WO2010140119A1 - COMBINATIONS OF PNEUMOCOCCAL RrgB CLADES - Google Patents

COMBINATIONS OF PNEUMOCOCCAL RrgB CLADES Download PDF

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Publication number
WO2010140119A1
WO2010140119A1 PCT/IB2010/052445 IB2010052445W WO2010140119A1 WO 2010140119 A1 WO2010140119 A1 WO 2010140119A1 IB 2010052445 W IB2010052445 W IB 2010052445W WO 2010140119 A1 WO2010140119 A1 WO 2010140119A1
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WIPO (PCT)
Prior art keywords
seq
acid sequence
ammo
ammo acid
rrgb
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PCT/IB2010/052445
Other languages
French (fr)
Inventor
Vega Masignani
Michele Anne Barocchi
Monica Moschioni
Paolo Ruggiero
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Novartis Ag
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Publication date
Priority to JP2012513718A priority Critical patent/JP6008741B2/en
Priority to SG2011088275A priority patent/SG176285A1/en
Priority to RU2011154363/10A priority patent/RU2011154363A/en
Priority to EP10723798.4A priority patent/EP2437777B1/en
Priority to BRPI1011857A priority patent/BRPI1011857A2/en
Priority to US13/375,759 priority patent/US20120076814A1/en
Priority to CN2010800310544A priority patent/CN102481354A/en
Priority to AU2010255356A priority patent/AU2010255356A1/en
Application filed by Novartis Ag filed Critical Novartis Ag
Priority to CA2764022A priority patent/CA2764022A1/en
Priority to MX2011012854A priority patent/MX2011012854A/en
Priority to ES10723798T priority patent/ES2531115T3/en
Publication of WO2010140119A1 publication Critical patent/WO2010140119A1/en
Priority to IL216696A priority patent/IL216696A0/en
Priority to ZA2011/09046A priority patent/ZA201109046B/en

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    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • RrgA is the major subumt that forms the backbone of the structure, while the other two pilms (RrgA, RrgC) are ancillary structural proteins [1-4]
  • RrgA is the major pilus- 1 adhesm, 10 bacte ⁇ a lacking RrgA are less adherent to epithelial cells than wild-type organisms
  • the present invention relates to an immunogenic composition
  • an immunogenic composition comprising at least two of
  • a second polypeptide comprising a second ammo acid sequence, where the second ammo acid sequence comp ⁇ ses an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
  • a third polypeptide comprising a third ammo acid sequence, where the third ammo acid sequence comp ⁇ ses an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3
  • the present invention also relates to a polypeptide comprising at least two of
  • a first ammo acid sequence comprising an ammo acid sequence (i) having at least 90% '5 sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 1,
  • a second ammo acid sequence comprising an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
  • a third ammo acid sequence comprising an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3
  • the present invention further relates to a polypeptide comprising ammo acid sequence
  • each X is an ammo acid sequence of first polypeptide, second polypeptide or third polypeptide as defined in claim 1
  • L is an optional linker ammo acid sequence
  • A is an optional N terminal ammo acid sequence
  • B is an optional C terminal ammo acid sequence
  • n is an integer of 2 or more
  • the polypeptide comp ⁇ ses at least two of a first, second and third polypeptide as defined in claim 1
  • polypeptides of the invention comprise an ammo acid sequence selected from the group consisting of SEQ ID NOs 11, 13, 15, 17, 19 and 21
  • the present invention also relates to a bacterium which expresses at least two of (a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 1,
  • a second polypeptide comprising a second ammo acid sequence, where the second ammo acid sequence comp ⁇ ses an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
  • a third polypeptide comprising a third ammo acid sequence, where the third ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3 BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows results of a bacteremia study with five RrgB chimeras and a control The figures are CFU/ml Each mark shows data for a single mouse
  • Figure 2 shows results of a mortality study with five RrgB chimeras and a control The figures are survival time in days
  • Figure 3 shows a gel with four lanes From left to right the lanes contain MW markers, a I-II-III chimera, a I-II-III chimera, and a BSA standard The arrow indicates a MW of 214kDa
  • Figure 4A shows passive protection data using foui mAbs raised against the TIGR4 RrgB, or a salme control
  • the y-axis shows CFU/ml for 24 hour bacteremia
  • Figure 4B shows results of a mortality study with the four mAbs raised against the TIGR4 RrgB
  • Figures 5 and 6 show western blots using mAbs raised against the TIGR4 sequence ( Figure 5) or the 6B sequence ( Figure 6) Lanes are, from left to ⁇ ght marker, RrgB I-II-III, RrgB H-I-III, RrgB H-III-I, RrgB HI-I-II, RrgB IH-II-I, RrgB TIGR4, RrgB 6B, RrgB 23F, BSA control Figure 7 shows (A) bacteremia and (B) mortality data after immunisation with alum-adjuvanted I-II-III chimera
  • Figure 8 shows OPKA results against TIGR4 strain, showing % OPKA killing against serum dilution 5 Diamonds show positive control sera, pre-immune sera are filled boxes, visible near the x-axis, the other five lines are for sera raised against the five chimeras
  • Figure 9 shows a tree based on % identity for SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID NOs 1-3 & 85-96 The label is the S
  • Figure 10 shows OPKA results against S pneumoniae serotype 6B, showing % killing against serum dilution
  • Figure 11 shows shows OPKA results against S pneumoniae serotype 6B, showing % killing against serum dilution up to a dilution of 1/131220
  • Figure 12 shows (A) bacteremia and (B) mortality data after immunisation with HI-II-I chimera at different doses
  • the data are CFU/ml and m Figure 12B the data are survival time in days
  • Figure 13 shows (A) bacteremia and (B) mortality data after immunisation with 20 ⁇ g RrgB HI-II-I 15 chimera
  • the data are CFU/ml and in Figure 13B the data are survival time in days
  • Figure 14 demonstrates that the IH-II-I RrgB chimera is protective using the MF59 adjuvant Diamonds show adjuvanted RrgB chimera, circles show MF59 alone
  • Figure 15 shows (A) bacteremia and (B) mortality data after subcutaneous immunisation with RrgB III-II-I chimera
  • the data are CFU/ml and in Figure 15B the data are survival time m 20 days
  • Figute 16 shows that RrgB III-II-I chimera elicits production of functional antibodies m a passive protection study, compared to a Normal Rabbit Serum (NRS) control, m a 24hour bacteremia assay
  • Figure 17 shows OPKA results against (A) TIGR4 and (B) ST35B, showing % killing against serum dilution Diamonds show Anti-T4, circles show RrgB III-II-I chimera and squares show NSK
  • Figure 19 shows that single RrgB domains confer protection in vivo
  • Triangles show RrgB chimera, diamonds show D 1 domain, squares show D4 domain and circles show Alum
  • Figure 20 shows western blot analysis of different RrgB domains (single domains Dl, D2, D3 and D4 >0 and multi-domain fragments Dl-3, D2-4, D3-4) tested for binding with each of four protective mAbs raised against TIGR4 RrgB
  • Figure 21 shows a western blot analysis performed with monoclonal antibody 23F8/C10 binding to trypsm-digested RrgB
  • Figure 22 is a model of RrgB domain D 1 ammo acid sequence onto the domain 1 crystal structure of S pyogenes pilus backbone SpyO128 (B) is S pneumoniae RrgB crystal structure (D2-D3) and modelled 5 Dl domain (C) is a 3D reconstruction electron density map of the S pneumoniae pilus
  • Figure 23 shows (A) 48 hour bacteremia and (B) mortality data against 6B-Finland strain (i v challenge) after i p immunisation with RrgB IH-II-I chimera when combined with different combinations of further polypeptide antigens (20 ⁇ g antigens)
  • the data are CFU/ml and in Figure 23B the data are survival time in days hi both (A) and (B)
  • column 1 shows a combination of spr0057, spr0096 and spr2021
  • column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA
  • column 3 shows RrgB III-II- I chimera
  • column 4 shows RrgB HI-II-I chimera combined with spr0057, spr0096 and spr2021
  • column 5 shows RrgB HI-II-I chimera combined with SP2216-1, SP 1732-3 and PsaA
  • Figure 24 shows (A) 48 hour bacteremia and (B) mortality data against 35B-SME15 strain (i v challenge) after i p immunisation with RrgB IH-II-I chimera when combined with different combinations of further polypeptide antigens (20 ⁇ g antigens)
  • the data are CFU/ml and in Figure 24B the data are survival time m days
  • column 1 shows a combination of spr0057, spr0096 and spr2021
  • column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA
  • column 3 shows RrgB HI-II-I chimera
  • column 4 shows RrgB HI-II-I chimera combined with spr0057, spr0096 and spr2021
  • column 5 shows RrgB III-II-I chimera combined with SP2216-1, SP 1732-3 and PsaA
  • column 6 shows an a
  • Figure 25 shows (A) a 24 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i p challenge with TIGR4 2 1E+02 CFU/mouse)
  • the data m (A) are CFU/ml and in (B) are survival time in days
  • Figure 26 shows a 24 hour bacteremia assay m BALB/c mice using a III-II-I chimera that contains a polyhistidme tag compared to (i) a tag-less III-II-I chimera, (n) a combination of spi0057, sprO096 and spr2021, (in) the combination of spr0057, s ⁇ rOO96 and spr2021 further combined with the tag-less III-II-I chimera, and (iv) an alum control (i p immunisation, i p challenge with TIGR4 1 6E+02 ) CFU/mouse)
  • Figure 27 shows (A) a 48 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i v challenge with 35B- SME 15 4 6E+07 CFU/mouse)
  • the data in (A) are CFU/ml and in (B) are survival time in days
  • Figure 28 shows (A) a 48 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i v challenge with 6BFmlandl2 9 4E+07 CFU/mouse)
  • the data in (A) are CFU/ml and in (B) are survival time m days
  • Figure 29 shows (A) a 48 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i v challenge with TIGR4 6 3E+05 CFU/mouse)
  • the data in (A) are CFU/ml and in (B) are survival time in days
  • Figure 30 shows (A) a 48 hour bacteremia assay and (B) mortality data after immunisation with 20 ⁇ g III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with TIGR4)
  • the data m (A) are CFU/ml and m (B) are survival time m days
  • Figure 31 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 20 ⁇ g III-II-I chimera, compared to an alum control (i p immunisation, i p challenge with TIGR4)
  • the data in (A) are CFU/ml and in (B) are survival time in days
  • Figure 32 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 20 ⁇ g III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with 35B-SME15)
  • the data in (A) are CFU/ml and in (B) are survival time m days
  • Figure 33 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 20 ⁇ g III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with 6B Finland 12)
  • the data m (A) are CFU/ml and in (B) are survival time m days
  • Figure 34 shows (A) a 48 hour bacteremia assay and (B) mortality data after immunisation with III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with TIGR4) when challenged with a TIGR4 strain overexpressmg pilus (T4+) compared to a TIGR4 tram expressing very low amounts of pilus (T4-)
  • the data m (A) are CFU/ml and in (B) are survival time in days
  • Figure 35 shows 48 hour bacteremia assays after immunisation with H-I-III and III-II-I chimeras (A) when challenged with a 6BFmI 12 strain overexpressmg pilus (i p immunisation, i v challenge with 6BFmlandl2 overexpressmg pilus 7 OE+09 CFU/mouse) and (B) when challenged with a 6BFmI 12 tram expressing only very low amounts of pilus (i p immunisation, i v challenge with 6BFmlandl2 underexpressing pilus 7 3E+09 CFU/mouse) Both (A) and (B) also show data for a combination of spr0057, spr0096 and spr2021, a 6BFinland-CRM197 conjugate, and alum The data m (A) are CFU/ml and m (B) are survival time in days
  • Figure 36 is an in silico analysis of the MLST database showing that, for
  • the RrgB pilus subumt has at least three clades Reference amino acid sequences for the three clades are SEQ ID NOs 1 , 2 and 3 herein
  • the clades are well conserved at their N- and C-termim but deviate in between SEQ ID NOs 1 and 2 are 46% identical, SEQ ID NOs 1 and 3 are 51% identical, SEQ ID NOs 2 and 3 are 65% identical
  • an immunogenic composition includes at least two different clades of RrgB These may be present m the immunogenic composition as separate polypeptides or may be fused as a single polypeptide chain
  • the inclusion of multiple RrgB clades as vaccine components improves the strain
  • an immunogenic composition comprising at least two of
  • a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence comp ⁇ ses an ammo acid sequence (i) having at least a% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least x contiguous ammo acids from SEQ ID NO 1
  • a second polypeptide comprising a second ammo acid sequence, where the second ammo acid sequence comprises an ammo acid sequence (i) having at least b% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least y contiguous ammo acids from SEQ ID NO 2, and/or
  • a third polypeptide comprising a third ammo acid sequence, where the third ammo acid sequence comprises an ammo acid sequence (i) having at least c% sequence identity to SEQ ID NO 3 and/or (ii) consisting of a fragment of at least z contiguous ammo acids from SEQ ID NO 3
  • the invention also provides a polypeptide comprising at least two of (a) a first ammo acid sequence comprising an ammo acid sequence (i) having at least a% sequence identity to SEQ ID NO 1 and/or (n) consisting of a fragment of at least x contiguous ammo acids from SEQ ID NO 1,
  • a second ammo acid sequence comprising an amino acid sequence (i) having at least b% sequence identity to SEQ ID NO 2 and/or (n) consisting of a fragment of at least y contiguous ammo acids from SEQ ID NO 2, and/or
  • a third ammo acid sequence comprising an ammo acid sequence (i) having at least c% sequence identity to SEQ ID NO 3 and/or (ii) consisting of a fragment of at least z contiguous ammo acids from SEQ ID NO 3
  • the invention also provides a polypeptide comprising ammo acid sequence
  • X is an ammo acid sequence of first polypeptide, second polypeptide or third polypeptide as defined above, L is an optional linker ammo acid sequence, A is an optional N-termmal ammo acid sequence, B is an optional C-terminal ammo acid sequence, n is an integer of 2 or more (e g 2, 3, 4, 5, 6, etc )
  • the polypeptide comp ⁇ ses at least two of a first, second and third polypeptide as defined m claim 1 Usually n is 2 or 3, and X moieties are selected from the following
  • the invention also provides a cell (typically a bacterium, such as a pneumococcus) which expresses at least two of
  • a first polypeptide comprising a first ammo acid sequence where the first ammo acid sequence compnses an ammo acid sequence (i) having at least a% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least x contiguous ammo acids from SEQ ID NO 1 ,
  • a second polypeptide composing a second ammo acid sequence, where the second ammo acid sequence comp ⁇ ses an ammo acid sequence (i) having at least b% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least y contiguous ammo acids from SEQ ID NO 2, and/or (c) a third polypeptide, comprising a third ammo acid sequence, where the third ammo acid sequence comp ⁇ ses an ammo acid sequence (i) having at least c% sequence identity to SEQ ID NO 3 and/or ( ⁇ ) consisting of a fragment of at least z contiguous ammo acids from SEQ ID NO 3
  • the first, second and third amino acid sequences The value of a is at least 15 eg 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more.
  • the value of b is at least 75 eg 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more
  • the value of c is at least 75 e g 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more
  • the values of a, b and c may be the same or different In some embodiments, a b and c are identical Typically, a, b and c axe at least 90 e g at least 95
  • the value ofx is at least 7 eg 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250)
  • the value of/ is at least 7 eg 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,
  • the value of z is at least l e g 8, 9, 10,
  • x, y and z may be the same or different In some embodiments, x y and z are identical
  • Fragments preferably comp ⁇ se an epitope from the respective SEQ ID NO sequence
  • Other useful fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of the respective SEQ ID NO while retaining at least one epitope thereof Truncation by 20-25 ammo acids at the N-termmus is convenient e g removal of aa 1 -23 of any of SEQ ID NOs 1 to 3 (or of any one of SEQ ID NOs 85 to 96)
  • the RrgB protein can be split into four domains (Dl to D4) between its leader peptide and its LPXTG anchor These four domains are as follows in SEQ ID NOs 1 to 3, and the positions m further RrgB sequences which correspond to these residues can readily be identified by alignment
  • useful fragments of RrgB may retain epitopes from at least domains Dl and/or D4 As shown in Figure 20, antibodies have been raised that bind to domain Dl, domain D4 and a fragment containing domains D2 to D4 Accordingly, preferred fragments include domain Dl, domain D4 and a fragment containing domains D2 to D4
  • a suitable fragment of SEQ ID NO 1 is SEQ ID NO 4
  • a suitable fragment of SEQ ID NO 2 is SEQ ID NO 5
  • a suitable fragment of SEQ ID NO 3 is SEQ ID NO 6
  • a fragment of SEQ ID NO 1 is preferably from between ammo acids 31 -614 of SEQ ID NO 1
  • a fragment of SEQ ID NO 2 is preferably from between ammo acids 31-593 of SEQ ID NO 2
  • a fragment of SEQ ID NO 3 is preferably from between ammo acids 31-603 of SEQ ID NO 3
  • the fragment of at least x contiguous ammo acids from SEQ ID NO 1 may also be present m any one of SEQ ID NOs 85, 88
  • a polypeptide comprising the first ammo acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 1 (strain TIGR4) In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 2 or to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 3
  • a polypeptide comprising the second ammo acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 2 (strain Finland 68 - 12) In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 1 or to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 3
  • a polypeptide comprising the third ammo acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild- type pneumococcus protein having ammo acid sequence SEQ ID NO 3 (strain Taiwan 23F -15) In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 1 or to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 2
  • first, second and third ammo acid sequences may share some sequences m common, overall they have different ammo acid sequences
  • composition or polypeptide can include both (a) a first ammo acid sequence as defined above, and (b) a second ammo acid sequence as defined above
  • composition includes both (a) a first ammo acid sequence as defined above, and (b) a third ammo acid sequence as defined above
  • composition includes both (a) a second ammo acid sequence as defined above, and (b) a third ammo acid sequence as defined above
  • Ammo acid sequences used with the invention may, compared to SEQ ID NOs 1, 2 or 3, include one or more (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc ) conservative ammo acid replacements i e replacements of one ammo acid with another which has a i elated side chain
  • Genetically-encoded ammo acids are generally divided into four families (1) acidic i e aspartate, glutamate, (2) basic i e lysine, argmine, histidine, (3) non-polar i e alanine, valine, leucine, isoleucme, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar i e glycine, asparagme, glutamine, cysteine, serine, threonine, tyrosine Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic ammo acids In general, substitution of single
  • (c) has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more) single ammo acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b), and
  • each moving window of x ammo acids from N-termmus to C-termmus (such that for an alignment that extends to p ammo acids, where p>x, there are p-x+1 such windows) has at least xy identical aligned ammo acids, where x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, y is selected from 0 50, 0 60, 0 70. 0 75.
  • deletions or substitutions may be at the N-termmus and/or C-termmus, or may be between the two termini
  • a truncation is an example of a deletion Truncations may involve deletion of up to 40 (or more) ammo acids at the N-termmus and/or C-terrmnus
  • a polypeptide of the invention comprises a sequence that is not identical to a complete pneumococcal sequence from SEQ ID NOs 1 to 3 (e g when it comprises a sequence listing with ⁇ 100% sequence identity thereto, or when it compnses a fragment thereof), it is preferred m each individual instance that the polypeptide can elicit an antibody that recognises the complete pneumococcal sequence
  • SEQ ID NOs 1 to 3 and 85 to 96 are 15 unique RrgB sequences which have been identified in 45 different strains Any of these sequences can be used for implementing the invention
  • a first polypeptide for use with the invention could comprise any one of SEQ ID NOs listed m group (1) below
  • a second polypeptide for use with the invention could comprise any one of SEQ ID NOs listed in group (2) below
  • a third polypeptide for use with the invention could comprise any one of SEQ ID NOs listed in group (3) below
  • Groups (1) to (3) are as follows
  • Hyb ⁇ d polypeptides offer two mam advantages first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable hyb ⁇ d partner that ovei comes the problem, second, commercial manufacture is simplified as only one expression and purification need to be employed in order to produce two polypeptides which are both antigenically useful
  • Hybrid polypeptides can include sequences from only RrgB antigens but in other embodiments can include non-RrgB antigens (usually pneumococcal non-RrgB antigens), such as other pilus subumts If non-RrgB antigens are present these may be to the N-termmus of any two RrgB sequences, to the C-termmus of any two RrgB sequences, or may be between two RrgB sequences
  • Hyb ⁇ ds may be mixed together m a single formulation Hyb ⁇ ds may be combined with non-hybrid RrgB antigens or other non-RrgB antigens
  • Hybrid polypeptides may be represented by the formula NH 2 -A- ⁇ -X-L- ⁇ ,,-B-COOH
  • a -X- moiety has a leader peptide sequence m its wild-type form, this may be included or omitted m the hyb ⁇ d protein Pn some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-termmus of the hybrid protein i e the leader peptide of Xi will be retained, but the leader peptides of X 2 X n will be omitted This is equivalent to deleting all leader peptides and0 using the leader peptide of X] as moiety -A-
  • linker ammo acid sequence -L- may be present or absent
  • the hyb ⁇ d may be NH 2 -Xi-L 1 -X 2 -L 2 -COOH, NH 2 -XrX 2 -COOH, NH 2 -X 1 -Li-X 2 -COOH, NH 2 -Xi-X 2 -L 2 -COOH, etc
  • -B- is an optional C-termmal ammo acid sequence
  • This will typically be short (e g 40 or fewer ammo acids i e 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1)
  • Other suitable C-termmal ammo acid sequences will be apparent to those skilled in the art, such as a glutathione-S-transferase, thioredoxm, 14kDa fragment of S aureus protein A, a biotmylated peptide, a0 maltose-bmdmg protein, an enterokmas
  • -A-, -B- and -L- sequences do not include a sequence that shares 10 or more contiguous amino acids in common with a human polypeptide sequence
  • a -L- moiety comprises a non-RrgB antigen
  • the -A- moiety compnses a non-RrgB antigen
  • the -B- moiety comprises a non-RrgB antigen 5
  • the invention also provides nucleic acid which encodes a hyb ⁇ d polypeptide of the invention
  • useful combinations include, but are not limited to
  • hyb ⁇ ds of the invention include polypeptides comprising an ammo acid sequence selected from the group consisting of SEQ ID NO 11 (encoded by SEQ ID NO 12), SEQ ID NO 13O (encoded by SEQ ID NO 14), SEQ ID NO 15 (encoded by SEQ ID NO 16), SEQ ID NO 17 (encoded by SEQ ID NO 18), SEQ ID NO 19 (encoded by SEQ ID NO 20), SEQ ID NO 21 (encoded by SEQ ID NO 22)
  • the invention provides a polypeptide comprising an ammo acid sequence having at least ⁇ % sequence identity to any one of SEQ ID NOs 11 , 13, 15, 17, 19 or 21
  • the value of i may be selected from 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99 or more Polypeptides
  • Polypeptides used with the invention can be prepared in many ways e g by chemical synthesis (in whole or in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e g from recombinant expression), from the organism itself (e g after bacte ⁇ al 5 culture, or direct from patients), etc
  • a preferred method for production of peptides ⁇ 40 ammo acids long involves in vitro chemical synthesis [7,8] Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [9] chemistry
  • Enzymatic synthesis [10] may also be used in part or m full
  • biological synthesis may be used e g the polypeptides may be produced by translation This may be earned out in vitro or in vivo
  • Biological methods are in general 10 restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e g of ammoacyl
  • Polypeptides can take various forms (e g native, fusions, glycosylated, non-glycosylated, lipi dated, non-hpidated, phosphorylated, non-phosphorylated, myristoylated, non-my ⁇ stoylated, monome ⁇ c, multime ⁇ c, particulate, denatured, etc )
  • Polypeptides are preferably provided in purified or substantially purified form i e substantially free from other polypeptides (e g free from naturally-occurring polypeptides), particularly from other 0 pneumococcal or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i e less than about 50%, and more preferably less than about 10% (e g 5% or less) of a composition is made up of other expressed polypeptides
  • Polypeptides may be attached to a solid support
  • Polypeptides may comp ⁇ se a detectable label (e g a radioactive or fluorescent label, or a biotin label)
  • polypeptide refers to ammo acid polymers of any length
  • the polymer may be linear or branched, it may comp ⁇ se modified ammo acids, and it may be interrupted by non-ammo acids
  • the terms also encompass an ammo acid polymer that has been modified naturally or by intervention, for example, disulfide bond formation, glycosylation, hpidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component Also included withm the
  • ⁇ 0 definition are, for example, polypeptides containing one or more analogs of an ammo acid (including, for example, unnatural ammo acids, etc ), as well as other modifications known in the art Polypeptides can occur as single chains or associated chains Polypeptides can be naturally or non-naturally glycosylated ( ⁇ e the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found m the co ⁇ espondmg naturally occurring polypeptide)
  • the invention provides a process for producing polypeptides of the invention, comprising cultunng a host cell of to the invention under conditions which induce polypeptide expression Although expression of the polypeptide may take place in a Streptococcus, the invention will usually use a heterologous host for expression
  • the heterologous host may be prokaryotic (e g & bacte ⁇ um) or eukaryotic It will usually 5 be E coli, but other suitable hosts include Bacillus subtihs, Vibri
  • the invention also provides a process for producing a polypeptide of the invention, wherein the polypeptide is synthesised m part or in whole using chemical means
  • the invention also provides a composition comprising two or more polypeptides of the invention
  • the invention also provides a nucleic acid comprising a nucleotide sequence encoding a hyb ⁇ d polypeptide of the invention
  • the invention also provides nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences
  • Such nucleic acids include those using 15 alternative codons to encode the same ammo acid
  • the invention also provides nucleic acid which can hybridize to these nucleic acids Hybridization reactions can be performed under conditions of different 'stringency" Conditions that increase stringency of a hybridization ieaction of widely known and published in the art Examples of relevant conditions include (m order of increasing stringency) incubation temperatures of 25°C, 37 0 C, 5O 0 C,
  • the invention includes nucleic acid comprising sequences complementary to these sequences (e g for antisense or probing, or for use as primers)
  • Nucleic acid according to the invention can take various forms (e g smgle-stranded, double-stranded, vectors, primers, probes, labelled etc ) Nucleic acids of the invention may be circular or branched, but >0 will generally be linear Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary smgle- stranded forms which make up the double- stranded form Primers and probes are generally smgle- stranded, as are antisense nucleic acids Nucleic acids of the invention are preferably provided in purified or substantially purified form i e substantially free from other nucleic acids (e g free from naturally-occumng nucleic acids), particularly from other pneumococcal or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90% pure Nucleic acids of the invention are preferably pneumococcal nucleic 5 acids
  • Nucleic acids of the invention may be prepared in many ways e g by chemical synthesis ⁇ e g phosphoramidite synthesis of DNA) m whole or in part, by digesting longer nucleic acids using nucleases ⁇ e g rest ⁇ ction enzymes), by joining shorter nucleic acids or nucleotides ⁇ e g using ligases or polymerases), from genomic or cDNA libraries, etc 0
  • Nucleic acid of the invention may be attached to a solid support (e g a bead, plate, filter, film, slide, microarray support, resin, etc )
  • Nucleic acid of the invention may be labelled e g with a radioactive or fluorescent label, or a biotin label This is particularly useful where the nucleic acid is to be used in detection techniques e g where the nucleic acid is a p ⁇ mer or as a probe
  • nucleic acid includes in general means a polyme ⁇ c form of nucleotides of any length, which 5 contain deoxy ⁇ bonucleotides, ribonucleotides, and/or their analogs It includes DNA, RNA, DNA/RNA hybrids It also includes DNA or RNA analogs, such as those containing modified backbones ⁇ e g peptide nucleic acids (PNAs) or phosphorothioates) or modified bases
  • PNAs modified backbones ⁇ e g peptide nucleic acids
  • the invention includes mRNA, tRNA, rRNA, ⁇ bozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc Where nucleic acid of the invention takes the form of RNA, it
  • .0 may or may not have a 5' cap
  • Nucleic acids of the invention may be part of a vector i e part of a nucleic acid construct designed for transduction/transfection of one or more cell types
  • Vectors may be, for example, "cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors” which are designed for expression of a nucleotide sequence m a host cell, "viral vectors" which are designed for expression of a nucleotide sequence m a host cell.
