mutants
Where the mutation is a deletion, the mutation may comprise deletion of part or all of the lid region of the wild-type sortase C enzyme. The lid region is typically around 15-18 amino acids long and the mutation may comprise deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 or more amino acids from the lid region, or deletion of all of the amino acids in the lid region.
The mutation may comprise deletion of amino acids at positions predicted to interact with the catalytic triad in the active site of the sortase C enzyme. For example, the mutation may comprise the deletion of amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of other sortase C enzymes. The mutation may thus comprise the deletion of: i) an amino acid at position 84; ii) an amino acid at position 85; iii) an amino acid at position 86; iv) two amino acids at positions 84 and 85; v) two amino acids at positions 84 and 86; vi) two amino acids at positions 85 and 86; or vii) three amino acids at positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme. Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) can readily be determined by alignment. Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) are found at:
- positions 90, 91 and 92 of the GBS sortase CI of PI-1 (SEQ ID NO: l),
- positions 84, 85 and 86 of the GBS sortase C2 of PI-1 (SEQ ID NO:2), - positions 88, 89 and 90 of the GBS sortase C2 of PI-2a (SEQ ID NO:4),
- positions 53, 54 and 55 of the GBS sortase CI of PI-2b (SEQ ID NO:5),
- positions 58, 59 and 60 of the pneumococcal sortase CI (SEQ ID NO: 6),
- positions 50, 51 and 52 of the pneumococcal sortase C2 (SEQ ID NO:7),
- positions 74, 75 and 76 of the pneumococcal sortase C3 (SEQ ID NO: 8), or - positions 46, 47 and 48 of the GAS sortase CI (SEQ ID NO:9), respectively.
Alternatively, the mutation may comprise the deletion of all of amino acids in the lid region. The deletion may comprise further changes at positions within the remaining sortase sequence. For example, the sortase may comprise substitutions, deletions or insertions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acid positions. By way of further example, the sortase may comprise substitutions, deletions or insertions at fewer than, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 additional amino acid positions or any range therebetween.
In particular, the mutation may additionally comprise deletion of part or all of the signal peptide and/or transmembrane domain of the wild-type sortase C enzyme which is N- terminal of the lid region in the wild-type enzyme. The transmembrane domain comprises two alpha- helices. The mutation may comprise deletion of one or both of these two alpha- helices and, optionally, may also comprise deletion of the signal peptide N-terminal of the transmembrane domain. For example, the mutation may comprise deletion of part or all of the lid region and the deletion of 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids N- terminal of the lid region. By way of further example, the mutation may comprise deletion of part or all of the lid region and the deletion of less than 10, 20, 30, 40, 50, 60, 70, 80, 90 amino acids N-terminal of the lid region or any range therebetween. In some embodiments, the mutation comprises the deletion of all of amino acids in the lid region and all of amino acids N-terminal of the lid region. The sortase C enzyme in this embodiment of the invention thus consists of the C-terminal/positively charged domain of the wild-type sortase C enzyme.
The mutation may consist of the deletions described above in the absence of any further mutations. For example, the mutation may consist of deletion of part or all of the lid region, deletion of part or all of the lid region and the signal peptide and/or transmembrane domain, or deletion of part or all of the lid region and the entre N-terminal region in the absence of any further mutations. Examples of sequences of sortase C enzymes where the mutation consists of a) deletion of all of the lid region and the signal peptide/transmembrane domain, b) deletion of all of the lid region and the entire N- terminal regions, and c) deletion of the signal peptide/transmembrane domain and amino acids in the catalytic triad which are suitable for use in the methods of the invention are provided in Table 2 below.
Table 2: Deletion mutants of sortase C enzymes
Figure imgf000017_0001
GAS sortase CI SEQ ID NO: 18 SEQ ID NO:27 SEQ ID NO:36
Mutant sortase enzymes used in the methods of the invention may thus comprise or consist of an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36. Mutant sortase enzymes used in the methods of the invention may also comprise or consist of an amino acid sequence selected from SEQ ID NO: 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 except for the substitution, deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
Substitution mutants
The mutation may comprise one or more amino acid substitutions in the lid region compared to the wild-type sortase C enzyme sequence. The substitution(s) may be at positions in the lid region predicted to interact with amino acids in the catalytic site such that the substitutions abolish normal lid function. The mutation may comprise the substitution of amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution deletion of amino acids at corresponding positions in the amino acid sequence of other sortase C enzymes. The mutation may thus comprise the substitution of: i) an amino acid at position 84; ii) an amino acid at position 85; iii) an amino acid at position 86; iv) two amino acids at positions 84 and 85; v) two amino acids at positions 84 and 86; vi) two amino acids at positions 85 and 86; or vii) three amino acids at positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme. Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) can readily be determined by alignment. Amino acids at positions corresponding to positions 84, 85 and 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) are found at:
- positions 90, 91 and 92 of the GBS sortase CI of PI-1 (SEQ ID NO: l),
- positions 84, 85 and 86 of the GBS sortase C2 of PI-1 (SEQ ID NO:2),
- positions 88, 89 and 90 of the GBS sortase C2 of PI-2a (SEQ ID NO:4), - positions 53, 54 and 55 of the GBS sortase CI of PI-2b (SEQ ID NO:5),
- positions 58, 59 and 60 of the pneumococcal sortase CI (SEQ ID NO: 6),
- positions 50, 51 and 52 of the pneumococcal sortase C2 (SEQ ID NO:7),
- positions 74, 75 and 76 of the pneumococcal sortase C3 (SEQ ID NO: 8), or - positions 46, 47 and 48 of the GAS sortase CI (SEQ ID NO:9), respectively.
The substitutions at positions corresponding to position 84 and/or position 85 and/or position 86 may comprise replacement of the wild-type residue at these positions with an alanine residue.
Where the sortase is GBS sortase CI of PI-1 (SEQ ID NO: l), the mutation may comprise replacement of the aspartate residue at position 90 with an alanine residue (D90A) and/or replacement of the proline residue at position 91 with an alanine residue (P91A), and/or replacement of the tyrosine residue at position 92 with an alanine residue (Y92A).
Where the sortase is GBS sortase C2 of PI-1 (SEQ ID NO:2), the mutation may comprise replacement of the aspartate residue at position 84 with an alanine residue (D84A) and/or replacement of the proline residue at position 85 with an alanine residue (P85A), and/or replacement of the phenylalanine residue at position 86 with an alanine residue (F86A).
Where the sortase is GBS sortase CI of PI-2a (SEQ ID NO:3), the mutation may comprise replacement of the aspartate residue at position 84 with an alanine residue (D84A) and/or replacement of the proline residue at position 85 with an alanine residue (P85A), and/or replacement of the tyrosine residue at position 86 with an alanine residue (Y86A).
Where the sortase is GBS sortase C2 of PI-2a (SEQ ID NO:4), the mutation may comprise replacement of the aspartate residue at position 88 with an alanine residue (D88A) and/or replacement of the proline residue at position 89 with an alanine residue (P89A), and/or replacement of the tyrosine residue at position 90 with an alanine residue (Y90A). Where the sortase is GBS sortase CI of PI-2b (SEQ ID NO:5), the mutation may comprise replacement of the methionine residue at position 53 with an alanine residue (M53A) and/or replacement of the lysine residue at position 54 with an alanine residue (K54A), and/or replacement of the tryptophan residue at position 55 with an alanine residue (W55A). Where the sortase is pneumococcal sortase CI (SEQ ID NO:6), the mutation may comprise replacement of the aspartate residue at position 58 with an alanine residue (D58A) and/or replacement of the proline residue at position 59 with an alanine residue (P59A), and/or replacement of the tryptophan residue at position 60 with an alanine residue (W55A). Where the sortase is pneumococcal sortase C2 (SEQ ID NO:7), the mutation may comprise replacement of the aspartate residue at position 50 with an alanine residue (D50A) and/or replacement of the proline residue at position 51 with an alanine residue (P51A), and/or replacement of the phenylalanine residue at position 52 with an alanine residue (F52A).
Where the sortase is pneumococcal sortase C2 (SEQ ID NO: 8), the mutation may comprise replacement of the aspartate residue at position 74 with an alanine residue (D74A) and/or replacement of the proline residue at position 75 with an alanine residue (P75A), and/or replacement of the phenylalanine residue at position 76 with an alanine residue (F76A).
Where the sortase is GAS sortase CI (SEQ ID NO: 9), the mutation may comprise replacement of the aspartate residue at position 46 with an alanine residue (D46A) and/or and/or replacement of the phenylalanine residue at position 48 with an alanine residue (F48A). The GAS sortase CI enzyme already comprises an alanine residue at position 47.
The mutation may comprise further amino acid changes at positions other than at positions corresponding to positions 84 and/or 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3). For example, the mutation may comprise substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acid positions. Alternatively or in addition to these further substitutions, the mutation may comprise deletions and/or insertions. In particular, the mutation may comprise substitutions at positions corresponding to positions 84 and/or 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) and deletion of a) the signal peptide and/or transmembrane domain, or b) deletion of the entire N- terminal region of the wild-type sortase enzyme.
The sortase may consist of substitutions at positions 84 and/or 85 and/or 86 in the absence of any further mutations. Examples of sequences of sortase C enzymes consisting of substitutions at positions that are equivalent to positions 84 and/or 86 of the lid region of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3) and also consisting of deletion of the signal peptide/transmembrane region which are suitable for use in the methods of the invention are provided in Table 3 below.
Table 3 : Substitution mutants of sortase C enzymes
Figure imgf000021_0001
Figure imgf000022_0001
Mutant sortase enzymes used in the methods of the invention may thus comprise or consist of an amino acid sequence selected from SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71. Mutant sortase enzymes used in the methods of the invention may also comprise or consist of an amino acid sequence selected from SEQ ID NO:37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71 except for the substitution, deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
The mutant sortase C enzymes suitable for use in the methods of the invention described above are also embodiments of the invention in their own right. Particularly sortase mutants are the SrtCly92A and SrtC2F86A because the stability of these enzymes is higher, they are better expressed and more soluble in comparison with, for example SrtCl-ΔΝΤ and SrtC2-ANT deletion mutants. This is surprising since the Vmax of the cleavage reaction for the Y92A and F86A mutants was lower than that of the SrtCl-ΔΝΤ and SrtC2- ΔΝΤ mutants which are also more difficult to purify.
Sortase action
Sortases cleave the LPXTG motif of, for example, pilin proteins and covalently join the C terminus of one moiety, such as a pilin subunit, to a Lys side-chain NH2 group on the next moiety or subunit. Two recognition events are involved in this sortase action. Firstly, the sortase recognition motif (LPXTG or a variant) of the substrate protein must be recognised and bound. Secondly, the acceptor substrate, to which the substrate protein will be transferred, must be recognised and bound, and a specific amino group brought into position to attack the thioacyl intermediate.
Bacterial polypeptides polymerised by the mutant sortase C enzymes The mutant sortase C enzymes described above may be used to polymerise one or more polypeptides. The mutant sortase C enzymes are brought into contact with the one or more polypeptides in vitro and following a period of incubation, polymerised polypeptides are detected, for example by identifying a pattern of high molecular weight bands on SDS gels.
Incubation may be carried out at 37°C. Incubation may be carried out for 1 , 2, 3, 4, 5, 6, 7 days or more. The polypeptides and the mutant sortase C enzymes may be incubated in the presence of a reducing agent, for example DTT lmM, to keep the catalytic cysteine of the mutant sortase C enzyme active. Incubation may be carried out at around pH 7-8.
In contrast to the mutant sortase C enzymes of the invention, the wild-type sortase C enzymes fail to polymerise polypeptides in vitro. For the avoidance of doubt, use of the term "in vitro" refers to the use of isolated and/or purified components of a cell, such as an enzyme, to effect pilus polymerisation without requiring the presence of the cell itself.
The polypeptides polymerised by the mutant sortase C enzymes of the invention typically comprise the LPxTG motif. They may further comprise a pilin motif (consensus WxxxVxVyPK) and/or an E-Box motif (consensus YxLxETxAPxGY) shown to be important for pilus assembly [6]. In particular, the polypeptides may comprise a conserved lysine (K) residues, for example, found in the pilin motif. In other embodiments the polypeptides do not comprise a conserved lysine (K) residue in the pilin motif, i.e. wherein the presence of the conserved lysine residue is excluded. In some embodiments, the polypeptides polymerised by the mutant sortase C enzymes of the invention may comprise an N-terminal glycine residue. Other sequence motifs will be apparent to one skilled in the art and may include, by way of non-limiting example: LPETGG, LPXT, LPXTG, LPKTG, LPATG, LPNTG, IPQTG, IQTGGIGT.
Examples of polypeptides that may be polymerised by the mutant sortase C enzymes of the invention include polypeptides from Gram-positive bacteria, such as the backbone proteins and ancillary proteins that are found in the pili of Gram-positive bacteria. In particular, the mutant sortase C enzyme may be brought into contact with a backbone protein found in a pilus from GBS, GAS or Streptoccoccus pneumoniae. For example, the mutant sortase C enzyme may be brought into contact with the backbone protein from GBS PI-1 (GBS80/SAG0645), the backbone protein from GBS PI-2a (GBS59/SAG1407), the backbone protein from GBS PI-2b (Spbl/SAN1518), the backbone protein from Streptococcus pneumoniae (RrgB), or the backbone protein from GAS (fee6, spyl28, orf80, eftLSLA).
Alternatively or in addition, the mutant sortase C enzyme may be brought into contact with an ancillary protein found in a pilus from GBS, GAS or Streptococcus pneumoniae. For example, the mutant sortase C enzyme may be brought into contact with the ancillary protein 1 (AP-1) from GBS PI-1 (GBS104), the AP-1 from GBS PI-2a (GBS67/SAG1408), the AP-1 from GBS PI-2b (SAN1519), the AP-1 from Streptococcus pneumoniae (RrgA) or the AP-1 from GAS (cpa), the ancillary protein 2 (AP-2) from GBS PI-1 (GBS52), the AP-2 from GBS PI-2a (GBS150/SAG1404), the AP-2 from GBS PI-2b (SAN1516), the AP-2 from Streptococcus pneumoniae (RrgC) or the AP-2 from GAS spyl30, orf82, orf2). The mutant sortase C enzymes of the invention may be used to polymerise homologues, fragments or variants of the wild-type backbone protein and ancillary protein sequences, provided that these homologues, fragments and variants retain the sequences described above necessary for polymerisation by mutant sortase C enzymes. For example, variants of these polypeptides that may be used in the methods of the invention include backbone proteins and/or ancillary protein sequences from which the transmembrane domain has been deleted compared to the wild-type sequence. In addition or instead of the deletion of the transmembrane domain, variants may comprise the additional of a glycine residue at the N-terminal to promote polymerisation.
By way of non-limiting example, the sequences some of these polypeptides which may be polymerised by the mutant sortase enzymes of the invention are provided below for reference. The sequences of additional polypeptides which may be polymerised by the mutant sortases of the invention can be readily determined by the skilled person. Further details of these polypeptides are provided in reference [7].
BP from PI-1 (GBS80 The amino acid sequence of full length GBS80 as found in the 2603 strain is given as SEQ ID NO: 72 herein. Wild-type GBS80 contains a N-terminal leader or signal sequence region at amino acids 1-37 of SEQ ID NO:72. One or more amino acids from the leader or signal sequence region of GBS80 can be removed, e.g. SEQ ID NO:73.
BP from PI-2b (GBS1523/SAN1518 The original 'GBS1523' (SAN1518; Spbl) sequence was annotated as a cell wall surface anchor family protein (see GI: 77408651). For reference purposes, the amino acid sequence of full length GBS 1523 as found in the COH1 strain is given as SEQ ID NO: 110 herein. Preferred GBS 1523 polypeptides for use with the invention comprise an amino acid sequence: (a) having 60% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 110; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 110, wherein 'η' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
The wild-type sequence contains an amino acid motif indicative of a cell wall anchor (LPSTG) at amino acids 468-472 of SEQ ID NO: 110. An E box containing a conserved glutamic residue has also been identified at amino acids 419-429 of SEQ ID NO: l 10, with a conserved glutamic acid at residue 423. The E box motif may be important for the formation of oligomeric pilus-like structures, and so useful fragments of GBS1523 may include the conserved glutamic acid residue. A mutant of GBS1523 has been identified in which the glutamine (Q) at position 41 of SEQ ID NO: l 10 is substituted for a lysine (K), as a result of a mutation of a codon in the encoding nucleotide sequence from CAA to AAA. This substitution may be present in the GBS1523 sequences and GBS1523 fragments (e.g. SEQ ID NO: 112). A further variant of GBS1523 COH1 without signal sequence region is provided as SEQ ID NO: 111.
BP from GBS PI-2a (GBS59
The amino acid sequence of full length GBS59 as found in the 2603 strain is given as SEQ ID NO: 74 herein. Variants of GBS59 exist in strains H36B, 515, CJBl l l, DK21 and CJB110. The amino acid sequence of full length GBS59 as found in the H36B, 515, CJB111, CJB110 and DK21 strains are given as SEQ ID NOs: 75, 76, 77, 78, and 79.
BP from GBS PI-2b (Spbl) The amino acid sequence of full length Sbpl as found in the COH1 strain is given as SEQ ID NO: 80 herein. Wild-type Spbl contains a N-terminal leader or signal sequence region. One or more amino acids from the leader or signal sequence region of Spbl can be removed, e.g. SEQ ID NO:81.
BP from Streptococcus pneumoniae (RrgB) The RrgB pilus subunit has at least three clades. Reference amino acid sequences for the three clades are SEQ ID NOs: 82, 83 and 84 herein.
AP-1 from GBS PI-1 (GBS104/SAG0649
The amino acid sequence of full length GBS 104 as found in the 2603 strain is given as SEQ ID NO: 85 herein. AP-1 from GBS PI-2a (GBS67 The amino acid sequence of full length GBS67 as found in the 2603 strain is given as SEQ ID NO: 86 herein. A variant of GBS67 (SAI1512) exists in strain H36B. The amino acid sequence of full length GBS67 as found in the H36B strain is given as SEQ ID NO: 87. Variants of GBS67 also exists in strains CJBl l l, 515, NEM316, DK21 and CJBl lO. The amino acid sequences of full length GBS67 as found in the C JB 111 , 515, NEM316, DK21 and CJB110 strains are given as SEQ ID NOS: 88, 89, 90, 91, and 92 herein.
AP-1 from GBS PI-2b (GBS1524/SAN1519
The amino acid sequence of full length GBS1524 (SAN1519) as found in the COH1 strain is given as SEQ ID NO: 93 herein. AP-1 from Streptococcus pneumoniae (RrgA)
The amino acid sequence of full length RrgA is given as SEQ ID NO: 94 herein.
AP-2 from GBS PI-1 (GBS052/SAG0646
The amino acid sequence of full length GBS052/SAG0646 as found in the 2603 strain is given as SEQ ID NO: 95 herein. AP-2 from GBS PI-2a (GBS150/SAG1404
The amino acid sequence of full length GBS150/SAG1404 as found in the 2603 strain is given as SEQ ID NO: 96 herein.
AP-2 from Streptococcus pneumonia (RrgC)
The amino acid sequence of full length RrgC is given as SEQ ID NO:97 herein. Polypeptides for use with the invention may thus comprise or consist of an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97 or to any other backbone or ancillary protein sequences described above; or (b) that is a fragment of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more). Alternatively, 'n' is less than 20 or less than 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or less than 150. The methods of the invention may involve polymerisation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 polypeptides having 50% identity to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97, or of or of fragments of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more).
The methods of the invention may involve polymerisation of 1, 2, 3, 4, 5 or 6 polypeptides having 50% identity e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 74, 75, 76, 77, 78 and 79, or of fragments of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more). The methods of the invention may involve polymerisation of 1, 2, or 3 polypeptides having 50% identity e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%o, 99%), 99.5%> or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 82, 83 and 84, or of fragments of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more).
Amino acid fragments of these backbone and ancillary proteins may comprise an amino acid sequence of e.g up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 125, up to 150, up to 175, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450, up to 500, up to 550, up to 600, up to 650, up to 700, up to 750, up to 800, up to 850, up to 900, up to 950, up to 1000, up to 1 100, up to 1200, up to 1300, up to 1400, up to 1500, consecutive amino acid residues of the sequences provided above. Other fragments omit one or more polypeptide domains, for example the transmembrane domain.