  • '5 is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the att ⁇ butes of more than one type of vector Preferred vectors are plasmids
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural,
  • Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention
  • a nucleic acid is DNA
  • U in a RNA sequence
  • T in the DNA
  • RNA RNA
  • T m a DNA sequence will be replaced by "U” in the RNA
  • complement or “complementary” when used in relation to nucleic acids refers to Watson- 5 Crick base pai ⁇ ng
  • the complement of C is G
  • the complement of G is C
  • the complement of A is T (or U)
  • the complement of T is A
  • bases such as I (the purine mosme) e g to complement pyrimidmes (C or T)
  • Nucleic acids of the invention can be used, for example to produce polypeptides in vitro or in vivo, as hybridization probes for the detection of nucleic acid in biological samples, to generate additional copies0 of the nucleic acids, to generate ribozymes or antisense oligonucleotides, as single- stranded DNA primers or probes, or as triple-strand forming oligonucleotides
  • the invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesised in part or in whole using chemical means
  • the invention provides vectors comprising nucleotide sequences of the invention ⁇ e g cloning or 5 expression vectors) and host cells transformed with such vectors
  • immunogenic compositions may be useful as vaccines
  • vaccines may either be piophylactic ( ⁇ e to prevent infection) or therapeutic ( ⁇ e to treat infection), but will typically beO prophylactic
  • 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 car ⁇ er(s) and/or excipient(s) A thorough discussion of such components is available m reference 234
  • compositions will generally be administered to a mammal in aqueous form P ⁇ or to administration, 5 however, the composition may have been m a non-aqueous form
  • 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, d however, that the vaccine should be substantially free from ( ⁇ 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 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
  • NaCl sodium chloride
  • Other salts that may be present m clude potassium chloride, potassium dihydrogen phosphate, disodmm phosphate dehydrate, magnesium chlo ⁇ de, calcium chlo ⁇ de, 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
  • compositions may mclude one or more buffers Typical buffers include a phosphate buffer, a T ⁇ s buffer, a borate buffer, a succinate buffer, a histidme buffer (particularly with an aluminum hydroxide adjuvant), or a citrate buffei Buffers will typically be included in the 5-2OmM 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-pyrogemc 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 mclude mate ⁇ al for a single immunisation, or may include material for multiple immunisations ( ⁇ e a 'multidose' kit)
  • the inclusion of a preservative is prefe ⁇ ed in multidose arrangements
  • the compositions may be contained m a container having an aseptic adaptor for removal of mate ⁇ al
  • Human vaccines are typically administered in a dosage volume of about 0 5ml, although a half dose ( ⁇ 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 mclude one or more adjuvants, for example two, three, four or more adjuvants
  • the adjuvants may mclude a THl adjuvant and/or a TH2 adjuvant, further discussed below
  • Adjuvants which may be used m compositions of the invention mclude, but are not limited to
  • Mineral containing compositions suitable for use as adjuvants in the invention mclude mineral salts, such as aluminium salts and calcium salts
  • the invention includes mineral salts such as hydroxides ⁇ e g oxyhydroxides), phosphates (e g hydroxyphosphates, orthophosphates), sulphates, etc [e g see chapters ) 8 & 9 of ref 13], or mixtures of different mineral compounds, with the compounds taking any suitable form (e g gel, crystalline, amorphous, etc ), and with adsorption being preferred
  • the mineral containing compositions may also be formulated as a particle of metal salt
  • the adjuvants known as "aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline Aluminium oxyhydroxide, which can be represented by the formula AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH) 3 , by infrared (IR) spectroscopy, m
  • the adjuvants known as "aluminium phosphate” are typically aluminium hydroxyphosphates, often also 5 containing a small amount of sulfate ( ⁇ e aluminium hydroxyphosphate sulfate) They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt Hydroxyphosphates generally have a PO 4 / Al molar ratio between 0 3 and 1 2 Hydroxyphosphates can be distinguished from strict AlPO 4 by the presence of hydroxyl groups For example, an IR spectrum band at 3164cm ' (e g when heated to 200 0 C) indicates
  • the PO 4 ZAl 3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0 3 and 1 2, preferably between 0 8 and 1 2, and more preferably 0 95+0 1
  • the aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts
  • a typical adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /AI molar ratio between 0 84 and 0 92, included at 0 6mg Al 3+ AnI
  • the '5 aluminium phosphate will generally be particulate (e g plate-like morphology as seen m transmission electron micrographs) Typical diameters of the particles are in the range 0 5-20 ⁇ m (e g about 5-10 ⁇ m) after any antigen adsorption
  • Adsorptive capacities of between 0 7-1 5 mg protein per mg Al +++ at pH 7 4 have been reported for aluminium phosphate adjuvants
  • Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e g a phosphate or a histidme or a T ⁇ s buffer), but this is not always necessary
  • the suspensions are 5 preferably ste ⁇ le and pyrogen- free
  • a suspension may include free aqueous phosphate ions e g present at a concentration between 1 0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM
  • the suspensions may also comp ⁇ se sodium chlo ⁇ de
  • an adjuvant component includes a mixture of both an aluminium hydroxide and an aluminium phosphate
  • the concentration of Al +++ m a composition for administration to a patient is pieferably less than 10mg/ml e g ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc
  • a preferred range is between 0 3 and lmg/ml
  • a maximum of ⁇ 0 85mg/dose is preferred
  • Oil emulsion compositions suitable for use as adjuvants m the invention include squalene- water emulsions, such as MF59 [Chapter 10 of ref 13, see also ref 14] (5% Squalene, 0 5% Tween 80, and 0 5% Span 85, formulated into submicron particles using a microfluidizer) Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used
  • oin-in- water emulsions are know n, and they typically include at least one oil and at least0 one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabohsable) and biocompatible
  • the oil droplets in the emulsion are generally less than 5 ⁇ m in diameter, and advantageously the emulsion comprises oil droplets with a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization
  • the invention can be used with oils such as those from an animal (such as fish) or vegetable source
  • oils such as those from an animal (such as fish) or vegetable source
  • Sources for vegetable oils include nuts, seeds and grams Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils
  • Jojoba oil can be used e g obtained from the jojoba bean Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like In the gram group, corn oil is the most readily available, but the oil of other cereal grams such as
  • 0 wheat, oats, rye, rice, teff, t ⁇ ticale and the like may also be used 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, while not occurring naturally m seed oils, may be prepaied by hydrolysis, separation and este ⁇ f ⁇ cation of the appropriate materials starting from the nut and seed oils
  • Fats and oils from mammalian milk are metabohzable and may therefore be used in the practice of this invention
  • the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from ammal sources are well known in the art
  • Most fish contain metabohzable oils which may be readily recovered
  • cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein
  • a number of branched chain oils are synthesized biochemically in 5 -carbon isoprene units and are generally referred to as terpenoids
  • Surfactants can be classified by their 'HLB' (hydrophile/hpophile balance) Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16
  • the invention can be used with surfactants including, but not limited to the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80, copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWF AXTM tradename, such as linear EO/PO block copolymers, octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypolyethoxyethanol) being of particular interest, (octylphenoxy)poly
  • surfactants can be used e g Tween 80/Span 85 mixtures
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable
  • Another useful combination comp ⁇ ses laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol
  • Preferred amounts of surfactants are polyoxyethylene sorbitan esters (such as Tween 80) 0 01 to 1%, m particular about 0 1 %, octyl- or nonylphenoxy polyoxyethanols (such as T ⁇ ton X-100, or other detergents in the T ⁇ ton series) 0 001 to 0 1 %, in particular 0 005 to 0 02%, polyoxyethylene ethers (such as laureth 9) 0 1 to 20 %, preferably 0 1 to 10 % and m particular 0 1 to 1 % or about 0 5%
  • oil-m-water emulsion adjuvants useful with the invention include, but are not limited to
  • a submicron emulsion of squalene, Tween 80, and Span 85 The composition of the emulsion by volume can be about 5% squalene, about 0 5% polysorbate 80 and about 0 5% Span 85 In weight terms, these ratios become 4 3% squalene, 0 5% polysorbate 80 and 0 48% Span 85
  • This adjuvant is known as 'MF59' [15-17], as desc ⁇ bed m more detail m Chapter 10 of ref 18 and chapter 12 of ref 19
  • the MF59 emulsion advantageously includes citrate ions e g 1OmM sodium citrate buffer
  • An emulsion comprising squalene, an ⁇ -tocopherol, and polysorbate 80
  • emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0 3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably ⁇ 1 (e g 0 90) as this provides a more stable emulsion
  • Squalene and Tween 80 may be present volume ratio of about 5 2, or at a weight ratio of about 1 1 5
  • One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL- ⁇ -tocopherol and 5ml squalene), then micro fluidismg the mixture
  • the resulting emulsion may have submicron oil droplets e g with an average diameter of between 100 and 250nm, preferably about 180nm
  • An emulsion of squalene, a tocopherol, and a Triton detergent e g Tnton X-100
  • the emulsion may also include a 3d-MPL (see below)
  • the emulsion may contain a phosphate buffer
  • An emulsion comprising a polysorbate (e g polysorbate 80), a Tnton detergent (e g Tnton X-100) and a tocopherol (e g an ⁇ -tocopherol succinate)
  • the emulsion may include these three components at a mass ratio of about 75 11 10 (e g 750 ⁇ g/ml polysorbate 80, l lO ⁇ g/ml Triton X- 100 and lOO ⁇ g/ml ⁇ -tocopherol succinate), and these concentrations should include any contribution of these components from antigens
  • the emulsion may also include squalene
  • the emulsion may also include a 3d-MPL (see below)
  • the aqueous phase may contain a phosphate buffer
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 (“PluromcTM L121")
  • the emulsion can be formulated in phosphate buffered saline, pH 7 4
  • This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-I" adjuvant [20] (0 05-1% Thr-MDP, 5% squalane, 2 5% Pluromc Ll 21 and 0 2% polysorbate 80) It can also be used without the Thr-MDP, as in the "AF” adjuvant [21] (5% squalane, 1 25% Pluromc L121 and
  • An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophihc nonionic surfactant (e g polyoxyethylene (12) cetostearyl ether) and a hydrophobic noniomc surfactant (e g a sorbitan ester or manmde ester, such as sorbitan monoleate or 'Span 80')
  • the emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [22]
  • the emulsion may also include one or more of alditol, a cryoprotective agent (e g a sugar, such as dodecylmaltoside and/or sucrose), and/or an alkylpolyglycoside
  • Such emulsions may be lyophihzed • An emulsion having from 0 5-50% of an oil, 0 1-10% of
  • Additives may be included, such as QuilA saponin, cholesterol, a saponm-lipophile conjugate (such as GPI-0100, desc ⁇ bed m reference 24, produced by addition of aliphatic amine to desacylsapomn via the carboxyl group of glucuronic acid), dimethyidioctadecylammomum bromide and/or N,N-dioctadecyl-N,N-bis (2- hydroxyethyl)pro ⁇ anediamme ⁇
  • An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-iomc hydrophilic surfactant (e g an ethoxylated fatty alcohol and
  • Antigens and adjuvants m a composition will typically be in admixture at the time of delivery to a patient
  • the emulsions may be mixed with antigen during manufacture, or extemporaneously, at the time of delivery
  • the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use
  • the antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids
  • the volume ratio of the two liquids for mixing can vary (e g between 5 1 and 1 5) but is generally about 1 1
  • Saponin formulations may also be used as adjuvants m the invention
  • Saponins are a heterogeneous group of sterol glycosides and t ⁇ terpenoid glycosides that are found m the bark, leaves, stems, roots and even flowers of a wide range of plant species Saponin from the bark of the Qu ⁇ laia saponana Molma tree have been widely studied as adjuvants Saponin can also be commercially obtained from Smilax ornata (sarsap ⁇ lla), Gypsophilla panicnlata (brides veil), and Saponana officianalis (soap root)
  • Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs QS21 is marketed as StimulonTM Saponin compositions have been purified using HPLC and RP-HPLC Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, Q
  • Virosomes and virus-like particles can also be used as adjuvants in the invention These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid They are generally non-pathogenic, non-replicatmg and generally do not contain any of the native viral genome
  • the viral proteins may be recombmantly produced or isolated from whole viruses
  • These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Smdbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q ⁇ -phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi) VLPs are discussed further m refs 34-39 Virosome
  • Adjuvants suitable for use m the invention include bacte ⁇ al or microbial derivatives such as non-toxic de ⁇ vatives of enterobacterial lipopolysaccha ⁇ de (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP- ⁇ bosylatmg toxins and detoxified de ⁇ vatives thereof
  • Non-toxic de ⁇ vatives of LPS include monophosphoryl lipid A (MPL) and 3-0-deacylated MPL (3dMPL) 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains A preferred "small particle" fo ⁇ n of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref 41 Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0 22 ⁇ m membrane [41]
  • Other non-toxic LPS de ⁇ vatives include monophosphoryl lipid A mimics, such as ammoalkyl glucosamimde phosphate de ⁇ vatives e g RC-529 [42,43]
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM- 174 OM- 174 is described for example m refs 44 & 45
  • Immunostimulatory oligonucleotides suitable for use as adjuvants m the invention include nucleotide sequences containing a CpG motif (a dmucleotide sequence containing an unmethylated cytosme linked by a phosphate bond to a guanosme) Double- stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory
  • the CpG' s can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single- stranded
  • References 46, 47 and 48 disclose possible analog substitutions e g replacement of guanosme with 2'-deoxy-7-deazaguanosme The adjuvant effect of CpG oligonucleotides is further discussed in refs 49-54
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [55]
  • the CpG0 sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN CpG-A and CpG-B ODNs are discussed in refs 56-58
  • the CpG is a CpG-A ODN
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form5 "mimunomers" See, for example, refs 55 & 59-61
  • an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e g between 15-40 nucleotides) including at least one (and preferably multiple) CpI motifs (z e a cytosme linked to an inosine to form a dmucleotide), and ( ⁇ ) a polycationic polymer, such as an oligopeptide (e g0 between 5-20 ammo acids) including at least one (and preferably multiple) Lys-Arg-Lys t ⁇ peptide sequence(s)
  • the oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5'-(IC)i 3 -3' (SEQ ID NO 80)
  • the polycationic polymer may be a peptide comprising 11-mer ammo acid sequence KLKLLLLLKLK (SEQ ID NO 80)
  • Bacterial ADP- ⁇ bosylatmg toxins and detoxified de ⁇ vatives thereof may be used as adjuvants m the
  • the protein is de ⁇ ved from E cob (E coli heat labile enterotoxin "LT”), cholera
  • toxin or toxoid is preferably in the form of a holotoxm, comprising both A and B subunits
  • the A subunit contains a detoxifying mutation, preferably the B subunit is not mutated
  • the adjuvant is a detoxified LT mutant
  • ADP- ⁇ bosylatmg toxms and detoxified de ⁇ vatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found m refs 65-72
  • a useful CT mutant is or CT-E29H [73]
  • Nume ⁇ cal reference for ammo acid substitutions is preferably based on the alignments of the A and B subumts of ADP- ⁇ bosylatmg toxins set forth in ref 74, specifically incorporated herein by reference m its entirety
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as mterleukms (e g IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL- 12 [75], etc ) [76], interferons (e g mterferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor
  • cytokines such as mterleukms (e g IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL- 12 [75], etc ) [76], interferons (e g mterferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor
  • cytokines such as mterleukms (e g IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL- 12 [75], etc ) [76], interferons (e g
  • Bioadhesives and mucoadhesives may also be used as adjuvants in the invention Suitable bioadhesives include este ⁇ fied hyaluronic acid microspheres [77] or mucoadhesives such as cross-linked de ⁇ vatives of poly(acryhc acid), polyvinyl alcohol, polyvinyl pyrolhdone, polysaccharides and carboxymethylcellulose Chitosan and de ⁇ vatives thereof may also be used as adjuvants in the invention [78]
  • Microparticles may also be used as adjuvants m the invention
  • Microparticles ( ⁇ e a particle of -lOOnm to ⁇ 150 ⁇ m m diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500nm to ⁇ 10 ⁇ m m diameter) formed from materials that are biodegradable and non-toxic (e g a poly( ⁇ -hydroxy acid), a polyhydroxybuty ⁇ c acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc ), with poly(lactide-co-glycohde) are preferred, optionally treated to have a negatively-charged surface (e g with SDS) or a positively-charged surface (e g with a catiomc detergent, such as CTAB)
  • a negatively-charged surface e g with SDS
  • a positively-charged surface e g with a catiomc detergent, such as CTAB
  • liposome formulations suitable for use as adjuvants are described m refs 79-81
  • Adjuvants suitable for use m the invention include polyoxyethylene ethers and polyoxyethylene esters [82] Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [83] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-iomc surfactant such as an octoxynol [84]
  • Preferred polyoxyethylene ethers are selected from the following group polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, ⁇ olyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether
  • PCPP Polyphosyhazene
  • PCPP formulations are desc ⁇ bed, for example, m refs 85 and 86 L Muramyl peptides
  • muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl- L-threonyl-D-isoglutamme (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamme (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutammyl-L-alanme-2-(r-2'-dipalmitoyl-5 «-glycero-3- hydroxyphosphoryloxy)-ethylamme MTP-PE)
  • 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-m-watei emulsion [89], (2) a saponin (e g QS21) + a non-toxic LPS derivative (e g 3dMPL)
  • Immunochem containing 2% squalene, 0 2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), tiehalose 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)
  • an aluminium hydroxide and/or aluminium phosphate adjuvant is useful, particularly m children, and antigens are generally adsorbed to these salts Squalene-m-water emulsions are also preferred, particularly m the elderly
  • Useful adjuvant combinations include combinations of ThI and Th2 adjuvants such as CpG & alum or resiquimod & alum
  • ThI and Th2 adjuvants such as CpG & alum or resiquimod & alum
  • a combination of aluminium phosphate and 3dMPL may be used
  • compositions of the invention may elicit both a cell mediated immune response as well as a humoral immune response
  • CD8 T cells can express a CD8 co-receptor and aie commonly referred to as Cytotoxic T lymphocytes (CTLs) CD8 T cells are able to lecogmzed 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 phenotypes which differ in their cytokine and effector function
  • Activated THl cells enhance cellular immunity (including an increase m 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 m responding to extracellular infections
  • Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-IO
  • a TH2 immune response may result m the production of IgG 1 , IgE, IgA and memory B cells for future protection
  • An enhanced immune response may include one or more of an enhanced THl immune iesponse and a TH2 immune response
  • a THl immune response may include one or more of an increase in CTLs, an increase m one or more of the cytokines associated with a THl immune response (such as IL-2, IFN- ⁇ , and TNF- ⁇ ), an increase in activated macrophages, an increase m NK activity, or an increase m the production of IgG2a
  • the enhanced THl immune response will include an increase m IgG2a production
  • a THl immune response may be elicited using a THl adjuvant
  • a THl adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant
  • THl adjuvants suitable for use in the invention may include for example saponin formulations, virosomes and virus like particles, non-toxic de ⁇ vatives of enterobacterial lipopolysaccha ⁇ de (LPS), immuno stimulatory oligonucleotides
  • LPS enterobacterial lipopolysaccha ⁇ de
  • Immunostimulatory oligonucleotides such as oligonucleotides containing a CpG motif, are preferred THl adjuvants for use in the invention
  • a TH2 immune response may include one or more of an increase m 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 m the production of IgG 1 , IgE, IgA and memory B cells
  • the enhanced TH2 immune response 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 m
  • the invention include, for example, mineral containing compositions, oil- emulsions, and ADP- ⁇ bosylatmg toxms and detoxified de ⁇ vatives thereof
  • Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use m the invention
  • a composition may include a combination of a THl adjuvant and a TH2 adjuvant
  • a composition elicits an enhanced THl 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 THl and a TH2 adjuvant elicits an increased THl and/or an increased TH2 immune response relative to immunization with a single adjuvant (z e , relative to immunization with a THl adjuvant alone or immunization with a TH2 adjuvant alone)
  • the immune response may be one or both of a THl immune response and a TH2 response
  • immune response provides for one or both of an enhanced THl 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 m the production of IgA
  • compositions of the invention may be prepared in va ⁇ ous forms
  • the compositions may be prepared as mjectables, 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 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
  • the immunogenic compositions described above include polypeptide antigens from S pneumoniae In all cases, however, the polypeptide antigens can be replaced by nucleic acids (typically DNA) encoding those polypeptides, to give compositions, methods and uses based on nucleic acid immunisation [93 to 100
  • the nucleic acid encoding the immunogen is expressed in vivo after delivery to a patient and the expressed immunogen then stimulates the immune system
  • the active ingredient will typically take the form of a nucleic acid vector comprising (i) a promotei, (u) a sequence encoding the immunogen, operably linked to the promoter, and optionally (in) a selectable marker Preferred vectois may further comp ⁇ se (iv) an o ⁇ gin of replication, and (v) a transcription terminator downstream of and operably linked to (n)
  • (i) & (v) will be eukaryotic and (in) & (iv) will be prokaryotic
  • Preferred promoters are viral promoters e g from cytomegalovirus (CMV)
  • CMV cytomegalovirus
  • the vector may also include transcriptional regulatory sequences (e g enhancers) in addition to the promoter and which interact functionally with the promoter
  • Preferred vectors include the immediate-early CMV enhancer/promoter, and more preferred vectors also include CMV intron A
  • the promoter is operably linked to a downstream sequence encoding an immunogen, such that expression of the lmmunogen-encodmg sequence is under the promoter's control
  • a marker preferably functions m a microbial host (e g m a prokaryote, m a bacteria, m a yeast)
  • the marker is preferably a prokaryotic selectable marker (e g transcribed under the control of a prokaryotic promoter)
  • typical markers are antibiotic resistance genes
  • the vector is preferably an autonomously replicating episomal or extrachromosomal vector, such as a plasmid
  • the vector preferably comprises an origin of replication It is preferred that the origin of replication is active m prokaryotes but not m eukaryotes Preferred vectors thus include a prokaryotic marker for selection of the vector, a prokaryotic origin of replication, but a eukaryotic promoter for driving transc ⁇ ption of the immunogen-encodmg sequence
  • the vectors will therefore (a) be amplified and selected in prokaryotic hosts without polypeptide expression, but (b) be expressed m eukaryotic hosts without being amplified This arrangement is ideal for nucleic acid immunization vectors
  • the vector may comp ⁇ se a eukaryotic transcnptional terminator sequence downstream of the coding sequence This can enhance transc ⁇ ption levels
  • the vector preferably comprises a polyadenylation sequence
  • a preferred polyadenylation sequence is from bovme growth hormone
  • the vector may comp ⁇ se a multiple cloning site
  • the vector may comp ⁇ se a second eukaryotic coding sequence
  • the vector may also comprise an IRES upstream of said second sequence in order to permit translation of a second eukaryotic polypeptide from the same transc ⁇ pt as the immunogen
  • the lmmunogen-codmg sequence may be downstream of an IRES
  • the vector may comp ⁇ se unmethylated CpG motifs e g unmethylated DNA sequences which have m common a cytosme preceding a guanosme, flanked by two 5' purines and two 3' py ⁇ midmes In their unmethylated form these DNA motifs have been demonstrated to be potent stimulators of several types of immune cell
  • Vectors may be delivered in a targeted way Receptor-mediated DNA delivery techniques are described in, for example, references 101 to 106
  • Therapeutic compositions containing a nucleic acid are administered m a range of about lOOng to about 200mg of DNA for local administration m a gene therapy protocol Concentration ranges of about 500 ng to about 50 mg, about l ⁇ g to about 2 mg, about
  • 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about lOO ⁇ g of DNA can also be used during a gene therapy protocol
  • Factors such as method of action (e g for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy Where greater expression is desired over a larger area of tissue, larger amounts of vector or the same amounts re-admmistered m a successive protocol of administrations, or several administrations to different adjacent or close tissue portions may be required to effect a positive therapeutic outcome In all cases, routine experimentation m clinical trials will determine specific ranges for optimal therapeutic effect
  • Vectors can be delivered using gene delivery vehicles
  • the gene delivery vehicle can be of viral or non- vii al o ⁇ gm (see generally references 107 to 1 10)
  • Viral-based vectors for delivery of a desired nucleic acid and expression in a desired cell are well known in the art
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (e g references 111 to 121), alphavirus-based vectors (e g Smdbis virus vectors, Semliki forest virus (ATCC VR-67, ATCC VR- 1247), Ross River virus (ATCC VR-373, ATCC VR- 1246) and Venezuelan equine encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR 1249, ATCC VR-532), hyb ⁇ ds or chimeras of these viruses may also be used), poxvirus vectors (e g vaccinia, fowlpox, canarypox, modified vaccinia Ankar
  • Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycatiomc condensed DNA linked or unlinked to killed adenovirus alone [e g 128], ligand-linked
  • DNA [129], eukaryotic cell delivery vehicles cells [e g refs 130 to 134] and nucleic charge neutralization or fusion with cell membranes Naked DNA can also be employed Exemplary naked
  • non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in ref 143
  • the coding sequence and the product of expression of such can be delivered through deposition of photopolymenzed hydrogel mate ⁇ als or use of ionizing radiation [e g refs 144 & 145]
  • Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun [146] or use of ionizing radiation for activating transferred genes [144 & 145]
  • PLG poly(lactide-co-glycolide) ⁇ microparticles
  • Delivery of DNA using PLG is a particularly preferred method e g by adsorption to the microparticles, which are optionally treated to have a negatively- charged surface (e g treated with SDS) or a positively-charged surface (e g treated with a cationic detergent, such as CTAB)
  • the invention also provides a method for raising an immune response m a mammal comprising the step of administering an effective amount of an immunogenic composition 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 at least two different RrgB clades for combined use as a medicament e g for use in raising an immune response m a mammal
  • the invention also provides the use of at least two different RrgB clades m 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 pneumococcal disease and/or infection e g against pneumococcal meningitis
  • the invention also provides a delivery device pre-filled with an immunogenic composition of the invention
  • the mammal is preferably a human Where the vaccine is for prophylactic use, the human is preferably a child (e g a toddler or infant) or a teenager, where the vaccine is for therapeutic use, the human is preferably a teenager or an adult A vaccine intended for children may also be administered to adults e g to assess safety, dosage, lmmunogemcity, etc
  • One way of checking efficacy of therapeutic treatment involves monitoring pneumococcal infection after administration of the compositions of the invention
  • One way of checking efficacy of prophylactic treatment involves testing post-immunisation sera in standard tests, for example, sera can be tested m an opsonophagocytic killing assay (OPKA), with the ability to opsonise bacteria indicating protective efficacy
  • Another way of checking efficacy of prophylactic treatment involves post-immunisation challenge in an animal model of pneumococcal infection, e g , guinea pigs or mice One such model is described m reference 147
  • Another way of assessing the lmmunogemcity of the compositions of the present invention is to express the polypeptides recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays A positive reaction between the polypeptide and the patient sample indicates that the patient has mounted an immune response to the polypeptide m question This method may also be used to identify
  • compositions of the invention will generally be administered directly to a patient
  • Direct delivery may be accomplished by parenteral injection (e g subcutaneously, lntrape ⁇ toneally, 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 m an enhanced TH 1 and/or TH2 immune response
  • the enhanced immune response includes an increase m 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
  • the va ⁇ ous doses may be given by the same or different routes e g & parenteral p ⁇ me and mucosal boost, a mucosal p ⁇ me 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 )
  • multiple doses may be administered approximately 6 weeks, 10 weeks and 14 weeks after birth, e g at an age of 6 weeks, 10 weeks and 14 weeks, as often used in the World Health Organisation's Expanded Program on Immunisation ("EPI")
  • two primary doses are admimstered about two months apart, e g about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6
  • 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, less than 5 years 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 m a population Vaccines produced by the invention may be admimstered to patients at substantially the same time as (e g du ⁇ ng 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,
  • composition useful for immunisation comp ⁇ ses at least two RrgB clades, either as a hybrid polypeptide or as separate polypeptides
  • a composition may include (i) one or more further polypeptides that elicit antibody responses against pneumococcal proteins, particularly against pneumococcal proteins other than RrgB, (n) a capsular saccha ⁇ d ⁇ from pneumococcus, and/or (in) one or more further immunogens that elicit antibody responses that recognise epitopes on non-pneumococcal organisms
  • compositions of the invention comprising combinations such as these can optionally comp ⁇ se one or more adjuvants, for example two or more adjuvants Suitable adjuvants include mineral salts such as aluminium salts, and squalene- water emulsions such as MF59
  • RrgB polypeptides from one or more clades may be combined with one or more ( ⁇ e 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13) polypeptide antigens selected from the group consisting of (1) a spr0057 antigen, (2) a sprO565 antigen, (3) a sprlO98 antigen, (4) a sprl416 antigen, (5) a sprl418 antigen, (6) a sprO867 antigen, (7) a sprl431 antigen, (8) a sprl739 antigen, (9) a spr2021 antigen, (10) a spr0096 antigen, (11) a sprl707 antigen, (12) a sprl875 antigen, and/or (13) a sprO884 antigen
  • RrgB polypeptides from one or more clades may be combined with one or more ( ⁇ e 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20) polypeptide antigens selected from the group consisting of (1) CIpP, (2) LytA, (3) PhtA, (4) PhtB, (5) PhtD, (6) PhtE, (7) ZmpB, (8) CbpD, (9) CbpG, (10) PvaA, (11) CPLl, (12) PspC, (13) PspA, (14) PsaA, (15) PrtA, (16) Spl33, (17) PiaA, (18) PmA, (19) CbiO, and/or (20) 30S ⁇ bosomal protein S8
  • antigens may be added as separate polypeptides
  • they may be added as hyb ⁇ ds e g a spr0057-spr0096 hybrid or a spr0096-spr2021 hybrid, a sprO565-PhtD hybrid, etc
  • they may be fused to a RrgB polypeptide sequence to provide a hyb ⁇ d polypeptide e g a RrgB-spr0057 hyb ⁇ d
  • a chimeric RrgB polypeptide including two or three RrgB clades may be combined with (a) a mixture of s ⁇ rOO57, spr0096 and spr2021 , (b) a mixture of spr0057, sprO565 and spr2021 , (c) a mixture of spr0057, spr0096 and sprO565, (d) a mixture of spr0057, spr0096, sprO565 and spr2021 , (e) a mixture of sprl418, sprO884 and spr0096, (f) a mixture of sprl418, sprO884 and spr2021, (g) a mixture of sprl418, sprO884, spr0096 and spr2021, (h) a mixture of sprl
  • a chime ⁇ c RrgB polypeptide including two or three RrgB clades may be combined with a pneumococcal immunogen comp ⁇ smg an spr2021 (also referred to as SP2216) antigen, an SP 1732 antigen and optionally a PsaA antigen
  • a pneumococcal immunogen of this sort is the immunogen disclosed m reference 159 that comprises the antigens "SP2216-1" (SEQ ID NO 1 in reference 159, SEQ ID NO 97 herein), "SP 1732-3" (SEQ ID NO 2 m reference 159, SEQ ID NO 98 herein) and, optionally, PsaA (SEQ ID NO 3 in reference 159, SEQ ID NO 99 herein)
  • Polypeptides comprising immunogenic fragments of these SEQ ID NOs can be used m place of the actual disclosed SEQ ID NOs e g composing at least one immunogenic fragment from each of SEQ ID NOs 97 & 98
  • any of these combinations may also include one or more pneumococcal capsular sacchaode(s), which will typically be conjugated to earner protem(s) Further information about such sacchaodes and conjugation is provided below
  • Preferred spr0057 polypeptides for use with the invention compose an ammo 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 23, and/or (b) composing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 23, wherein 'n' 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
  • sprO565 polypeptides for use with the invention compose an ammo 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 25, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 25, wherein 'n' 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)
  • a variant form of sprO565 is SEQ ID NO 45 herein
  • Useful sprO565 polypeptides may thus comprise an ammo 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 45, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 45, wherein 'n' 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)
  • These polypeptides include va ⁇ ants of SEQ ID NO 45
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO 45
  • Other preferred fragments lack
  • sprlO98 polypeptides for use with the invention comprise an ammo 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 26, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 26, wherein 'n' 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)
  • sprlO98 proteins include va
  • ammo acid sequence of full length sprl418 as found m the R6 strain is given as SEQ ID NO 29 herein
  • Preferred s ⁇ rl418 polypeptides for use with the invention compnse an ammo 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 29, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 29, wherein 'n' 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)
  • ammo acid sequence of full length sprO867 as found in the R6 strain is given as SEQ ID NO 30 herein
  • Preferred sprO867 polypeptides for use with the invention compnse an ammo 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 30, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 30, wherein 'n' is 7 or more (e g 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,
  • sprl431 polypeptides for use with the invention comprise an ammo 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 31, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 31, wherein 'n' is 7 or more (e g 8, 10, 12, 14, 16,
  • the 's ⁇ rl739' polypeptide is pneumolysm (e g see GI 15903781)
  • the ammo acid sequence of full length sprl 739 as found in the R6 strain is given as SEQ ID NO 32 herein
  • Preferred sprl739 polypeptides for use with the invention comp ⁇ se an ammo 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 32, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 32, wherein 'n 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)
  • These sprl 739 proteins include
  • spr2021 polypeptides for use with the invention comp ⁇ se an ammo 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 33, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 33, wherein 'n' 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) These sp
  • spr0096 polypeptides for use with the invention comprise an ammo 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 34, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 34, wherein 'n' 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)
  • a va ⁇ ant form of s ⁇ r0096 is SEQ ID NO 54 herein
  • the use of this variant for immunisation is reported in reference 150 (SEQ ID NO 150 therein), where it is annotated as a LysM domain protein
  • a spr0096 for use with the invention may comprise an ammo 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 54, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 54, wherein 'n' 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)
  • These polypeptides include va ⁇ ants of
  • a spr0096 polypeptide may be used m the form of a dimer eg a homodimer
  • sprl707 polypeptides for use with the invention comprise an ammo 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 36, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 36, wherein 'n' is 7 or more (e g 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
  • R6 strain is given as SEQ ID NO 35 herein Preferred sprl875 polypeptides for use with the invention comp ⁇ se an ammo acid sequence (a) having 60% or more identity (e g 60%, 65%, 70%, 75%, 80%,
  • SEQ ID NO 35 comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 35, wherein 'n' 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)
  • sprl875 proteins include va ⁇ ants of SEQ ID NO 35
  • Preferred fragments of (b) comprise an epitope from SEQ ID NO 35
  • Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5,
  • the 'sprO884' protein is a peptidylprolyl isomerase, also known as protease maturation protein
  • the ammo acid sequence of full length sprO884 is SEQ ID NO 37 herein
  • Preferred sprO884 polypeptides for use with the invention comp ⁇ se an ammo 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 37, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 37, wherein 'n' 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)
  • These sprO884 proteins include va ⁇ ants of
  • LytA is the N-acetylmuramoyl-L-alanme amidase (autolysm)
  • the ammo acid sequence of full length LytA is SEQ ID NO 59 herein
  • LytA is sprl754
  • Preferred LytA polypeptides for use with the invention comp ⁇ se an ammo 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 59, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 59, wherein 'n' 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)
  • LytA proteins include
  • PhtA is the Pneumococcal histidme t ⁇ ad protein A
  • the ammo acid sequence of full length PhtA precursor is SEQ ID NO 60 herein
  • PhtA is sprlO ⁇ l
  • Preferred PhtA polypeptides for use with the invention comprise an ammo 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 60, and/or (b) comp ⁇ sing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 60, wherein 'n' 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)
  • These PhtA proteins include variants of SEQ ID NO 60 Preferred
  • PhtB is the pneumococcal histidme t ⁇ ad protein B
  • the ammo acid sequence of full length PhtB precursor is SEQ ID NO 61 herein Xaa at residue 578 can be Lysine Preferred PhtB polypeptides for use with the invention comp ⁇ se an ammo 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 61, and/or (b) comp ⁇ sing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 61, wherein 'n' is 7 or more (eg 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more)
  • These PhtB proteins include va ⁇ ants of SEQ ID NO 61 Preferred
  • PhtD is the Pneumococcal histidme triad protein D
  • the ammo acid sequence of full length PhtD precursor is SEQ ID NO 62 herein
  • PhtD is spr0907 [149]
  • Prefe ⁇ ed PhtD polypeptides for use with the invention comprise an ammo 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 62, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 62, wherein 'n' 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)
  • These PhtD proteins include va ⁇ ants of SEQ ID NO
  • ZmpB is the zmc metalloprotease
  • the ammo acid sequence of full length ZmpB is SEQ ID NO 64 herein hi the R6 genome ZmpB is sprO581
  • Preferred ZmpB polypeptides for use with the invention comprise an ammo 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 64, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 64, wherein W 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)
  • These ZmpB proteins include va ⁇ ants of SEQ ID NO 64 Preferred fragments of (b) comp ⁇ se an epitope from SEQ
  • CbpD is the Choline binding protein D
  • the ammo acid sequence of full length CbpD is SEQ ID NO 65 herein
  • CbpD is spr2006 [149]
  • Preferred CbpD polypeptides for use with the invention comp ⁇ se an ammo acid sequence (a) having 60% or more identity (e g 60%,
  • CbpD proteins include variants of SEQ ID NO 65 (e g SEQ ID NO 66, see below) Prefe ⁇ ed fragments of (b) comp ⁇ se an epitope from SEQ ID NO 65 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 65 while retaining at least one epitope of SEQ ID NO 65 Other fragments omit one or more protein domains The use of CbpD for immunisation is reported in reference 171
  • a variant of SEQ ID NO 65 is SEQ ID NO 66 herein
  • SEQ ID NO 66 A variant of SEQ ID NO 65
  • a CbpD polypeptide for use with the invention may comp ⁇ se an ammo 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 66, and/or (b) composing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 66, wherein 'n' 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)
  • These CbpD proteins include va ⁇ ants of SEQ ID NO 66 Preferred fragments of (b) comp ⁇ se an epitope
  • CbpG is the Choline binding protein G
  • the ammo acid sequence of full length CbpG is SEQ ID NO 67 herein
  • CbpG is sprO35O [149]
  • Preferred CbpG polypeptides for use with the invention comp ⁇ se an ammo acid sequence (a) having 60% or more identity (e g 60%,
  • SEQ ID NO 67 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 5% or more) to SEQ ID NO 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 67, and/or (b
  • CbpG proteins include variants of SEQ ID NO 67 Prefen ed fragments of
  • P ⁇ aA (Streptococcus pneumoniae pneumococcal vaccine antigen A) is also known as splOl
  • the ammo acid sequence of full length PvaA is SEQ ID NO 68 herein
  • PvaA is spr0930
  • Preferred PvaA polypeptides for use with the invention comprise an ammo 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 68, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 68, wherein 'n' 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)
  • PvaA proteins include
  • PspC is the pneumococcal surface protein C [170] and is also known as cholme-bmdmg protein A (CbpA) Its use for immunisation is reported m references 168 and 171 In the R6 strain it is sprl995 and, for reference, the ammo acid sequence of foil length sprl995 is SEQ ID NO 57 herein
  • Preferred PspC polypeptides for use with the invention comprise an ammo 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 57, and/or (b) comprising a ftagment of at least 'n' consecutive ammo acids of SEQ ID NO 57, wherein 'n' is 7 or more (e g 8, 10, 12, 14, 16, 18, 20, 25,
  • a va ⁇ ant of PspC is known as 'Hie' It is similar to PspC, as shown m Figure 1 of reference 172, where it is reported to bind to factor H (fH)
  • the ammo acid sequence of full length Hie is SEQ ID NO 71 herein
  • a Hie protein may be used with the invention in addition to or m place of a PspC polypeptide
  • Preferred Hie polypeptides for use with the invention comprise an ammo 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 71, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 71 , wherein 'n' is 7 or more (e g 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,
  • PspA is the Pneumococcal surface protein A
  • the ammo acid sequence of full length PspA is SEQ ID NO 72 herein
  • PspA is sprO121
  • Preferred PspA polypeptides for use with the invention comp ⁇ se an ammo 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 72, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 72, wherein 'n' 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)
  • These PspA proteins include variants of SEQ ID NO 72 Preferred fragments of (b)
  • PiaA is the membrane permease involved m iron acquisition by pneumococcus
  • the ammo acid sequence of full length PiaA is SEQ ID NO 76 herein
  • PiaA is sprO935
  • Preferred PiaA polypeptides for use with the invention comprise an ammo 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 76, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 76, wherein 'n' 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)
  • These PiaA proteins include va ⁇ ants of SEQ ID NO 76 Prefe ⁇
  • PmA is the ABC transporter substrate-bmdmg protein for ferric iron transport It is also known as FatB
  • the ammo acid sequence of full length PmA is SEQ ID NO 77 herein
  • Preferred PmA polypeptides for use with the invention comprise an ammo 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 77, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 77, wherein 'n' 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)
  • These PmA proteins include variants of SEQ ID NO
  • CbiO is annotated as a cobalt transporter ATP-bmdmg subunit
  • the ammo acid sequence of full length CbiO is SEQ ID NO 78 herein
  • CbiO is spr2025 [149]
  • the use of CbiO for immunisation is reported m reference 181 ('ID2' therein)
  • Preferred CbiO polypeptides for use with the invention comprise an ammo acid sequence (a) having 60% or more identity (e g 60%,
  • SEQ ID NO 78 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 5% or more) to SEQ ID NO 78, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 78, wherein 'n 1 is 7 or more (e g 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,
  • CbiO proteins include va ⁇ ants of SEQ ID NO 78 Preferred fragments of
  • ammo acid sequence of 30S ⁇ bosomal protein S8 is SEQ ID NO 79 herein
  • the S8 subumt is spr0203
  • Preferred S8 polypeptides for use with the invention comprise an ammo 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 79, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 79, wherein 'n' 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)
  • These S 8 proteins include va ⁇ ants of SEQ ID NO 79 Preferred fragments of (b) comp ⁇ se an epitope from SEQ
  • SP 1732 is a membrane-associated serme/threonme kinase, StkP
  • the sequence of SP 1732, comprising 659 ammo acids, is identified in reference 150 as SEQ ID NO 214 as SEQ ID NO 214
  • An exemplary fragment of this sequence, referred to as "SP 1732-3", is identified m reference 159 as SEQ ID NO 2
  • the ammo acid sequence of SP 1732-3 is provided as SEQ ID NO 98 herein
  • Preferred SPl 732 polypeptides for use with the invention comprise an ammo 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 98, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 98, wherein 'n' is 7 or
  • RrgB polypeptides from one or more clades may be combined with one or more pneumococcal capsular saccha ⁇ de(s), which will typically be conjugated to carrier protem(s)
  • an immunogenic composition comprising a combination of
  • a sacchande used in component (2) of this combination is ideally present as a conjugate comprising a saccharide moiety and a carrier protein moiety
  • the carrier moiety in the conjugate may be a single RrgB ) polypeptide, a hybnd RrgB polypeptide, a non-RrgB pneumococcal polypeptide, or a non-pneumococcal polypeptide
  • the sacchande is from the capsular sacchande of a pneumococcus
  • the sacchande may be a polysacchande having the size that anses during purification of the sacchande from bacteria, or it may be an oligosaccharide achieved by fragmentation of such a polysacchande
  • 6 of the saccharides are presented as intact polysaccha ⁇ des while one (the 18C serotype) is presented as an oligosaccharide
  • a composition may include a capsular saccha ⁇ de from one 01 more of the following pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 1OA, HA, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F
  • a composition may include multiple serotypes e g 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes 7-valent, 9-valent, 10-valent, 11-valent and 13- valent conjugate combinations are already known m the art, as is a 23-valent unconjugated combination
  • a 10-valent combination may include saccha ⁇ de from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F
  • An 11-valent combination may further include saccha ⁇ de from serotype 3
  • a 12-valent combination may add to the 10-valent mixture serotypes 6A and 19A, 6A and 22F, 19A and 22F, 6A and 15B, 19A and 15B, r 22F and 15B
  • a 13-valent combination may add to the 11-valent mixture serotypes 19A and 22F, 8 and 12F, 8 and 15B, 8 and 19A, 8 and 22F, 12F and 15B, 12F and 19A, 12F and 22F, 15B and 19A, 15B and 22F etc
  • One useful 13-valent combination includes capsular saccha ⁇ de from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19, 19F and 23F If saccharides are enclosed then it is preferred to include 1, 2 or 3 of serotypes 1, 5
  • a carrier protein m a conjugate may or may not be one of the RrgB antigens of (1) It it is not a RrgB antigen it may instead be a different pneumococcal antigen, such as spr0057, spr0096 and spr2021 , etc or pneumolysm [182] or its non-toxic denvatives [183], or pneumococcal surface protein PspA [184], In some embodiments, though, the carrier is not a pneumococcal antigen, and may be e g a bacterial toxm or toxoid Typical earner proteins are diphthe ⁇ a or tetanus toxoids or mutants thereof The CRM 1 Qy diphthe ⁇ a toxm mutant [185] is useful, and is the earner in the PREVNARTM product
  • Other suitable carrier proteins include N meningitidis outer membrane protein complex [186], synthetic peptides [187,188], heat shock proteins [189,
  • each conjugate may use the same carrier piotem or a different earner protein Reference 202 describes potential advantages when using different earner proteins in multivalent pneumococcal conjugate vaccines
  • a single conjugate may carry saccharides from multiple serotypes [203] Usually, however, each conjugate will include saccharide from a single serotype Conjugates may have excess earner (w/w) or excess saccharide (w/w) In some embodiments, a conjugate may include equal weights of each
  • the carrier molecule may be covalently conjugated to the earner directly or via a linker
  • Direct linkages to the protein may be achieved by, for instance, reductive animation between the sacchande and the carrier, as described in, for example, references 204 and 205
  • the sacchande may first need to be activated e g by oxidation
  • Linkages via a linker group may be made using any known procedure, for example, the procedures descnbed in references 206 and 207
  • a preferred type of linkage is an adipic acid linker, which may be formed by coupling a free -NKb group (e g introduced to a glucan by animation) with adipic acid (using, for example, dnmide activation), and then coupling a protein to the resulting sacchande-adipic acid intermediate [208,209]
  • Another preferred type of linkage is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a sacchande CDI [210,
  • the RrgB clade combinations may be used in combination with non-pneumococcal antigens
  • the invention provides an immunogenic composition comprising a combination of
  • Diphtheria toxoid can be obtained by treating (e g using formaldehyde) diphthena toxin from Cor ⁇ nebactenum diphthenae Diphthena toxoids are disclosed in more detail in, for example, chapter 13 of reference 220
  • Tetanus toxoid can be obtained by treating (e g using formaldehyde) tetanus toxin from Clostridium tetani Tetanus toxoids are disclosed in more detail in chapter 27 of reference 220
  • Pertussis antigens in vaccines are either cellular (whole cell, Pw) or acellular (Pa)
  • the invention can use either sort of pertussis antigen
  • Preparation of cellular pertussis antigens is well documented (e g see chapter 21 of reference 220) e g it may be obtained by heat inactivation of phase I culture of B pertussis
  • Acellular pertussis antigen(s) compose specific purified B pertussis antigens, either purified from the native bacterium or purified after expression in a recombinant host
  • a composition may include one, two or three of the following well-known and well-characterized B pertussis antigens (1) detoxified pertussis toxm (pertussis toxoid, or 'PT'), (2) filamentous hemagglutinin ('FHA'), (3) pertactin (also known as the '69 kiloDalton outer membrane protein') FHA
  • Hepatitis B virus surface antigen (HBsAg) is the major component of the capsid of hepatitis B virus It is conveniently produced by recombinant expression m a yeast, such as a Saccharomyces cerevisiae