The mutant sortase C enzymes of the invention polymerise these polypeptides in a manner that is analogous to the polymerisation of backbone proteins and accessory proteins by wild-type Streptococcal sortase C enzymes in vivo to form a pilus. The polymerised polypeptides produced according to these methods are thus structurally similar to a pilus produced by a Streptococcal bacterium in vivo. Pili in Gram-positive bacteria are constructed from either two or three types of pilin subunits. In two-component pili the shaft of the pilus is formed by multiple copies of a major pilin subunit, while the tip of the pilus contains a single copy of a minor 'tip' pilin subunit that typically functions as an adhesin. Three-component pili are similar, but they also contain a minor 'basal' pilin subunit that is covalently attached to the cell wall. Several transmission electron microscopy (EM) and immuno-gold labelling studies have led to the conclusion that the minor 'basal' pilin subunits are also interspersed throughout the shaft of the pilus, presumably because the sortase enzymes are promiscuous in the substrates they recognize. The mutant sortase C enzymes may be brought into contact with 1 polypeptide, leading to the formation of a monomeric pilus. For example, the mutant sortase enzyme may be brought into contact with GBS80, GBS59 or RrgB, leading to the formation of a monomeric pilus comprising subunits of GBS80, GBS59 or RrgB respectively. Where the polypeptide is from a Gram positive bacterium, the mutant sortase enzyme that is used to polymerise that polypeptide need not be from the same Gram positive bacterium. Thus, a mutant sortase C enzyme derived from GBS can be used to polymerise proteins not just from GBS but also from Streptococcus pneumoniae and/or GAS. Variants of some pilus proteins, such as GBS59 are not generally cross-protective. Therefore, the ability to polymerise combinations of at least 2, 3, 4, 5, 6 or more of these variants within an individual pilus is advantageous, for example avoiding the need for more complex compositions or use of protein fusions to achieve cross-protection. Particularly, pili polymerised in vitro may include a combination of GBS59 variants from GBS strains 515, CJB111, H36B, 2603, DK21 and 090, more particularly a combination of GBS59 variants from GBS strains 515, CJB111, H36B and 2603. Such pili comprising two or more variants of GBS59 are not found in nature because strains of wild type bacteria express only one variant of back-bone protein (BP-2a/GBS59).
Alternatively, the mutant sortase C enzymes may be brought into contact with 2, 3, 4, 5 or more different polypeptides which may be from 1, 2, 3, 4, 5 or more Gram positive bacteria, leading to the formation of a chimeric pilus. The mutant sortase C enzymes may be brought into contact with the backbone and accessory proteins from a single Gram positive bacterium which are found in combination in a natural Streptococcal pilus from that bacterium, resulting in a chimeric pilus that is equivalent in structure to a naturally- occurring pilus. Such chimeric pili are a useful tool to enable the study of pilus properties without the laborious purification process currently used to isolate pili from Gram positive bacteria.
In addition, as discussed above, the three-dimensional structures of the monomeric and chimeric pili produced by the methods of the invention make them particularly convenient and effective for immunisation purposes compared to the administration of individual recombinant proteins. Indeed, protection assays have shown that these pili are more effective at inducing protection against the Streptococcus bacteria from which they are derived than monomeric recombinant proteins. It is postulated that this may be because the pili contain epitopes present in pili in vivo that are not replicated in monomeric recombinant proteins, particularly such epitopes are structural epitopes.
The invention includes pili obtained or obtainable using the methods of the invention. In some aspects, the combinations of polypeptides found in these pili differ from the combination of polypeptides found in naturally-occurring pili in Streptococcal bacteria. Examples of pili that may be produced according to the methods of the invention include pili comprising or consisting of the backbone proteins and/or the ancillary proteins from Streptococcus described above. In some embodiments, these pili do not contain the combinations of polypeptides found in naturally-occurring pili found in GBS, GAS or Streptococcal pneumoniae. Particularly, pili polymerised in vitro differ from naturally- occuring pili in terms of their composition, for example, because the acyl enzyme intermediate is not attached to a wild type sortase but is attached to a mutant sortase of the invention. In other cases, pili polymerised in vitro do not comprise cell wall/membrane components such as lipid II or precursors of peptidoglycan such as MurNAc-N-acetyl- muramic acid. In yet other cases, pili polymerised in vitro comprise combinations of pilus proteins not found in nature. Thus, pili polymerised in vitro can be differentiated from those occurring naturally. Thus, the term "artificial" refers to a synthetic, or non-cell derived composition, particularly a structure which is synthesized in vitro and which is not identical to structures found in native bacteria such as Streptococcus.
Immunogenic compositions comprising pili The invention provides immunogenic compositions comprising the pili described above, which may be obtained or obtainable by the methods of the invention. The term
"immunogenic" is used to mean that the pilus is capable of eliciting an immune response, such as a cell-mediated and/or an antibody response, against the polypeptide or polypeptides making up the pilus when used to immunise a subject (preferably a mammal, more preferably a human or a mouse). Particularly, the immune response is a protective immune response which provides protective immunity. Immunogenic compositions of the invention may be useful as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Prophylactic vaccines do not guarantee complete protection from disease because even if the patient develops antibodies, there may be a lag or delay before the immune system is able to fight off the infection. Therefore, and for the avoidance of doubt, the term prophylactic vaccine may also refer to vaccines that ameliorate the effects of a future infection, for example by reducing the severity or duration of such an infection.
The terms "protection against infection" and/or "provide protective immunity" means that the immune system of a subject has been primed (e.g by vaccination) to trigger an immune response and repel infection. Particularly, the immune response triggered is capable of repelling infection against a number of different strains of bacteria. A vaccinated subject may thus get infected, but is better able to repel the infection than a control subject.
Compositions may thus be pharmaceutically acceptable. They will usually include components in addition to the antigens e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in reference [8].
Compositions will generally be administered to a mammal in aqueous form. Prior to administration, however, the composition may have been in a non-aqueous form. For instance, although some vaccines are manufactured in aqueous form, then filled and distributed and administered also in aqueous form, other vaccines are lyophilised during manufacture and are reconstituted into an aqueous form at the time of use. Thus a composition of the invention may be dried, such as a lyophilised formulation.
The composition may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the vaccine should be substantially free from (i.e. less than 5μg/ml) mercurial material e.g. thiomersal-free. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly preferred. To improve thermal stability, a composition may include a temperature protective agent. Further details of such agents are provided below.
To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml e.g. about 10+2mg/ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290-310 mOsm/kg.
Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20mM range. The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.
The composition is preferably sterile. The composition is preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The composition is preferably gluten free. The composition may include material for a single immunisation, or may include material for multiple immunisations {i.e. a 'multidose' kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material. Human vaccines are typically administered in a dosage volume of about 0.5ml, although a half dose {i.e. about 0.25ml) may be administered to children.
Immunogenic compositions of the invention may also comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents include one or more adjuvants. The adjuvants may include a TH1 adjuvant and/or a TH2 adjuvant, further discussed below. Adjuvants which may be used in compositions of the invention include, but are not limited to:
• mineral salts, such as aluminium salts and calcium salts, including hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates) and sulphates, etc. [e.g. see chapters 8 & 9 of ref. 9];
• oil-in-water emulsions, such as squalene-water emulsions, including MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer) [Chapter 10 of ref.9, see also ref. 10-13, chapter 10 of ref. 14 and chapter 12 of ref. 15], complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A);
• saponin formulations [chapter 22 of ref. 9], such as QS21 [16] and ISCOMs [chapter 23 of ref. 9];
• virosomes and virus-like particles (VLPs) [17-23];
• bacterial or microbial derivatives, such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives [24, 25], immunostimulatory oligonucleotides [26-31], such as IC-31™ [32] (deoxynucleotide comprising 26- mer sequence 5'-(IC)i3-3' (SEQ ID NO: 46) and polycationic polymer peptide comprising 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 47)) and ADP-ribosylating toxins and detoxified derivatives thereof [33 - 42];
• human immunomodulators, including cytokines, such as interleukins (e.g. IL-1 , IL- 2, IL-4, IL-5, IL-6, IL-7, IL-12 [43, 44], interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor;
• bioadhesives and mucoadhesives, such as chitosan and derivatives thereof, esterified hyaluronic acid microspheres [45] or mucoadhesives, such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulos [46];
• microparticles (i.e. a particle of -lOOnm to ~150um in diameter, more preferably ~200nm to ~30um in diameter, and most preferably ~500nm to ~10μιη in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(a-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycapro lactone, etc.);
• liposomes [Chapters 13 & 14 of ref. 9, ref. 47-49];
• polyoxy ethylene ethers and polyoxy ethylene esters [50];
• PCPP formulations [51 and 52];
• muramyl peptides, including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-normuramyl-l-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2-( -2'-dipalmitoyl-5/7- glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE); and
• imidazoquinolone compounds, including Imiquamod and its homologues {e.g.
"Resiquimod 3M") [53 and 54].
Immunogenic compositions and vaccines of the invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil- in- water emulsion [55]; (2) a saponin {e.g. QS21) + a non-toxic LPS derivative {e.g. 3dMPL) [56]; (3) a saponin {e.g. QS21) + a non-toxic LPS derivative {e.g. 3dMPL) + a cholesterol; (4) a saponin {e.g. QS21) + 3dMPL + IL-12 (optionally + a sterol) [57]; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [58]; (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL). Other substances that act as immunostimulating agents are disclosed in chapter 7 of ref. 9.
The use of an aluminium hydroxide and/or aluminium phosphate adjuvant is particularly preferred, and antigens are generally adsorbed to these salts. Calcium phosphate is another preferred adjuvant. Other preferred adjuvant combinations include combinations of Thl and Th2 adjuvants such as CpG & alum or resiquimod & alum. A combination of aluminium phosphate and 3dMPL may be used (this has been reported as effective in pneumococcal immunisation [59]).
The compositions of the invention may elicit both a cell mediated immune response as well as a humoral immune response. This immune response will preferably induce long lasting (e.g. neutralising) antibodies and a cell mediated immunity that can quickly respond upon exposure to infection.
Two types of T cells, CD4 and CD8 cells, are generally thought necessary to initiate and/or enhance cell mediated immunity and humoral immunity. CD8 T cells can express a CD8 co-receptor and are commonly referred to as Cytotoxic T lymphocytes (CTLs). CD8 T cells are able to recognized or interact with antigens displayed on MHC Class I molecules.
CD4 T cells can express a CD4 co-receptor and are commonly referred to as T helper cells. CD4 T cells are able to recognize antigenic peptides bound to MHC class II molecules. Upon interaction with a MHC class II molecule, the CD4 cells can secrete factors such as cytokines. These secreted cytokines can activate B cells, cytotoxic T cells, macrophages, and other cells that participate in an immune response. Helper T cells or CD4+ cells can be further divided into two functionally distinct subsets: THl phenotype and TH2 pheno types which differ in their cytokine and effector function.
Activated THl cells enhance cellular immunity (including an increase in antigen- specific CTL production) and are therefore of particular value in responding to intracellular infections. Activated THl cells may secrete one or more of IL-2, IFN-γ, and TNF-β. A THl immune response may result in local inflammatory reactions by activating macrophages, NK (natural killer) cells, and CD8 cytotoxic T cells (CTLs). A THl immune response may also act to expand the immune response by stimulating growth of B and T cells with IL-12. THl stimulated B cells may secrete IgG2a. Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections. Activated TH2 cells may secrete one or more of IL-4, IL-5, IL- 6, and IL-10. A TH2 immune response may result in the production of IgGl , IgE, IgA and memory B cells for future protection.
An enhanced immune response may include one or more of an enhanced THl immune response and a TH2 immune response. A TH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFN-γ, and TNF-β), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. Preferably, the enhanced TH1 immune response will include an increase in IgG2a production.
A TH1 immune response may be elicited using a TH1 adjuvant. A TH1 adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant. TH1 adjuvants suitable for use in the invention may include for example saponin formulations, virosomes and virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides. Immunostimulatory oligonucleotides, such as oligonucleotides containing a CpG motif, are preferred TH1 adjuvants for use in the invention.
A TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgGl, IgE, IgA and memory B cells. Preferably, the enhanced TH2 immune resonse will include an increase in IgGl production.
A TH2 immune response may be elicited using a TH2 adjuvant. A TH2 adjuvant will generally elicit increased levels of IgGl production relative to immunization of the antigen without adjuvant. TH2 adjuvants suitable for use in the invention include, for example, mineral containing compositions, oil-emulsions, and ADP-ribosylating toxins and detoxified derivatives thereof. Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use in the invention.
Preferably, the invention includes a composition comprising a combination of a TH1 adjuvant and a TH2 adjuvant. Preferably, such a composition elicits an enhanced TH1 and an enhanced TH2 response, i.e., an increase in the production of both IgGl and IgG2a production relative to immunization without an adjuvant. Still more preferably, the composition comprising a combination of a TH1 and a TH2 adjuvant elicits an increased TH1 and/or an increased TH2 immune response relative to immunization with a single adjuvant (i.e., relative to immunization with a TH1 adjuvant alone or immunization with a TH2 adjuvant alone). The immune response may be one or both of a TH1 immune response and a TH2 response. Preferably, immune response provides for one or both of an enhanced TH1 response and an enhanced TH2 response.
The enhanced immune response may be one or both of a systemic and a mucosal immune response. Preferably, the immune response provides for one or both of an enhanced systemic and an enhanced mucosal immune response. Preferably the mucosal immune response is a TH2 immune response. Preferably, the mucosal immune response includes an increase in the production of IgA.
The compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared as a solid dosage form for parenteral or needleless administration, for example intra-dermal administration. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
Where a composition is to be prepared extemporaneously prior to use (e.g. where a component is presented in lyophilised form) and is presented as a kit, the kit may comprise two vials, or it may comprise one ready- filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the pilus, as well as any other components, as needed. By 'immunologically effective amount', it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Examples of an immunologically effective amount are around 0. ^g-l(^g pilus, for example 0^g-l(^g pilus.
As mentioned above, a composition may include a temperature protective agent, and this component may be particularly useful in adjuvanted compositions (particularly those containing a mineral adjuvant, such as an aluminium salt). As described in reference 60, a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point e.g. to reduce the freezing point to below 0°C. Thus the composition can be stored below 0°C, but above its freezing point, to inhibit thermal breakdown. The temperature protective agent also permits freezing of the composition while protecting mineral salt adjuvants against agglomeration or sedimentation after freezing and thawing, and may also protect the composition at elevated temperatures e.g. above 40°C. A starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1-80% by volume of the final mixture. Suitable temperature protective agents should be safe for human administration, readily miscible/soluble in water, and should not damage other components {e.g. antigen and adjuvant) in the composition. Examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG). Suitable PEGs may have an average molecular weight ranging from 200-20,000 Da. In a preferred embodiment, the polyethylene glycol can have an average molecular weight of about 300 Da ('PEG-300').
Methods of treatment, and administration of the vaccine The invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention, or a pilus of the invention. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. The method may raise a booster response. The invention also provides immunogenic combinations or compositions for use as a medicament e.g. for use in raising an immune response in a subject, such as a mammal. The invention also provides the use of the pilus of the invention in the manufacture of a medicament for raising an immune response in a mammal.
By raising an immune response in the mammal by these uses and methods, the mammal can be protected against diseases caused by the bacteria from which the polypeptides in the pilus are derived. In particular, the mammal can be protected against disease caused by Streptococcal bacteria, including GAS, GBS and Streptococcus pneumoniae. The invention also provides a delivery device pre-filled with an immunogenic composition of the invention.
The mammal is preferably a human, a large veterinary mammal (e.g. horses, cattle, deer, goats, pigs) and/or a domestic pet (e.g. dogs, cats, gerbils, hamsters, guinea pigs, chinchillas). Most preferably, the mammal is a human, e.g. human patient. Where the vaccine is for prophylactic use, the human may be a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human may be a teenager or an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. A mammal (e.g. human, e.g. a patient) may either be at risk from the disease themselves or may be a pregnant female, e.g. woman ('maternal immunisation'). Vaccination of pregnant females may be advantageous as a means of providing antibody mediated passive protection to new born mammals. Maternal passive immunity is a type of naturally acquired passive immunity, and refers to antibody- mediated immunity conveyed to a fetus by its mother during pregnancy. Maternal antibodies (MatAb) are passed through the placenta to the fetus by an FcRn receptor on placental cells. This occurs around the third month of gestation. Particularly the antibodies are Immunoglobulin G (IgG) or Immunoglobulin A (IgA). IgGy antibody isotypes can pass through the placenta during pregancy. Passive immunity may also provided through the transfer of IgA antibodies found in breast milk that are transferred to the gut of the infant, protecting against bacterial infections, until the newborn can synthesize its own antibodies.
One way of checking efficacy of therapeutic treatment involves monitoring infection after administration of the compositions of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses, systemically (such as monitoring the level of IgGl and IgG2a production) and/or mucosally (such as monitoring the level of IgA production), against the antigen(s) in the pilus of the invention after administration of the composition. Typically, antigen-specific serum antibody responses are determined post-immunisation but pre-challenge whereas antigen-specific mucosal antibody responses are determined post-immunisation and post-challenge.
Another way of assessing the immunogenicity of the compositions of the present invention is to express the proteins recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays. A positive reaction between the protein and the patient sample indicates that the patient has mounted an immune response to the protein in question. This method may also be used to identify immunodominant antigens and/or epitopes within antigens. The efficacy of compositions of the invention can also be determined in vivo by challenging animal models of infection, e.g., guinea pigs or mice, with the vaccine compositions.
Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection {e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or mucosally, such as by rectal, oral {e.g. tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration.
The invention may be used to elicit systemic and/or mucosal immunity, preferably to elicit an enhanced systemic and/or mucosal immunity. Preferably the enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response. Preferably, the enhanced immune response includes an increase in the production of IgGl and/or IgG2a and/or IgA.
Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart {e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). Vaccines prepared according to the invention may be used to treat both children and adults.
Thus a human patient may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients for receiving the vaccines are the elderly (e.g. >50 years old, >60 years old, and preferably >65 years), the young (e.g. <5 years old), hospitalised patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population.
Vaccines produced by the invention may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre) other vaccines e.g. at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzae type b vaccine, an inactivated polio virus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Y vaccine), a respiratory syncytial virus vaccine, etc.
Further antigenic components of compositions of the invention
The invention also provides compositions further comprising at least one further antigen.
In particular, the invention also provides a composition comprising a polypeptide of the invention and one or more of the following further antigens:
- a saccharide antigen from N. meningitidis serogroup A, C, W135 and/or Y (preferably all four). - a saccharide or polypeptide antigen from Streptococcus pneumoniae [e.g. 61, 62, 63].
- an antigen from hepatitis A virus, such as inactivated virus [e.g. 64, 65].
- an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 65, 66]. - a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 67] or the CRMi97 mutant [e.g. 68].
- a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 67].
- an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 69 & 70]. - a saccharide antigen from Haemophilus influenzae B [e.g. 71].
- polio antigen(s) [e.g. 72, 73] such as IPV.
- measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 67].
- influenza antigen(s) [e.g. chapter 19 of ref. 67], such as the haemagglutinin and/or neuraminidase surface proteins.
- an antigen from Moraxella catarrhalis [e.g. 74].
- an protein antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 15, 76].
- a saccharide antigen from Streptococcus agalactiae (group B streptococcus). - an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 76, 77, 78].
- an antigen from Staphylococcus aureus [e.g. 79].
- an antigen from E. coli
The composition may comprise one or more of these further antigens.
Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [70]).
Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
Saccharide antigens are preferably in the form of conjugates. Carrier proteins for the conjugates include diphtheria toxin, tetanus toxin, the N. meningitidis outer membrane protein [80], synthetic peptides [81,82], heat shock proteins [83,84], pertussis proteins [85,86], protein D from H. influenzae [87], cytokines [88], lymphokines [88], streptococcal proteins, hormones [88], growth factors [88], toxin A or B from C. difficile [89], iron- uptake proteins [90], etc. A preferred carrier protein is the CRM 197 mutant of diphtheria toxin [91]. Antigens in the composition will typically be present at a concentration of at least ^g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
As an alternative to using proteins antigens in the immunogenic compositions of the invention, nucleic acid (preferably DNA e.g. in the form of a plasmid) encoding the antigen may be used.
Antigens are preferably adsorbed to an aluminium salt.