  • IPV antigens are prepared from viruses grown on cell culture and then inactivated (e g using formaldehyde) Because poliomyelitis can be caused by one of three types of poliovirus, as explained in chapter 24 of reference 220, a composition may include three poliovirus antigens poliovirus Type 1 (e g Mahoney strain), poliovirus Type 2 (e g MEF-I strain), and poliovirus Type 3 (e g Saukett strain)
  • composition when a composition includes one of diphtheria toxoid, tetanus toxoid or an acellular pertussis antigen in component (2) then it will usually include all three of them i e component (2) will include a D-T-Pa combination
  • composition when a composition includes one of diphthe ⁇ a toxoid, tetanus toxoid or a cellular pertussis antigen m component (2) then it will usually include all three of them i e component (2) will include a D-T-Pw combination
  • Immunogenic compositions of particular interest comprise (i) a combination of at least two RrgB clades as discussed above as a mixture or hyb ⁇ d, diphtheria toxoid, tetanus toxoid, whole cell pertussis antigens, a conjugate of Haemophilus influenzae type B capsular saccharide, and HBsAg, ( ⁇ ) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, diphthe ⁇ a toxoid, tetanus toxoid, acellular pertussis antigen(s), a conjugate of Haemophilus influenzae type B capsular saccharide, and HBsAg, (in) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, and conjugate(s) from one or more of meningococcal serogroups A, C, Wl 35 and Y, (iv) a combination
  • Antibodies against pneumococcal antigens can be used for passive immunisation [223]
  • the invention provides a combination of antibodies for simultaneous, separate or sequential administration, wherein the combination includes at least two of (a) an antibody which recognises a first ammo acid sequence as defined above, (b) an antibody which recognises a second ammo acid sequence as defined above, and/or (c) an antibody which recognises a third ammo acid sequence as defined above,
  • the invention also provides the use of such antibody combinations in therapy
  • the invention also provides the use of such antibody combinations in the manufacture of a medicament
  • the invention also provides a method for treating a mammal composing the step of administering to the mammal an effective amount of such a combination
  • these methods and uses allow a mammal to be protected against pneumococcal infection
  • antibody includes intact immunoglobulin molecules, as well as fragments thereof which are capable of binding an antigen These include hybrid (chimeric) antibody molecules [224, 225], F(ab')2 and F(ab) fragments and Fv molecules, non-covalent heterodimers [226, 227], smgle-chain Fv molecules
  • the antibodies are monoclonal antibodies
  • Methods of obtaining monoclonal antibodies are well known m the art Humanised or fully-human antibodies are preferred
  • GI numbering is used above A GI number, or "Genlnfo Identifier", is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases The GI number bears no resemblance to the accession number of the sequence record When a sequence is updated (e g for correction, or to add more annotation or information) then it receives a new GI number Thus the sequence associated with a given GI number is never changed
  • this epitope may be a B-cell epitope and/or a T-cell epitope
  • Such epitopes can be identified empirically (e g using PEPSCAN [242,243] or similar methods), or they can be predicted (e g using the Jameson- Wolf antigenic index [244], matrix-based approaches [245], MAPITOPE [246], TEPITOPE [247,248], neural networks [249], OptiMer & EpiMer [250, 251], ADEPT [252], Tsites [253], hydrophilicity [254], antigenic index [255] or the methods disclosed in references 256-260, etc )
  • Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as "antigenic determinants"
  • composition “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
  • a process comprising a step of mixing two or more components does not require any specific order of mixing
  • components can be mixed m any order Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc
  • Antibodies will generally be specific for their target Thus they will have a higher affinity for the target than for an irrelevant control protein, such as bovine serum albumin
  • references to a percentage sequence identity between two ammo acid sequences means that, when aligned, that percentage of ammo 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 m the art, for example those desc ⁇ bed in section 7 7 18 of ref 261
  • 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 m ref 262
  • PI-I RrgB protein has three different clades Fifteen different RrgB ammo acid sequences were found m 45 different strains and Figure 9 shows their relationship The wild-type sequences are >98% conserved withm each clade RrgB protein was found to elicit immune responses which are protective against homologous strains (mtra-clade), but which fail to protect against strains having RrgB from a different clade (mter-clade) Thus it was decided to combine multiple RrgB clades into a single composition, thereby increasing the spectrum of strain coverage SEQ ID NOs 1, 2 and 3 are the full-length encoded sequences for RrgB from strains TIGR4, Fmland 6B - 12 and Taiwan 2jF -15 To construct chimeras of these three proteins their N- and C-te ⁇ mm were truncated to give SEQ ID NOs 4, 5 and 6 Restriction enzymes Nhel BamHI and Xhol were used in this procedure To join these fragments to
  • RTgB I-II-III SEQ ID NO 11
  • RTgB I-III-II SEQ ID NO 13
  • RTgB III-II-I SEQ ID NO 15
  • RrgB III-I-II SEQ ID NO 17
  • RTgB II-III-I SEQ ID NO 19
  • RTgB II-I-III SEQ ID NO 21 Except for the I-III-II chimera (SEQ ID NO 13) the expressed chimeras had a molecular weight of 205kDa, could be expressed m E cob in soluble form, and were pu ⁇ fied from the soluble proteins For example, figure 3 shows a gel of the I-II-III chimera at 1 6mg/ml with 90% purity
  • Figure 2 shows mortality after the challenge Median survival times m days weie as follows
  • Figures 30 to 33 show the results of bacteremia and mortality assays for mice immunised mtrape ⁇ toneally with 20 ⁇ g of the III-II-I chimera
  • Figure 30 shows data for i v challenge with TIGR4
  • Figure 31 shows data for i p challenge with TIGR4
  • Figure 32 shows data for i v challenge with 35B-SME15
  • Figure 33 shows data for i v challenge with 6B Finland 12
  • Figure 12 shows a 48 hoar bacteremia (Figure 12A) and mortality (Figure 12B) assay using a III-II-I chimera (immunised i p and challenged i p with 35B-SME15) is comparable when using different chimera doses (2 ⁇ g and 20 ⁇ g)
  • Figure 14 shows that RrgB IH-II-I chimera is protective using MF59 adjuvant in BalB/c mice, intraperitoneal immunisation with 20 ⁇ g chimera (0-14-28 days) and challenged mtransally
  • Figure 15 shows that RrgB HI-II-I chimera is protective upon subcutaneous immunization in BalB/c mice, immunized subcutaneously and challenged mtrape ⁇ toneally with TIGR4 (130CFU/mouse)
  • Figure 15A shows a 24hour bacteremia assay and Figure 15B shows a mortality assay
  • Figure 16 shows that RrgB IH-II-I chimera elicits production of functional antibodies in a passive protection study, compared to a Normal Rabbit Serum (NRS) control, in a 24hour bacteremia assay
  • Figure 17 shows that antibodies are functional m OPA against strains of the three clades and Figure 18 shows that the OPA activity is specifically due to the antibodies against RrgB IH-II-I chimera
  • Figure 19 shows that sible RrgB domains confer protection in vivo Specifically, the data show % survival of BalB/c mice immunised with the RrgB D 1 domain or the RrgB D4 domain (i p immunization 20 ⁇ g, 0-14-28 days, i p challenge with TIGR4 lOOCFU)
  • Figure 23 shows a 48 hour bacteremia (Figure 23A) and mortality (Figure 23B) assay using a IH-II-I chimera when combined with different combinations of further polypeptide antigens (20 ⁇ g antigens with alum, immunised i p and challenged i v with 6B-Fmland 1 2E+08 CFU/mouse)
  • column 1 shows a combination of spiOO57, spr0096 and spr2021
  • column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA
  • column 3 shows RrgB IH-II-I chimera
  • column 4 shows RrgB IH-II-I chimera combined with spr0057, spr0096 and spr2021
  • column 5 shows RrgB IH-II-I chimera combined with SP2216-1 , SP 1732-3 and PsaA
  • column 6 shows an alum control
  • Figure 24 shows a 48 hour bacteremia (Figure 24A) and mortality (Figure 24B) assay using a HI-II-I chimera when combined with different combinations of further polypeptide antigens (20 ⁇ g antigens with alum, immunised i p and challenged i v with 35B-SME15 5 2E+07 CFU/mouse)
  • column 1 shows a combination of spr0057, spr0096 and spr2021
  • column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA
  • column 3 shows RrgB HI-II-I chimera
  • column 4 shows RrgB IH-II-I chimera combined with spr0057, spr0096 and spr2021
  • column 5 shows RrgB IH-II-I chimera combined with SP2216-1, SPl 732-3 and PsaA
  • column 6 shows an alum control
  • Figure 25 shows (A) a 24 hour bacteremia assay and (B) mortality data in BALB/c mice using a IH-II-I chimera that contains a polyhistidine tag compared to a tag-less IH-II-I chimera and an alum control (i p immunisation, i p challenge with TIGR4 2 1E+02 CFU/mouse)
  • Figure 26 shows similar data i e a 24 hour bacteremia assay in BALB/c mice using a IH-II-I chimera that contains a polyhistidme tag compared to a tag-less IH-II-I chimera and an alum control, further compared to a combination of 5 sprOO57, spr0096 and spr2021
  • Figure 34 shows the results of 48 hour bacteremia and mortality assays for III-II-I chimera comparing a TIGR4 challenging strain over-expressmg pilus to a challenging strain that only expresses very low amounts of pilus These data show that protection is very good when the pilus is overexpressed and also when the pilus is only present at very low levels
  • Figure 35 shows similar bacteremia data for both 5 III-II-I and H-I-III chimeras compa ⁇ ng a 6BFmll2 challenging strain over-expressmg pilus ( Figure 35A) to a 6BFmI 12 challenging strain under-expressing pilus ( Figure 35B) The chimeras show significant protection against both the strain overexpressmg and the strain underexpressmg pilus
  • Figure 36 shows that pilus- 1 is more prevalent m pneumococcal strains that are iesistant to antiobioticsO (erythromycm-resistance, penicillin-resistance and multiple-drug- resistance) compared to strains that aie susceptible to antibiotics There is a significant association between pilus- 1 presence and antibiotic resistance An increase in the presence of pilus- 1 in antibiotic-resistant strains has also been observed in the multi-resistant PMEN strain collection (data not shown) These data suggest that immunising against pilus- 1 using an immunogenic composition including multiple RrgB clades will have the additional
  • each of the four tested anti-TIGR4 mAbs was able to reduce bacteremia in a passive protection test, with the best results coming from 23F8/10
  • the different RrgB domains were cloned, as single domains (Dl, D2, D3, D4) or as multi-domain fragments (Dl-3, D2-4, D3-4), expressed in E CoIi as His-tagged polypeptides and successfully purified in a soluble form by affinity chiomatography on His-trap high performance columns (GE Healthcare)
  • the recombinant proteins were then probed m western blot analysis against the MAbs by using FL RrgB clade 1 and BSA as positive and negative controls respectively
  • the RrgB chimeras are suitable for use as earner proteins m saccha ⁇ de- carrier conjugates
  • the I-II-III and III-II-I chimeras were conjugated to a saccharide immunogen and IgG responses (GMT) against the saccharide were then measured by ELISA Results were compared to a number of other pneumococcal proteins, and also to Nl 9 and CRM 197 as positive controls Results from study VFVII were as follows
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Abstract

Pneumococcal pilus subunit RrgB has at least three clades. Serum raised against a given clade is active against pneumococci which express that RrgB clade, but is not active against strains which express one of the other two clades i.e. there is intra-clade cross-protection, but not inter-clade cross-protection. Thus an immunogenic composition can include at least two different clades of RrgB to improve strain coverage against pilus-containing pneumococci. These multiple clades may be present in the immunogenic composition as separate polypeptides or may be fused as a single polypeptide chain.

Description

COMBINATIONS OF PNEUMOCOCCAL RrgB CLADES TECHNICAL FIELD This invention is in the field of immunising against Streptococcus pneumoniae (pneumococcus)
BACKGROUND OF THE INVENTION
5 S pneumoniae has a pilus known as pilus-1 encoded by a 14-kb islet (PI-I) having seven genes encoding the RIrA transcπptional regulator, three pilus subunits with LPXTG-type cell wall sorting signals, and three sortase enzymes involved in synthesis of the pilus polymer and in the incorporation of ancillary pilus components RrgB is the major subumt that forms the backbone of the structure, while the other two pilms (RrgA, RrgC) are ancillary structural proteins [1-4] RrgA is the major pilus- 1 adhesm, 10 bacteπa lacking RrgA are less adherent to epithelial cells than wild-type organisms
SUMMARY OF THE INVENTION
The present invention relates to an immunogenic composition comprising at least two of
(a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 1
15 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 1 ,
(b) a second polypeptide, comprising a second ammo acid sequence, where the second ammo acid sequence compπses an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
-0 (c) a third polypeptide, comprising a third ammo acid sequence, where the third ammo acid sequence compπses an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3
The present invention also relates to a polypeptide comprising at least two of
(a) a first ammo acid sequence comprising an ammo acid sequence (i) having at least 90% '5 sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 1,
(b) a second ammo acid sequence comprising an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
0 (c) a third ammo acid sequence comprising an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3 The present invention further relates to a polypeptide comprising ammo acid sequence
A-{-X-L-}n-B wherein each X is an ammo acid sequence of first polypeptide, second polypeptide or third polypeptide as defined in claim 1, L is an optional linker ammo acid sequence, A is an optional N terminal ammo acid sequence, B is an optional C terminal ammo acid sequence, n is an integer of 2 or more Optionally, the polypeptide compπses at least two of a first, second and third polypeptide as defined in claim 1
In a particular embodiment, the polypeptides of the invention comprise an ammo acid sequence selected from the group consisting of SEQ ID NOs 11, 13, 15, 17, 19 and 21
The present invention also relates to a bacterium which expresses at least two of (a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 1,
(b) a second polypeptide, comprising a second ammo acid sequence, where the second ammo acid sequence compπses an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
(c) a third polypeptide, comprising a third ammo acid sequence, where the third ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3 BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows results of a bacteremia study with five RrgB chimeras and a control The figures are CFU/ml Each mark shows data for a single mouse
Figure 2 shows results of a mortality study with five RrgB chimeras and a control The figures are survival time in days Each mark shows data for a single mouse Figure 3 shows a gel with four lanes From left to right the lanes contain MW markers, a I-II-III chimera, a I-II-III chimera, and a BSA standard The arrow indicates a MW of 214kDa
Figure 4A shows passive protection data using foui mAbs raised against the TIGR4 RrgB, or a salme control The y-axis shows CFU/ml for 24 hour bacteremia Figure 4B shows results of a mortality study with the four mAbs raised against the TIGR4 RrgB Figures 5 and 6 show western blots using mAbs raised against the TIGR4 sequence (Figure 5) or the 6B sequence (Figure 6) Lanes are, from left to πght marker, RrgB I-II-III, RrgB H-I-III, RrgB H-III-I, RrgB HI-I-II, RrgB IH-II-I, RrgB TIGR4, RrgB 6B, RrgB 23F, BSA control Figure 7 shows (A) bacteremia and (B) mortality data after immunisation with alum-adjuvanted I-II-III chimera, HI-II-I chimera, TIGR4 or alum alone In Figure 7 A the data are CFU/ml and in Figure 7B the data are survival time m days
Figure 8 shows OPKA results against TIGR4 strain, showing % OPKA killing against serum dilution 5 Diamonds show positive control sera, pre-immune sera are filled boxes, visible near the x-axis, the other five lines are for sera raised against the five chimeras
Figure 9 shows a tree based on % identity for SEQ ID NOs 1-3 & 85-96 The label is the SEQ ID
Figure 10 shows OPKA results against S pneumoniae serotype 6B, showing % killing against serum dilution
10 Figure 11 shows shows OPKA results against S pneumoniae serotype 6B, showing % killing against serum dilution up to a dilution of 1/131220
Figure 12 shows (A) bacteremia and (B) mortality data after immunisation with HI-II-I chimera at different doses In Figure 12A the data are CFU/ml and m Figure 12B the data are survival time in days
Figure 13 shows (A) bacteremia and (B) mortality data after immunisation with 20μg RrgB HI-II-I 15 chimera In Figure 13A the data are CFU/ml and in Figure 13B the data are survival time in days
Figure 14 demonstrates that the IH-II-I RrgB chimera is protective using the MF59 adjuvant Diamonds show adjuvanted RrgB chimera, circles show MF59 alone
Figure 15 shows (A) bacteremia and (B) mortality data after subcutaneous immunisation with RrgB III-II-I chimera In Figure 15A the data are CFU/ml and in Figure 15B the data are survival time m 20 days
Figute 16 shows that RrgB III-II-I chimera elicits production of functional antibodies m a passive protection study, compared to a Normal Rabbit Serum (NRS) control, m a 24hour bacteremia assay
Figure 17 shows OPKA results against (A) TIGR4 and (B) ST35B, showing % killing against serum dilution Diamonds show Anti-T4, circles show RrgB III-II-I chimera and squares show NSK
>5 Figure 18 shows OPKA results against TIGR4 strain, showimg that the OPA activity is specifically due to antibodies against RrgB III-II-I chimeras
Figure 19 shows that single RrgB domains confer protection in vivo Triangles show RrgB chimera, diamonds show D 1 domain, squares show D4 domain and circles show Alum
Figure 20 shows western blot analysis of different RrgB domains (single domains Dl, D2, D3 and D4 >0 and multi-domain fragments Dl-3, D2-4, D3-4) tested for binding with each of four protective mAbs raised against TIGR4 RrgB Figure 21 shows a western blot analysis performed with monoclonal antibody 23F8/C10 binding to trypsm-digested RrgB
Figure 22 (A) is a model of RrgB domain D 1 ammo acid sequence onto the domain 1 crystal structure of S pyogenes pilus backbone SpyO128 (B) is S pneumoniae RrgB crystal structure (D2-D3) and modelled 5 Dl domain (C) is a 3D reconstruction electron density map of the S pneumoniae pilus
Figure 23 shows (A) 48 hour bacteremia and (B) mortality data against 6B-Finland strain (i v challenge) after i p immunisation with RrgB IH-II-I chimera when combined with different combinations of further polypeptide antigens (20μg antigens) In Figure 23A the data are CFU/ml and in Figure 23B the data are survival time in days hi both (A) and (B) column 1 shows a combination of spr0057, spr0096 and spr2021, column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA, column 3 shows RrgB III-II- I chimera, column 4 shows RrgB HI-II-I chimera combined with spr0057, spr0096 and spr2021, column 5 shows RrgB HI-II-I chimera combined with SP2216-1, SP 1732-3 and PsaA, and column 6 shows an alum control
Figure 24 shows (A) 48 hour bacteremia and (B) mortality data against 35B-SME15 strain (i v challenge) after i p immunisation with RrgB IH-II-I chimera when combined with different combinations of further polypeptide antigens (20μg antigens) In Figure 24A the data are CFU/ml and in Figure 24B the data are survival time m days In both (A) and (B) column 1 shows a combination of spr0057, spr0096 and spr2021, column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA, column 3 shows RrgB HI-II-I chimera, column 4 shows RrgB HI-II-I chimera combined with spr0057, spr0096 and spr2021, column 5 shows RrgB III-II-I chimera combined with SP2216-1, SP 1732-3 and PsaA, and column 6 shows an alum control
Figure 25 shows (A) a 24 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i p challenge with TIGR4 2 1E+02 CFU/mouse) The data m (A) are CFU/ml and in (B) are survival time in days
Figure 26 shows a 24 hour bacteremia assay m BALB/c mice using a III-II-I chimera that contains a polyhistidme tag compared to (i) a tag-less III-II-I chimera, (n) a combination of spi0057, sprO096 and spr2021, (in) the combination of spr0057, sρrOO96 and spr2021 further combined with the tag-less III-II-I chimera, and (iv) an alum control (i p immunisation, i p challenge with TIGR4 1 6E+02 ) CFU/mouse)
Figure 27 shows (A) a 48 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i v challenge with 35B- SME 15 4 6E+07 CFU/mouse) The data in (A) are CFU/ml and in (B) are survival time in days
Figure 28 shows (A) a 48 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i v challenge with 6BFmlandl2 9 4E+07 CFU/mouse) The data in (A) are CFU/ml and in (B) are survival time m days
Figure 29 shows (A) a 48 hour bacteremia assay and (B) mortality data using a III-II-I chimera that contains a polyhistidme tag compared to a tag-less III-II-I chimera and an alum control (i p immunisation, i v challenge with TIGR4 6 3E+05 CFU/mouse) The data in (A) are CFU/ml and in (B) are survival time in days
Figure 30 shows (A) a 48 hour bacteremia assay and (B) mortality data after immunisation with 20μg III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with TIGR4) The data m (A) are CFU/ml and m (B) are survival time m days
Figure 31 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 20μg III-II-I chimera, compared to an alum control (i p immunisation, i p challenge with TIGR4) The data in (A) are CFU/ml and in (B) are survival time in days
Figure 32 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 20μg III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with 35B-SME15) The data in (A) are CFU/ml and in (B) are survival time m days Figure 33 shows (A) a 24 hour bacteremia assay and (B) mortality data after immunisation with 20μg III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with 6B Finland 12) The data m (A) are CFU/ml and in (B) are survival time m days
Figure 34 shows (A) a 48 hour bacteremia assay and (B) mortality data after immunisation with III-II-I chimera, compared to an alum control (i p immunisation, i v challenge with TIGR4) when challenged with a TIGR4 strain overexpressmg pilus (T4+) compared to a TIGR4 tram expressing very low amounts of pilus (T4-) The data m (A) are CFU/ml and in (B) are survival time in days
Figure 35 shows 48 hour bacteremia assays after immunisation with H-I-III and III-II-I chimeras (A) when challenged with a 6BFmI 12 strain overexpressmg pilus (i p immunisation, i v challenge with 6BFmlandl2 overexpressmg pilus 7 OE+09 CFU/mouse) and (B) when challenged with a 6BFmI 12 tram expressing only very low amounts of pilus (i p immunisation, i v challenge with 6BFmlandl2 underexpressing pilus 7 3E+09 CFU/mouse) Both (A) and (B) also show data for a combination of spr0057, spr0096 and spr2021, a 6BFinland-CRM197 conjugate, and alum The data m (A) are CFU/ml and m (B) are survival time in days Figure 36 is an in silico analysis of the MLST database showing that, for a collection of 113 Acute Otitis Media S pneumoniae isolates, pilus-1 is more prevalent in strains that are resistant to antibiotics (erythromycm-resistance, penicillin-resistance and multiple-drug-resistance) compared to strains that are susceptible to antibiotics
DETAILED DESCRIPTION OF THE INVENTION
The RrgB pilus subumt has at least three clades Reference amino acid sequences for the three clades are SEQ ID NOs 1 , 2 and 3 herein The clades are well conserved at their N- and C-termim but deviate in between SEQ ID NOs 1 and 2 are 46% identical, SEQ ID NOs 1 and 3 are 51% identical, SEQ ID NOs 2 and 3 are 65% identical It has been found that serum raised against a given RrgB clade is active against pneumococci which express that clade, but is not active against strains which express one of the other two clades i e there is mtra-clade cross-protection, but not mter-clade cross-protection According to the invention, therefore, an immunogenic composition includes at least two different clades of RrgB These may be present m the immunogenic composition as separate polypeptides or may be fused as a single polypeptide chain The inclusion of multiple RrgB clades as vaccine components improves the strain coverage of the immunogenic composition against pilus-contammg pneumococci Furthermore, it has been observed that there is a significant association between pilus-1 presence and antibiotic resistance, this observation suggests that immunising against pilus-1 using an immunogenic composition including multiple RrgB clades will have the additional advantage of protecting against pneumococci that are resistant to antibiotic treatment
Thus the invention provides an immunogenic composition comprising at least two of
(a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence compπses an ammo acid sequence (i) having at least a% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least x contiguous ammo acids from SEQ ID NO 1 , (b) a second polypeptide, comprising a second ammo acid sequence, where the second ammo acid sequence comprises an ammo acid sequence (i) having at least b% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least y contiguous ammo acids from SEQ ID NO 2, and/or
(c) a third polypeptide, comprising a third ammo acid sequence, where the third ammo acid sequence comprises an ammo acid sequence (i) having at least c% sequence identity to SEQ ID NO 3 and/or (ii) consisting of a fragment of at least z contiguous ammo acids from SEQ ID NO 3
The invention also provides a polypeptide comprising at least two of (a) a first ammo acid sequence comprising an ammo acid sequence (i) having at least a% sequence identity to SEQ ID NO 1 and/or (n) consisting of a fragment of at least x contiguous ammo acids from SEQ ID NO 1,
(b) a second ammo acid sequence comprising an amino acid sequence (i) having at least b% sequence identity to SEQ ID NO 2 and/or (n) consisting of a fragment of at least y contiguous ammo acids from SEQ ID NO 2, and/or
(c) a third ammo acid sequence comprising an ammo acid sequence (i) having at least c% sequence identity to SEQ ID NO 3 and/or (ii) consisting of a fragment of at least z contiguous ammo acids from SEQ ID NO 3
The invention also provides a polypeptide comprising ammo acid sequence
-A-{-X-L-}B-B- wherem X is an ammo acid sequence of first polypeptide, second polypeptide or third polypeptide as defined above, L is an optional linker ammo acid sequence, A is an optional N-termmal ammo acid sequence, B is an optional C-terminal ammo acid sequence, n is an integer of 2 or more (e g 2, 3, 4, 5, 6, etc ) Optionally, the polypeptide compπses at least two of a first, second and third polypeptide as defined m claim 1 Usually n is 2 or 3, and X moieties are selected from the following
Figure imgf000008_0001
The invention also provides a cell (typically a bacterium, such as a pneumococcus) which expresses at least two of
(a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence compnses an ammo acid sequence (i) having at least a% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least x contiguous ammo acids from SEQ ID NO 1 ,