Surprisingly, the Inventors have discovered that the pilin motif is not required for polymerisation by mutant sortases of the invention in contrast to the wild type sortases (from which the mutants are derived) wherein the presence of this motif is essential. In addition, mutant sortases of the invention can use different nucleophile/s to resolve the acyl-intermediate between the enzyme and the LPXTG-like sorting signal. In contrast, wild type sortases from which the mutant sortases are derived require the presence of a lysine residue. Mutant sortases of the invention are effective in vitro at catalysing transpeptidation reactions and forming polymers of GBS pilus proteins. Mutant sortases of the invention are further useful in a variety of protein engineering applications. The structural differences between the sortases of the present invention and other pilus-related sortases in gram positive bacteria may provide new functionality and enable new in vitro methods to be performed, or may allow polymerisation and ligation reactions to be performed more efficiently.
The mutant sortase enzymes of the invention are useful for performing ligation reactions between any moiety that comprises the LPXTG recognition motif (or those listed above) and any moiety that comprises an amino acid residue that can provide the nucleophile to complete the transpeptidation reaction. As shown in the Examples, mutant sortases of the invention are able to cleave and polymerise backbone proteins and ancillary proteins comprising the LPXTG motif. Previous work has demonstrated that bacterial sortases require only a single amino acid to provide the nucleophile to complete the transpeptidation reaction (Proft., Biotechnology Letters, 2010, 32: 1-10; Popp et al, Current Protocols in Protein Science, 2009, 15, WO2010/087994). In certain embodiments of the methods of the invention, either the first moiety or the second moiety in the ligation is a polypeptide and the other moiety is a protein or glycoprotein on the surface of a cell. The sortases of the invention can be used to attach polypeptides to proteins on the cell surface. This can be particularly useful for, for example, labelling specific proteins on the cell surface. In certain embodiments, the cell has been transfected to express the surface protein of interest with a LPXTG motif. This motif can then be targeted for ligation using a sortase of the invention. Alternatively, the protein label may comprise the motif.
Use of Sortases for ligation of substrates other than pilus proteins
In other embodiments of the invention, mutant sortases of the invention are used to ligate proteins to a solid support and either the first moiety or the second moiety is a polypeptide and the other moiety comprises amino acids conjugated to a solid support. Such covalent attachment allows extensive washing to be carried out. In certain such embodiments, the protein comprises the LPXTG motif and the solid support has amino acids, such as lysine, conjugated to it. In certain embodiments the solid support is a bead, such as a polystyrene bead or gold bead or particle such as a nanoparticle. Similarly, the methods of the invention allow circularisation of polypeptide chains. In such embodiments the first moiety and the second moiety are the N-terminus and C-terminus of a polypeptide chain, and ligation results in the formation of a circular polypeptide.
Bacterial sortases are also of significant interest for protein modification and engineering applications. Sortases promote pilin formation in vivo by catalysing a transpepditation reaction between backbone and ancillary proteins. Sortases recognise and cleave a recognition motif (for example, LPXTG) and form an amide linkage with a target protein. By utilising the recognition motif, a variety of protein engineering functions can be performed. Ligation reactions performed using sortases are flexible, efficient and require fewer steps than comparable chemical ligation techniques. Therefore, another object of the invention is to provide improved sortases for protein engineering applications. The techniques of Sortagging are known in the art.
In addition to the sortase mutants described above, other sortase enzymes may be used for ligation.For example, the sortases SrtCl and SrtC2 from GBS pathogenicity island PI-2b.
The amino acid sequence of wild type SrtCl from PI-2b is presented in SEQ ID NO:5. Particularly, SrtCl as used in the methods of the invention does not comprise a signal peptide or N-terminal transmembrane domain (as in SEQ ID NO:98, SEQ ID NO:99 or SEQ ID NO: 100). In certain preferred embodiments, SrtCl as used in the methods of the invention comprises SEQ ID NO: 101, which corresponds to the cloned soluble domain. SrtCl comprising SEQ ID NO: 101, which corresponds to the cloned soluble domain. In certain embodiments, SrtCl may have a W55F mutation (as in SEQ ID NO: 102). W55 may be important in regulating the activity of SrtCl , because it is located in the region that the canonical sortases lid motif is normally found in Streptococcal sortases. W55 may mimic the function of the lid found in other sortases. In certain embodiments, the SrtCl as used in the methods of the invention may have a C188A mutation (as in SEQ ID NO: 103). CI 88 may be a catalytic cysteine. The amino acid sequence of wild type SrtC2 from PI-2b is presented in SEQ ID NO: 105. In certain embodiments, the SrtC2 as used in the methods of the invention may have its cysteines substituted with alanines (as in SEQ ID NO: 106). In certain embodiments of the invention, SrtC2 as used in the methods of the invention does not comprise a signal peptide or N-terminal transmembrane domain (as in SEQ ID NO: 108 or SEQ ID NO: 109). The skilled person is capable of identifying any signal peptide or N-terminal transmembrane domain.
PI-2b sortase CI and sortase C2 enzymes for use with the invention may thus comprise or consist of an amino acid sequence: (a) having 70% or more identity (e.g. 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs:5, 98,100, 101 , 102, 103, 105, 106, 108 and 109; or (b) that is a fragment of at least 'n' consecutive amino acids of one of these sequences wherein 'n' is 100 or more (e.g. 120, 150, 170 or 190 or more). PI-2b sortase CI and sortase C2 enzymes for use with the invention retain the ability to perform ligation and polymerisation reactions. The nucleotide sequences encoding SrtCl and SrtC2 are provided in SEQ ID NO: 104 and SEQ ID NO: 107. Particular recognition motifs may include LPETGG, LPXTG, LPXT, LPKTG, LPATG, LPNTG, LPET, VPDT, IPQT, YPR , LPMT, LAFT, LPQT, NSKT, NPQT, NAKT, NPQS, LPKT, LPIT, LPDT, SPKT, LAET, LAAT, LAET, LAST, LPLT, LSRT.
General The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 92-99, etc. The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.
The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do no materially alter the basic and novel characteristics of the claimed composition, method or structure. The term "consisting of is generally taken to mean that the invention as claimed is limited to those elements specifically recited in the claim (and may include their equivalents, insofar as the doctrine of equivalents is applicable). The term "about" in relation to a numerical value x means, for example, x+10%.
References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 100. A preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith- Waterman homology search algorithm is disclosed in ref. 101. The percent identity of a first polypeptide and a second polypeptide is generally determined by counting the number of matched positions between the first and second polypeptides and dividing that number by the total length of the shortest polypeptide followed by multiplying the resulting value by 100. For fragments of polypeptides this value is usually around 100% and therefore has little meaning. Therefore, in the context of fragments of the present invention, the term "proportion of reference polypeptide" (expressed as a percentage) is used. Proportion of reference polypeptide is calculated by counting the number of matched positions between the fragment and reference polypeptides and dividing that number by the total length of the reference polypeptide followed by multiplying the resulting value by 100. Particularly, fragments will comprise less than 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25 or less than 20% of the sequence of the reference polypeptide. MODES FOR CARRYING OUT THE INVENTION Example 1: Functional regulation of GBS SrtCl: a single mutation in the lid region enhances BP polymerization in vitro.
Summary
Cell-surface pili are important virulence factors and promising vaccine candidates. Gram- positive bacteria elaborate pili via a sortase C-catalyzed transpeptidation mechanism from backbone and ancillary pilin substrates. For the covalent crosslinking of individual subunits, specific residues and/or motifs, such as the pilin motif and the conserved LPxTG sorting signal are absolutely necessary. Site-directed mutagenesis of GBS sortase CI of Pi- a (SrtCl) reveals the specific involvement of Tyr86 in the lid-regulatory site in the activation of recombinant SrtCl . This example shows that recombinant BP high molecular weight pili structures can be obtained in vitro using catalytic enzyme concentrations. This provides direct evidence of self-inhibition of sortase C enzymes by the presence of the lid and opens a field for studying pili assembly by using recombinant pili polymerized by a sortase-active mutant, reducing the necessity to purify high amount of wild type pili from pathogenic bacteria.
Background
Group B Streptococcus (GBS), or Streptococcus agalactiae, is the leading cause of life- threatening diseases in newborn and is also becoming a common cause of invasive disease in nonpregnant, elderly or immune-compromised adults [102]. Pili, long filamentous fibers protruding from bacterial surface, have been discovered in Gram-Positive pathogens as important virulence factors and potential vaccine candidates. From the analysis of the eight sequenced genomes of GBS, two genomic islands, each coding for three different pili, have been identified [103; 1]. Moreover, the srtA locus that encodes the "housekeeping' sortase A (SrtA) is present in a different genome region in all analyzed GBS strains [103]. Each pilus genomic island codes for three LPXTG proteins: the backbone protein (BP) representing the main pilus subunit, and two ancillary proteins (API and AP2). Moreover, each island encodes at least two class C sortases, each having specificity for one of the ancillary proteins [1; 104]. The crystal structures of several pilin related sortases, SrtCl -3 from S. pneumoniae [4], AcSrtC-1 from Actinomyces oris [105] and SrtCl from S.suis (106) have been recently solved, with only the S.suis SrtCl in an open active-site conformation Moreover, the crystal structure of S. pyogenes Spy_0129 has been solved, showing that it belongs to the class B sortase family, different to the other characterized pilin-specific sortases, which belong to class C. We have previously reported a structural and functional characterization of GBS SrtCl-2a. The crystal structure of the soluble core of GBS SrtCl-2a, containing its catalytic domain indicates that SrtCl employs a catalytic triad composed of Hisl57-Cys219-Arg228, essential for pilus fiber formation and covered by a loop, known as "lid", which is dispensable for sortase activity in vivo [3]. Moreover, the crystal structure suggests that SrtCl is folded as an auto -inactivated enzyme, by the presence of the lid that sterically blocks the active site. The function of the lid region in enzyme regulation and activity is still unclear, but is supposed to have a role in selecting the proper pilus proteins for polymerization. In this work we show, for the first time, efficient recombinant BP high molecular weight structures by using catalytic enzyme concentrations.
Methods
Bacterial strains and growth conditions
GBS 515 strain and mutants were grown in Todd Hewitt Broth (THB) or in Trypticase soy agar (TSA) supplemented with 5% sheep blood at 37°C.
Cloning, expression and purification of recombinant proteins
The proteins SrtC I 43-292, (SEQ ID NO:3 without signal and transmembrane domains), SrtClY86A (SEQ ID NO:48) and SrtC 1 ALID (SEQ ID NO: 12) were expressed as His-MBP, TEV cleavable, fusion proteins and purified as previously described [3]. Recombinant BP30-649, containing both the pilin motif and the sorting signal, was cloned in speedET vector and expressed as previously described [107], and BPKI 89A was generated by PIPE site-directed mutagenesis using wild type BP30-649. Recombinant BP30-64o, lacking the C- terminal LPxTG motif was cloned in speedET vector and expressed and purified as N terminal His-tag, TEV cleavable, fusion protein using the same protocol used for wild type BP.
Antisera
Antisera specific for the BP-2a and API -2a proteins were produced by immunizing CD1 mice with the purified recombinant proteins [107, 108].
Construction of complementation vectors and site-specific mutagenesis GBS knock-out (KO) mutant strain for BP was generated as previously reported [1]. For the generation of complementation vectors DNA fragments corresponding to wild type BP (SAL 1486), gene was PCR amplified from GBS 515 genome and the product was cloned into the E. co/z'-streptococcal shuttle vector pAM401/gbs80P+T, previously described [11, 27] and containing the promoter and terminator regions of the gbs80 gene (TIGR annotation SAG 0645). Site-directed mutagenesis of pAM BP was performed using the PIPE (Polymerase Incomplete Primer Extension) method [19]. The complementation vectors PAM BPALPXTG and pAM_BPKi 89A were electroporated into the KO strain ΔΒΡ. Complementation was confirmed by checking BP expression by Western Blotting. Western Blotting Analysis
Mid-exponential phase bacterial cells were resuspended in 50mM Tris-HCl containing 400U of mutanolysin (Sigma-Aldrich) and COMPLETE protease inhibitors (Roche). The mixtures were then incubated at 37°C for lh and cells lysed by three cycles of freeze- thawing. Cellular debris were removed by centrifugation and protein concentration was determined using BCA protein assay (Pierce, Rockford, IL). Total protein extracts (20 μg) or recombinant pili were resolved on 3-8% or 4-12% NuPAGE precast gels (Invitrogen) by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with mouse antiserum directed against BP and API proteins (1 :1,000 dilution) followed by a rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). Bands were then visualized using an Opti-4CN substrate kit (Bio-Rad).
Results
Lysine 189 in the putative pilin motif and IPQTG sorting signal of BP-2a are essential for pilus formation by wild-type sortase C.
In the backbone protein of GBS pilus 2a, BP-2a (strain 515, TIGR annotation SAL 1486) we identified a putative pilin motif containing a highly conserved lysine residue (Lysl89) and the IPQTGG motif at residue 641-646 as the C terminus sorting motif (Fig. 3 A). In order to investigate the specific contribution in pilus assembly of each residue/motif we used site-specific mutagenesis and complementation studies using the PIPE (Polymerase Incomplete Primer Extension) mutagenesis method to the vector pAM401 previously used in complementation studies of GBS knock-out (KO) mutant strains. As template for the introduction by PCR of specific mutations/deletions we used the complementation vector carrying the BP-2a gene (pAM BP).
To evaluate the role of the Lysl 89 in the pilin motif and the IPQTGG motif in the cell wall sorting signal (CWSS) of BP-2a we generated a plasmid (pAM_BPKi 89A) expressing a mutated backbone protein carrying a substitution of the pilin motif lysine residue with an alanine and a second plasmid (PAM BPAIPQTG) carrying the entire deletion of the IPQTG sorting signal. Both the K189 and the C terminus sorting signal of BP-2a were absolutely required for pilus polymerization and ancillary proteins incorporation into the high molecular weight structures (Fig. 3B). When the K189 was mutated into an alanine, only the monomer form of the BP could be identified, whereas when the sorting signal IPQTG was deleted in the BP, in addition to the monomeric form of BP a higher molecular weight band was also observed (Fig. 3C). Immunoblotting performed with antibodies raised against BP and API showed that this higher molecular weight band, resistant to SDS treatment, contained both the backbone protein (BP) and the major ancillary protein (API) (Fig. 3C). The polymerization of the BP cannot occur as its sorting signal is deleted, but the pilin motif of the BP is still available for forming a covalent bond between the BP pilin motif and the API sorting signal.
The LPXTG-like sorting signal is essential for the transpeptidation reaction mediated in vitro by the SrtCly86A mutant but the pilin motif is NOT.
To investigate the specific contribution of the Lysl 89 in the pilin motif and the IPQTG sorting signal in the in vitro polymerization reaction, we expressed in E. coli and purified mutated forms of the BP-2a protein, BPAIPQTG and BPKI89A, carrying the deletion of the IPQTG region and the substitution of the Lysl 89 with an alanine, respectively. After mixing the active SrtCly86A with the recombinant BPAIPQTG mutant, HMW polymers could not be detected, confirming that the polymerization reaction occurs through the cleavage of the sorting signal and the formation of the acyl- intermediate between SrtCly86A and the IPQTG motif (Fig. 14A). On the contrary, in the reactions in which the active SrtCly86A was incubated with BPKI 89A HMW polymers could be observed, indicating that the Lys residue of the pilin motif (K189), differently from what happens in GBS, is not essential for in vitro polymerization (Fig. 14B). Moreover, when SrtCly86A was mixed with recombinant forms of the ancillary proteins (API -2a and AP2-2a), that in vivo can be polymerized only in the presence of the BP-2a protein (data not shown), some HMW structures were formed (Fig. 14C). These data demonstrate that SrtCly86A can use different nucleophile/s to resolve the acyl-intermediate between the enzyme and the LPXTG-like sorting signal. Therefore, since the pilin motif is not required, surprisingly this finding suggests that the mutant enzyme may be used in a broader range of reactions and is able to catalyse reactions with proteins to which an LPXTG motif has been added.
Wild-type SrtCl-2a is not able to induce recombinant BP polymerization in vitro.
The presence of pili on GBS surface is characterized by a ladder of high-molecular- weight bands on SDS-PAGE by immunoblotting analysis of cell-wall preparations, in which GBS BP monomers are covalently linked forming the pilus backbone [1]. To test the hypothesis that it is the interaction with the backbone-protein substrate that induce the lid-open-active conformation of SrtCl, we tested the functional activity in vitro of recombinant SrtCl (r- SrtCl) and recombinant backbone protein (r-BP) (107), by searching for a pattern of high- molecular- weight bands on gradient SDS gels. Recombinant GBS major pilin subunit BP carrying the pilin motif Kl 89 and the C-terminal LPxTG recognition site, was mixed with WT SrtCl, at various ratios and incubated at 37°C for different times reaching also the high enzyme amounts used for S. pneumoniae SrtCl [4]. SDS-page analysis of these samples, however, showed no formation of high molecular weight bands that could represent pilus polymers (Fig. 4A), but only the formation of a complex compatible with the formation of a hetero-dimer formed by rSrtCl and rBP, as previously described for S. pneumoniae [4] and a dimer BP-BP that is formed also in absence of SrtCl (Fig. 4B).
BP high molecular weight structures can be assembled in vitro by recombinant SrtCl lid mutant.
To confirm our hypothesis that the catalytic cysteine is locked by the aromatic ring of Tyr86, we performed the same experiment by mixing recombinant SrtC-ly86A [3], with recombinant purified BP and we tested the ability of this sortase mutant to polymerize GBS BP monomers. The typical pili pattern of bands with molecular weights above 260 kDa, visible by SDS-page, could be generated when monomeric r-BP was incubated with rSrtC ly86A (Fig. 5 A). The reaction after 48 h was quenched and analyzed by Western Blotting using BP antibodies, checking for the typical ladder of BP polymerization compared to wild type pili of GBS 515 strain (Fig. 5B). As part of the BP monomer still remains unprocessed after 10 days of reaction, we tested if higher enzyme amounts could achieve a complete conversion of monomeric BP in polymeric structures.
We found that enzyme concentrations from 10 to 100 μΜ mixed with a fixed BP concentration did not change the rate of recombinant BP polymers formation (Fig. 5C). Using a fixed enzyme concentration of 25 μΜ for the polymerization reaction, varying concentration of monomeric BP were also tested (Fig. 5D).
BP high molecular weight structures formation in vitro by recombinant SrtCly86A mutant is mediated by LPXTG and pilin motives.
To confirm that the polymerization of the BP occurs through the correct motives, the polymerization in vitro was tested by incubating Γ-BPALPXTG and r-BPKi 89A with SrtCly86A confirming that the polymerization occurs through the cleavage of the LPXTG sorting signal and the subsequent linking to the pilin motif of the next subunit.
Large-scale recombinant BP HMW structures production and purification. Pili purification from gram positive pathogens is very challenging and time consuming and allows the purification of low amounts of material only. As we could achieve BP polymerization in a 50 μΐ reaction volume, we tried to scale-up the production of recombinant pili production. We found that the best reaction conditions were achieved by using the enzyme at 25 μΜ and the BP at 100 μΜ. The reaction volume is also important, as using up to 100 μΐ of the reaction decreases the efficiency of BP polymerization. We performed 10 reactions using these concentrations of substrate and enzyme in 100 μΐ each, for a total amount of 6.5 mg of pure BP, and we incubated the reaction for 7 days in presence of reducing agent. After this time, the pool of the reactions (1 ml total) was separated by gel filtration. Two fractions, containing mostly high molecular weight pili, were isolated from the monomeric BP and SrtCl, and were quantified to contain 0.5 mg of pili (Fig. 6). Fig. 7 shows that mutant sortase enzymes polymerize pilus proteins from a variety of gram positive bacteria. SrtCly86A (GBS sortase CI of PI-2a) was incubated with backbone protein PI-1 of GBS (also referred to as GBS 80) (Fig. 7A) or with pilus protein from Streptococcus pneumoniae (also referred to as RrgB) (Fig. 7B). Conclusion In Gram-positive bacteria the covalent association of pili requires the action of specific sortases. The pilus 2a biosynthesis in GBS is promoted by two sortase enzymes (SrtC-1 and SrtC-2) that polymerize the BP and display ancillary-proteins substrate specificity. Previously, we have shown that a triad composed of His, Cys and Arg residues is essential for SrtC-1 activity. Moreover, the crystal structure clearly indicates that GBS SrtCl is auto -inhibited by the presence of the lid in the catalytic pocket. Recently, our group measured the catalytic activity for GBS SrtCl by using a self-quenched fluorescent peptide mixed with recombinant GBS SrtCl WT and lid mutants to monitor substrate cleavage, and we found that the lid-mutants are even more active than the WT. These data, in accordance with in vivo experiments with lid mutants, suggested that the activation of sortases C might occur by a conformational change that results in the movement of the lid away from the catalytic site that could be induced by the protein substrates.