(b) a second polypeptide, composing a second ammo acid sequence, where the second ammo acid sequence compπses an ammo acid sequence (i) having at least b% sequence identity to SEQ ID NO 2 and/or (ii) consisting of a fragment of at least y contiguous ammo acids from SEQ ID NO 2, and/or (c) a third polypeptide, comprising a third ammo acid sequence, where the third ammo acid sequence compπses an ammo acid sequence (i) having at least c% sequence identity to SEQ ID NO 3 and/or (π) consisting of a fragment of at least z contiguous ammo acids from SEQ ID NO 3
The first, second and third amino acid sequences The value of a is at least 15 eg 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more The value of b is at least 75 eg 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more The value of c is at least 75 e g 80, 85, 90, 92, 94, 95, 96, 97, 98, 99 or more The values of a, b and c may be the same or different In some embodiments, a b and c are identical Typically, a, b and c axe at least 90 e g at least 95
The value ofx is at least 7 eg 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250) The value of/ is at least 7 eg 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,
45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250) The value of z is at least l e g 8, 9, 10,
1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 120, 140, 160, 180, 200, 225, 250) The values of x, y and z may be the same or different In some embodiments, x y and z are identical
Fragments preferably compπse an epitope from the respective SEQ ID NO sequence Other useful fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of the respective SEQ ID NO while retaining at least one epitope thereof Truncation by 20-25 ammo acids at the N-termmus is convenient e g removal of aa 1 -23 of any of SEQ ID NOs 1 to 3 (or of any one of SEQ ID NOs 85 to 96)
The RrgB protein can be split into four domains (Dl to D4) between its leader peptide and its LPXTG anchor These four domains are as follows in SEQ ID NOs 1 to 3, and the positions m further RrgB sequences which correspond to these residues can readily be identified by alignment
Figure imgf000009_0001
Based on passive protection studies, useful fragments of RrgB may retain epitopes from at least domains Dl and/or D4 As shown in Figure 20, antibodies have been raised that bind to domain Dl, domain D4 and a fragment containing domains D2 to D4 Accordingly, preferred fragments include domain Dl, domain D4 and a fragment containing domains D2 to D4 A suitable fragment of SEQ ID NO 1 is SEQ ID NO 4 A suitable fragment of SEQ ID NO 2 is SEQ ID NO 5 A suitable fragment of SEQ ID NO 3 is SEQ ID NO 6
The fragment of at least x contiguous ammo acids from SEQ ID NO 1 should not also be present withm SEQ ID NO 2 or withm SEQ ID NO 3 Similarly, the fragment of at least y contiguous ammo acids from SEQ ID NO 2 should not also be present withm SEQ ID NO 1 or withm SEQ ID NO 3 Similarly, the fragment of at least z contiguous ammo acids from SEQ ID NO 3 should not also be present withm SEQ ID NO 1 or withm SEQ ID NO 2 In some embodiments, therefore a fragment of SEQ ID NO 1 is preferably from between ammo acids 31 -614 of SEQ ID NO 1 , a fragment of SEQ ID NO 2 is preferably from between ammo acids 31-593 of SEQ ID NO 2, and a fragment of SEQ ID NO 3 is preferably from between ammo acids 31-603 of SEQ ID NO 3 The fragment of at least x contiguous ammo acids from SEQ ID NO 1 may also be present m any one of SEQ ID NOs 85, 88 and/or 89 Similarly, the fragment of at least y contiguous ammo acids from SEQ ID NO 2 may also be present in any one of SEQ ID NOs 86, 90, 91, 94 and/or 96 Similarly, the fragment of at least z contiguous ammo acids from SEQ ID NO 3 may also be present m any one of SEQ ID NOs 87, 92, 93 and/or 95 In some embodiments, when a fragment from one of SEQ ID NOs 1 to 3 is aligned as a contiguous sequence against the other two SEQ ID NOs, the identity between the fragment and each of the other two SEQ ID NOs is less than 75% e g less than 60%, less than 50%, less than 40%, less than 30% Based on epitope mapping studies, an epitope of SEQ ID NO 1 has been identified between residues 32 and 141 of SEQ ID NO 1, more specifically between residues 55 and 89 of SEQ ID NO 1 Useful fragments of SEQ ID NO 1 therefore include residues 32 to 141 of SEQ ID NO 1 and residues 55 to 89 of SEQ ID NO 1
A polypeptide comprising the first ammo acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 1 (strain TIGR4) In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 2 or to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 3
A polypeptide comprising the second ammo acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 2 (strain Finland68- 12) In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 1 or to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 3 A polypeptide comprising the third ammo acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild- type pneumococcus protein having ammo acid sequence SEQ ID NO 3 (strain Taiwan23F-15) In some embodiments these antibodies do not bind to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 1 or to the wild-type pneumococcus protein having ammo acid sequence SEQ ID NO 2
Although the first, second and third ammo acid sequences may share some sequences m common, overall they have different ammo acid sequences
Where the invention uses only two RrgB clades a composition or polypeptide can include both (a) a first ammo acid sequence as defined above, and (b) a second ammo acid sequence as defined above In an alternative embodiment the composition includes both (a) a first ammo acid sequence as defined above, and (b) a third ammo acid sequence as defined above In an alternative embodiment the composition includes both (a) a second ammo acid sequence as defined above, and (b) a third ammo acid sequence as defined above
Ammo acid sequences used with the invention, may, compared to SEQ ID NOs 1, 2 or 3, include one or more (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc ) conservative ammo acid replacements i e replacements of one ammo acid with another which has a i elated side chain Genetically-encoded ammo acids are generally divided into four families (1) acidic i e aspartate, glutamate, (2) basic i e lysine, argmine, histidine, (3) non-polar i e alanine, valine, leucine, isoleucme, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar i e glycine, asparagme, glutamine, cysteine, serine, threonine, tyrosine Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic ammo acids In general, substitution of single ammo acids within these families does not have a major effect on the biological activity The polypeptides may have one or more (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc ) single ammo acid deletions relative to a reference sequence The polypeptides may also include one or more (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc ) insertions (e g each of 1, 2, 3, 4 or 5 ammo acids) relative to a reference sequence A polypeptide used with the invention may comprise an ammo acid sequence that
(a) is identical (ι e 100% identical) to SEQ ID NO 1, 2 or 3,
(b) shares sequence identity SEQ ID NO 1, 2 or 3,
(c) has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more) single ammo acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of (a) or (b), and
(d) when aligned SEQ ID 1 , 2 or 3 using a pairwise alignment algorithm, each moving window of x ammo acids from N-termmus to C-termmus (such that for an alignment that extends to p ammo acids, where p>x, there are p-x+1 such windows) has at least xy identical aligned ammo acids, where x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, y is selected from 0 50, 0 60, 0 70. 0 75. 0 80, 0 85, 0 90, 0 91, 0 92, 0 93, 0 94, 0 95, 0 96, 0 97, 0 98, 0 99, and if x y is not an integer then it is rounded up to the nearest integer The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [5], using default parameters (e g with Gap opening penalty = 10 0, and with Gap extension penalty = 0 5, using the EBLOSUM62 scoring matπx) This algorithm is conveniently implemented in the needle tool in the EMBOSS package [6]
Withm group (c), deletions or substitutions may be at the N-termmus and/or C-termmus, or may be between the two termini Thus a truncation is an example of a deletion Truncations may involve deletion of up to 40 (or more) ammo acids at the N-termmus and/or C-terrmnus
In general, when a polypeptide of the invention comprises a sequence that is not identical to a complete pneumococcal sequence from SEQ ID NOs 1 to 3 (e g when it comprises a sequence listing with <100% sequence identity thereto, or when it compnses a fragment thereof), it is preferred m each individual instance that the polypeptide can elicit an antibody that recognises the complete pneumococcal sequence
For reference, SEQ ID NOs 1 to 3 and 85 to 96 are 15 unique RrgB sequences which have been identified in 45 different strains Any of these sequences can be used for implementing the invention Thus, for example a first polypeptide for use with the invention could comprise any one of SEQ ID NOs listed m group (1) below, a second polypeptide for use with the invention could comprise any one of SEQ ID NOs listed in group (2) below, and a third polypeptide for use with the invention could comprise any one of SEQ ID NOs listed in group (3) below Groups (1) to (3) are as follows
(1) SEQ ID NOs 1, 85, 88, 89
(2) SEQ ID NOs 2, 86, 90, 91, 94, 96
(3) SEQ ID NOs 3, 87, 92, 93, 95
Hybrid polypeptides
Different RrgB clades used m the invention do not have to be present as separate polypeptides but can instead be expressed as a single polypeptide chain (a 'hybrid' polypeptide or 'chimera') Hybπd polypeptides offer two mam advantages first, a polypeptide that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybπd partner that ovei comes the problem, second, commercial manufacture is simplified as only one expression and purification need to be employed in order to produce two polypeptides which are both antigenically useful
Hybrid polypeptides can include sequences from only RrgB antigens but in other embodiments can include non-RrgB antigens (usually pneumococcal non-RrgB antigens), such as other pilus subumts If non-RrgB antigens are present these may be to the N-termmus of any two RrgB sequences, to the C-termmus of any two RrgB sequences, or may be between two RrgB sequences
Different hybπd polypeptides may be mixed together m a single formulation Hybπds may be combined with non-hybrid RrgB antigens or other non-RrgB antigens
5 Hybrid polypeptides may be represented by the formula NH2-A- {-X-L-},,-B-COOH
If a -X- moiety has a leader peptide sequence m its wild-type form, this may be included or omitted m the hybπd protein Pn some embodiments, the leader peptides will be deleted except for that of the -X- moiety located at the N-termmus of the hybrid protein i e the leader peptide of Xi will be retained, but the leader peptides of X2 Xn will be omitted This is equivalent to deleting all leader peptides and0 using the leader peptide of X] as moiety -A-
For each n instances of {-X-L-}, linker ammo acid sequence -L- may be present or absent For instance, when «=2 the hybπd may be NH2-Xi-L1-X2-L2-COOH, NH2-XrX2-COOH, NH2-X1-Li-X2-COOH, NH2-Xi-X2-L2-COOH, etc Linker ammo acid sequence(s) -L- will typically be short (e g 20 or fewer ammo acids 1 e 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples compπse5 short peptide sequences which facilitate cloning, poly-glycme linkers (1 e compπsmg GIyn where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidme tags (1 e His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable linker ammo acid sequences will be apparent to those skilled in the art A useful linker is GSGGGG (SEQ ID NO 7) or GSGSGGGG (SEQ ID NO 8), with the Gly-Ser dφeptide being formed from a BamHl restπction site, thus aiding cloning and manipulation, and the (GIy)4 tetrapeptide being a
'0 typical poly-glycme linker Other suitable linkers, particularly for use as the final Ln are a Leu-Glu dipeptide or Gly-Ser Linkers will usually contain at least one glycine residue to facilitate structural flexibility eg a -L- moiety may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycine residues Such glycines may be arranged to include at least two consecutive glycines m a Gly-Gly dipeptide sequence, or a longer oligo-Gly sequence 1 e GIyn where n = 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
5 -A- is an optional N-termmal ammo acid sequence This will typically be short (e g 40 or fewer ammo acids i e 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e g histidme tags 1 e His,, where n = 3, 4, 5, 6, 7, 8, 9, 10 or more) Other suitable N-termmal ammo acid sequences will be d apparent to those skilled in the art If Xi lacks its own N-termmus methionine, -A- is preferably an oligopeptide (e g with 1, 2, 3, 4, 5, 6, 7 or 8 ammo acids) which provides a N-termmus methionine e g Met-Ala-Ser, or a single Met residue In a nascent polypeptide the -A- moiety can provide the polypeptide's N-termmal methionine (formyl-methionme, fMet, m bacteria) One or more ammo acids may be cleaved from the N-termmus of a nascent -A- moiety, however, such that the -A- moiety m a mature polypeptide of the invention does not necessanly include a N-termmal methionine
-B- is an optional C-termmal ammo acid sequence This will typically be short (e g 40 or fewer ammo acids i e 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e g comprising histidme tags i e HiSn where n = 3, 4, 5, 6, 7, 8, 9, 10 or more, such as SEQ ID NO 9), or sequences which enhance protein stability Other suitable C-termmal ammo acid sequences will be apparent to those skilled in the art, such as a glutathione-S-transferase, thioredoxm, 14kDa fragment of S aureus protein A, a biotmylated peptide, a0 maltose-bmdmg protein, an enterokmase flag, etc
It is preferred that -A-, -B- and -L- sequences do not include a sequence that shares 10 or more contiguous amino acids in common with a human polypeptide sequence
In some embodiments, a -L- moiety comprises a non-RrgB antigen In some embodiments, the -A- moiety compnses a non-RrgB antigen, and in some the -B- moiety comprises a non-RrgB antigen 5 The invention also provides nucleic acid which encodes a hybπd polypeptide of the invention Of the various A, B, X, and L moieties, useful combinations include, but are not limited to
Figure imgf000014_0001
* Number indicates SEQ ID NO
Thus examples of hybπds of the invention include polypeptides comprising an ammo acid sequence selected from the group consisting of SEQ ID NO 11 (encoded by SEQ ID NO 12), SEQ ID NO 13O (encoded by SEQ ID NO 14), SEQ ID NO 15 (encoded by SEQ ID NO 16), SEQ ID NO 17 (encoded by SEQ ID NO 18), SEQ ID NO 19 (encoded by SEQ ID NO 20), SEQ ID NO 21 (encoded by SEQ ID NO 22)
The invention provides a polypeptide comprising an ammo acid sequence having at least ι% sequence identity to any one of SEQ ID NOs 11 , 13, 15, 17, 19 or 21 The value of i may be selected from 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99 or more Polypeptides
Polypeptides used with the invention can be prepared in many ways e g by chemical synthesis (in whole or in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e g from recombinant expression), from the organism itself (e g after bacteπal 5 culture, or direct from patients), etc A preferred method for production of peptides <40 ammo acids long involves in vitro chemical synthesis [7,8] Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [9] chemistry Enzymatic synthesis [10] may also be used in part or m full As an alternative to chemical synthesis, biological synthesis may be used e g the polypeptides may be produced by translation This may be earned out in vitro or in vivo Biological methods are in general 10 restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e g of ammoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural ammo acids, such as iodotyrosme or methylphenylalanme, azidohomoalanme, etc ) [11] Where D-ammo acids are included, however, it is preferred to use chemical synthesis Polypeptides may have covalent modifications at the C-termmus and/or N-termmus
15 Polypeptides can take various forms (e g native, fusions, glycosylated, non-glycosylated, lipi dated, non-hpidated, phosphorylated, non-phosphorylated, myristoylated, non-myπstoylated, monomeπc, multimeπc, particulate, denatured, etc )
Polypeptides are preferably provided in purified or substantially purified form i e substantially free from other polypeptides (e g free from naturally-occurring polypeptides), particularly from other 0 pneumococcal or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i e less than about 50%, and more preferably less than about 10% (e g 5% or less) of a composition is made up of other expressed polypeptides
Polypeptides may be attached to a solid support Polypeptides may compπse a detectable label (e g a radioactive or fluorescent label, or a biotin label)
.5 The term "polypeptide" refers to ammo acid polymers of any length The polymer may be linear or branched, it may compπse modified ammo acids, and it may be interrupted by non-ammo acids The terms also encompass an ammo acid polymer that has been modified naturally or by intervention, for example, disulfide bond formation, glycosylation, hpidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component Also included withm the
Ϊ0 definition are, for example, polypeptides containing one or more analogs of an ammo acid (including, for example, unnatural ammo acids, etc ), as well as other modifications known in the art Polypeptides can occur as single chains or associated chains Polypeptides can be naturally or non-naturally glycosylated (ι e the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found m the coπespondmg naturally occurring polypeptide) The invention provides a process for producing polypeptides of the invention, comprising cultunng a host cell of to the invention under conditions which induce polypeptide expression Although expression of the polypeptide may take place in a Streptococcus, the invention will usually use a heterologous host for expression The heterologous host may be prokaryotic (e g & bacteπum) or eukaryotic It will usually 5 be E coli, but other suitable hosts include Bacillus subtihs, Vibrio cholerae, Salmonella typhi, Salmonella typhimunum, Neissena lactamica, Neisseria cinerea, Mycobacteria {e g M tuberculosis), yeasts, etc
The invention also provides a process for producing a polypeptide of the invention, wherein the polypeptide is synthesised m part or in whole using chemical means
10 The invention also provides a composition comprising two or more polypeptides of the invention
Nucleic acids
The invention also provides a nucleic acid comprising a nucleotide sequence encoding a hybπd polypeptide of the invention The invention also provides nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences Such nucleic acids include those using 15 alternative codons to encode the same ammo acid
The invention also provides nucleic acid which can hybridize to these nucleic acids Hybridization reactions can be performed under conditions of different 'stringency" Conditions that increase stringency of a hybridization ieaction of widely known and published in the art Examples of relevant conditions include (m order of increasing stringency) incubation temperatures of 25°C, 370C, 5O0C,
20 55°C and 68°C, buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0 1 x SSC (where SSC is 0 15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems, formamide concentrations of 0%, 25%, 50%, and 75%, incubation times from 5 minutes to 24 hours, 1 , 2, or more washing steps, wash incubation times of 1, 2, or 15 minutes, and wash solutions of 6 x SSC, 1 x SSC, 0 1 x SSC, or de- ionized water Hybridization techniques and their optimization are well known in the
>5 art [e g see refs 12 & 239, etc ]
The invention includes nucleic acid comprising sequences complementary to these sequences (e g for antisense or probing, or for use as primers)
Nucleic acid according to the invention can take various forms (e g smgle-stranded, double-stranded, vectors, primers, probes, labelled etc ) Nucleic acids of the invention may be circular or branched, but >0 will generally be linear Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary smgle- stranded forms which make up the double- stranded form Primers and probes are generally smgle- stranded, as are antisense nucleic acids Nucleic acids of the invention are preferably provided in purified or substantially purified form i e substantially free from other nucleic acids (e g free from naturally-occumng nucleic acids), particularly from other pneumococcal or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90% pure Nucleic acids of the invention are preferably pneumococcal nucleic 5 acids
Nucleic acids of the invention may be prepared in many ways e g by chemical synthesis {e g phosphoramidite synthesis of DNA) m whole or in part, by digesting longer nucleic acids using nucleases {e g restπction enzymes), by joining shorter nucleic acids or nucleotides {e g using ligases or polymerases), from genomic or cDNA libraries, etc 0 Nucleic acid of the invention may be attached to a solid support (e g a bead, plate, filter, film, slide, microarray support, resin, etc ) Nucleic acid of the invention may be labelled e g with a radioactive or fluorescent label, or a biotin label This is particularly useful where the nucleic acid is to be used in detection techniques e g where the nucleic acid is a pπmer or as a probe
The term "nucleic acid" includes in general means a polymeπc form of nucleotides of any length, which 5 contain deoxyπbonucleotides, ribonucleotides, and/or their analogs It includes DNA, RNA, DNA/RNA hybrids It also includes DNA or RNA analogs, such as those containing modified backbones {e g peptide nucleic acids (PNAs) or phosphorothioates) or modified bases Thus the invention includes mRNA, tRNA, rRNA, πbozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc Where nucleic acid of the invention takes the form of RNA, it
.0 may or may not have a 5' cap
Nucleic acids of the invention may be part of a vector i e part of a nucleic acid construct designed for transduction/transfection of one or more cell types Vectors may be, for example, "cloning vectors" which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors" which are designed for expression of a nucleotide sequence m a host cell, "viral vectors" which
'5 is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attπbutes of more than one type of vector Preferred vectors are plasmids A "host cell" includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural,
0 accidental, or deliberate mutation and/or change Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention Where a nucleic acid is DNA, it will be appreciated that "U" in a RNA sequence will be replaced by "T" in the DNA Similarly, where a nucleic acid is RNA, it will be appreciated that "T" m a DNA sequence will be replaced by "U" in the RNA
The term "complement" or "complementary" when used in relation to nucleic acids refers to Watson- 5 Crick base paiπng Thus the complement of C is G, the complement of G is C, the complement of A is T (or U), and the complement of T (or U) is A It is also possible to use bases such as I (the purine mosme) e g to complement pyrimidmes (C or T)
Nucleic acids of the invention can be used, for example to produce polypeptides in vitro or in vivo, as hybridization probes for the detection of nucleic acid in biological samples, to generate additional copies0 of the nucleic acids, to generate ribozymes or antisense oligonucleotides, as single- stranded DNA primers or probes, or as triple-strand forming oligonucleotides
The invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesised in part or in whole using chemical means
The invention provides vectors comprising nucleotide sequences of the invention {e g cloning or 5 expression vectors) and host cells transformed with such vectors
Immunogenic compositions
Mixtures and hybrid polypeptides of the invention are useful as active ingredients in immunogenic compositions Such immunogenic compositions may be useful as vaccines These vaccines may either be piophylactic (ι e to prevent infection) or therapeutic (ι e to treat infection), but will typically beO prophylactic
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 carπer(s) and/or excipient(s) A thorough discussion of such components is available m reference 234
Compositions will generally be administered to a mammal in aqueous form Pπor to administration, 5 however, the composition may have been m a non-aqueous form For instance, although some vaccines are manufactured m aqueous form, then filled and distπbuted and administered also in aqueous form, other vaccines aie lyophilised during manufacture and are reconstituted mto 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, d however, that the vaccine should be substantially free from (ι 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 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 mclude potassium chloride, potassium dihydrogen phosphate, disodmm phosphate dehydrate, magnesium chloπde, calcium chloπde, 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 withm the range of 290-310 mOsm/kg
Compositions may mclude one or more buffers Typical buffers include a phosphate buffer, a Tπs buffer, a borate buffer, a succinate buffer, a histidme buffer (particularly with an aluminum hydroxide adjuvant), or a citrate buffei Buffers will typically be included in the 5-2OmM 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-pyrogemc 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 mclude mateπal for a single immunisation, or may include material for multiple immunisations (ι e a 'multidose' kit) The inclusion of a preservative is prefeπed in multidose arrangements As an alternative (or m addition) to including a preservative m multidose compositions, the compositions may be contained m a container having an aseptic adaptor for removal of mateπal
Human vaccines are typically administered in a dosage volume of about 0 5ml, although a half dose (ι 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 mclude one or more adjuvants, for example two, three, four or more adjuvants The adjuvants may mclude a THl adjuvant and/or a TH2 adjuvant, further discussed below Adjuvants which may be used m compositions of the invention mclude, but are not limited to
A Minei al-containing compositions
Mineral containing compositions suitable for use as adjuvants in the invention mclude mineral salts, such as aluminium salts and calcium salts The invention includes mineral salts such as hydroxides {e g oxyhydroxides), phosphates (e g hydroxyphosphates, orthophosphates), sulphates, etc [e g see chapters ) 8 & 9 of ref 13], or mixtures of different mineral compounds, with the compounds taking any suitable form (e g gel, crystalline, amorphous, etc ), and with adsorption being preferred The mineral containing compositions may also be formulated as a particle of metal salt The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline Aluminium oxyhydroxide, which can be represented by the formula AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH)3, by infrared (IR) spectroscopy, m particular by the presence of an adsorption band at 5 1070cm ' and a strong shoulder at 3090-3100cm ' [chapter 9 of ref 13] The degree of crystallimty of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystallme particles showing greater lme broadening due to smaller crystallite sizes The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption A fibrous morphology (e g as seen in transmission electron 0 micrographs) is typical for aluminium hydroxide adjuvants The pi of aluminium hydroxide adjuvants is typically about 11 i e the adjuvant itself has a positive surface charge at physiological pH Adsorptive capacities of between 1 8-2 6 mg protein per mg Al+++ at pH 7 4 have been reported for aluminium hydroxide adjuvants
The adjuvants known as "aluminium phosphate" are typically aluminium hydroxyphosphates, often also 5 containing a small amount of sulfate (ι e aluminium hydroxyphosphate sulfate) They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt Hydroxyphosphates generally have a PO4/ Al molar ratio between 0 3 and 1 2 Hydroxyphosphates can be distinguished from strict AlPO4 by the presence of hydroxyl groups For example, an IR spectrum band at 3164cm ' (e g when heated to 2000C) indicates
.0 the presence of structural hydroxyls [ch 9 of ref 13]
The PO4ZAl3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0 3 and 1 2, preferably between 0 8 and 1 2, and more preferably 0 95+0 1 The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts A typical adjuvant is amorphous aluminium hydroxyphosphate with PO4/AI molar ratio between 0 84 and 0 92, included at 0 6mg Al3+AnI The '5 aluminium phosphate will generally be particulate (e g plate-like morphology as seen m transmission electron micrographs) Typical diameters of the particles are in the range 0 5-20μm (e g about 5-10μm) after any antigen adsorption Adsorptive capacities of between 0 7-1 5 mg protein per mg Al+++ at pH 7 4 have been reported for aluminium phosphate adjuvants
The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution 0 of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PZC) or by adding a buffer such as a histidme buffer (makes PZC more basic) Aluminium phosphates used according to the invention will generally have a PZC of between 4 0 and 7 0, more preferably between 5 0 and 6 5 β g about 5 7
Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e g a phosphate or a histidme or a Tπs buffer), but this is not always necessary The suspensions are 5 preferably steπle and pyrogen- free A suspension may include free aqueous phosphate ions e g present at a concentration between 1 0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM The suspensions may also compπse sodium chloπde
In one embodiment, an adjuvant component includes a mixture of both an aluminium hydroxide and an aluminium phosphate In this case there may be more aluminium phosphate than hydroxide e g a weight0 ratio of at least 2 1 e g >5 1, >6 1, >7 1, >8 1, >9 I, etc
The concentration of Al+++ m a composition for administration to a patient is pieferably less than 10mg/ml e g <5 mg/ml, <4 mg/ml, <3 mg/ml, <2 mg/ml, <1 mg/ml, etc A preferred range is between 0 3 and lmg/ml A maximum of <0 85mg/dose is preferred
B Oil Emulsions 5 Oil emulsion compositions suitable for use as adjuvants m the invention include squalene- water emulsions, such as MF59 [Chapter 10 of ref 13, see also ref 14] (5% Squalene, 0 5% Tween 80, and 0 5% Span 85, formulated into submicron particles using a microfluidizer) Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used
Various suitable oin-in- water emulsions are know n, and they typically include at least one oil and at least0 one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabohsable) and biocompatible The oil droplets in the emulsion are generally less than 5μm in diameter, and advantageously the emulsion comprises oil droplets with a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization
'5 The invention can be used with oils such as those from an animal (such as fish) or vegetable source Sources for vegetable oils include nuts, seeds and grams Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils Jojoba oil can be used e g obtained from the jojoba bean Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like In the gram group, corn oil is the most readily available, but the oil of other cereal grams such as
0 wheat, oats, rye, rice, teff, tπticale and the like may also be used 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, while not occurring naturally m seed oils, may be prepaied by hydrolysis, separation and esteπfϊcation of the appropriate materials starting from the nut and seed oils Fats and oils from mammalian milk are metabohzable and may therefore be used in the practice of this invention The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from ammal sources are well known in the art Most fish contain metabohzable oils which may be readily recovered For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein A number of branched chain oils are synthesized biochemically in 5 -carbon isoprene units and are generally referred to as terpenoids Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10, 15, 19,23-hexamethyl- 2,6,10,14,18,22-tetracosahexaene Other preferred oils are the tocopherols (see below) Oil in water emulsions comprising sqlauene are particularly preferred Mixtures of oils can be used
Surfactants can be classified by their 'HLB' (hydrophile/hpophile balance) Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16 The invention can be used with surfactants including, but not limited to the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80, copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWF AX™ tradename, such as linear EO/PO block copolymers, octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypolyethoxyethanol) being of particular interest, (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40), phospholipids such as phosphatidylcholine (lecithin), polyoxyethylene fatty ethers deπved from lauryl, cetyl, steal yl and oleyl alcohols (known as Bπj surfactants), such as tπethyleneglycol monolauryl ether (Bπj 30), and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate Preferred surfactants for including m the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-IOO As mentioned above, detergents such as Tween 80 may contribute to the thermal stability seen in the examples below
Mixtures of surfactants can be used e g Tween 80/Span 85 mixtures A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable Another useful combination compπses laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol
Preferred amounts of surfactants (% by weight) are polyoxyethylene sorbitan esters (such as Tween 80) 0 01 to 1%, m particular about 0 1 %, octyl- or nonylphenoxy polyoxyethanols (such as Tπton X-100, or other detergents in the Tπton series) 0 001 to 0 1 %, in particular 0 005 to 0 02%, polyoxyethylene ethers (such as laureth 9) 0 1 to 20 %, preferably 0 1 to 10 % and m particular 0 1 to 1 % or about 0 5%
Specific oil-m-water emulsion adjuvants useful with the invention include, but are not limited to
• A submicron emulsion of squalene, Tween 80, and Span 85 The composition of the emulsion by volume can be about 5% squalene, about 0 5% polysorbate 80 and about 0 5% Span 85 In weight terms, these ratios become 4 3% squalene, 0 5% polysorbate 80 and 0 48% Span 85 This adjuvant is known as 'MF59' [15-17], as descπbed m more detail m Chapter 10 of ref 18 and chapter 12 of ref 19 The MF59 emulsion advantageously includes citrate ions e g 1OmM sodium citrate buffer
• An emulsion comprising squalene, an α-tocopherol, and polysorbate 80 These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0 3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably <1 (e g 0 90) as this provides a more stable emulsion Squalene and Tween 80 may be present volume ratio of about 5 2, or at a weight ratio of about 1 1 5 One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL-α-tocopherol and 5ml squalene), then micro fluidismg the mixture The resulting emulsion may have submicron oil droplets e g with an average diameter of between 100 and 250nm, preferably about 180nm
• An emulsion of squalene, a tocopherol, and a Triton detergent (e g Tnton X-100) The emulsion may also include a 3d-MPL (see below) The emulsion may contain a phosphate buffer
• An emulsion comprising a polysorbate (e g polysorbate 80), a Tnton detergent (e g Tnton X-100) and a tocopherol (e g an α-tocopherol succinate) The emulsion may include these three components at a mass ratio of about 75 11 10 (e g 750μg/ml polysorbate 80, l lOμg/ml Triton X- 100 and lOOμg/ml α-tocopherol succinate), and these concentrations should include any contribution of these components from antigens The emulsion may also include squalene The emulsion may also include a 3d-MPL (see below) The aqueous phase may contain a phosphate buffer
• An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluromc™ L121") The emulsion can be formulated in phosphate buffered saline, pH 7 4 This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-I" adjuvant [20] (0 05-1% Thr-MDP, 5% squalane, 2 5% Pluromc Ll 21 and 0 2% polysorbate 80) It can also be used without the Thr-MDP, as in the "AF" adjuvant [21] (5% squalane, 1 25% Pluromc L121 and
0 2% polysorbate 80) Microfluidisation is preferred
• An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophihc nonionic surfactant (e g polyoxyethylene (12) cetostearyl ether) and a hydrophobic noniomc surfactant (e g a sorbitan ester or manmde ester, such as sorbitan monoleate or 'Span 80') The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [22] The emulsion may also include one or more of alditol, a cryoprotective agent (e g a sugar, such as dodecylmaltoside and/or sucrose), and/or an alkylpolyglycoside Such emulsions may be lyophihzed • An emulsion having from 0 5-50% of an oil, 0 1-10% of a phospholipid, and 0 05-5% of a non-ionic surfactant As descπbed m reference 23, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamme, phosphatidylseπne, phosphatidylmositol, phosphatidylglycerol, phosphatide acid, sphingomyelin and cardiohpm Submicron droplet sizes are advantageous
• A submicron oil-m-water emulsion of a non-metabohsable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80) Additives may be included, such as QuilA saponin, cholesterol, a saponm-lipophile conjugate (such as GPI-0100, descπbed m reference 24, produced by addition of aliphatic amine to desacylsapomn via the carboxyl group of glucuronic acid), dimethyidioctadecylammomum bromide and/or N,N-dioctadecyl-N,N-bis (2- hydroxyethyl)proρanediamme β An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-iomc hydrophilic surfactant (e g an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [25] • An emulsion comprising a mineral oil, a non-iomc hydrophilic ethoxylated fatty alcohol, and a non-iomc lipophilic surfactant (e g an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [25]
• An emulsion in which a saponin (e g QuilA or QS21) and a sterol (e g a cholesterol) are associated as helical micelles [26] Antigens and adjuvants m a composition will typically be in admixture at the time of delivery to a patient The emulsions may be mixed with antigen during manufacture, or extemporaneously, at the time of delivery Thus the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use The antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids The volume ratio of the two liquids for mixing can vary (e g between 5 1 and 1 5) but is generally about 1 1
C Saponin formulations [chapter 22 ofref 13]
Saponin formulations may also be used as adjuvants m the invention Saponins are a heterogeneous group of sterol glycosides and tπterpenoid glycosides that are found m the bark, leaves, stems, roots and even flowers of a wide range of plant species Saponin from the bark of the Quύlaia saponana Molma tree have been widely studied as adjuvants Saponin can also be commercially obtained from Smilax ornata (sarsapπlla), Gypsophilla panicnlata (brides veil), and Saponana officianalis (soap root) Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs QS21 is marketed as Stimulon™ Saponin compositions have been purified using HPLC and RP-HPLC Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C Preferably, the saponin is QS21 A method of production of QS21 is disclosed in ref 27 Saponin formulations may also compπse a sterol, such as cholesterol [28] Combinations of saponins and cholesterols can be used to form unique particles called immunostimulatmg complexs (ISCOMs) [chapter 23 of ref 13] ISCOMs typically also include a phospholipid such as phosphatidylethanolamme or phosphatidylcholine Any known saponin can be used in ISCOMs Preferably, the ISCOM includes one or more of QuilA, QHA & QHC ISCOMs are further descπbed in refs 28-30 Optionally, the ISCOMS may be devoid of additional detergent [31] A review of the development of saponin based adjuvants can be found in refs 32 & 33
D Virosomes and virus-like particles
Virosomes and virus-like particles (VLPs) can also be used as adjuvants in the invention These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid They are generally non-pathogenic, non-replicatmg and generally do not contain any of the native viral genome The viral proteins may be recombmantly produced or isolated from whole viruses These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Smdbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi) VLPs are discussed further m refs 34-39 Virosomes are discussed further m, for example, ref 40
E Bacterial or microbial derivatives
Adjuvants suitable for use m the invention include bacteπal or microbial derivatives such as non-toxic deπvatives of enterobacterial lipopolysacchaπde (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-πbosylatmg toxins and detoxified deπvatives thereof
Non-toxic deπvatives of LPS include monophosphoryl lipid A (MPL) and 3-0-deacylated MPL (3dMPL) 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains A preferred "small particle" foπn of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref 41 Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0 22μm membrane [41] Other non-toxic LPS deπvatives include monophosphoryl lipid A mimics, such as ammoalkyl glucosamimde phosphate deπvatives e g RC-529 [42,43]
Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM- 174 OM- 174 is described for example m refs 44 & 45 Immunostimulatory oligonucleotides suitable for use as adjuvants m the invention include nucleotide sequences containing a CpG motif (a dmucleotide sequence containing an unmethylated cytosme linked by a phosphate bond to a guanosme) Double- stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory
5 The CpG' s can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single- stranded References 46, 47 and 48 disclose possible analog substitutions e g replacement of guanosme with 2'-deoxy-7-deazaguanosme The adjuvant effect of CpG oligonucleotides is further discussed in refs 49-54
The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [55] The CpG0 sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN CpG-A and CpG-B ODNs are discussed in refs 56-58 Preferably, the CpG is a CpG-A ODN
Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form5 "mimunomers" See, for example, refs 55 & 59-61
A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-31™ [62] Thus an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e g between 15-40 nucleotides) including at least one (and preferably multiple) CpI motifs (z e a cytosme linked to an inosine to form a dmucleotide), and (π) a polycationic polymer, such as an oligopeptide (e g0 between 5-20 ammo acids) including at least one (and preferably multiple) Lys-Arg-Lys tπpeptide sequence(s) The oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5'-(IC)i3-3' (SEQ ID NO 80) The polycationic polymer may be a peptide comprising 11-mer ammo acid sequence KLKLLLLLKLK (SEQ ID NO 81)
Bacterial ADP-πbosylatmg toxins and detoxified deπvatives thereof may be used as adjuvants m the
'5 invention Preferably, the protein is deπved from E cob (E coli heat labile enterotoxin "LT"), cholera
("CT"), or pertussis ("PT") The use of detoxified ADP-πbosylatmg toxins as mucosal adjuvants is described m rel 63 and as parenteral adjuvants in ref 64 The toxin or toxoid is preferably in the form of a holotoxm, comprising both A and B subunits Preferably, the A subunit contains a detoxifying mutation, preferably the B subunit is not mutated Preferably, the adjuvant is a detoxified LT mutant
0 such as LT-K63, LT-R72, and LT-G192 The use of ADP-πbosylatmg toxms and detoxified deπvatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found m refs 65-72 A useful CT mutant is or CT-E29H [73] Numeπcal reference for ammo acid substitutions is preferably based on the alignments of the A and B subumts of ADP-πbosylatmg toxins set forth in ref 74, specifically incorporated herein by reference m its entirety
F_ Human immunomodulators
Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as mterleukms (e g IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL- 12 [75], etc ) [76], interferons (e g mterferon-γ), macrophage colony stimulating factor, and tumor necrosis factor A preferred immunomodulator is IL-
12
G Bioadhesives and Miicoadhesives
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention Suitable bioadhesives include esteπfied hyaluronic acid microspheres [77] or mucoadhesives such as cross-linked deπvatives of poly(acryhc acid), polyvinyl alcohol, polyvinyl pyrolhdone, polysaccharides and carboxymethylcellulose Chitosan and deπvatives thereof may also be used as adjuvants in the invention [78]
H Microparticles Microparticles may also be used as adjuvants m the invention Microparticles (ι e a particle of -lOOnm to ~150μm m diameter, more preferably ~200nm to ~30μm in diameter, and most preferably ~500nm to ~10μm m diameter) formed from materials that are biodegradable and non-toxic (e g a poly(α-hydroxy acid), a polyhydroxybutyπc acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc ), with poly(lactide-co-glycohde) are preferred, optionally treated to have a negatively-charged surface (e g with SDS) or a positively-charged surface (e g with a catiomc detergent, such as CTAB)
/ Liposomes (Chapters 13 & 14 of ref 13)
Examples of liposome formulations suitable for use as adjuvants are described m refs 79-81
J_ Polyoxyethylene ether and poly oxy ethylene ester formulations
Adjuvants suitable for use m the invention include polyoxyethylene ethers and polyoxyethylene esters [82] Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [83] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-iomc surfactant such as an octoxynol [84] Preferred polyoxyethylene ethers are selected from the following group polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, ρolyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether
K Polyphosyhazene (PCPP)
PCPP formulations are descπbed, for example, m refs 85 and 86 L Muramyl peptides
Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl- L-threonyl-D-isoglutamme (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamme (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutammyl-L-alanme-2-(r-2'-dipalmitoyl-5«-glycero-3- hydroxyphosphoryloxy)-ethylamme MTP-PE)
M Imidazoqinnolone Compounds
Examples of lmidazoqumolone compounds suitable for use adjuvants in the invention include
Imiquamod and its homologues (e g "Resiquimod 3M"), descπbed further m refs 87 and 88
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-m-watei emulsion [89], (2) a saponin (e g QS21) + a non-toxic LPS derivative (e g 3dMPL)
[90], (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) [91], (5) combinations of 3dMPL with, for example, QS21 and/or oil-m- water emulsions [92], (6) SAF, containing 10% squalane, 0 4% Tween 80™, 5% pluronic-block polymer L121, and thr-MDP, either miciofluidized 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), tiehalose 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 immunostimulatmg agents are disclosed m chapter 7 of ref 13
The use of an aluminium hydroxide and/or aluminium phosphate adjuvant is useful, particularly m children, and antigens are generally adsorbed to these salts Squalene-m-water emulsions are also preferred, particularly m the elderly Useful adjuvant combinations include combinations of ThI and Th2 adjuvants such as CpG & alum or resiquimod & alum A combination of aluminium phosphate and 3dMPL may be used
The compositions of the invention may elicit both a cell mediated immune response as well as a humoral immune response
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 aie commonly referred to as Cytotoxic T lymphocytes (CTLs) CD8 T cells are able to lecogmzed 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 phenotypes which differ in their cytokine and effector function
Activated THl cells enhance cellular immunity (including an increase m 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 m responding to extracellular infections Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-IO A TH2 immune response may result m the production of IgG 1 , IgE, IgA and memory B cells for future protection
An enhanced immune response may include one or more of an enhanced THl immune iesponse and a TH2 immune response
A THl immune response may include one or more of an increase in CTLs, an increase m one or more of the cytokines associated with a THl immune response (such as IL-2, IFN-γ, and TNF-β), an increase in activated macrophages, an increase m NK activity, or an increase m the production of IgG2a Preferably, the enhanced THl immune response will include an increase m IgG2a production
A THl immune response may be elicited using a THl adjuvant A THl adjuvant will generally elicit increased levels of IgG2a production relative to immunization of the antigen without adjuvant THl adjuvants suitable for use in the invention may include for example saponin formulations, virosomes and virus like particles, non-toxic deπvatives of enterobacterial lipopolysacchaπde (LPS), immuno stimulatory oligonucleotides Immunostimulatory oligonucleotides, such as oligonucleotides containing a CpG motif, are preferred THl adjuvants for use in the invention
A TH2 immune response may include one or more of an increase m 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 m the production of IgG 1 , IgE, IgA and memory B cells Preferably, the enhanced TH2 immune response 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 m the invention include, for example, mineral containing compositions, oil- emulsions, and ADP-πbosylatmg toxms and detoxified deπvatives thereof Mineral containing compositions, such as aluminium salts are preferred TH2 adjuvants for use m the invention
A composition may include a combination of a THl adjuvant and a TH2 adjuvant Preferably, such a composition elicits an enhanced THl 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 THl and a TH2 adjuvant elicits an increased THl and/or an increased TH2 immune response relative to immunization with a single adjuvant (z e , relative to immunization with a THl adjuvant alone or immunization with a TH2 adjuvant alone)
The immune response may be one or both of a THl immune response and a TH2 response Preferably, immune response provides for one or both of an enhanced THl 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 m the production of IgA
Pneumococcal infections can affect various areas of the body and so the compositions of the invention may be prepared in vaπous forms For example, the compositions may be prepared as mjectables, 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 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 pπor 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 pπor to injection Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), 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 seπes, 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 pπmate, pπmate, 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
Nucleic acid immunisation
The immunogenic compositions described above include polypeptide antigens from S pneumoniae In all cases, however, the polypeptide antigens can be replaced by nucleic acids (typically DNA) encoding those polypeptides, to give compositions, methods and uses based on nucleic acid immunisation [93 to 100 The nucleic acid encoding the immunogen is expressed in vivo after delivery to a patient and the expressed immunogen then stimulates the immune system The active ingredient will typically take the form of a nucleic acid vector comprising (i) a promotei, (u) a sequence encoding the immunogen, operably linked to the promoter, and optionally (in) a selectable marker Preferred vectois may further compπse (iv) an oπgin of replication, and (v) a transcription terminator downstream of and operably linked to (n) In general, (i) & (v) will be eukaryotic and (in) & (iv) will be prokaryotic
Preferred promoters are viral promoters e g from cytomegalovirus (CMV) The vector may also include transcriptional regulatory sequences (e g enhancers) in addition to the promoter and which interact functionally with the promoter Preferred vectors include the immediate-early CMV enhancer/promoter, and more preferred vectors also include CMV intron A The promoter is operably linked to a downstream sequence encoding an immunogen, such that expression of the lmmunogen-encodmg sequence is under the promoter's control
Where a marker is used, it preferably functions m a microbial host (e g m a prokaryote, m a bacteria, m a yeast) The marker is preferably a prokaryotic selectable marker (e g transcribed under the control of a prokaryotic promoter) For convenience, typical markers are antibiotic resistance genes The vector is preferably an autonomously replicating episomal or extrachromosomal vector, such as a plasmid
The vector preferably comprises an origin of replication It is preferred that the origin of replication is active m prokaryotes but not m eukaryotes Preferred vectors thus include a prokaryotic marker for selection of the vector, a prokaryotic origin of replication, but a eukaryotic promoter for driving transcπption of the immunogen-encodmg sequence The vectors will therefore (a) be amplified and selected in prokaryotic hosts without polypeptide expression, but (b) be expressed m eukaryotic hosts without being amplified This arrangement is ideal for nucleic acid immunization vectors
The vector may compπse a eukaryotic transcnptional terminator sequence downstream of the coding sequence This can enhance transcπption levels Where the coding sequence does not have its own, the vector preferably comprises a polyadenylation sequence A preferred polyadenylation sequence is from bovme growth hormone The vector may compπse a multiple cloning site
In addition to sequences encoding the immunogen and a marker, the vector may compπse a second eukaryotic coding sequence The vector may also comprise an IRES upstream of said second sequence in order to permit translation of a second eukaryotic polypeptide from the same transcπpt as the immunogen Alternatively, the lmmunogen-codmg sequence may be downstream of an IRES The vector may compπse unmethylated CpG motifs e g unmethylated DNA sequences which have m common a cytosme preceding a guanosme, flanked by two 5' purines and two 3' pyπmidmes In their unmethylated form these DNA motifs have been demonstrated to be potent stimulators of several types of immune cell
Vectors may be delivered in a targeted way Receptor-mediated DNA delivery techniques are described in, for example, references 101 to 106 Therapeutic compositions containing a nucleic acid are administered m a range of about lOOng to about 200mg of DNA for local administration m a gene therapy protocol Concentration ranges of about 500 ng to about 50 mg, about lμg to about 2 mg, about
5μg to about 500μg, and about 20μg to about lOOμg of DNA can also be used during a gene therapy protocol Factors such as method of action (e g for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy Where greater expression is desired over a larger area of tissue, larger amounts of vector or the same amounts re-admmistered m a successive protocol of administrations, or several administrations to different adjacent or close tissue portions may be required to effect a positive therapeutic outcome In all cases, routine experimentation m clinical trials will determine specific ranges for optimal therapeutic effect
Vectors can be delivered using gene delivery vehicles The gene delivery vehicle can be of viral or non- vii al oπgm (see generally references 107 to 1 10) Viral-based vectors for delivery of a desired nucleic acid and expression in a desired cell are well known in the art Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (e g references 111 to 121), alphavirus-based vectors (e g Smdbis virus vectors, Semliki forest virus (ATCC VR-67, ATCC VR- 1247), Ross River virus (ATCC VR-373, ATCC VR- 1246) and Venezuelan equine encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR 1249, ATCC VR-532), hybπds or chimeras of these viruses may also be used), poxvirus vectors (e g vaccinia, fowlpox, canarypox, modified vaccinia Ankara, etc ), adenovirus vectors, and adeno-associated virus (AAV) vectors (e g see refs 122 to 127) Administration of DNA linked to killed adenovirus [128] can also be employed
Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycatiomc condensed DNA linked or unlinked to killed adenovirus alone [e g 128], ligand-linked