Starting from these observations, we performed in vitro experiments using recombinant GBS SrtCl WT and lid mutants mixed with recombinant backbone pilus protein and we observed that WT SrtCl enzyme was not able to induce recombinant BP protein polymerization. Enzyme activation was achieved, in vitro, through a single mutation in the lid region of recombinant SrtCl -2a that enhances BP polymerization in vitro and recombinant pili formation. These experiments suggest that for SrtC, the mechanism behind recognition and polymerization of pilus subunits could not depend only on the interaction between the fimbrial shaft protein and the sortase, as the enzyme activation could not be achieved in vitro simply by mixing SrtClWT with the BP. The experiments with the lid mutants indicate that the presence of the lid, and in particular of the Tyr86 in this loop, prevent BP polymerization. Our work provides the first direct evidence of self- inhibition of sortase C enzymes by the presence of the lid and opens a field for studying pili assembly by using recombinant pili polymerized by a sortase-active mutant, reducing the necessity to purify high amount of wild type pili from pathogenic bacteria. Moreover, the anchoring of many surface virulence factors on Gram-positive bacteria is mediated by sortase-activity and, therefore, these enzymes are attractive targets for the design of novel anti-infective therapeutics. Example 2: Immunisation studies using in vitro polymerized pili.
The in vitro polymerized pili structures may be used in immunisation studies in mice. For example, 10 μg of purified recombinant pili may be mixed with an adjuvant (e.g. alum) and injected into mice in a final volume of 200 μΐ. This may be followed by one or more booster immunisations. The mice may then be analysed for an immune response to the pili structures. This immune response may be protective against the bacteria from which the monomeric pilus proteins were originally derived. An immunisation study has been conducted in which mice were immunised with monomeric pili comprising GBS59 generated according to the methods of the invention in combination with alum, and the protective immune response was assessed following subsequent challenge with GBS. The results were compared to immunisation using a similar protocol with recombinant GBS59 not in pilus form and alum, the SrtCMl (Y86A) mutant and alum, Crmla and alum. The results of the immunisation experiment are provided in Table 4 below.
Table 4: immunisation with GBS59 pili
Figure imgf000053_0001
These results show that the GBS59 pili generated using the mutant sortase C enzymes according to the methods of the invention are significantly more effective at generating a protective immune response to GBS than the recombinant monomeric protein and are equivalent to the use of CRM la.
Example 3: Polymerisation of BP-2a (GBS59) variants in vitro.
We tested the ability of the sortase mutant to polymerize variants of GBS BP monomers of GBS59 corresponding to SEQ IDs: 74, 75, 76, 77, 78 and 79. Bacterial strains and growth conditions
The GBS strains used in this work were 2603 V/R (serotype V), 515 (la), CJBl l l (V), H36B (serotype lb), 5401 (II) and 3050 (II). Bacteria were grown at 37°C in Todd Hewitt Broth (THB; Difco Laboratories) or in trypticase soy agar supplemented with 5% sheep blood.
Cloning, expression, purification of recombinant proteins and antisera.
Genomic DNA was isolated by a standard protocol for gram-positive bacteria using a NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer's instructions. The full length recombinant BP-2a proteins, corresponding to 515, CJB111 and 2603 allelic variants (TIGR annotation SAL1486, SAM1372 and SAG1407, respectively), were produced as reported in Margarit et al, Journal of Infectious Diseases, 2009, 199: 108-115, whilst the full length H36B variant (TIGR annotation SAI 1511) was cloned in pET24b+ (Novagen) using strain H36B as source of DNA. Primers were designed to amplify the coding regions without the signal peptide and the 3' terminal sequence starting from the LPXTG motif.
For recombinant protein expression, the cultures were maintained at 25°C for 5h after induction with ImM IPTG for the pET clones or with 0.2% arabinose for the SpeedET clones. All recombinant proteins were purified by affinity chromatography and gel filtration. Briefly, cells were harvested by centrifugation and lysed in "lysis buffer", containing lOmM imidazole, lmg\ml lysozyme, 0.5 mg\ml DNAse and COMPLETE inhibitors cocktail (Roche) in PBS. The lysate was clarified by centrifugation and applied onto His-Trap HP column (Armesham Biosciences) pre-equilibrated in PBS containing lOmM imidazole. Protein elution was performed using the same buffer containing 250mM imidazole, after two wash steps using 20mM and 50mM imidazole buffers. The eluted proteins were then concentrated and loaded onto HiLoad 16/60 Superdex 75 (Amersham Biosciences) pre-equilibrated in PBS.
Antisera specific for each protein were produced by immunizing CD1 mice with the purified recombinant proteins as previously described (WO90/07936). Protein-specific immune responses (total Ig) in pooled sera were monitored by ELISA.
As before, we found that enzyme concentrations from 10 to 100 μΜ mixed with a fixed BP concentration did not change the rate of recombinant GBS59 polymer formation. GBS59 variant monomers were mixed at a 1 : 1 : 1 : 1 :1 : 1 ratio. Using a fixed enzyme concentration of 25μΜ for the polymerization reaction, varying concentrations of the mixture of variants of monomeric BP GBS59 were also tested. In vitro polymerization with three variants of BP-2a (H36B, 515, CJB111) :
BP-2a (variants H36B, 515, CJB111) concentrations: 35μΜ each- 105μΜ tot. SrtCly86A concentration: 25μΜ
Buffer: 25mM Tris-HCl pH 7,5 - lOOmM NaCl- lmM DTT Total volume of reaction ΙΟΟμΙ Incubation at 37°C for 48 h
The typical pili pattern of bands with molecular weights above 260 kDa, visible by SDS- page, could be generated when the mixture of variants of monomeric r-BPs was incubated with rSrtC ly86A (Figure 11). The reaction after 48 h was quenched and analyzed by Western Blotting using BP antibodies, checking for the typical ladder of BP polymerization compared to wild type pili. Pili comprising each of the GBS59 variants were created and used for immunisation. Vaccination of mice following the procedures described above was successful in protecting against challenge with each of the three GBS strains. In contrast, mice vaccinated with only one variant form were only protected against challenge with that particular strain. Surprisingly, these artificial pili were more effective at generating a protective immune response to GBS than the recombinant monomeric protein
Example 4: In vitro polymerization with two type of backbone proteins (BP-2a + pilus 1 BP (BP-1) and/or Pneumococcus RrgB) : Following the procedures outlined above, chimeric pili comprising backbone proteins from both Streptococcus agalactiae and Pneumococcus were prepared:
BP concentrations: 50μΜ each- ΙΟΟμΜ tot.
SrtCly86A concentration: 25μΜ
Buffer: 25mM Tris-HCl pH 7,5 - lOOmM NaCl- lmM DTT Total volume of reaction ΙΟΟμΙ Incubation at 37°C for 48 h
As shown in Figure 12A and Figure 12B, the presence of HMW bands demonstrates the ability of mutant sortase C enzymes to polymerise proteins from other strains/types of bacteria. Vaccination of mice following the procedures described above was successful in protecting against challenge with both Group B Streptococcus and Streptococcus pneumonia (data not shown). Sortases of the invention were also able to polymerise combinations of GBS67 and GBS59.
Example 4: Mutant SrtC can polymerize GFP-IPQTG The "IQTGGIGT" sequence was added at the C-terminus of the GFP protein DNA sequence using mutagenesis:.
Pimers used:
GFP-lpxtg_F attccacaaacaggtggtattggtacaTAACGCGACTTAATTAAACGG GFP-lpxtg_Rl
TGTACCAATACCACCTGTTTGTGGAATCTTGTACAGCTCGTCCATGCC Mutagenesis DNA template: SpeedET vector + GFP EGFP DNA sequence below (from pSpeedET):
CTTTAAGAAGGAGATATACATACCCATGGGATCTGATAAAATTCATCATCATC ATCATCACGAAAACCTGTACTTCCAGGGCatggtgagcaagggcgaggagctgttcaccggggtggt gcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggc gtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagc gcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatc gagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctat atcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgc cgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccc tgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacg agctgtacaagTAACGCGACTTAATTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGAT GAGAGAAGATTTTCAGCCTGATACAGATTAAATC
EGFP amino acid sequence below (from pSpeedET):
MGSDKIHHHHHHENLYFQGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE
GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP
EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY LSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
Nucleic acid sequence after mutagenesis:
CTTTAAGAAGGAGATATACATACCCATGGGATCTGATAAAATTCATCATCATC ATCATCACGAAAACCTGTACTTCCAGGGCatggtgagcaagggcgaggagctgttcaccggggtggt gcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggc gtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagc gcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatc gagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctat atcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgc cgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccc tgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacg agctgtacaagattccacaaacaggtggtattggtacaTAACGCGACTTAATTAAACGGTCTCCAGCT TGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATAC AGATTAAATC
Amino acid sequence after mutagenesis:
MGSDKIHHHHHHENLYFQGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE GDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY NSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHY LSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKIPQTGGIGT-
Production of recombinant "GFP-IQTGGIGT": expression and purification
GFP-IPQTGGIGT Expression in HK100 in LB + kanamicine 30 ug\ml using biosilta media at 30°C, induction with arabinose 0.15% final. Purification: standard IMAC GFP-IQTGGIGT and SrtClY86A polymerization reaction: mix 25 uM SrtClY86A with 25-50 or 100 uM GFP-IPQTGGIGT in buffer 25 mM Tris pH7.5, 150 mM NaCl, DTT1 mM for 72h at 37°C in termomixer. Reaction volume 50 ul.
As shown in Figure 13, the SrtCly86A mutant was able to polymerise GFP-IPQTG.
Example 5: Recombinant PI-2b SrtCl and SrtC2 proteins are active in vitro and are able to cleave fluorescent peptides carrying the LPXTG-like motif of pilus proteins Full-length SrtCl and C2 were cloned (using strain COH1 as template) in fusion with a His-MBP-tag. Recombinant enzymes were then expressed in E.coli and purified with IMAC or IMAC and MBP-trap column. FRET assays with purified sortases were carried out using synthetic fluorescent peptides carrying the LPXTG sorting motif of PI-1 backbone protein and of PI-1 minor ancillary protein in order to assess the catalytic activity. The PI-2b SrtCl and SrtC2 enzymes are able to cleave the fluorescent peptides. These data demonstrate that thePI-2b SrtCl and SrtC2 enzymes are active in vitro and are suitable for use in ligating and polymerising proteins.
The following protocols and conditions were used: Purification of SrtCl enzyme - IMAC
- 3 litre culture of Rosetta cells expressing the SrtCl -MBP-His construct
- pellets collected and lysed
- lOmM Imidazole added to 30ml lysate
- column: 5 ml and 4 flow (approximately 5 ml/min) - lysate loaded and through flow collected
- washed with 15 ml of buffer with 10 mM Imidazole (the first 3 ml are the dead volume of the column)
- washed with 15 ml of buffer with 20 mM Imidazole
- eluted with 300 mM Imidazole buffer 10 ml - 1 mM DTT added
- protein concentrated with amicon at 6000rpm to 4° C for 20 minutes
- the final protein concentration was 1.78 mg/ml Purification of SrtC2 enzyme - IMAC
- 3 litre culture of Rosetta cells expressing the SrtC2-MBP-His construct - 2 columns (30 ml of pellets with cell lysate + 20 ml of Buffer 10 mM) 50ml FT
- pre-flushed with 20ml of buffer lOmM
- washed with 50ml of buffer lOmM - washed with 150ml of buffer 20mM
- elution buffer 300 mM: 5 ml dead volume, 10ml elute2 + 20μ1 DTT 1M, 10 ml elute3
- elutesl and 3 were combined, whereas 10ml of 300mM NaCl, 50 mM and Tris pH8 were added to 10 ml of elute 2 Purification of SrtC2 enzyme - MBP-trap
- 2 columns were used MBP-trap
- the column was washed with 50ml of buffer with maltose (Tris 50mM, 150 mM NaCl, pH8 Maltose 100 mM)
- washed with 50ml Urea 8 m pH8 - Tris 50 mM - washed with distilled water (80 ml)
- balanced with 25ml of Tris buffer 50mM pH8, 300 mM NaCl diluted with water 1 : 2
- elute2 loaded
- elutes 1 + 3 loaded
- washed - eluted with buffer containing maltose FRET analysis
Closed plate with termofluor plastic.
1) 50μ1 buffer (300mM NaCl + 50mM Tris pH8) + 50μ1 1515 + Ιμΐ BP peptide
2) 50μ1 buffer (300mM NaCl + 50mM Tris pH8) + 50μ1 1515 + Ιμΐ AP2 peptide 3) 1 ΟΟμΙ 1515 + 1 μΐ BP peptide
4) ΙΟΟμΙ 1515 + Ιμΐ AP2 peptide
5) ΙΟΟμΙ elution buffer (300mM Imidazole) + Ιμΐ BP peptide
6) ΙΟΟμΙ elution buffer (300mM Imidazole) + Ιμΐ AP2 peptide
We used 200μ1 [1.78 mg/ml] of concentrated protein + 2μ1 LPXTG peptide of BP and AP2, and as control used the elution buffer 300 mM Imidazole instead of protein. Tecan plate reader - 300 cycles with a measurement every 10 minutes, temperature [34- 37.5° C°] with 37° C for optimum and wavelength [400nm-600 nm] have been obtained with maximum absorption provided to 500nm.
Example 6: SrtCl is effective for polymerising BP
The activity of SrtCl was further assayed by using a mutant GBS strain that does not express any pili (515A2a). This strain was transformed with complementation vectors PAMp80/t80 carrying genes coding for BP alone or BP with PI-2b SrtCl . The ability of the complementation vectors to restore pili polymerisation was analysed by western blot. As shown in Figure 10, transfection with BP alone did not result in any polymerisation. However, transfection with BP and SrtCl resulted in the formation of high molecular weight polymers. Strain A909, which expresses pilus 2b, was used as a positive control.
Figure 10 provides a western blot of the membrane preparation from the 515A2a mutant strain and from the wild type A909 strain complemented by a plasmid containing SrtCl and BP genes or BP gene alone. Antibodies against SrtCl were used. Expected signals at 30 kDa confirm the expression and correct localization of SrtC 1.
These data demonstrate that PI-2b SrtCl is effective at polymerising pili.
The following protocols and conditions were used:
Electroporation
ΙΟΟμΙ of the A909 and 515A2a strains were transformed with 3/7 μΐ of Spbl (BP - PI-2b) or Spbl + SrtCl (PI-2b).
Inoculation
In 10 ml THB + elm glycerol.