DNA [129], eukaryotic cell delivery vehicles cells [e g refs 130 to 134] and nucleic charge neutralization or fusion with cell membranes Naked DNA can also be employed Exemplary naked
DNA introduction methods are described in refs 135 and 136 Liposomes (e g lmmunohposomes) that can act as gene delivery vehicles are described in refs 137 to 141 Additional approaches are descπbed m references 142 & 143
Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in ref 143 Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymenzed hydrogel mateπals or use of ionizing radiation [e g refs 144 & 145] Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun [146] or use of ionizing radiation for activating transferred genes [144 & 145]
Delivery of DNA using PLG (poly(lactide-co-glycolide)} microparticles is a particularly preferred method e g by adsorption to the microparticles, which are optionally treated to have a negatively- charged surface (e g treated with SDS) or a positively-charged surface (e g treated with a cationic detergent, such as CTAB)
Methods of treatment, and administration of the vaccine
The invention also provides a method for raising an immune response m a mammal comprising the step of administering an effective amount of an immunogenic composition 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 at least two different RrgB clades for combined use as a medicament e g for use in raising an immune response m a mammal The invention also provides the use of at least two different RrgB clades m 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 pneumococcal disease and/or infection e g against pneumococcal meningitis The invention also provides a delivery device pre-filled with an immunogenic composition of the invention
The mammal is preferably a human Where the vaccine is for prophylactic use, the human is preferably a child (e g a toddler or infant) or a teenager, where the vaccine is for therapeutic use, the human is preferably a teenager or an adult A vaccine intended for children may also be administered to adults e g to assess safety, dosage, lmmunogemcity, etc
One way of checking efficacy of therapeutic treatment involves monitoring pneumococcal infection after administration of the compositions of the invention One way of checking efficacy of prophylactic treatment involves testing post-immunisation sera in standard tests, for example, sera can be tested m an opsonophagocytic killing assay (OPKA), with the ability to opsonise bacteria indicating protective efficacy Another way of checking efficacy of prophylactic treatment involves post-immunisation challenge in an animal model of pneumococcal infection, e g , guinea pigs or mice One such model is described m reference 147 Another way of assessing the lmmunogemcity of the compositions of the present invention is to express the polypeptides recombinantly for screening patient sera or mucosal secretions by immunoblot and/or microarrays A positive reaction between the polypeptide and the patient sample indicates that the patient has mounted an immune response to the polypeptide m question This method may also be used to identify immunodominant antigens and/or epitopes within antigens
Compositions of the invention will generally be administered directly to a patient Direct delivery may be accomplished by parenteral injection (e g subcutaneously, lntrapeπtoneally, 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 m an enhanced TH 1 and/or TH2 immune response Preferably, the enhanced immune response includes an increase m 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 vaπous doses may be given by the same or different routes e g & parenteral pπme and mucosal boost, a mucosal pπme 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 ) In one embodiment, multiple doses may be administered approximately 6 weeks, 10 weeks and 14 weeks after birth, e g at an age of 6 weeks, 10 weeks and 14 weeks, as often used in the World Health Organisation's Expanded Program on Immunisation ("EPI") In an alternative embodiment, two primary doses are admimstered about two months apart, e g about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second pπmary dose, e g about 6, 8, 10 or 12 months after the second pπmary dose In a further embodiment, three primary doses are administered about two months apart, e g about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the third pπmary dose, e g about 6, 8, 10, or 12 months after the third pπmary dose
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, less than 5 years 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 m a population Vaccines produced by the invention may be admimstered to patients at substantially the same time as (e g duπng 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 diphthena vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H influenzae type b vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-Wl 35-Y vaccine), a respiratory syncytial virus vaccine, etc
Combinations
A composition useful for immunisation compπses at least two RrgB clades, either as a hybrid polypeptide or as separate polypeptides In addition, a composition may include (i) one or more further polypeptides that elicit antibody responses against pneumococcal proteins, particularly against pneumococcal proteins other than RrgB, (n) a capsular sacchaπdε from pneumococcus, and/or (in) one or more further immunogens that elicit antibody responses that recognise epitopes on non-pneumococcal organisms As detailed above, compositions of the invention comprising combinations such as these can optionally compπse one or more adjuvants, for example two or more adjuvants Suitable adjuvants include mineral salts such as aluminium salts, and squalene- water emulsions such as MF59
Combinations with further polypeptide antigens [148]
RrgB polypeptides from one or more clades may be combined with one or more (ι e 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13) polypeptide antigens selected from the group consisting of (1) a spr0057 antigen, (2) a sprO565 antigen, (3) a sprlO98 antigen, (4) a sprl416 antigen, (5) a sprl418 antigen, (6) a sprO867 antigen, (7) a sprl431 antigen, (8) a sprl739 antigen, (9) a spr2021 antigen, (10) a spr0096 antigen, (11) a sprl707 antigen, (12) a sprl875 antigen, and/or (13) a sprO884 antigen
Similarly, RrgB polypeptides from one or more clades may be combined with one or more (ι e 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20) polypeptide antigens selected from the group consisting of (1) CIpP, (2) LytA, (3) PhtA, (4) PhtB, (5) PhtD, (6) PhtE, (7) ZmpB, (8) CbpD, (9) CbpG, (10) PvaA, (11) CPLl, (12) PspC, (13) PspA, (14) PsaA, (15) PrtA, (16) Spl33, (17) PiaA, (18) PmA, (19) CbiO, and/or (20) 30S πbosomal protein S8
These further antigens may be added as separate polypeptides As an alternative, they may be added as hybπds e g a spr0057-spr0096 hybrid or a spr0096-spr2021 hybrid, a sprO565-PhtD hybrid, etc As a further alternative, they may be fused to a RrgB polypeptide sequence to provide a hybπd polypeptide e g a RrgB-spr0057 hybπd
For example, a chimeric RrgB polypeptide including two or three RrgB clades may be combined with (a) a mixture of sρrOO57, spr0096 and spr2021 , (b) a mixture of spr0057, sprO565 and spr2021 , (c) a mixture of spr0057, spr0096 and sprO565, (d) a mixture of spr0057, spr0096, sprO565 and spr2021 , (e) a mixture of sprl418, sprO884 and spr0096, (f) a mixture of sprl418, sprO884 and spr2021, (g) a mixture of sprl418, sprO884, spr0096 and spr2021, (h) a mixture of sprO884, sprl416 and spr0057, (h) a mixture of spr0884, sprl416 and spr0096, (h) a mixture of sprO884, sprl416, spr0057 and spr0096, or (i) a mixture of sprl418, sprl431 and sprO565 Where these mixtures include both spr0057 and spr0096, a hybrid protein can be used e g comprising SEQ ID NO 82 (see SEQ ID NO 200 of ref 148) or comprising SEQ ID NO 83 Where these mixtures include both spr0096 and spr2021, a hybπd protein can be used e g comprising SEQ ID NO 84 (see SEQ ID NO 205 of ref 148)
In a further example, a chimeπc RrgB polypeptide including two or three RrgB clades may be combined with a pneumococcal immunogen compπsmg an spr2021 (also referred to as SP2216) antigen, an SP 1732 antigen and optionally a PsaA antigen A suitable pneumococcal immunogen of this sort is the immunogen disclosed m reference 159 that comprises the antigens "SP2216-1" (SEQ ID NO 1 in reference 159, SEQ ID NO 97 herein), "SP 1732-3" (SEQ ID NO 2 m reference 159, SEQ ID NO 98 herein) and, optionally, PsaA (SEQ ID NO 3 in reference 159, SEQ ID NO 99 herein) Polypeptides comprising immunogenic fragments of these SEQ ID NOs can be used m place of the actual disclosed SEQ ID NOs e g composing at least one immunogenic fragment from each of SEQ ID NOs 97 & 98 Polypeptides composing variants of spr2021 (SP2216), SP1732 and optionally PsaA can also be used m place of the actual disclosed SEQ ID NOs e g composing at least one vaoant from each of SEQ ID NOs 97 and 98 Examples of this combination include the combination of a pneumococcal immunogen as disclosed in reference 159 with a chimeoc RrgB polypeptide composing chimera II-I-III (e g SEQ ID NO 21) or chimeia IH-II-I (e g SEQ ID NO 15) as detailed below The further antigens may be added as separate polypeptides As an alternative, they may be added as hybrids e g a spr2021-SP1732 hybod or a spr2021-SP1732-PsaA hybod As a further alternative, they may be fused to a RrgB polypeptide sequence, e g a chimeoc RrgB polypeptide, to provide a hybrid polypeptide e g a RrgB-spr2021-SP1732 hybod As detailed above, compositions of the invention composing combinations such as these can optionally comprise one or more adjuvants Suitable adjuvants include mineral salts such as aluminium salts, and squalene- water emulsions such as MF59
Any of these combinations may also include one or more pneumococcal capsular sacchaode(s), which will typically be conjugated to earner protem(s) Further information about such sacchaodes and conjugation is provided below
The original 'sprOO57' sequence was annotated in reference 149 as 'Beta-N-acetyl-hexosamimdase precursor' (see GI 15902101) For reference purposes, the ammo acid sequence of full length sprOO57 as found m the R6 strain is given as SEQ ID NO 23 herein Preferred spr0057 polypeptides for use with the invention compose an ammo 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 23, and/or (b) composing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 23, wherein 'n' 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) These spr0057 proteins include variants of SEQ ID NO 23 Preferred fragments of (b) compose an epitope from SEQ ID NO 23 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 23 while retaining at least one epitope of SEQ ID NO 23 Other fragments omit one or more protein domains One suitable fragment is SEQ ID NO 38, which omits the natural leader peptide and sortase recognition sequences Another suitable fragment is SEQ ID NO 24, which has N-termmal and C-termmal truncations SEQ ID NO 27 is a variant of SEQ ID NO 24 based on a different wild-type strain and is a useful spr0057 sequence for use with the invention
The original 'sρrO565' sequence was annotated m reference 149 as 'beta-galactosidase precursor' (see GI 15902609) For reference purposes, the ammo acid sequence of full length sprO565 as found in the R6 strain is given as SEQ ID NO 25 herein Preferred sprO565 polypeptides for use with the invention compose an ammo 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 25, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 25, wherein 'n' 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) These sprO565 proteins include vaπants of SEQ ID NO 25 (e g SEQ ID NO 45, see below) Preferred fragments of (b) compnse an epitope from SEQ ID NO 25 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 25 while retaining at least one epitope of SEQ ID NO 25 Other fragments omit one or more protein domains One suitable fragment is SEQ ID NO 42, which omits the natural leader peptide and sortase recognition sequences Other suitable fragments are SEQ ID NOs 43 and 44 These shortened versions of sprO565 are particularly useful because the natural polypeptide is very long (>2000 aa)
A variant form of sprO565 is SEQ ID NO 45 herein The use of this variant form for immunisation is reported in reference 150 (SEQ ID NO 178 therein) Useful sprO565 polypeptides may thus comprise an ammo 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 45, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 45, wherein 'n' 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) These polypeptides include vaπants of SEQ ID NO 45 Preferred fragments of (b) comprise an epitope from SEQ ID NO 45 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 45 while retaining at least one epitope of SEQ ID NO 45 Other fragments omit one or more protein domains Immunogenic fragments of SEQ ID NO 45 are identified m table 1 of reference 150
The original 'sprlO98' sequence was annotated in reference 149 as 'Sortase' (see GI 15903141) For reference purposes, the ammo acid sequence of full length spi 1098 as found m the R6 strain is given as SEQ ID NO 26 herein Preferred sprlO98 polypeptides for use with the invention comprise an ammo 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 26, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 26, wherein 'n' 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) These sprlO98 proteins include vaπants of SEQ ID NO 26 Preferred fragments of (b) comprise an epitope from SEQ ID NO 26 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 26 while retaining at least one epitope of SEQ ID NO 26 Other fragments omit one or more protein domains One suitable fragment is SEQ ID NO 46, which omits the natural leader peptide sequence The original fsprl416' sequence was annotated m reference 149 as 'hypothetical protein1 (see GI 15903459) For reference purposes, the ammo acid sequence of full length sρrl416 as found m the R6 strain is given as SEQ ID NO 28 herein Preferred sprl416 polypeptides for use with the invention comprise an ammo 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 28, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 28, wherein 'n' 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) These sprl416 proteins include variants of SEQ ID NO 28 Preferred fragments of (b) comprise an epitope from SEQ ID NO 28 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 28 while retaining at least one epitope of SEQ ID NO 28 Other fragments omit one or more protein domains
The original 'sprl418' sequence was annotated in reference 149 as 'hypothetical protein' (see GI 15903461) For reference purposes, the ammo acid sequence of full length sprl418 as found m the R6 strain is given as SEQ ID NO 29 herein Preferred sρrl418 polypeptides for use with the invention compnse an ammo 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 29, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 29, wherein 'n' 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) These sprl418 proteins include variants of SEQ ID NO 29 Preferred fragments of (b) comprise an epitope from SEQ ID NO 29 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 29 while retaining at least one epitope of SEQ ID NO 29 Other fragments omit one or more protein domains
The original 'sprO867' sequence was annotated m reference 149 as 'Endo-beta-N-acetylglucosamimdase' (see GI 15902911) For reference purposes, the ammo acid sequence of full length sprO867 as found in the R6 strain is given as SEQ ID NO 30 herein Preferred sprO867 polypeptides for use with the invention compnse an ammo 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 30, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 30, wherein 'n' 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) These sρrO867 proteins include variants of SEQ ID NO 30 Preferred fragments of (b) compπse an epitope from SEQ ID NO 30 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 30 while retaining at least one epitope of SEQ ID NO 30 Other fragments omit one or more protein domains One suitable fragment is SEQ ID NO 48, which omits the natural leader peptide sequence
The original 'sprl431' sequence was annotated in reference 149 as T ,4-beta-N-acetylmuramidase1 (see GI 15903474) It is also known as 'LytC, and its use for immunisation is reported in reference 171 For reference purposes, the ammo acid sequence of full length sprl431 as found in the R6 strain is given as SEQ ID NO 31 herein Preferred sprl431 polypeptides for use with the invention comprise an ammo 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 31, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 31, wherein 'n' 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) These sprl431 proteins include variants of SEQ ID NO 31 Preferred fragments of (b) comprise an epitope fiom SEQ ID NO 31 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one oi more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 31 while retaining at least one epitope of SEQ ID NO 31 Other fragments omit one or more protem domains One suitable fragment is SEQ ID NO 49, which omits the natural leader peptide sequence
The 'sρrl739' polypeptide is pneumolysm (e g see GI 15903781) For reference purposes, the ammo acid sequence of full length sprl 739 as found in the R6 strain is given as SEQ ID NO 32 herein Preferred sprl739 polypeptides for use with the invention compπse an ammo 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 32, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 32, wherein 'n 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) These sprl 739 proteins include variants of SEQ ID NO 32 Preferred fragments of (b) compπse an epitope from SEQ ID NO 32 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 32 while retaining at least one epitope of SEQ ID NO 32 Other fragments omit one or more protem domains Mutant forms of pneumolysm for vaccination use are known m the art [183, 151-156], and these mutant forms may be used with the invention Detoxification can be achieved by C-termmal truncation (e g see ref 157) e g deleting 34 amino acids, 45 ammo acids, 7 ammo acids [158], etc Further mutations, numbered according to SEQ ID NO 32, include Pro325-→Leu {e g SEQ ID NO 50) and/or Trp433→Phe {e g SEQ ID NO 51) These mutations may be combined with C-termmal truncations e g to combine a Pro325→Leu mutation with a 7-mer truncation (eg SEQ ID NO 52)
The original 'spr2021' sequence was annotated m reference 149 as 'General stress protein GSP-781' (see GI 15904062) For reference purposes, the ammo acid sequence of full length spr2021 as found m the R6 strain is given as SEQ ID NO 33 herein Preferred spr2021 polypeptides for use with the invention compπse an ammo 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 33, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 33, wherein 'n' 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) These spr2021 proteins include vaπants of SEQ ID NO 33 Preferred fragments of (b) compπse an epitope from SEQ ID NO 33 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 33 while retaining at least one epitope of SEQ ID NO 33 Other fragments omit one or more protein domains One suitable fragment is SEQ ID NO 53, which omits the natural leader peptide sequence Reference 150 annotates spr2021 as a secreted 45kDa protein with homology to GbpB and discloses its use as an immunogen (SEQ ID NO 243 therein, SP2216) Immunogenic fragments ot spr2021 are identified m table 1 of reference 150 (page 73) Another useful fragment of spr2021 is disclosed as SEQ ID NO 1 of reference 159 (ammo acids 28-278 of SEQ ID NO 33 herein, this useful fragment of spr2021 is provided as SEQ ID NO 97 herein, SP2216-1)
The original 'spr0096' sequence was annotated m reference 149 as 'hypothetical protein' (see GI 15902140) For refeience purposes, the ammo acid sequence of full length spr0096 as found m the R6 strain is given as SEQ ID NO 34 herein Preferred spr0096 polypeptides for use with the invention comprise an ammo 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 34, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 34, wherein 'n' 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) These spr0096 proteins include vaπants of SEQ ID NO 34 (e g SEQ ID NO 54, see below) Preferred fragments of (b) compπse an epitope from SEQ ID NO 34 Othei preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 34 while retaining at least one epitope of SEQ ID NO 34 Other fragments omit one or more protein domains
A vaπant form of sρr0096, with an insert near its C-termmus relative to SEQ ID NO 34, is SEQ ID NO 54 herein The use of this variant for immunisation is reported in reference 150 (SEQ ID NO 150 therein), where it is annotated as a LysM domain protein Thus a spr0096 for use with the invention may comprise an ammo 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 54, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 54, wherein 'n' 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) These polypeptides include vaπants of SEQ ID NO 54 Preferred fragments of (b) compose an epitope from SEQ ID NO 54 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 54 while retaining at least one epitope of SEQ ID NO 54 Other fragments omit one or more protein domains Immunogenic fragments of SEQID NO 54 are identified m table 1 of refeience 150
A spr0096 polypeptide may be used m the form of a dimer eg a homodimer
The original 'sprl707' sequence was annotated m reference 149 as 'ABC transporter substrate-bmdmg protein - oligopeptide transport' (see GI 15903749) For reference purposes, the ammo acid sequence of full length sprl707 as found in the R6 strain is given as SEQ ID NO 36 herein Preferred sprl707 polypeptides for use with the invention comprise an ammo 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 36, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 36, wherein 'n' 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) These sprl707 proteins include variants of SEQ ID NO 36 (e g SEQ ID NO 55, see below) Preferred fragments of (b) compπse an epitope from SEQ ID NO 36 Other preferred fragments lack one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus ol SEQ ID NO 36 while retaining at least one epitope of SEQ ID NO 36 Other fragments omit one or more protein domains A variant form of sprl707, differing from SEQ ID NO 14 by 4 ammo acids, is SEQ ID NO 55 herein The use of SEQ ID NO 55 for immunisation is reported m reference 150 (SEQ ID NO 220 therein) Thus a sprl707 polypeptide for use with the invention may compose an ammo 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 55, and/or (b) composing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 55, wherein 'n' 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) These polypeptides include vaπants of SEQ ID NO 55 Preferred fragments of (b) compπse an epitope from SEQ ID NO 55 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 55 while retaining at least one epitope of SEQ ID NO 55 Other fragments omit one or more protein domains Immunogenic fragments of SEQ ID NO 55 are identified in table 1 of reference 150
The original 'sprl875' sequence was annotated m reference 149 as 'hypothetical protein' (see GI 15903916) For reference purposes, the ammo acid sequence of full length sprl875 as found in the
R6 strain is given as SEQ ID NO 35 herein Preferred sprl875 polypeptides for use with the invention compπse an ammo 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 35, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 35, wherein 'n' 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)
These sprl875 proteins include vaπants of SEQ ID NO 35 Preferred fragments of (b) comprise an epitope from SEQ ID NO 35 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO 35 while retaining at least one epitope of SEQ ID NO 35 Other fragments omit one or more protein domains
The 'sprO884' protein is a peptidylprolyl isomerase, also known as protease maturation protein For reference purposes, the ammo acid sequence of full length sprO884 is SEQ ID NO 37 herein Preferred sprO884 polypeptides for use with the invention compπse an ammo 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 37, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 37, wherein 'n' 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) These sprO884 proteins include vaπants of SEQ ID NO 37 Preferred fragments of (b) compπse an epitope from SEQ ID NO 37 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/oi one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 37 while retaining at least one epitope of SEQ ID NO 37 Other fragments omit one or more protein domains One suitable fragment is SEQ ID NO 56, which omits the natural leader peptide sequence The use of sprO884 for immunisation is reported in reference 160 CIpP is the ATP-dependent CIp protease proteolytic subumt For reference purposes, the ammo acid sequence of full length CIpP is SEQ ID NO 58 herein In the R6 genome CIpP is sprO656 [149] Preferred CIpP polypeptides for use with the invention compπse 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 58, and/or (b) compπsing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 58, wherein 'n1 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) These CIpP proteins include variants of SEQ ID NO 58 Preferred fragments of (b) comprise an epitope from SEQ ID NO 58 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- terminus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 58 while retaining at least one epitope of SEQ ID NO 58 Other fragments omit one or more protein domains The use of CIpP for immunisation is reported m references 161 and 162 It may advantageously be used in combination with PspA and PsaA and/or PspC [161]
LytA is the N-acetylmuramoyl-L-alanme amidase (autolysm) For reference purposes, the ammo acid sequence of full length LytA is SEQ ID NO 59 herein In the R6 genome LytA is sprl754 [149] Preferred LytA polypeptides for use with the invention compπse an ammo 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 59, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 59, wherein 'n' 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) These LytA proteins include variants of SEQ ID NO 59 (e g GI 18568354) Preferred fragments of (b) comprise an epitope from SEQ ID NO 59 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO 59 while retaining at least one epitope of SEQ ID NO 59 Other fragments omit one or more protein domains The use of LytA for immunisation is reported m reference 163, particularly in a form comprising the LytA choline binding domain fused to a heterologous promiscuous T helper epitope
PhtA is the Pneumococcal histidme tπad protein A For reference purposes, the ammo acid sequence of full length PhtA precursor is SEQ ID NO 60 herein In the R6 genome PhtA is sprlOόl [149] Preferred PhtA polypeptides for use with the invention comprise an ammo 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 60, and/or (b) compπsing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 60, wherein 'n' 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) These PhtA proteins include variants of SEQ ID NO 60 Preferred fragments of (b) comprise an epitope from SEQ ID NO 60 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 60 while retaining at least one epitope of SEQ ID NO 60 Other fragments omit one or more protein domains The use of PhtA for immunisation is reported in references 164 and 165
PhtB is the pneumococcal histidme tπad protein B For reference purposes, the ammo acid sequence of full length PhtB precursor is SEQ ID NO 61 herein Xaa at residue 578 can be Lysine Preferred PhtB polypeptides for use with the invention compπse an ammo 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 61, and/or (b) compπsing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 61, wherein 'n' is 7 or more (eg 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more) These PhtB proteins include vaπants of SEQ ID NO 61 Preferred fragments of (b) comprise an epitope from SEQ ID NO 61 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 61 while retaining at least one epitope of SEQ ID NO 61 Other fragments omit one or more protein domains The use of PhtB for immunisation is reported in references 164, 165 and 166