Cells were pelleted and washed with 25ml PBS
- 940μ1 TRIS pH 6.8, 50 mM + 60μ (lOU/μΙ) - 2 hours at 37° C, shaking
Gel and western blot GBS extracts
- 10 extracts centrifuged for 10 minutes at maximum speed
- 30μ1 supernatant + 15μ1 of LDS + 5μ1 of reducing - pellets were resuspended in 2% buffer TRIS 50mM SDS pH8 300 mm NaCl
- western blot and membrane washed
- washed with water
- 2 hours stirring with milk 5%
- rinsed with PBS
-on every membrane 5 ml of milk 1% + antibody (anti-Spbl on culture supematants and anti SrtC 1 on pellets)
- left over night in with shaking in cold room
-washed with 10 ml of PBS-Tween 0.05% for 10 minutes 3 times -washed with PBS for 5 minutes
- 20 ml of 1% milk + P161 anti-mouse antibody and left for 1 hour with stirring
- washed with PBS
- development solution prepared (10 ml = 9 ml water +lml diluent + 200μ1 sample substrate +) - 5ml per membrane
SEQUE] \CES
SEQ ID NO: Polypeptide Sequence
MGQKSKISLATNIRIWIFRLIFLAGFLVLAFPIVSQVMYFQASHANINAFKEAVTKIDRVEINRRLE
1 LAYAYNASIAGAKTNGEYPALKDPYSAEQKQAGWEYARMLEVKEQIGHVI IPRINQDIPIYAGSAE
ENLQRGVGHLEGTSLPVGGESTHAVLTAHRGLPTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIK VIAPDQLEDLYVIQGEDHVTLLTCTPYMINSHRLLVRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWV WGLILLSLLIWFKKTKQKKRRKNEKAASQNSHNNSK
2 MKKRLVKIV I IRNNKIRTLIFVMGSLILLFPIVSQVSYYLASHQNINQFKREVAKIDTNTVERRIA
LANAYNETLSRNPLLIDPFTSKQKEGLREYARMLEVHEQIGHVAIPSIGVDIPIYAGTSETVLQKGS GHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQIFYVTNIKETLAYKWSIKWDPTAL SEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEAEKHKEQTVQDYRLSLVLKILLVLLIG LFIVIMMRRWMQHRQ
3 MKTKKI IKKTKKKKSNLPFI ILFLIGLSILLYPWSRFYY IESNNQTQDFERAAKKLSQKEINRRM
ALAQAYNDSLNNVHLEDPYEKKRIQKGIAEYARMLEVSEKIGI ISVPKIGQKLPIFAGSSQEVLSKG AGHLEGTSLPIGGNSTHTVITAHSGIPDKELFSNLKKLKKGDKFYIQNIKETIAYQVDQIKWTPDN FSDLLWPGHDYATLLTC PIMVNTHRLLVRGHRIPYKGPIDEKLIKDGHLN IYRYLFYISLVI IA WLLWLIKRQRQKNRLSSVRKGIES
4 MRGKFQKNLKKSWLNRWMNIGLILLFLVGLLITSYPFISNWYYNIKANNQVTNFDNQTQKLNAKEI
NRRFELAKAYNRTLDPSRLSDPYTEKEKKGIAEYAHMLEITEMIGYIDIPSIKQKLPIYAGTTSSVL EKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKIFYIHNIKEVLAYKVDQISWK PDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKEKNYLMKELQTHYKLYFLLSIL VILILVALLLYLKRKFKERKRKGNQK
5 MAYPSLANYWNSFHQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFK SGMKWHM SQERLDYNS
QLAIDKTGNMGYISIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRL FSDLDKLKVGDHWTVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVR GHRVPNDNGNALWAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
6 MAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGDPWSEEMKKK
GRAEYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGL PTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTH RLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAA RKEVKVEDGQQ
7 MSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANM
LKVHERIGYVEIPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQ LDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKR IPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
8 MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLA
SQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDG PLPLDG TGIRSVIAGHRAEPSHVFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIM TLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLWLAFLG ILFVLWKLARLLRGK
9 MECYRDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPDAFSFRDGIHDKNYESLLQIEN
NDIMGYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNL VKKGDTFYFRVLNKVLAYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAY HYKKYQQAKKAMKLVDKSRMWAEWCAAFGWIAI ILVFMYSRVSAKKSK
10 IVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYEYARMLEVKEQIGH
VI IPRINQDIPIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGLPTAKLFTNLDKVTVGDR FYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSHRLLVRGKRIPYVEKTVQ KDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNSHNNSK
11 ASHQNINQFKREVAKIDTNTVERRIALANAYNETLSREYARMLEVHEQIGHVAIPSIGVDIPIYAGT
SETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQIFYVTNIKETLAYKWS IKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEAEKHKEQTVQDYRLSLVL KILLVLLIGLFIVIMMRRWMQHRQ
12 ESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNVEYARMLEVSEKIGI ISVPKIGQKLPIFAG
SSQEVLSKGAGHLEGTSLPIGGNSTHTVITAHSGIPDKELFSNLKKLKKGDKFYIQNIKETIAYQVD QIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLLVRGHRIPYKGPIDEKLIKDGHLNTIYRYL FYI SLVI IAWLLWLIKRQRQKNRLSSVRKGIES KANNQVTNFDNQTQKLNAKEINRRFELAKAYNRTLDPEYAHMLEITEMIGYIDIPSIKQKLPIYAGT TSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKIFYIHNIKEVLAYKVDQ ISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKEKNYLMKELQTHYKLYF LLSILVILILVALLLYLKRKFKERKRKGNQK
HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFNSQLAIDKTGNMGYISIPKINIKLPLYHGTSE KVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWTVSILNETYTYQVDQIR TVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALWAEAIQIEPIYIAPFIA IFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
ESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLEYARMLEIHERMGHVEIPVIDVDLPVYAGTAE EVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVK VIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYV AVGLIVILLWI IRRLRKKKKQPEKALKALKAARKEVKVEDGQQ
ESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKEYANMLKVHERIGYVEIPAIDQEIPMYVGTS EDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQI VTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRAVRERGQFWLWLLL GAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
QSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQEVNYQVSDDPDAVYGYLSIPSLEIMEPVY LGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDALYYDNGQEIVEYQ MMDTEI ILPSEWEKLESVSSKNIMTLI CDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKE GQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK
RDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLNYESLLQIENNDIMGYVEVPSIKVTLPIYHYT TDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNLVKKGDTFYFRVLNKVLAYKVDQ ILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKKYQQAKKAMKLVDKSRMWA EWCAAFGWIA11LVFMYSRVSAKKSK
EYARMLEVKEQIGHVI IPRINQDIPIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGLPTA KLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSHRLL VRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNSHNN SK
EYARMLEVHEQIGHVAIPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTA RLFTDLNKVKKGQIFYVTNIKETLAYKWSIKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLL VKGERIPYDSTEAEKHKEQTVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ
EYARMLEVSEKIGI ISVPKIGQKLPIFAGSSQEVLSKGAGHLEG SLPIGGNSTHTVI AHSGIPDK ELFSNLKKLKKGDKFYIQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLL VRGHRIPYKGPIDEKLIKDGHLN IYRYLFYISLVI IAWLLWLIKRQRQKNRLSSVRKGIES
EYAHMLEITEMIGYIDIPSIKQKLPIYAGTTSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKA KLFTDLDKLKKGKIFYIHNIKEVLAYKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLL VRGHRIKYVPPVKEKNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
NSQLAIDKTGNMGYISIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSS RLFSDLDKLKVGDHWTVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLL VRGHRVPNDNGNALWAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENN DL
EYARMLEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTA KMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLL VRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAARKE VKVEDGQQ
EYANMLKVHERIGYVEIPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTA ELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLL VRGKRIPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
EVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDG PLPLDGTGIRSVIAGHRAEP SHVFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLI CDPIPTFNKR LLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRG K
HDKNYESLLQIENNDIMGYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHR GLPSAEMFTNLNLVKKGDTFYFRVLNKVLAYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVN TKRLLVRGHRIAYHYKKYQQAKKAMKLVDKSRMWAEWCAAFGWIAI ILVFMYSRVSAKKSK
IVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYPALKSAEQKQAGW EYARMLEVKEQIGHVI I PRINQDI PIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGLPTA KLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSHRLL VRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNSHNN SK
ASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLITSKQKEGLREYARMLEVHEQIGHVA IPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQIFY VTNIKETLAYKWSIKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEAEKH KEQTVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ
ESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNVHLEEKKRIQKGIAEYARMLEVSEKIGI IS VPKIGQKLPIFAGSSQEVLSKGAGHLEGTSLPIGGNSTHTVITAHSGIPDKELFSNLKKLKKGDKFY IQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLLVRGHRIPYKGPIDEKL IKDGHLN IYRYLFYISLVI IAWLLWLIKRQRQKNRLSSVRKGIES
KANNQVTNFDNQTQKLNAKEINRRFELAKAYNRTLDPSRLSTEKEKKGIAEYAHMLEITEMIGYIDI PSIKQKLPIYAGTTSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKIFYI HNIKEVLAYKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKEKNY LMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFKTSGHMTSQERLDYNSQLAIDKTGNMGYISIP KINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWTVS ILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALWAE AIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
ESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGSEEMKKKGRAEYARMLEIHERMGHVE IPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGDKFY VHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEF IAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAARKEVKVEDGQQ
ESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILTEQEKKKGVSEYANMLKVHERIGYVE IPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGDIFY LHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERN RAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
QSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVLAEGYEVNYQVSDDPDAVYGYLSIP SLEIMEPVYLGADYHHLGMGLAHVDG PLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDALYYD NGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLI CDPIPTFNKRLLVNFERVAVYQKSDPQTAA VARVAFTKEGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK
RDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPSFRDGIHDKNYESLLQIENNDIMGYV EVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNLVKKGDTF YFRVLNKVLAYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKKYQQ AKKAMKLVDKSRMWAEWCAAFGWIA11LVFMYSRVSAKKSK
IVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYPALKAPYSAEQKQA GWEYARMLEVKEQIGHVI IPRINQDIPIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGL PTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSH RLLVRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNS HNNSK
ASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLIAPFTSKQKEGLREYARMLEVHEQIG HVAIPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQ IFYVTNIKETLAYKWSIKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEA EKHKEQTVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ
ESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNVHLEAPYEKKRIQKGIAEYARMLEVSEKIG I ISVPKIGQKLPIFAGSSQEVLSKGAGHLEG SLPIGGNSTHTVI AHSGIPDKELFSNLKKLKKGD KFYIQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLLVRGHRIPYKGPID EKLIKDGHLN IYRYLFYISLVI IAWLLWLIKRQRQKNRLSSVRKGIES
KANNQVTNFDNQTQKLNAKEINRRFELAKAYNRTLDPSRLSAPYTEKEKKGIAEYAHMLEITEMIGY IDIPSIKQKLPIYAGTTSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKI FYIHNIKEVLAYKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKE KNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFKTSGAKWHMTSQERLDYNSQLAIDKTGNMGYI SIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHW TVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALV VAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
ESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGAPWSEEMKKKGRAEYARMLEIHERMG HVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGD KFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVE EEFIAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAARKEVKVEDGQQ ESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILAPFTEQEKKKGVSEYANMLKVHERIG YVEIPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGD IFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIA ERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
QSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVAPFLAEGYEVNYQVSDDPDAVYGYL SIPSLEIMEPVYLGADYHHLGMGLAHVDG PLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDAL YYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLI CDPIPTFNKRLLVNFERVAVYQKSDPQ TAAVARVAFTKEGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK
RDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPAAFSFRDGIHDKNYESLLQIENNDIM GYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNLVKKG DTFYFRVLNKVLAYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKK YQQAKKAMKLVDKSRMWAEWCAAFGWIA11LVFMYSRVSAKKSK
IVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYPALKDPASAEQKQA GWEYARMLEVKEQIGHVI IPRINQDIPIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGL PTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSH RLLVRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNS HNNSK
ASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLIDPATSKQKEGLREYARMLEVHEQIG HVAIPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQ IFYVTNIKETLAYKWSIKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEA EKHKEQTVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ
ESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNVHLEDPAEKKRIQKGIAEYARMLEVSEKIG I ISVPKIGQKLPIFAGSSQEVLSKGAGHLEG SLPIGGNSTHTVI AHSGIPDKELFSNLKKLKKGD KFYIQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLLVRGHRIPYKGPID EKLIKDGHLN IYRYLFYISLVI IAWLLWLIKRQRQKNRLSSVRKGIES
KANNQVTNFDNQTQKLNAKEINRRFELAKAYNRTLDPSRLSDPATEKEKKGIAEYAHMLEITEMIGY IDIPSIKQKLPIYAGTTSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKI FYIHNIKEVLAYKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKE KNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFK SGMKAHM SQERLDYNSQLAIDKTGNMGYI SIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHW TVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALV VAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
ESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGDPASEEMKKKGRAEYARMLEIHERMG HVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGD KFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVE EEFIAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAARKEVKVEDGQQ
ESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPATEQEKKKGVSEYANMLKVHERIG YVEIPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGD IFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIA ERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
QSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVDPALAEGYEVNYQVSDDPDAVYGYL SIPSLEIMEPVYLGADYHHLGMGLAHVDG PLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDAL YYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLITCDPIPTFNKRLLVNFERVAVYQKSDPQ TAAVARVAFTKEGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK
RDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPDAASFRDGIHDKNYESLLQIENNDIM GYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNLVKKG DTFYFRVLNKVLAYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKK YQQAKKAMKLVDKSRMWAEWCAAFGWIA11LVFMYSRVSAKKSK
IVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYPALKAPASAEQKQA GWEYARMLEVKEQIGHVI I PRINQDI PIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGL PTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSH RLLVRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNS HNNSK
ASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLIAPA SKQKEGLREYARMLEVHEQIG HVAIPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQ IFYVTNIKETLAYKWSIKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEA EKHKEQTVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ ESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNVHLEAPAEKKRIQKGIAEYARMLEVSEKIG I ISVPKIGQKLPIFAGSSQEVLSKGAGHLEG SLPIGGNSTHTVI AHSGIPDKELFSNLKKLKKGD KFYIQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLLVRGHRIPYKGPID EKLIKDGHLN IYRYLFYISLVI IAWLLWLIKRQRQKNRLSSVRKGIES
KANNQVTNFDNQTQKLNAKEINRRFELAKAYNRTLDPSRLSAPATEKEKKGIAEYAHMLEITEMIGY IDIPSIKQKLPIYAGTTSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKI FYIHNIKEVLAYKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKE KNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFK SGAKAHM SQERLDYNSQLAIDKTGNMGYI SIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHW TVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALV VAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
ESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGAPASEEMKKKGRAEYARMLEIHERMG HVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGD KFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVE EEFIAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAARKEVKVEDGQQ
ESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILAPATEQEKKKGVSEYANMLKVHERIG YVEIPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGD IFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIA ERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
QSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVAPALAEGYEVNYQVSDDPDAVYGYL SIPSLEIMEPVYLGADYHHLGMGLAHVDG PLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDAL YYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLI CDPIPTFNKRLLVNFERVAVYQKSDPQ TAAVARVAFTKEGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK
RDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPAAASFRDGIHDKNYESLLQIENNDIM GYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHRGLPSAEMFTNLNLVKKG DTFYFRVLNKVLAYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKK YQQAKKAMKLVDKSRMWAEWCAAFGWIA11LVFMYSRVSAKKSK
IVSQVMYFQASHANINAFKEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYPALKAAASAEQKQA GWEYARMLEVKEQIGHVI IPRINQDIPIYAGSAEENLQRGVGHLEGTSLPVGGESTHAVLTAHRGL PTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQIKVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSH RLLVRGKRIPYVEKTVQKDSKTFRQQQYLTYAMWVWGLILLSLLIWFKKTKQKKRRKNEKAASQNS HNNSK
ASHQNINQFKREVAKIDTNTVERRIALANAYNETLSRNPLLIAAATSKQKEGLREYARMLEVHEQIG HVAIPSIGVDIPIYAGTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKKGQ IFYVTNIKETLAYKWSIKWDPTALSEVKIVNGKDYITLLTCTPYMINSHRLLVKGERIPYDSTEA EKHKEQTVQDYRLSLVLKILLVLLIGLFIVIMMRRWMQHRQ
ESNNQTQDFERAAKKLSQKEINRRMALAQAYNDSLNNVHLEAAAEKKRIQKGIAEYARMLEVSEKIG I ISVPKIGQKLPIFAGSSQEVLSKGAGHLEG SLPIGGNSTHTVI AHSGIPDKELFSNLKKLKKGD KFYIQNIKETIAYQVDQIKWTPDNFSDLLWPGHDYATLLTCTPIMVNTHRLLVRGHRIPYKGPID EKLIKDGHLN IYRYLFYISLVI IAWLLWLIKRQRQKNRLSSVRKGIES
KANNQVTNFDNQTQKLNAKEINRRFELAKAYNRTLDPSRLSAAATEKEKKGIAEYAHMLEI EMIGY IDIPSIKQKLPIYAGTTSSVLEKGSGHLEGTSLPIGGKSSHTVITAHRGLPKAKLFTDLDKLKKGKI FYIHNIKEVLAYKVDQISWKPDNFSKLLWKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKE KNYLMKELQTHYKLYFLLSILVILILVALLLYLKRKFKERKRKGNQK
HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFKTSGAAAHMTSQERLDYNSQLAIDKTGNMGYI SIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHW TVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALV VAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
ESNQQIADFDKEKATLDEADIDERMKLAQAFNDSLNNWSGAAASEEMKKKGRAEYARMLEIHERMG HVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGD KFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVE EEFIAANKLSHLYRYLFYVAVGLIVILLWI IRRLRKKKKQPEKALKALKAARKEVKVEDGQQ
ESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILAAATEQEKKKGVSEYANMLKVHERIG YVEIPAIDQEIPMYVGTSEDILQKGAGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGD IFYLHVLDQVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIA ERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
QSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRLASQNRIVAAALAEGYEVNYQVSDDPDAVYGYL SIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSHVFFRHLDQLKVGDAL YYDNGQEIVEYQMMDTEI ILPSEWEKLESVSSKNIMTLI CDPIPTFNKRLLVNFERVAVYQKSDPQ TAAVARVAFTKEGQSVSRVATSQWLYRGLWLAFLGILFVLWKLARLLRGK
72 MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTVNIYKLQADSYKSEI SN
GGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLP QKTNAQGLWDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKNV VTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITDKFADGLTYKSVGKIKIGS KTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPV ASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFD LLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDANAEGTAVTYKL KETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIG AAVMAFAVKGMKRRTKDN
73 AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYKVKTD
ISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLWDALDSKSNVRYLYVEDLKNSPSNITKA YAVPFVLELPVANSTGTGFLSEINIYPKNWTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPA NLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFKEIAEL LKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYDHTPDKADNPKPSNP PRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKL KSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSA DATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRRTKDN
74 MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGKDYKG
GAISDLAGYFGEGSKEIEGAFFALALKEDKSGKVQYVKAKEGNKLTPALINKDGTPEITVNIDEAVS GLTPEGDTGLVFNTKGLKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGWADAHVYP KNTEEKPEIDKNFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAEIGKWPYEVKTKIHKGSKY ENLVWTDIMSNGLTMGSTVSLKASGTTETFAKDTDYELSIDARGFTLKFTADGLGKLEKAAKTADIE FTLTYSATVNGQAI IDNPESNDIKLSYGNKPGKDLTELPV PSKGEVTVAKTWSDGIAPDGVNWYT LKDKDKTVASVSLTKTSKGTIDLGNGIKFEVSGNFSGKFTGLENKSYMISERVSGYGSAINLENGKV TITNTKDSDNPTPLNPTEPKVETHGKKFVKTNEQGDRLAGAQFWKNSAGKYLALKADQSEGQKTLA AKKIALDEAIAAYNKLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYTSND QGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFWNQGSYITGGNIDYVANSNQKDATRVENKKVT IPQTGGIG ILF I IGLSIMLGAWIMKRRQSKEA
75 MKKINKYFAVFSALLLTVTSLFSVAPVFAEEAKTTDTVTLHKIVMPRTAFDGFTAGTKGKDNTDYVG
KQIEDLKTYFGSGEAKEIAGAYFAFKNEAGTKYITENGEEVDTLDTTDAKGCAVLKGLTTDNGFKFN TSKLTGTYQIVELKEKSTYNNDGSILADSKAVPVKITLPLVNDNGWKDAHVYPKNTETKPQVDKNF ADKELDYANNKKDKGTVSASVGDVKKYHVGTKILKGSDYKKLIWTDSMTKGLTFNNDIAVTLDGATL DATNYKLVADDQGFRLVLTDKGLEAVAKAAKTKDVEIKITYSATLNGSAWEVLETNDVKLDYGNNP IENEPKEGI PVDKKI VNK WAVDGNEVNKADE VDAVFTLQVKDGDKWVNVDSAKA AATSFKHT FENLDNAKTYRVIERVSGYAPEYVSFVNGWTIKNNKDSNEPTPINPSEPKWTYGRKFVKTNKDGK ERLAGATFLVKKDGKYLARKSGVATDAEKAAVDSTKSALDAAVKAYNDLTKEKQEGQDGKSALATVS EKQKAYNDAFVKANYSYEWVEDKNAKNWKLISNDKGQFEITGLTEGQYSLEETQAPTGYAKLSGDV SFNVNA SYSKGSAQDIEYTQGSKTKDAQQVINKKV IPQTGGIG IFF I IGLSIMLGAWIMKRR QSEEV
76 MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFDNFTEGTKGKNDSDYVGK
QINDLKSYFGSTDAKEIKGAFFVFKNETGTKFITENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNT AKLKGIYQIVELKEKSNYDNNGSILADSKAVPVKITLPLVNNQGWKDAHIYPKNTETKPQVDKNFA DKDLDYTDNRKDKGWSATVGDKKEYIVGTKILKGSDYKKLVWTDSMTKGLTFNNNVKVTLDGEDFP VLNYKLVTDDQGFRLALNATGLAAVAAAAKDKDVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPT EESEPQEGTPANQEIKVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEHTF TGLDNAKTYRWERVSGYTPEYVSFKNGWTIKNNKNSNDPTPINPSEPKWTYGRKFVKTNQANTE RLAGATFLVKKEGKYLARKAGAATAEAKAAVKTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQ KQKAYNDAFVKANYSYEWVADKKADNWKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSGDVN FEVTA SYSKGATTDIAYDKGSVKKDAQQVQNKKV IPQTGGIG ILF I IGLSIMLGAWIMKKRQ SEEA
77 MKRINKYFAMFSALLLILTSLLSVAPVFAAEMGNITKTVTLHKIVQTSDNLAKPNFPGINGLNGTKY
MGQKLTDISGYFGQGSKEIAGAFFAVMNESQTKYITESGTEVESIDAAGVLKGLTTENGITFNTANL KGTYQIVELLDKSNYKNGDKVLADSKAVPVKITLPLYNEEGIWDAEVYPKNTEEAPQIDKNFAKAN KLLNDSDNSAIAGGADYDKYQAEKAKATAEIGQEIPYEVKTKIQKGSKYKNLAWVDTMSNGLTMGNT VNLEASSGSFVEGTDYNVERDDRGFTLKFTDTGLTKLQKEAETQAVEFTLTYSATVNGAAIDDKPES NDIKLQYGNKPGKKVKEIPVTPSNGEITVSKTWDKGSDLENANWYTLKDGGTAVASVSLTKTTPNG EINLGNGIKFTVTGAFAGKFSGLTDSKTYMISERIAGYGNTITTGAGSAAITNTPDSDNPTPLNPTE PKWTHGKKFVKTSSTETERLQGAQFWKDSAGKYLALKSSATISAQTTAYTNAKTALDAKIAAYNK LSADDQKGTKGETAKAEIKTAQDAYNAAFIVARTAYEWVTNKEDANWKVTSNADGQFEVSGLATGD YKLEETQAPAGYAKLAGDVDFKVGNSSKADDSGNIDYTASSNKKDAQRIENKKVTIPQTGGIGTILF TI IGLSIMLGAVI IMKRRQSEEA
78 MKKINKYFAVFSALLLTVTSLLSVAPAFADEATTNTVTLHKILQTESNLNKSNFPGTTGLNGDDYKG
ESISDLAEYFGSGSKEIDGAFFALALEEEKDGWQYVKAKANDKLTPDLITKGTPATTTKVEEAVGG LTTGTGIVFNTAGLKGNFKI IELKDKSTYNNNGSLLAASKAVPVKI LPLVSKDGWKDAHVYPKNT ETKPEVDKNFAKTNDLTALKDATLLKAGADYKNYSATKATVTAEIGKVIPYEVKTKVLKGSKYEKLV WTDTMSNGLTMGDDVNLAVSGTTTTFIKDIDYTLSIDDRGFTLKFKATGLDKLEEAAKASDVEFTLT YKATVNGQAI IDNPEVNDIKLDYGNKPGTDLSEQPV PEDGEVKVTKTWAAGANKADAKWYTLKNA TKQWASVALTAADTKGTINLGKGMTFEI GAFSGTFKGLQNKAYTVSERVAGYTNAINVTGNAVAI TNTPDSDNPTPLNPTQPKVETHGKKFVKVGDADARLAGAQFWKNSAGKFLALKEDAAVSGAQTELA TAKTDLDNAIKAYNGLTKAQQEGADGTSAKELINTKQSAYDAAFIKARTAYTWVDEKTKAITFTSNN QGQFEVTGLEVGSYKLEETLAPAGYAKLSGDIEFTVGHDSYTSGDIKYKTDDASNNAQKVFNKKVTI PQTGGIG ILF I IGLSIMLGAWIMKRRQSEEA
79 MKKINKFFVAFSALLLILTSLLSVAPAFAEEERTTETVTLHKILQTETNLKNSAFPGTKGLDGTEYD
GKAIDKLDSYFGNDSKDIGGAYFILANSKGEYIKANDKNKLKPEFSGNTPKTTLNISEAVGGLTEEN AGIKFETTGLRGDFQI IELKDKSTYNNGGAILADSKAVPVKI LPLINKDGWKDAHVYPKNTETKP QIDKNFADKNLDYINNQKDKGTISATVGDVKKYTVGTKILKGSDYKKLVWTDSMTKGLTFNNDVTVT LDGANFEQSNYTLVADDQGFRLVLNATGLSKVAEAAKTKDVEIKINYSATVNGSTWEKSENNDVKL DYGNNPTTENEPQTGNPVNKEITVRKTWAVDGNEVNKGDEKVDAVFTLQVKDSDKWVNVDSATATAA TDFKYTFKNLDNAKTYRWERVSGYAPAYVSFVGGWTIKNNKNSNDPTPINPSEPKWTYGRKFVK TNQDGSERLAGATFLVKNSQSQYLARKSGVATNEAHKAVTDAKVQLDEAVKAYNKLTKEQQESQDGK AALNLIDEKQTAYNEAFAKANYSYEWWDKNAANWKLISNTAGKFEITGLNAGEYSLEETQAPTGY AKLSSDVSFKVNDTSYSEGASNDIAYDKDSGKTDAQKWNKKVTIPQTGGIGTILFTI IGLSIMLGA WIMKRRQSEEA
80 MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYKVFDAEIDNANVS
DSNKDGASYLIPQGKEAEYKASTDFNSLFTTTTNGGRTYVTKKDTASANEIATWAKSISANT TPVSTVTESNNDGTEVINVSQYGYYYVSSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGG KTVDQKTYSVGDTVKYTITYKNAVNYHGTEKVYQYVIKDTMPSASWDLNEGSYEVTITDGS GNITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFYKGINTITVTY TGVLKSGAKPGSADLPENTNIATINPNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIF VLKNATGQFLNFNDTNNVEWGTEANATEYTTGADGI ITITGLKEGTYYLVEKKAPLGYNLLD NSQKVILGDGATDTTNSDNLLVNPTVENNKGTELPSTGGIGTTIFYI IGAILVIGAGIVLVA RRRLRS
81 AETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKDGASYLIPQGKEAEYKASTDFNSLF
TTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYYVSS TVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNYHGT EKVYQYVIKDTMPSASWDLNEGSYEVTITDGSGNITTLTQGSEKATGKYNLLEENNNFTIT IPWAATNTPTGNTQNGANDDFFYKGINTITVTYTGVLKSGAKPGSADLPENTNIATINPNTS NDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTEANATEY TTGADGI ITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTNSDNLLVNPTVENN KGTE
82 MKSINKFLTMLAALLLTASSLFSAATVFAAGTTTTSVTVHKLLATDGDMDKIANELETGN
YAGNKVGVLPANAKEIAGVMFVWTNTNNEI IDENGQTLGVNIDPQTFKLSGAMPATAMKK LTEAEGAKFNTANLPAAKYKIYEIHSLSTYVGEDGATLTGSKAVPIEIELPLNDWDAHV YPKNTEAKPKIDKDFKGKANPDTPRVDKDTPVNHQVGDWEYEIVTKIPALANYATANWS DRMTEGLAFNKGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKTWVDATGAPIP AGAEATFDLVNAQTGKWQTVTLTTDKNTVTVNGLDKNTEYKFVERSIKGYSADYQEITT AGEIAVKNWKDENPKPLDPTEPKWTYGKKFVKVNDKDNRLAGAEFVIANADNAGQYLAR KADKVSQEEKQLWTTKDALDRAVAAYNALTAQQQTQQEKEKVDKAQAAYNAAVIAANNA FEWVADKDNENWKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY SATGQGIEYTAGSGKDDATKWNKKITIPQTGGIGTI IFAVAGAAIMGIAVYAYVKNNKD EDQLA
83 MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLLLSEDDLKTWD
TNGPKGYDGTQSSLKDLTGWAEEIPNVYFELQKYNLTDGKEKENLKDDSKWTTVHGGLT TKDGLKIETSTLKGVYRIREDRTKTTYVGPNGQVLTGSKAVPALVTLPLVNNNGTVIDAH VFPKNSYNKPWDKRIADTLNYNDQNGLSIGTKIPYWNTTIPSNATFATSFWSDEMTEG LTYNEDVTITLNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPEGVKATVQLVN AKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGYSAEYTVESKGKLGVKNWKDN NPAPINPEEPRVKTYGKKFVKVDQKDTRLENAQFWKKADSNKYIAFKSTAQQAADEKAA ATAKQKLDAAVAAYTNAADKQAAQALVDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSN TDGQFQISGLAAGTYKLEEIKAPEGFAKIDDVEFWGAGSWNQGEFNYLKDVQKNDATKV VNKKI IPQTGGIG I IFAVAGAAIMGIAVYAYVKNNKDEDQLA
84 MKSINKFLTILAALLLTVSSLFSAATVFAAEQKTKTLTVHKLLMTDQELDAWNSDAITTA
GYDGSQNFEQFKQLQGVPQGVTEISGVAFELQSYTGPQGKEQENLTNDAVWTAVNKGVTT ETGVKFDTEVLQGTYRLVEVRKESTYVGPNGKVLTGMKAVPALITLPLVNQNGWENAHV YPKNSEDKPTATKTFDTAAGFVDPGEKGLAIGTKVPYIVTTTIPKNSTLATAFWSDEMTE GLDYNGDVWNYNGQPLDNSHYTLEAGHNGFILKLNEKGLEAINGKDAEA I LKYTATL NALAVADVPEANDVTFHYGNNPGHGNTPKPNKPKNGELTITKTWADAKDAPIAGVEVTFD LVNAQTGEWKVPGHETGIVLNQTNNWTFTATGLDNNTEYKFVERTIKGYSADYQTITET GKIAVKNWKDENPEPINPEEPRVKTYGKKFVKVDQKDERLKEAQFWKNEQGKYLALKSA AQQAVNEKAAAEAKQALDAAIAAYTNAADKNAAQAWDAAQKTYNDNYRAARFGYVEVER KEDALVLTSNTDGQFQISGLAAGSYTLEETKAPEGFAKLGDVKFEVGAGSWNQGDFNYLK DVQKNDATKWNKKI IPQTGGIG I IFAVAGAVIMGIAVYAYVKNNKDEDQLA
85 MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKS
E SHETVEGSGEATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGA I IEGMDADKAEKRKEV LNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNKY KIELTVEGKTTVETKELNQPLDVWLLDNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVA LVTYAS IFDGTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRI PKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYISTS YQNQFNSFLNKIPDRSGILQEDFI INGDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLS VMSNEGYAINSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGI GVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMGEMI EFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQK WLTYDVRLKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVE FIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEISSPDG YIEVKTKPWTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYI LVGSTFMILTICSFRRKQL
86 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHP
ESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQ NQEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVG DLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDI LGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKF IQTENYSHKQLTNNAEEI IKRIP TEAPKAKWGSTTNGL PEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKI IVHVTDGVPTRSY AINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQI ISGNLQKLHY LDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQK LKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDP MGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGE GQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGE IEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVS PEDYQKI NKPILTFEWKGSIKNI IAVNKQISEYHEEGDKHLI NTHIPPKGI IPMTGGKGILSFI LIGGAMMSIAGGIYIWKRYKKSSDMSIKKD
87 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKPTSHS
ESKVEKVTTEVTGEATFDNL PGDYTLSEETAPEGYKKTTQTWQVKVESNGKT IQNSDDKKSI IEQ RQEELDKQYPLTGAYEDTKESYNLEHVKNSIPNGKLEAKAVNPYSSEGEHIREIQEGTLSKRISEVN DLDHNKYKIELTVSGKSI IK INKDEPLDWFVLDNSNSMKNNGKNNKAKKAGEAVE I IKDVLGAN VENRAALVTYGSDIFDGRTVKVIKGFKEDPYYGLE SFTVQTNDYSYKKFTNIAADI IKKIPKEAPE AKWGG SLGL PEKKREYDLSKVGETFTMKAFMEADTLLSSIQRKSRKI IVHLTDGVPTRSYAINSF VKGSTYANQFERIKEKGYLDKNNYFI DDPEKIKGNGESYFLFPLDSYQTQI ISGNLQKLHYLDLNL NYPKGTIYRNGPVREHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEDYKKNQDGTFQKLKEEA FELSDGEITELMNSFSSKPEYYTPIVTSADVSNNEILSKIQQQFEKILTKENSIVNGTIEDPMGDKI NLHLGNGQTLQPSDYTLQGNDGSIMKDSIATGGPNNDGGILKGVKLEYIKNKLYVRGLNLGEGQKVT LTYDVKLDDSFISNKFYDTNGRTTLNPKSEEPDTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFTK VDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPKDYQ KITNKPILTFEWKGSIQNI IAVNKQISEYHEEGDKHLITNTHIPPKGI IPMTGGKGILSFILIGGA MMSIAGGIYIWKRHKKSSDASIEKD
88 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHP
ESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQ NQEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVG DLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDI LGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKF IQTENYSHKQLTNNAEEI IKRIP TEAPKAKWGSTTNGL PEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKI IVHVTDGVPTRSY AINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQI ISGNLQKLHY LDLNLNYPKGTFYRNGPVREHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEDYKKNQDGTFQK LKEEAFELSDGEITELMKSFSSKPEYYTPIVTSSDASNNEILSKIQQQFEKILTKENSIVNGTIEDP MGDKINLQLGNGQTLQPSDYTLQGNDGSIMKDSIATGGPNNDGGILKGVKLEYIKNKLYVRGLNLGE GQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGE IEFTKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVS PKDYQKI NKPILTFEWKGSIQNI IAVNKQISEYHEEGDKHLI NTHIPPKGI IPMTGGKGILSFI LIGGSMMSIAGGIYIWKRYKKSSDISREKD
89 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHP
ESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQ NQEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVG DLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDI LGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLTNNAEEI IKRIP TEAPKAKWGSTTNGLTPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKI IVHVTDGVPTRSY AINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPDDIKGNGESYFLFPLDSYQTQI ISGNLQKLHY LDLNLNYPKGTIYRNGPVKEHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQK LKEEAFKLSDGEITELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTIEDP MGDKINLQLGNGQILQPSDYTLQGNDGSVMKDGIATGGPNNDGGILKGVKLEYIGNKLYVRGLNLGE GQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGE IEFIKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVS PEDYQKITNKPILTFEWKGSIKNI IAVNKQISEYHEEGDKHLITNTHIPPKGI IPKTGGKGILSFI LIGGAMMSIAGGIYIWKRYKKSSDMSIKKD
90 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHP
ESKIEKVTAELTGEATFDNLIPGDYTLSEETAPEGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQ NHEELDKQYPPTGIYEDTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEVG DLAHNKYKIELTVSGKTIVKPVDKQKPLDWFVLDNSNSMNNDGPNFQRHNKAKKAAEALGTAVKDI LGANSDNRVALVTYGSDIFDGRSVDWKGFKEDDKYYGLQTKFTIQTENYSHKQLTNNAEEI IKRIP TEAPRAKWGSTTNGLTPEQQKQYYLSKVGETFTMKAFMEADDILSQVDRNSQKI IVHITDGVPTRSY AINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLFPLDSYQTQI ISGNLQKLHY LDLNLNYPKGTIYRNGPVREHGTPTKLYINSLKQKNYDIFNFGIDISAFRQVYNEDYKKNQDGTFQK LKEEAFELSDGEITELMKSFSSKPEYYTPIVTSSDASNNEILSKIQQQFEKVLTKENSIVNGTIEDP MGDKINLQLGNGQTLQPSDYTLQGNDGSIMKDSIATGGPNNDGGILKGVKLEYIKNKLYVRGLNLGE GQKVTLTYDVKLDDSFISNKFYDTNGRTTLNPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGE IEFTKVDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVS PKDYQKITNKPILTFEWKGSIQNI IAVNKQISEYHEEGDKHLITNTHIPPKGI IPMTGGKGILSFI LIGGSMMSIAGGIYIWKRYKKSSDISREKD
91 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKPTSHS
ESKVEKVTTEVTGEATFDNLTPGDYTLSEETAPEGYKKTTQTWQVKVESNGKTTIQNSDDKKSI IEQ RQEELDKQYPLTGAYEDTKESYNLEHVKNSIPNGKLEAKAVNPYSSEGEHIREIQEGTLSKRISEVN DLDHNKYKIELTVSGKSI IKTINKDEPLDWFVLDNSNSMKNNGKNNKAKKAGEAVETI IKDVLGAN VENRAALVTYGSDIFDGRTVKVIKGFKEDPYYGLETSFTVQTNDYSYKKFTNIAADI IKKIPKEAPE AKWGGTSLGLTPEKKREYDLSKVGETFTMKAFMEADTLLSSIQRKSRKI IVHLTDGVPTRSYAINSF VTGSTYANQFERIKEKGYLDKNNYFITDDPEKIKGNGESYFLFPLDSYQTQI ISGNLQKLHYLDLNL NYPKGTIYRNGPVREHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEDYKKNQDGTFQKLKEEA FELSDGEITELMNSFSSKPEYYTPIVTSADVSNNEILSKIQQQFEKILTKENSIVNGTIEDPMGDKI NLQLGNGQTLQPSDYTLQGNDGSIMKDSIATGGPNNDGGILKGVKLEYIKNKLYVRGLNLGEGQKVT LTYDVKLDDSFISNKFYDTNGRTTLNPKSEEPDTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFTK VDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPKDYQ KITNKPILTFEWKGSIQNI IAVNKQISEYHEEGDKHLITNTHIPPKGI IPMTGGKGILSFILIGGA MMSIAGGIYIWKRHKKSSDASIEKD
92 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLWKKTDDQNKPLSKATFVLKTTAHP
ESKIEKVTAEVTGEATFDNLTPGDYTLSEETAPEGYKKTTQTWQVKVESNGKTTIQNSDDKKSI IEQ RQEELDKQYPLTGAYEDTKESYNLEHVKNSIPNGKLEAKAVNPYSSEGEHIREIQEGTLSKRISEVN DLDHNKYKIELTVSGKSI IK INKDEPLDWFVLDNSNSMKNNGKNNKAKKAGEAVE I IKDVLGAN VENRAALVTYGSDIFDGRTVKVIKGFKEDPYHGLE SFTVQTNDYSYKKFTNIAADI IKKIPKEAPE AKWGG SLGL PEKKREYDLSKVGETFTMKAFMEADTLLSSIQRKSRKI IVHLTDGVPTRSYAINSF VTGSTYANQFERIKEKGYLDKNNYFI DDPEKIKGNGESYFLFPLDSYQTQI ISGNLQKLHYLDLNL NYPKGTIYRNGPVREHGTPTKLYINSLKQKNYDIFNFGIDISGFRQVYNEDYKKNQDGTFQKLKEEA FELSGGEITELMKSFSSKPEYYTPIVTSADVSNNEILSKIQQQFEKILTKENSIVNGTIEDPMGDKI NLQLGNGQTLQPSDYTLQGNDGSIMKDSIATGGPNNDGGILKGVKLEYIKNKLYVRGLNLGEGQKVT LTYDVKLDDSFISNKFYDTNGRTTLNPKSEEPDTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFTK VDKDNNKLLLKGATFELQEFNEDYKLYLPIKNNNSKWTGENGKISYKDLKDGKYQLIEAVSPKDYQ KI NKPILTFEWKGSIQNI IAVNKQISEYHEEGDKHLI NTHIPPKGI IPMTGGKGILSFILIGGA MMSIAGGIYIWKKHKKSSDASIEKD
93 MLKKCQTFI IESLKKKKHPKEWKI IMWSLMILTTFLTTYFLILPAI VEETKTDDVGI LENKNSSQ
VTSSTSSSQSSVEQSKPQTPASSVTETSSSEEAAYREEPLMFRGADYTVTVTLTKEAKIPKNADLKV TELKDNSATFKDYKKKALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDEN LKWHFKDDGQTEVLKSKDTAETKNTSSDVAFKTDSFSIYAIVQEDNTEVPRLTYHFQNNDGTDYDF LTASGMQVHHQI IKDGESLGEVGIP IKAGEHFNGWYTYDPTTGKYGDPVKFGEPI VTETKEICVR PFMSKVATVTLYDDSAGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLSES EIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPASTIKPNDPTRPGYTFAGWY TAASGGAAFDFNQVLTKDTTLYAHWSPAQTTYTINYWQQSATDNKNATDAQKTYEYAGQVTRSGLSL SNQTLTQQDINDKLPTGFKVNNTRTETSVMIKDDGSSWNVYYDRKLITIKFAKYGGYSLPEYYYSY NWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSYLGEFILPNDTVDSDVIKLFP KGNIVQTYRFFKQGLDGTYSLADTGGGAGADEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAG VKVPISDEYYDRYGAYHKDYLNLWWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYAPDTT TVQPKPSRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYAGWQKVTYRVNIDPNGGRLSKTDDTY LDLHYGDRIPDYTDITRDYIQDPSGTYYYKYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDA YKLVGWYYVNPDGSIRPYNFSGAVTQDINLRAIWRKAGDYHI IYSNDAVGTDGKPALDASGQQLQ S NEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSIDIDAHLADANKNI IKPVI IPV GDIKLEDTSIKYNGNGGTRVENGNWTQVETPRMELNSTTTIPENQYFTRTGYNLIGWHHDKDLADT GRVEFTAGQSIGIDNNPDATNTLYAVWQPKEYTVRVSKTWGLDEDKTKDFLFNPSETLQQENFPLR DGQTKEFKVPYGTSISIDEQAYDEFKVSESITEKNLATGEADKTYDATGLQSLTVSGDVDISFTNTR IKQKVRLQKVNVENDNNFLAGAVFDIYESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKY YLTETKAPPGYLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGTELPSTGG IGTHIYILVGLALALPSGLILYYRKKI
94 MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKWIKETGEGGALLGDAVFELKNNTD
GTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEAL SDQYPQTGTYPDVQ PYQI IKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIEL TVSGKTVYEQKDKSVPLDWILLDNSNSMSNIRNKNARRAERAGEATRSLIDKITSDPENRVALVTY ASTIFDGTEFTVEKGVADKNGKRLNDSLFWNYDQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEA EDHDGNRLMYQFGATFTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSYQN QLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVYEKGAPAAFPVKPEKYSEMK AAGYAVIGDPINGGYIWLNWRESILAYPFNSNTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGING DPGTDEATATSFMQSISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELIDLQL GTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVLYDTTEKRIRVTGLYLGTDEKVTLT YNVRLNDEFVSNKFYDTNGRTTLHPKEVEQNTVRDFPIPKIRDVRKYPEITISKEKKLGDIEFIKVN KNDKKPLRDAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYK PVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPKREYPRTGGIGMLPFYLIG CMMMGGVLLYTRKHP
95 MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQLEIAPKEGTPIEGVLYQL
YQLKSTEDGDLLAHWNSLTITELKKQAQQVFEATTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSA FLVDLSEDKVIYPKI IWSTGELDLLKVGVDGDTKKPLAGWFELYEKNGR PIRVKNGVHSQDIDAA KHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVTIEKSKTVTVTIENKKVPTPKVPSRG GLIPKTGEQQAMALVI IGGILIALALRLLSKHRKHQNKD
96 MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTEEGISYRLWTVTDNLKVD
LLSQMTDSELNQKYKSILTSPTDTNGQTKIALPNGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQL QINPKRKVETGRLKLIKYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKGE IEVEGLLPGKYIFREAKALTGYRISMKDAWAWANKTQEVEVENEKETPPPTNPKPSQPLFPQSFL PKTGMI IGGGLTILGCI ILGILFIFLRKTKNSKSERNDTV 97 MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEWSQLPSRDGHRLQVWKLDD SYSYDDRVQIVRDLHSWDENKLSSFKK SFEMTFLENQIEVSHIPNGLYYVRSI IQTDAVSYPAEFL FEMTDQTVEPLVIVAKKTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSSS GQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITWNQKLPRGNV DFMKVDGRTNTSLQGAMFKVMKEESGHYTPVLQNGKEVWTSGKDGRFRVEGLEYGTYYLWELQAPT GYVQLTSPVSFTIGKDTRKELVTWKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN
98 HQSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFK SGMKWHM SQERLDYNSQLAIDKTGNMGYI
SIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHW TVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALV VAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
99 NSQLAIDKTGNMGYISIPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSS
RLFSDLDKLKVGDHWTVSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLL VRGHRVPNDNGNALWAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENN DL
100 QSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFK SGMKWHM SQERLDYNSQLAIDKTGNMGYIS
IPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWT VSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALW AEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
101 QSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFKTSGMKWHMTSQERLDYNSQLAIDKTGNMGYIS
IPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWT VSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGN
102 QSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFKTSGMKAHMTSQERLDYNSQLAIDKTGNMGYIS
IPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWT VSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLVRGHRVPNDNGNALW AEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
103 QSRAIMDYQDRVTHMDENDYKKI INRAKEYNKQFKTSGMKWHMTSQERLDYNSQLAIDKTGNMGYIS
IPKINIKLPLYHGTSEKVLQTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWT VSILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTATPYGVNTHRLLVRGHRVPNDNGNALW AEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRARQRKKILKQAMRKEENNDL
104 ATGGCTTATCCTTCACTTGCTAATTATTGGAATTCATTTCACCAATCTCGAGCGATTATGGATTACC
AAGACCGCGTAACGCATATGGATGAAAACGATTATAAAAAAATTATTAACCGAGCCAAAGAATATAA TAAGCAATTTAAAACTTCAGGAATGAAGTGGCACATGACTAGCCAAGAGCGTTTGGATTATAATTCA CAACTGGCTATCGATAAAACGGGTAATATGGGTTATATTTCAATTCCAAAGATAAACATAAAATTAC CACTTTATCATGGTACAAGTGAAAAAGTGCTTCAAACTTCTATTGGTCATTTAGAAGGAAGTAGTCT TCCAATTGGAGGAGACTCAACTCATTCTATTTTATCAGGACATAGAGGTTTACCCTCTTCAAGGCTT TTTTCTGATTTGGATAAGTTAAAAGTTGGAGACCACTGGACAGTCAGTATCTTAAATGAAACATATA CTTATCAAGTGGATCAAATCAGAACAGTTAAACCGGATGATTTGAGGGATTTACAAATTGTTAAAGG TAAAGACTACCAAACTTTGGTGACGTGTACACCATATGGCGTTAATACCCATCGGTTACTAGTGAGA GGACATCGTGTACCAAACGATAATGGTAACGCTTTGGTAGTAGCAGAGGCAATACAAATAGAGCCTA TTTATATCGCACCATTTATCGCTATTTTCCTTACTTTGATTTTACTTTTAATCTCTTTAGAAGTAAC TAGGAGAGCACGTCAACGTAAGAAAATTTTAAAACAAGCAATGAGAAAGGAAGAGAACAATGATTTA TAA
105 MIRRYSANFLAILGI ILVSSGIYWGWYNINQAHQADLTSQHIVKVLDKSITHQVKGSENGELPVKKL
DKTDYLGTLDIPNLKLHLPVAANYSFEQLSKTPTRYYGSYLTNNMVICAHNFPYHFDALKNVDMGTD VYFTTTTGQIYHYKISNREI IEPTAIEKVYKTATSDNDWDLSLFTCTKAGVARVLVRCQLIDVKN
106 MIRRYSANFLAILGI ILVSSGIYWGWYNINQAHQADLTSQHIVKVLDKSITHQVKGSENGELPVKKL
DKTDYLGTLDIPNLKLHLPVAANYSFEQLSKTPTRYYGSYLTNNMVIAAHNFPYHFDALKNVDMGTD VYFTTTTGQIYHYKISNREI IEPTAIEKVYKTATSDNDWDLSLFTATKAGVARVLVRAQLIDVKN
107 GTGATTAGAAGATATTCAGCAAATTTTTTAGCTATACTCGGAATTATTCTGGTAAGTTCTGGAATCT
ATTGGGGTTGGTATAATATTAATCAGGCGCATCAAGCTGATTTAACTTCTCAGCATATTGTCAAGGT GCTTGATAAATCTATTACGCATCAAGTAAAGGGTTCAGAAAATGGAGAATTACCTGTAAAAAAGTTG GATAAAACAGATTACTTGGGAACTCTGGATATTCCGAACTTAAAACTGCATTTACCGGTAGCTGCTA ATTATAGTTTTGAACAACTGTCTAAGACGCCTACAAGGTATTATGGTTCTTATTTAACTAATAACAT GGTGATTTGTGCGCATAATTTTCCTTATCATTTTGATGCTTTAAAAAATGTAGATATGGGAACGGAT GTTTATTTTACAACTACAACAGGGCAAATCTATCACTACAAAATCAGTAATAGAGAAATTATTGAAC CAACAGCGATTGAAAAAGTTTATAAAACTGCCACATCAGACAATGATTGGGACTTAAGCTTGTTTAC TTGTACAAAGGCAGGAGTAGCTAGAGTATTAGTGCGCTGTCAATTAATTGATGTTAAAAATTAA
108 QAHQADLTSQHIVKVLDKSITHQVKGSENGELPVKKLDKTDYLGTLDIPNLKLHLPVAANYSFEQLS
KTPTRYYGSYLTNNMVICAHNFPYHFDALKNVDMGTDVYFTTTTGQIYHYKISNREI IEPTAIEKVY KTATSDNDWDLSLFTCTKAGVARVLVRCQLIDVKN
109 LAILGI ILVSSGIYWGWYNINQAHQADL SQHIVKVLDKSI HQVKGSENGELPVKKLDKTDYLGTL
DIPNLKLHLPVAANYSFEQLSKTPTRYYGSYLTNNMVICAHNFPYHFDALKNVDMGTDVYFTTTTGQ IYHYKISNREI IEPTAIEKVYKTATSDNDWDLSLFTCTKAGVARVLVRCQLIDVKN
110 MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKD
GASYLIPQGKEAEYKASTDFNSLFTTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESN NDGTEVINVSQYGYYYVSSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVK YTITYKNAVNYHGTEKVYQYVIKDTMPSASWDLNEGSYEVTITDGSGNITTLTQGSEKATGKYNLL EENNNF I IPWAATNTPTGNTQNGANDDFFYKGIN I VTYTGVLKSGAKPGSADLPENTNIA IN PNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTEANATEYT TGADGI ITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTNSDNLLVNPTVENNKGTELP STGGIGT IFYI IGAILVIGAGIVLVARRRLRS
111 AETGTITVQDTQKGATYKAYKVFDAEIDNANVSDSNKDGASYLI PQGKEAEYKASTDFNSL
FTTTTNGGRTYVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYYV SSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNY HGTEKVYQYVIKDTMPSASWDLNEGSYEVTITDGSGNITTLTQGSEKATGKYNLLEENNN FTITI PWAATNTPTGNTQNGANDDFFYKGINTITVTYTGVLKSGAKPGSADLPENTNIATI NPNTSNDDPGQKVTVRDGQITIKKI DGSTKASLQGAI FVLKNATGQFLNFNDTNNVEWGTE ANATEYTTGADGI ITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTNSDNLLV NPTVENNKGTE
112 AETGTITVQDTKKGATYKAYKVFDAEIDNANVSDSNKDGASYLI PQGKEAEYKASTDFNSL
FTTTTNGGRTYVTKKDTASANEIATWAKSI SANTTPVSTVTESNNDGTEVINVSQYGYYYV SSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTVKYTITYKNAVNY HGTEKVYQYVIKDTMPSASWDLNEGSYEVTITDGSGNITTLTQGSEKATGKYNLLEENNN FTITI PWAATNTPTGNTQNGANDDFFYKGINTITVTYTGVLKSGAKPGSADLPENTNIATI NPNTSNDDPGQKVTVRDGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTE ANATEYTTGADGI ITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTNSDNLLV NPTVENNKGTE
113 attccacaaacaggtggtattggtacaTAACGCGACTTAATTAAACGG
114 TGTACCAATACCACCTGTTTGTGGAATCTTGTACAGCTCGTCCATGCC
115 CTTTAAGAAGGAGATATACATACCCATGGGATCTGATAAAATTCATCATCATCATCATCAC
GAAAACCTGTACTTCCAGGGCatggtgagcaagggcgaggagctgttcaccggggtggtgc ccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgaggg cgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctg cccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgct accccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtcca ggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggca acatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccga caagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagc gtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgc ccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcga tcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctg tacaagTAACGCGACTTAATTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAG ATTTTCAGCCTGATACAGATTAAATC
116 MGSDKIHHHHHHENLYFQGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK
LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGI DFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV TAAGITLGMDELYK
117 CTTTAAGAAGGAGATATACATACCCATGGGATCTGATAAAATTCATCATCATCATCATCAC
GAAAACCTGTACTTCCAGGGCatggtgagcaagggcgaggagctgttcaccggggtggtgc ccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgaggg cgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctg cccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgct accccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtcca ggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggca acatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccga caagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagc gtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgc ccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcga tcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctg tacaagattccacaaacaggtggtattggtacaTAACGCGACTTAATTAAACGGTCTCCAG CTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATC
118 MGSDKIHHHHHHENLYFQGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK
LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV TAAGITLGMDELYKIPQTGGIGT
REFERENCES
[I] Rosini et al, Molecular Microbiology, 2006, 61(1): 126-141
[2] Margarit et al, Journal of Infectious Diseases, 2009, 199: 108-115
[3] Cozzi et al, The FASEBJ, 201 1, 25: 1874-1886
[4] Manzano et al, 2008, Structure, 16: 1838-1848
[5] Neiers et al, 2009, J. Mol. Biol. 393, 704-716
[6] Cozzi et al, 2012, FASEBJ, 26: 1-11
[7 ] Telford et al, 2006, Nature Reviews Microbiology 4: 509-519
[8] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.
[9] Vaccine Design (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.
[10] WO90/14837.
[I I] WO90/14837.
[12] Podda & Del Giudice (2003) Expert Rev Vaccines 2: 197-203.
[13] Podda (2001) Vaccine 19: 2673-2680.
[14] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).
[15] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.
[16] US 5,057,540.
[17] Niikura et al. (2002) Virology 293:273-280.
[18] Lenz et al. (2001) J Immunol 166:5346-5355.
[19] Pinto et al. (2003) J Infect Dis 188:327-338.
[20] Gerber et al. (2001) J Virol 75:4752-4760.
[21] WO03/024480.
[22] WO03/024481.
[23] Gluck et al. (2002) Vaccine 20:B10-B16.
[24] Meraldi et al. (2003) Vaccine 21 :2485-2491.
[25] Pajak et al. (2003) Vaccine 21 :836-842.
[26] Krieg (2003) Nature Medicine 9:831-835.
[27] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32: 179-185. [29] US 6,207,646.
[30] US 6,239,116.
[31] US 6,429,199.
[32] Schellack et al. (2006) Vaccine 24:5461-72.
[33] Johnson et al. (1999) BioorgMed Chem Lett 9:2273-2278.
[34] Evans et al. (2003) Expert Rev Vaccines 2:219-229.
[35] Beignon et al. (2002) Infect Immun 70:3012-3019.
[36] Pizza et al. (2001) Vaccine 19:2534-2541.
[37] Pizza et al. (2000) Int J Med Microbiol 290:455-461.
[38] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313.
[39] Ryan et al. (1999) Infect Immun 67:6270-6280.
[40] Partidos et al. (1999) Immunol Lett 67:209-216.
[41] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293.
[42] Pine et al. (2002) J Control Release 85:263-270.
[43] WO99/40936.
[44] W099/44636.
[45] Singh et al] (2001) J Cont Release 70:267-276.
[46] WO99/27960.
[47] US 6,090,406.
[48] US 5,916,588.
[49] EP-A-0626169.
[50] W099/52549.
[51] Andrianov et al. (1998) Biomaterials 19: 109-115.
[52] Payne et al. (1998) Adv Drug Delivery Review 31 : 185-196.
[53] Stanley (2002) Clin Exp Dermatol 27:571-577.
[54] Jones (2003) Curr Opin Investig Drugs 4:214-218.
[55] W099/11241.
[56] WO94/00153.
[57] W098/57659.
[58] European patent applications 0835318, 0735898 and 0761231.
[59] Ogunniyi et al. (2001) Infect Immun 69:5997-6003.
[60] WO2006/110603.
[61] Watson (2000) Pediatr Infect Dis J 19:331-332.
[62] Rubin (2000) Pediatr Clin North Am 47:269-285, v.
[63] Jedrzejas (2001) Microbiol Mol Biol Rev 65: 187-207.
[64] Bell (2000) Pediatr Infect Dis J 19: 1187-1188.
[65] Iwarson (1995) APMIS 103:321-326.
[66] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.
[67] Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.
[68] Del Guidice et al. (1998) Molecular Aspects of Medicine 19: 1-70.
[69] Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.
[70] Rappuoli et al. (1991) TIBTECH 9:232-238.
[71] Costantino et al. (1999) Vaccine 17: 1251-1263.
[72] Sutter et al. (2000) Pediatr Clin North Am 47:287-308.
[73] Zimmerman & Spann (1999) Am Fam Physician 59: 113-118, 125-126.
[74] McMichael (2000) Vaccine 19 Suppl 1 :S101-107. [75] Schuchat (1999) Lancet 353(9146):51-6.
[76] WO02/34771.
[77] Dale (1999) Infect Dis Clin North Am 13:227-43, viii.
[78] Ferretti et al. (2001) PNAS USA 98: 4658-4663.
[79] Kuroda et al. (2001) Lancet 357(9264): 1225-1240; see also pages 1218-1219.
[80] EP-A-0372501
[81] EP-A-0378881
[82] EP-A-0427347
[83] W093/17712
[84] WO94/03208
[85] W098/58668
[86] EP-A-0471177
[87] WO00/56360
[88] WO91/01146
[89] WO00/61761
[90] WO01/72337
[91] Research Disclosure, 453077 (Jan 2002)
[92] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.
[93] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [94] Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds, 1986, Blackwell Scientific Publications)
[95] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press).
[96] Handbook of Surface and Colloidal Chemistry (Birdi, K.S. ed., CRC Press, 1997) [97] Ausubel et al. (eds) (2002) Short protocols in molecular biology, 5th edition (Current Protocols).
[98] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press)
[99] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag)
[100] Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) Supplement 30
[101] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.
[102] Sendi P et al, (2008) Infection. 36(2): 100-11
[103] Dramsi S et al, (2006), Mol Microbiol. 60(6): 1401-13.
[104 ]Nobbs AH, et al (2008) Infect Immun. ;76(8):3550-60.
[105] Persson K.(2011) Acta Crystallogr D Biol Crystallogr. 67(Pt 3):212-7.
[106] Lu G, et al, (2011) Proteins. 79(9):2764-9.
[107] Nuccitelli A, et al (2011) Proc Natl Acad Sci USA. 21;108(25): 10278-83.
[108] Maione D, et al (2005) Science 309(5731): 148-50

Claims

1. A method of ligating at least two moieties comprising contacting the at least two moieties with a pilus-related sortase C enzyme in vitro under conditions suitable for a sortase mediated transpeptidation reaction to occur, wherein the pilus-related sortase C enzyme comprises an exposed active site.
2. A method according to claim 1 wherein the pilus-related sortase C enzyme is from Streptococcus.
3. A method according to claim 2 wherein the Streptococcus is selected from the group consisting of Streptococcus agalactiae (GBS), Streptococcus pneumonia (pneumococcus) and Streptococcus pyogenes (GAS).
4. A method according to claim 3 wherein the pilus-related sortase C enzyme is a sortase CI enzyme (srtCl), sortase C2 enzyme (SrtC2) or a sortase C3 enzyme (SrtC3).
5. A method according to any one of claims 1 to 4 wherein the pilus-related sortase C enzyme mutation comprises a deletion of part or all of the lid.
6. A method according to claim 5 wherein the mutation comprises a deletion of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the deletion of amino acids at corresponding positions in the amino acid sequence of another pilus-related sortase C enzyme.
7. A method according to any one of claims 1 to 4 wherein the mutation comprises substitution of the amino acids at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase CI enzyme of PI-2a (SEQ ID NO:3), or the substitution of amino acids at corresponding positions in the amino acid sequence of another sortase C enzyme.
8. A method according to any one of claims 1 to 4 wherein the pilus-related sortase C enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70 and 71.
9. A method according to any one of claims 1 to 8 wherein the at least two moieties comprise an LPxTG motif and a pilin motif.
10. A method according to any of claims 1 to 8 wherein the at least two moieties are from Gram-positive bacteria.
11. A method according to claim 10 wherein the at least two moieties are from the same Gram-positive bacteria or from different Gram positive bacteria.
12. A method according to claim 9 or claim 10 wherein the at least two moieties are Streptococcal polypeptides.
13. A method according to claim 12 wherein the at least two moieties are Streptococcal backbone proteins and/or ancillary proteins.
14. A method according to claim 13 wherein the at least two moieties comprise or consist of an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%), 99.5%) or more) to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97; or (b) that is a fragment of at least *n* consecutive amino acids of one of these sequences wherein 'n' is 20 or more (e.g. 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150 or more; e.g. 20 or more; or e.g. 50 or more; or e.g. 80 or more).
15. An artificial pilus obtained or obtainable from the method of any one of claims 1 to 14.
16. An artificial pilus which comprises at least two variants of backbone protein
GBS59 and wherein the at least two variants are selected from the group consisting of Group B Streptococcus strains 2603, H36B, 515, CJB111, CJB110 and DK21.
17. An artificial pilus according to claim 15 or 16 for use in medicine.
18. An artificial pilus according to claim 15 or 16 for use in preventing or treating Streptococcal infection.
19. A method of treating or preventing Streptococcal infection in a patient in need thereof comprising administering an effective amount of an artificial pilus according to claim 15 or 16 to said patient.
20. A method according to any one of claims 1 to 8 wherein the at least two moieties comprise a first moiety comprising the amino acid motif LPXTG, wherein X is any amino acid, and a second moiety comprising at least one amino acid.
21. The method according to claim 20, wherein the first moiety is a first polypeptide and the second moiety is a second polypeptide.
22. The method according to 21, wherein the first polypeptide and the second
polypeptide are from Gram-positive bacteria.
23. A method according to claim 22 wherein the first polypeptide and the second
polypeptide are from the same Gram-positive bacteria or from different Gram positive bacteria.
24. A method according to claim 22 or claim 23, wherein first polypeptide and the second polypeptide are Streptococcal polypeptides.
25. A method according to claim 24, wherein first polypeptide and the second polypeptide are Streptococcal backbone proteins and/or ancillary proteins.
26. The method according to claim 20, wherein either the first moiety or the second moiety comprises a detectable label.
27. The method according to claim 26, wherein the detectable label is a fluorescent label, a radiolabel, a chemiluminescent label, a phosphorescent label, a biotin label, or a streptavidin label.
28. The method according to claim 20, wherein either the first moiety or the second moiety is a polypeptide and the other moiety is a protein or glycoprotein on the surface of a cell.
29. The method according to claim 20, wherein either the first moiety or the second moiety is a polypeptide and the other moiety comprises amino acids conjugated to a solid support.
30. The method according to claim 20, wherein either the first moiety or the second moiety is a polypeptide and the other moiety comprises at least one amino acid conjugated to a polynucleotide.
31. The method according to any claim 20, wherein the first moiety and the second moiety are the N-terminus and C-terminus of a polypeptide chain, and ligation results in the formation of a circular polypeptide.
32. The method according to claim 9, wherein the pilin motif comprises the amino acids YPAN.
33. A kit comprising a PI-2b sortase CI or a PI-2b sortase C2 enzyme from
Streptococcus agalactiae and a moiety comprising the amino acid motif LPXTG, wherein X is any amino acid.
34. A conjugate obtained or obtainable from the method of any one of claims 20 to 31.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293814A (en) * 2014-01-09 2015-01-21 河南科技大学 Diapause hormone fusion gene, fusion protein, preparation method and application
WO2016020413A1 (en) * 2014-08-05 2016-02-11 Glaxosmithkline Biologicals S.A. Carrier molecule for antigens
WO2018229708A1 (en) 2017-06-16 2018-12-20 Glaxosmithkline Biologicals Sa Method of treatment
WO2021099982A1 (en) 2019-11-22 2021-05-27 Glaxosmithkline Biologicals Sa Dosage and administration of a bacterial saccharide glycoconjugate vaccine

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10738338B2 (en) 2016-10-18 2020-08-11 The Research Foundation for the State University Method and composition for biocatalytic protein-oligonucleotide conjugation and protein-oligonucleotide conjugate
GB201705750D0 (en) * 2017-04-10 2017-05-24 Univ Oxford Innovation Ltd Peptide ligase and use therof

Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0372501A2 (en) 1988-12-07 1990-06-13 BEHRINGWERKE Aktiengesellschaft Synthetic antigens, method for their preparation and their use
EP0378881A1 (en) 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Synthetic peptides and their use as universal carriers for the preparation of immunogenic conjugates suitable for the development of synthetic vaccines
WO1990007936A1 (en) 1989-01-23 1990-07-26 Chiron Corporation Recombinant therapies for infection and hyperproliferative disorders
WO1990014837A1 (en) 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
WO1991001146A1 (en) 1989-07-14 1991-02-07 Praxis Biologics, Inc. Cytokine and hormone carriers for conjugate vaccines
EP0427347A1 (en) 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Synthetic peptides useful as universal carriers for the preparation of immunogenic conjugates and their use in the development of synthetic vaccines
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
EP0471177A2 (en) 1990-08-13 1992-02-19 American Cyanamid Company Filamentous hemagglutinin of bordetella pertussis as a carrier molecule for conjugate vaccines
WO1993017712A2 (en) 1992-03-06 1993-09-16 Biocine Spa Conjugates formed from heat shock proteins and oligo- or polysaccharides
WO1994000153A1 (en) 1992-06-25 1994-01-06 Smithkline Beecham Biologicals (S.A.) Vaccine composition containing adjuvants
WO1994003208A1 (en) 1992-07-30 1994-02-17 Yeda Research And Development Company Ltd. Conjugates of poorly immunogenic antigens and synthetic peptide carriers and vaccines comprising them
EP0626169A2 (en) 1988-08-25 1994-11-30 The Liposome Company, Inc. A dosage form comprising an antigen and a salt form of an organic acid derivative of a sterol
EP0735898A1 (en) 1993-12-23 1996-10-09 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines
EP0835318A2 (en) 1995-06-29 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines against hepatitis c
WO1998040100A1 (en) 1997-03-10 1998-09-17 Ottawa Civic Loeb Research Institute USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE AS AN ADJUVANT
WO1998057659A1 (en) 1997-06-14 1998-12-23 Smithkline Beecham Biologicals S.A. Adjuvant compositions for vaccines
WO1998058668A2 (en) 1997-06-20 1998-12-30 Microbiological Research Authority Bordetella pertussis antigens as carriers in vaccinating conjugates and oral vaccines comprising bordetella pertussis fimbriae
WO1999011241A1 (en) 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Oil in water emulsions containing saponins
WO1999027960A1 (en) 1997-11-28 1999-06-10 West Pharmaceutical Services Vaccine compositions for mucosal administration comprising chitosan
US5916588A (en) 1984-04-12 1999-06-29 The Liposome Company, Inc. Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use
WO1999040936A2 (en) 1998-02-12 1999-08-19 American Cyanamid Company Pneumococcal and meningococcal vaccines formulated with interleukin-12
WO1999044636A2 (en) 1998-03-05 1999-09-10 The Medical College Of Ohio Il-12 enhancement of immune responses to t-independent antigens
WO1999052549A1 (en) 1998-04-09 1999-10-21 Smithkline Beecham Biologicals S.A. Adjuvant compositions
US6090406A (en) 1984-04-12 2000-07-18 The Liposome Company, Inc. Potentiation of immune responses with liposomal adjuvants
WO2000056360A2 (en) 1999-03-19 2000-09-28 Smithkline Beecham Biologicals S.A. Vaccine against antigens from bacteriae
WO2000061761A2 (en) 1999-04-09 2000-10-19 Techlab, Inc. Recombinant clostridium toxin a protein carrier for polysaccharide conjugate vaccines
WO2000062804A2 (en) * 1999-04-15 2000-10-26 The Regents Of The University Of California Identification of sortase gene
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO2001072337A1 (en) 2000-03-27 2001-10-04 Microbiological Research Authority Proteins for use as carriers in conjugate vaccines
WO2002034771A2 (en) 2000-10-27 2002-05-02 Chiron Srl Nucleic acids and proteins from streptococcus groups a & b
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
WO2003024481A2 (en) 2001-09-14 2003-03-27 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
WO2003024480A2 (en) 2001-09-14 2003-03-27 Cytos Biotechnology Ag In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles
WO2005051976A2 (en) * 2003-11-20 2005-06-09 Ansata Therapeutics, Inc. Protein and peptide ligation processes and one-step purification processes
WO2006110603A1 (en) 2005-04-11 2006-10-19 Program For Appropriate Technology In Health Stabilization and preservation of temperature-sensitive vaccines
WO2010087994A2 (en) 2009-01-30 2010-08-05 Whitehead Institute For Biomedical Research Methods for ligation and uses thereof
WO2011056911A2 (en) * 2009-11-04 2011-05-12 Alnylam Pharmaceuticals, Inc. Compositions and methods for enhancing production of a biological product

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2824074A1 (en) * 2001-04-26 2002-10-31 Pasteur Institut SEQUENCE OF THE GENOME STREPTOCOCCUS AGALACTIAE, APPLICATION TO THE DEVELOPMENT OF VACCINES, DIAGNOSIS TOOLS, AND THE IDENTIFICATION OF THERAPEUTIC TARGETS
WO2004020609A2 (en) * 2002-08-30 2004-03-11 Tufts University Streptococcus pneumoniae antigens for diagnosis, treatment and prevention of active infection
EP2333114A1 (en) * 2003-04-15 2011-06-15 Intercell AG S. pneumoniae antigens
MX2007007033A (en) * 2004-12-22 2007-08-03 Novartis Vaccines & Diagnostic Group b streptococcus.
EP2063911A2 (en) * 2006-07-26 2009-06-03 Novartis Ag Immunogenic compositions for gram positive bacteria

Patent Citations (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6090406A (en) 1984-04-12 2000-07-18 The Liposome Company, Inc. Potentiation of immune responses with liposomal adjuvants
US5916588A (en) 1984-04-12 1999-06-29 The Liposome Company, Inc. Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
EP0626169A2 (en) 1988-08-25 1994-11-30 The Liposome Company, Inc. A dosage form comprising an antigen and a salt form of an organic acid derivative of a sterol
EP0372501A2 (en) 1988-12-07 1990-06-13 BEHRINGWERKE Aktiengesellschaft Synthetic antigens, method for their preparation and their use
EP0378881A1 (en) 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Synthetic peptides and their use as universal carriers for the preparation of immunogenic conjugates suitable for the development of synthetic vaccines
WO1990007936A1 (en) 1989-01-23 1990-07-26 Chiron Corporation Recombinant therapies for infection and hyperproliferative disorders
WO1990014837A1 (en) 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
WO1991001146A1 (en) 1989-07-14 1991-02-07 Praxis Biologics, Inc. Cytokine and hormone carriers for conjugate vaccines
EP0427347A1 (en) 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Synthetic peptides useful as universal carriers for the preparation of immunogenic conjugates and their use in the development of synthetic vaccines
EP0471177A2 (en) 1990-08-13 1992-02-19 American Cyanamid Company Filamentous hemagglutinin of bordetella pertussis as a carrier molecule for conjugate vaccines
WO1993017712A2 (en) 1992-03-06 1993-09-16 Biocine Spa Conjugates formed from heat shock proteins and oligo- or polysaccharides
WO1994000153A1 (en) 1992-06-25 1994-01-06 Smithkline Beecham Biologicals (S.A.) Vaccine composition containing adjuvants
EP0761231A1 (en) 1992-06-25 1997-03-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccine composition containing adjuvants
WO1994003208A1 (en) 1992-07-30 1994-02-17 Yeda Research And Development Company Ltd. Conjugates of poorly immunogenic antigens and synthetic peptide carriers and vaccines comprising them
EP0735898A1 (en) 1993-12-23 1996-10-09 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
EP0835318A2 (en) 1995-06-29 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines against hepatitis c
WO1998040100A1 (en) 1997-03-10 1998-09-17 Ottawa Civic Loeb Research Institute USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE AS AN ADJUVANT
WO1998057659A1 (en) 1997-06-14 1998-12-23 Smithkline Beecham Biologicals S.A. Adjuvant compositions for vaccines
WO1998058668A2 (en) 1997-06-20 1998-12-30 Microbiological Research Authority Bordetella pertussis antigens as carriers in vaccinating conjugates and oral vaccines comprising bordetella pertussis fimbriae
WO1999011241A1 (en) 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Oil in water emulsions containing saponins
WO1999027960A1 (en) 1997-11-28 1999-06-10 West Pharmaceutical Services Vaccine compositions for mucosal administration comprising chitosan
WO1999040936A2 (en) 1998-02-12 1999-08-19 American Cyanamid Company Pneumococcal and meningococcal vaccines formulated with interleukin-12
WO1999044636A2 (en) 1998-03-05 1999-09-10 The Medical College Of Ohio Il-12 enhancement of immune responses to t-independent antigens
WO1999052549A1 (en) 1998-04-09 1999-10-21 Smithkline Beecham Biologicals S.A. Adjuvant compositions
WO2000056360A2 (en) 1999-03-19 2000-09-28 Smithkline Beecham Biologicals S.A. Vaccine against antigens from bacteriae
WO2000061761A2 (en) 1999-04-09 2000-10-19 Techlab, Inc. Recombinant clostridium toxin a protein carrier for polysaccharide conjugate vaccines
WO2000062804A2 (en) * 1999-04-15 2000-10-26 The Regents Of The University Of California Identification of sortase gene
WO2001072337A1 (en) 2000-03-27 2001-10-04 Microbiological Research Authority Proteins for use as carriers in conjugate vaccines
WO2002034771A2 (en) 2000-10-27 2002-05-02 Chiron Srl Nucleic acids and proteins from streptococcus groups a & b
WO2003024481A2 (en) 2001-09-14 2003-03-27 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
WO2003024480A2 (en) 2001-09-14 2003-03-27 Cytos Biotechnology Ag In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles
WO2005051976A2 (en) * 2003-11-20 2005-06-09 Ansata Therapeutics, Inc. Protein and peptide ligation processes and one-step purification processes
WO2006110603A1 (en) 2005-04-11 2006-10-19 Program For Appropriate Technology In Health Stabilization and preservation of temperature-sensitive vaccines
WO2010087994A2 (en) 2009-01-30 2010-08-05 Whitehead Institute For Biomedical Research Methods for ligation and uses thereof
WO2011056911A2 (en) * 2009-11-04 2011-05-12 Alnylam Pharmaceuticals, Inc. Compositions and methods for enhancing production of a biological product

Non-Patent Citations (85)

* Cited by examiner, † Cited by third party
Title
"Methods in Molecular Medicine series", vol. 42, article "Vaccine Adjuvants: Preparation Methods and Research Protocols"
ANDRIANOV ET AL., BIOMATERIALS, vol. 19, 1998, pages 109 - 115
AUSUBEL ET AL.: "Short protocols in molecular biology, 5th edition", 2002
BEIGNON ET AL., INFECT IMMUN, vol. 70, 2002, pages 3012 - 3019
BELL, PEDIATR INFECT DIS J, vol. 19, 2000, pages 1187 - 1188
BIOTECHNOLOGY LETTERS, vol. 32, 2010, pages 1 - 10
BIRDI, K.S.: "Handbook of Surface and Colloidal Chemistry", 1997, CRC PRESS
COSTANTINO ET AL., VACCINE, vol. 17, 1999, pages 1251 - 1263
COZZI ET AL., FASEBJ, vol. 26, 2012, pages 1 - 11
COZZI ET AL., THE FASEBJ, vol. 25, 2011, pages 1874 - 1886
D.M. WEIR AND C.C. BLACKWELL,: "Handbook of Experimental Immunology", vol. I-IV, 1986, BLACKWELL SCIENTIFIC PUBLICATIONS
DALE, INFECT DIS CLIN NORTH AM, vol. 13, 1999, pages 227 - 43
DAVID A LEVARY ET AL: "Protein-Protein Fusion Catalyzed by Sortase A", vol. 6, no. 4, 1 April 2011 (2011-04-01), pages e18342.1 - e18342.6, XP002686608, ISSN: 1932-6203, Retrieved from the Internet <URL:http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018342> [retrieved on 20110406], DOI: 10.1371/JOURNAL.PONE.0018342 *
DEL GUIDICE ET AL., MOLECULAR ASPECTS OF MEDICINE, vol. 19, 1998, pages 1 - 70
DRAMSI S ET AL., MOL MICROBIOL., vol. 60, no. 6, 2006, pages 1401 - 13
EVANS ET AL., EXPERT REV VACCINES, vol. 2, 2003, pages 219 - 229
F.M. AUSUBEL ET AL.,: "Current Protocols in Molecular Biology", 1987
FERRETTI ET AL., PNAS USA, vol. 98, 2001, pages 4658 - 4663
GENNARO, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 2000
GENNARO: "Remington: The Science and Practice of Pharmacy. 20th edition", 2000
GERBER ET AL., J VIROL, vol. 75, 2001, pages 4752 - 4760
GERLICH ET AL., VACCINE, vol. 8, no. 1, 1990, pages S63 - 68,79-80
GLUCK ET AL., VACCINE, vol. 20, 2002, pages B10 - B16
GUSTAFSSON ET AL., N. ENGL. J. MED., vol. 334, 1996, pages 349 - 355
HONGYUAN MAO ET AL: "Sortase-Mediated Protein Ligation: A New Method for Protein Engineering", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 126, no. 9, 1 March 2004 (2004-03-01), pages 2670 - 2671, XP055060278, ISSN: 0002-7863, DOI: 10.1021/ja039915e *
IWARSON, APMIS, vol. 103, 1995, pages 321 - 326
JEDRZEJAS, MICROBIOL MOL BIOL REV, vol. 65, 2001, pages 187 - 207
JOHNSON ET AL., BIOORG MED CHEM LETT, vol. 9, 1999, pages 2273 - 2278
JONES, CURR OPIN INVESTIG DRUGS, vol. 4, 2003, pages 214 - 218
KRIEG, NATURE MEDICINE, vol. 9, 2003, pages 831 - 835
KURODA ET AL., LANCET, vol. 357, no. 9264, 2001, pages 1225 - 1240
LENZ ET AL., J IMMUNOL, vol. 166, 2001, pages 5346 - 5355
LU G ET AL., PROTEINS, vol. 79, no. 9, 2011, pages 2764 - 9
MAIONE D ET AL., SCIENCE, vol. 309, no. 5731, 2005, pages 148 - 50
MANZANO ET AL., STRUCTURE, vol. 16, 2008, pages 1838 - 1848
MARGARIT ET AL., JOURNAL OF INFECTIOUS DISEASES, vol. 199, 2009, pages 108 - 115
MARGARIT IMMACULADA ET AL: "Preventing bacterial infections with pilus-based vaccines: the group B streptococcus paradigm", JOURNAL OF INFECTIOUS DISEASES, UNIVERSITY OF CHICAGO PRESS, CHICAGO, IL, vol. 199, no. 1, 1 January 2009 (2009-01-01), pages 108 - 115, XP008106292, ISSN: 0022-1899, [retrieved on 20081215], DOI: 10.1086/595564 *
MCCLUSKIE ET AL., FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, vol. 32, 2002, pages 179 - 185
MCMICHAEL, VACCINE, vol. 19, no. 1, 2000, pages 101 - 107
MERALDI ET AL., VACCINE, vol. 21, 2003, pages 2485 - 2491
NEIERS ET AL., J. MOL. BIOL., vol. 393, 2009, pages 704 - 716
NEIERS F ET AL: "Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 393, no. 3, 30 October 2009 (2009-10-30), pages 704 - 716, XP026676227, ISSN: 0022-2836, [retrieved on 20090831], DOI: 10.1016/J.JMB.2009.08.058 *
NEWTON & GRAHAM: "PCR, 2nd ed.", 1997, SPRINGER VERLAG, article "Introduction to Biotechniques Series"
NIIKURA ET AL., VIROLOGY, vol. 293, 2002, pages 273 - 280
NOBBS AH ET AL., INFECT IMMUN., vol. 76, no. 8, 2008, pages 3550 - 60
NUCCITELLI A ET AL., PROC NATL ACAD SCI USA. 21, vol. 108, no. 25, 2011, pages 10278 - 83
OGUNNIYI ET AL., INFECT IMMUN, vol. 69, 2001, pages 5997 - 6003
PAJAK ET AL., VACCINE, vol. 21, 2003, pages 836 - 842
PARTHASARATHY R ET AL: "Sortase A as a Novel Molecular Stapler for Sequence-Specific Protein Conjugation", BIOCONJUGATE CHEMISTRY, ACS, WASHINGTON, DC, US, vol. 18, 16 February 2007 (2007-02-16), pages 469 - 476, XP002459581, ISSN: 1043-1802, DOI: 10.1021/BC060339W *
PARTIDOS ET AL., IMMUNOL LETT, vol. 67, 1999, pages 209 - 216
PAYNE ET AL., ADV DRUG DELIVERY REVIEW, vol. 31, 1998, pages 185 - 196
PEPPOLONI ET AL., EXPERT REV VACCINES, vol. 2, 2003, pages 285 - 293
PERSSON K., ACTA CRYSTALLOGR D BIOL CRYSTALLOGR., vol. 67, no. PT 3, 2011, pages 212 - 7
PINE ET AL., J CONTROL RELEASE, vol. 85, 2002, pages 263 - 270
PINTO ET AL., J INFECT DIS, vol. 188, 2003, pages 327 - 338
PIZZA ET AL., INT J MED MICROBIOL, vol. 290, 2000, pages 455 - 461
PIZZA ET AL., VACCINE, vol. 19, 2001, pages 2534 - 2541
PLOTKIN & MORTIMER: "Vaccines", 1988
PODDA, VACCINE, vol. 19, 2001, pages 2673 - 2680
PODDA; DEL GIUDICE, EXPERT REV VACCINES, vol. 2, 2003, pages 197 - 203
POPP ET AL., CURRENT PROTOCOLS IN PROTEIN SCIENCE, 2009, pages 15
POWELL & NEWMAN.: "Vaccine Design", 1995, PLENUM
POWELL & NEWMAN: "Vaccine Design: The Subunit and Adjuvant Approach", 1995, PLENUM PRESS
RAPPUOLI ET AL., TIBTECH, vol. 9, 1991, pages 232 - 238
REAM ET AL.,: "Molecular Biology Techniques: An Intensive Laboratory Course", 1998, ACADEMIC PRESS
RESEARCH DISCLOSURE, January 2002 (2002-01-01), pages 453077
ROBERTA COZZI ET AL: "Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity", PLOS ONE, vol. 7, no. 11, 1 January 2012 (2012-01-01), pages e49048, XP055060362, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0049048 *
ROSINI ET AL., MOLECULAR MICROBIOLOGY, vol. 61, no. 1, 2006, pages 126 - 141
ROSINI ROBERTO ET AL: "IDENTIFICATION OF NOVEL GENOMIC ISLANDS CODING FOR ANTIGENIC PILUS-LIKE STRUCTURES IN STREPTOCOCCUS AGALACTIAE", MOLECULAR MICROBIOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 61, no. 1, 1 July 2006 (2006-07-01), pages 126 - 141, XP009077023, ISSN: 0950-382X, DOI: 10.1111/J.1365-2958.2006.05225.X *
RUBIN, PEDIATR CLIN NORTH AM, vol. 47, 2000, pages 269 - 285
RYAN ET AL., INFECT IMMUN, vol. 67, 1999, pages 6270 - 6280
S. COLOWICK AND N. KAPLAN,: "Methods In Enzymology", ACADEMIC PRESS, INC.
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 3rd edition", 2001, COLD SPRING HARBOR LABORATORY PRESS
SCHARTON-KERSTEN ET AL., INFECT IMMUN, vol. 68, 2000, pages 5306 - 5313
SCHELLACK ET AL., VACCINE, vol. 24, 2006, pages 5461 - 72
SCHUCHAT, LANCET, vol. 353, no. 9146, 1999, pages 51 - 6
See also references of EP2817320A1
SENDI P ET AL., INFECTION, vol. 36, no. 2, 2008, pages 100 - 11
SINGH ET AL., J CONT RELEASE, vol. 70, 2001, pages 267 - 276
SMITH; WATERMAN: "Adv. Appl. Math.", vol. 2, 1981, pages: 482 - 489
STANLEY, CLIN EXP DERMATOL, vol. 27, 2002, pages 571 - 577
SUTTER ET AL., PEDIATR CLIN NORTH AM, vol. 47, 2000, pages 287 - 308
TELFORD ET AL., NATURE REVIEWS MICROBIOLOGY, vol. 4, 2006, pages 509 - 519
WATSON, PEDIATR INFECT DIS J, vol. 19, 2000, pages 331 - 332
ZIMMERMAN; SPANN, AM FAM PHYSICIAN, vol. 59, 1999, pages 113 - 118,125-126

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