PhtD is the Pneumococcal histidme triad protein D For reference purposes, the ammo acid sequence of full length PhtD precursor is SEQ ID NO 62 herein In the R6 genome PhtD is spr0907 [149] Prefeπed PhtD polypeptides for use with the invention comprise an ammo 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 62, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 62, wherein 'n' 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) These PhtD proteins include vaπants of SEQ ID NO 62 Preferred fragments of (b) compπse an epitope from SEQ ID NO 62 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 62 while retaining at least one epitope of SEQ ID NO 62 Other fragments omit one or more protein domains The use of PhtD for immunisation is reported in references 164, 165 and 167 PhtE is the Pneumococcal histidme tπad protein E For reference purposes, the ammo acid sequence of full length PhtE precursor is SEQ ID NO 63 herein In the R6 genome PhtE is sprO9O8 [149] Preferred PhtE polypeptides for use with the invention comprise an ammo 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 63, and/or (b) compπsmg a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 63, wherein 'n' 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) These PhtE proteins include vaπants of SEQ ID NO 63 Preferred fragments of (b) comprise an epitope from SEQ ID NO 63 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 63 while retaining at least one epitope of SEQ ID NO 63 Other fragments omit one or more protein domains The use of PhtE for immunisation is reported in references 164 and 165
ZmpB is the zmc metalloprotease For reference purposes, the ammo acid sequence of full length ZmpB is SEQ ID NO 64 herein hi the R6 genome ZmpB is sprO581 [149] Preferred ZmpB polypeptides for use with the invention comprise an ammo 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 64, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 64, wherein W 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) These ZmpB proteins include vaπants of SEQ ID NO 64 Preferred fragments of (b) compπse an epitope from SEQ ID NO 64 Other preferred fragments lack one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 64 while retaining at least one epitope of SEQ ID NO 64 Other fragments omit one or more protein domains
CbpD is the Choline binding protein D For reference purposes, the ammo acid sequence of full length CbpD is SEQ ID NO 65 herein In the R6 genome CbpD is spr2006 [149] Preferred CbpD polypeptides for use with the invention compπse an ammo 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 65, and/or (b) compπsmg a fragment of at least 'n' consecutive ammo acids of SEQ ID
NO 65, wherein 'n' 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) These CbpD proteins include variants of SEQ ID NO 65 (e g SEQ ID NO 66, see below) Prefeπed fragments of (b) compπse an epitope from SEQ ID NO 65 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 65 while retaining at least one epitope of SEQ ID NO 65 Other fragments omit one or more protein domains The use of CbpD for immunisation is reported in reference 171
A variant of SEQ ID NO 65 is SEQ ID NO 66 herein The use of SEQ ID NO 66 for immunisation is reported m reference 150 (SEQ ID NO 241 therein) Thus a CbpD polypeptide for use with the invention may compπse an ammo 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 66, and/or (b) composing a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 66, wherein 'n' 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) These CbpD proteins include vaπants of SEQ ID NO 66 Preferred fragments of (b) compπse an epitope from SEQ ID NO 66 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 66 while retaining at least one epitope of SEQ ID NO 66 Other fragments omit one or more protein domains Immunogenic fragments of SEQ ID NO 66 are identified in table 1 of ref 150
CbpG is the Choline binding protein G For reference purposes, the ammo acid sequence of full length CbpG is SEQ ID NO 67 herein In the R6 genome CbpG is sprO35O [149] Preferred CbpG polypeptides for use with the invention compπse an ammo 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 67, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID
NO 67, wherein 'n' 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) These CbpG proteins include variants of SEQ ID NO 67 Prefen ed fragments of
(b) comprise an epitope from SEQ ID NO 67 Other preferred fragments lack one oi more ammo acids
(e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids
(e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 67 while retaining at least one epitope of SEQ ID NO 67 Other fragments omit one or more protein domains The use of CbpG for immunisation is reported m reference 171
P\aA (Streptococcus pneumoniae pneumococcal vaccine antigen A) is also known as splOl For reference purposes, the ammo acid sequence of full length PvaA is SEQ ID NO 68 herein In the R6 genome PvaA is spr0930 [149] Preferred PvaA polypeptides for use with the invention comprise an ammo 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 68, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 68, wherein 'n' 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) These PvaA proteins include variants of SEQ ID NO 68 Preferred fragments of (b) comprise an epitope from SEQ ID NO 68 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 68 while retaining at least one epitope of SEQ ID NO 68 Other fragments omit one or more protein domains The use of PvaA for immunisation is reported in references 168 and 169 CPLl is the pneumococcal phage CPl lysozyme For reference purposes, the ammo acid sequence of foil length CPLl is SEQ ID NO 69 herein Preferred CPLl polypeptides for use with the invention compπse an ammo 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 69, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 69, wherein 'n' 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) These CPLl proteins include variants of SEQ ID NO 69 Preferred fragments of (b) compπse an epitope from SEQ ID NO 69 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 69 while retaining at least one epitope of SEQ ID NO 69 Other fragments omit one or more protein domains The use of CPLl for immunisation is reported in reference 163, particularly in a form comprising the CPLl choline binding domain fused to a heterologous promiscuous T helper epitope
PspC is the pneumococcal surface protein C [170] and is also known as cholme-bmdmg protein A (CbpA) Its use for immunisation is reported m references 168 and 171 In the R6 strain it is sprl995 and, for reference, the ammo acid sequence of foil length sprl995 is SEQ ID NO 57 herein Preferred PspC polypeptides for use with the invention comprise an ammo 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 57, and/or (b) comprising a ftagment of at least 'n' consecutive ammo acids of SEQ ID NO 57, wherein 'n' 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) These sprl995 proteins include variants of SEQ ID NO 57 (e g SEQ ID NO 71, see below) Preferred fragments of (b) compπse an epitope from SEQ ID NO 57 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 57 while retaining at least one epitope of SEQ ID NO 57 Other fragments omit one or more protein domains
A vaπant of PspC is known as 'Hie' It is similar to PspC, as shown m Figure 1 of reference 172, where it is reported to bind to factor H (fH) For reference purposes, the ammo acid sequence of full length Hie is SEQ ID NO 71 herein A Hie protein may be used with the invention in addition to or m place of a PspC polypeptide Preferred Hie polypeptides for use with the invention comprise an ammo 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 71, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 71 , wherein 'n' 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) These Hie proteins include variants of SEQ ID NO 71 Preferred fragments of (b) compπse an epitope from SEQ ID NO 71 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 71 while retaining at least one epitope of SEQ ID NO 71 Other fragments omit one or more protein domains PspC and/or Hie can advantageously be used in combination with PspA and/or PsaA
PspA is the Pneumococcal surface protein A For reference purposes, the ammo acid sequence of full length PspA is SEQ ID NO 72 herein In the R6 genome PspA is sprO121 [149] Preferred PspA polypeptides for use with the invention compπse an ammo 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 72, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 72, wherein 'n' 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) These PspA proteins include variants of SEQ ID NO 72 Preferred fragments of (b) comprise an epitope from SEQ ID NO 72 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 72 while retaining at least one epitope of SEQ ID NO 72 Other fragments omit one or more protein domains The use of PspA for immunisation is reported intei aha m reference 173 It can advantageously be administered in combination with PspC PsaA is the Pneumococcal surface adhesm For reference purposes, the ammo acid sequence of full length PsaA is SEQ ID NO 73 herein Preferred PsaA polypeptides for use with the invention comprise an ammo 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 73, and/or (b) compnsmg a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 73, wherein 'n' 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) These PsaA proteins include variants of SEQ ID NO 73 Preferred fragments of (b) comprise an epitope from SEQ ID NO 73 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 73 while retaining at least one epitope of SEQ ID NO 73 Other fragments omit one or moi e protein d omams A useful ft agment of PsaA is disclosed as SEQ ID NO 3 m reference 159 (corresponding to ammo acids 21-309 of SEQ ID NO 73 herein, this useful fragment of PsaA is provided as SEQ ID No 99 herein) The use of PsaA for immunisation is reported in reference 174 It can be used m combination with PspA and/oi PspC PrtA is the cell wall-associated seπne proteinase It has also been known as spl28 and sρl30, and is m a subtilism-hke seπne protease For reference purposes, the ammo acid sequence of full length PrtA precursor is SEQ ID NO 74 herein In the R6 genome PrtA is sprO561 [149] Preferred PrtA polypeptides for use with the invention compπse an ammo 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 74, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 74, wherein 'n' 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) These PrtA proteins include vaπants of SEQ ID NO 74 Preferred fragments of (b) comprise an epitope from SEQ ID NO 74 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 74 while retaining at least one epitope of SEQ ID NO 74 Other fragments omit one or more protein domains The use of PrtA for immunisation is reported m references 175 & 176, and also m reference 168 Spl33 is a conserved pneumococcal antigen For reference purposes, the ammo acid sequence of full length Spl33 is SEQ ID NO 75 herein In the R6 genome Spl33 is sprO931 [149] Preferred Spl33 polypeptides for use with the invention compπse an ammo 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 75, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 75, wherein 'n1 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) These Spl33 proteins include variants of SEQ ID NO 75 Preferred fragments of (b) compπse an epitope from SEQ ID NO 75 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 75 while retaining at least one epitope of SEQ ID NO 75 Other fragments omit one or more protein domains The use of Spl33 for immunisation is reported m reference 177
PiaA is the membrane permease involved m iron acquisition by pneumococcus For reference purposes, the ammo acid sequence of full length PiaA is SEQ ID NO 76 herein In the R6 genome PiaA is sprO935 [149] Preferred PiaA polypeptides for use with the invention comprise an ammo 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 76, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 76, wherein 'n' 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) These PiaA proteins include vaπants of SEQ ID NO 76 Prefeπed fragments of (b) comprise an epitope from SEQ ID NO 76 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- termmus of SEQ ID NO 76 while retaining at least one epitope of SEQ ID NO 76 Other fragments omit one or more protein domains The use of PiaA for immunisation is reported in references 178, 179 and 180, particularly m combination with PmA
PmA is the ABC transporter substrate-bmdmg protein for ferric iron transport It is also known as FatB For reference purposes, the ammo acid sequence of full length PmA is SEQ ID NO 77 herein In the R6 genome PmA is sprl687 [149] Preferred PmA polypeptides for use with the invention comprise an ammo 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 77, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 77, wherein 'n' 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) These PmA proteins include variants of SEQ ID NO 77 Preferred fragments of (b) comprise an epitope from SEQ ID NO 77 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO 77 while retaining at least one epitope of SEQ ID NO 77 Other fragments omit one or more protein domains The use of PmA for immunisation is reported in refs 178 to 180, particularly m combination with PiaA
CbiO is annotated as a cobalt transporter ATP-bmdmg subunit For reference purposes, the ammo acid sequence of full length CbiO is SEQ ID NO 78 herein In the R6 genome CbiO is spr2025 [149] The use of CbiO for immunisation is reported m reference 181 ('ID2' therein) Preferred CbiO polypeptides for use with the invention comprise an ammo 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 78, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 78, wherein 'n1 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) These CbiO proteins include vaπants of SEQ ID NO 78 Preferred fragments of
(b) compnse an epitope from SEQ ID NO 78 Other preferred fragments lack one or more ammo acids
(e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids
(e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 78 while retaining at least one epitope of SEQ ID NO 78 Other fragments omit one or more protein domains
For reference purposes, the ammo acid sequence of 30S πbosomal protein S8 is SEQ ID NO 79 herein In the R6 genome the S8 subumt is spr0203 [149] Preferred S8 polypeptides for use with the invention comprise an ammo 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 79, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 79, wherein 'n' 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) These S 8 proteins include vaπants of SEQ ID NO 79 Preferred fragments of (b) compπse an epitope from SEQ ID NO 79 Other preferred fragments lack one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 5 9, 10, 15, 20, 25 or more) from the C-termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-termmus of SEQ ID NO 79 while retaining at least one epitope of SEQ ID NO 79 Other fragments omit one or more protein domains
SP 1732 is a membrane-associated serme/threonme kinase, StkP The sequence of SP 1732, comprising 659 ammo acids, is identified in reference 150 as SEQ ID NO 214 An exemplary fragment of this sequence, referred to as "SP 1732-3", is identified m reference 159 as SEQ ID NO 2 For reference purposes, the ammo acid sequence of SP 1732-3 is provided as SEQ ID NO 98 herein Preferred SPl 732 polypeptides for use with the invention comprise an ammo 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 98, and/or (b) comprising a fragment of at least 'n' consecutive ammo acids of SEQ ID NO 98, wherein 'n' 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) These SP1732 proteins include vaπants of SEQ ID NO 98 Preferred fragments of (b) compπse an epitope from SEQ ID NO 98 Other preferred fragments lack one or more ammo acids (e g 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C- termmus and/or one or more ammo acids (e g 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- terminus of SEQ ID NO 98 while retaining at least one epitope of SEQ ID NO 98 Other fragments omit one or more protein domains
Combinations with pneumococcal saccharides
RrgB polypeptides from one or more clades may be combined with one or more pneumococcal capsular sacchaπde(s), which will typically be conjugated to carrier protem(s) Thus the invention provides an immunogenic composition comprising a combination of
(1) a combination of at least two RrgB clades as discussed above, as a mixture or hybnd, and
(2) one or more pneumococcal capsular saccharides
A sacchande used in component (2) of this combination is ideally present as a conjugate comprising a saccharide moiety and a carrier protein moiety The carrier moiety in the conjugate may be a single RrgB ) polypeptide, a hybnd RrgB polypeptide, a non-RrgB pneumococcal polypeptide, or a non-pneumococcal polypeptide
The sacchande is from the capsular sacchande of a pneumococcus The sacchande may be a polysacchande having the size that anses during purification of the sacchande from bacteria, or it may be an oligosaccharide achieved by fragmentation of such a polysacchande In the 7-valent PREVNAR™ product, for instance, 6 of the saccharides are presented as intact polysacchaπdes while one (the 18C serotype) is presented as an oligosaccharide
A composition may include a capsular sacchaπde from one 01 more of the following pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 1OA, HA, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F A composition may include multiple serotypes e g 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes 7-valent, 9-valent, 10-valent, 11-valent and 13- valent conjugate combinations are already known m the art, as is a 23-valent unconjugated combination
For example, a 10-valent combination may include sacchaπde from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F An 11-valent combination may further include sacchaπde from serotype 3 A 12-valent combination may add to the 10-valent mixture serotypes 6A and 19A, 6A and 22F, 19A and 22F, 6A and 15B, 19A and 15B, r 22F and 15B, A 13-valent combination may add to the 11-valent mixture serotypes 19A and 22F, 8 and 12F, 8 and 15B, 8 and 19A, 8 and 22F, 12F and 15B, 12F and 19A, 12F and 22F, 15B and 19A, 15B and 22F etc One useful 13-valent combination includes capsular sacchaπde from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19, 19F and 23F If saccharides are enclosed then it is preferred to include 1, 2 or 3 of serotypes 1, 5 and 14
A carrier protein m a conjugate may or may not be one of the RrgB antigens of (1) It it is not a RrgB antigen it may instead be a different pneumococcal antigen, such as spr0057, spr0096 and spr2021 , etc or pneumolysm [182] or its non-toxic denvatives [183], or pneumococcal surface protein PspA [184], In some embodiments, though, the carrier is not a pneumococcal antigen, and may be e g a bacterial toxm or toxoid Typical earner proteins are diphtheπa or tetanus toxoids or mutants thereof The CRM1Qy diphtheπa toxm mutant [185] is useful, and is the earner in the PREVNAR™ product Other suitable carrier proteins include N meningitidis outer membrane protein complex [186], synthetic peptides [187,188], heat shock proteins [189,190], pertussis proteins [191,192], cytokines [193], lymphokmes [193], hormones [193], growth factors [193], artificial proteins compπsmg multiple human CD4+ T cell epitopes from vanous pathogen-derived antigens [194] such as N19 [195], protein D from H influenzae [196-198], iron-uptake proteins [199], toxm A or B from C difficile [200], recombinant P aei uginosa exoprotem A (rEPA) [201], etc
Where a composition includes more than one conjugate, each conjugate may use the same carrier piotem or a different earner protein Reference 202 describes potential advantages when using different earner proteins in multivalent pneumococcal conjugate vaccines
In some embodiments, a single conjugate may carry saccharides from multiple serotypes [203] Usually, however, each conjugate will include saccharide from a single serotype Conjugates may have excess earner (w/w) or excess saccharide (w/w) In some embodiments, a conjugate may include equal weights of each
The carrier molecule may be covalently conjugated to the earner directly or via a linker Direct linkages to the protein may be achieved by, for instance, reductive animation between the sacchande and the carrier, as described in, for example, references 204 and 205 The sacchande may first need to be activated e g by oxidation Linkages via a linker group may be made using any known procedure, for example, the procedures descnbed in references 206 and 207 A preferred type of linkage is an adipic acid linker, which may be formed by coupling a free -NKb group (e g introduced to a glucan by animation) with adipic acid (using, for example, dnmide activation), and then coupling a protein to the resulting sacchande-adipic acid intermediate [208,209] Another preferred type of linkage is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a sacchande CDI [210, 211] followed by reaction with a protein to form a carbamate linkage Other linkers include β-propionamido [212], mtrophenyl-ethylamme [213], haloacyl halides [214], glycosidic linkages [215], 6-ammocaproic acid [216], ADH [217], C4 to Ci2 moieties [218], etc Carbodiimide condensation can also be used [219] Combinations with non-pneiimococcal antigens
The RrgB clade combinations may be used in combination with non-pneumococcal antigens Thus the invention provides an immunogenic composition comprising a combination of
(1) a combination of at least two RrgB clades as discussed above, as a mixture or hybrid, and
(2) one or more antigen(s) selected from the group consisting of diphtheria toxoid, tetanus toxoid, one or more pertussis antigens, hepatitis B virus surface antigen, an inactivated poliovirus antigen, a conjugate of the capsular sacchande antigen from Haemophilus influenzae type B, a conjugate of the capsular sacchande antigen from serogroup C of Neisseria meningitidis, a conjugate of the capsular sacchande antigen from serogroup Y of Neisseria meningitidis, a conjugate of the capsular sacchande antigen from serogroup W135 of Neisseria meningitidis, and a conjugate of the capsular sacchande antigen from serogroup A of Neisseria meningitidis
Diphtheria toxoid can be obtained by treating (e g using formaldehyde) diphthena toxin from Corγnebactenum diphthenae Diphthena toxoids are disclosed in more detail in, for example, chapter 13 of reference 220
Tetanus toxoid can be obtained by treating (e g using formaldehyde) tetanus toxin from Clostridium tetani Tetanus toxoids are disclosed in more detail in chapter 27 of reference 220
Pertussis antigens in vaccines are either cellular (whole cell, Pw) or acellular (Pa) The invention can use either sort of pertussis antigen Preparation of cellular pertussis antigens is well documented (e g see chapter 21 of reference 220) e g it may be obtained by heat inactivation of phase I culture of B pertussis Acellular pertussis antigen(s) compose specific purified B pertussis antigens, either purified from the native bacterium or purified after expression in a recombinant host It is usual to use more than one acellular antigen, and so a composition may include one, two or three of the following well-known and well-characterized B pertussis antigens (1) detoxified pertussis toxm (pertussis toxoid, or 'PT'), (2) filamentous hemagglutinin ('FHA'), (3) pertactin (also known as the '69 kiloDalton outer membrane protein') FHA and pertactm may be treated with formaldehyde pπor to use according to the invention PT may be detoxified by treatment with formaldehyde and/or glutaraldehyde but, as an alternative to this chemical detoxification procedure, it may be a mutant PT in which enzymatic activity has been reduced by mutagenesis [221] Further acellular pertussis antigens that can be used include fimbriae {e g agglutinogens 2 and 3)
Hepatitis B virus surface antigen (HBsAg) is the major component of the capsid of hepatitis B virus It is conveniently produced by recombinant expression m a yeast, such as a Saccharomyces cerevisiae
Inactivated poliovirus (IPV) antigens are prepared from viruses grown on cell culture and then inactivated (e g using formaldehyde) Because poliomyelitis can be caused by one of three types of poliovirus, as explained in chapter 24 of reference 220, a composition may include three poliovirus antigens poliovirus Type 1 (e g Mahoney strain), poliovirus Type 2 (e g MEF-I strain), and poliovirus Type 3 (e g Saukett strain)
When a composition includes one of diphtheria toxoid, tetanus toxoid or an acellular pertussis antigen in component (2) then it will usually include all three of them i e component (2) will include a D-T-Pa combination
When a composition includes one of diphtheπa toxoid, tetanus toxoid or a cellular pertussis antigen m component (2) then it will usually include all three of them i e component (2) will include a D-T-Pw combination
Immunogenic compositions of particular interest comprise (i) a combination of at least two RrgB clades as discussed above as a mixture or hybπd, diphtheria toxoid, tetanus toxoid, whole cell pertussis antigens, a conjugate of Haemophilus influenzae type B capsular saccharide, and HBsAg, (π) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, diphtheπa toxoid, tetanus toxoid, acellular pertussis antigen(s), a conjugate of Haemophilus influenzae type B capsular saccharide, and HBsAg, (in) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, and conjugate(s) from one or more of meningococcal serogroups A, C, Wl 35 and Y, (iv) a combination of at least two RrgB clades as discussed above as a mixture or hybπd, and conjugates from all of meningococcal serogroups A, C, Wl 35 and Y, and (v) a combination of at least two RrgB clades as discussed above as a mixture or hybrid, and a meningococcal serogroup B antigen, such as an outer membrane vesicle antigen and/or the combination disclosed m ref 222
Antibodies
Antibodies against pneumococcal antigens can be used for passive immunisation [223] Thus the invention provides a combination of antibodies for simultaneous, separate or sequential administration, wherein the combination includes at least two of (a) an antibody which recognises a first ammo acid sequence as defined above, (b) an antibody which recognises a second ammo acid sequence as defined above, and/or (c) an antibody which recognises a third ammo acid sequence as defined above,
The invention also provides the use of such antibody combinations in therapy The invention also provides the use of such antibody combinations in the manufacture of a medicament The invention also provides a method for treating a mammal composing the step of administering to the mammal an effective amount of such a combination As described above for immunogenic compositions, these methods and uses allow a mammal to be protected against pneumococcal infection
The term "antibody" includes intact immunoglobulin molecules, as well as fragments thereof which are capable of binding an antigen These include hybrid (chimeric) antibody molecules [224, 225], F(ab')2 and F(ab) fragments and Fv molecules, non-covalent heterodimers [226, 227], smgle-chain Fv molecules
(sFv) [228], dimeπc and trimeπc antibody fragment constructs, mimbodies [229, 230], humanized antibody molecules [231-233], and any functional fragments obtained from such molecules, as well as antibodies obtained through non-conventional processes such as phage display Preferably, the antibodies are monoclonal antibodies Methods of obtaining monoclonal antibodies are well known m the art Humanised or fully-human antibodies are preferred
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 m the literature See, e g , references 234-241 , etc
"GI" numbering is used above A GI number, or "Genlnfo Identifier", is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases The GI number bears no resemblance to the accession number of the sequence record When a sequence is updated (e g for correction, or to add more annotation or information) then it receives a new GI number Thus the sequence associated with a given GI number is never changed
Where the invention concerns an "epitope", this epitope may be a B-cell epitope and/or a T-cell epitope Such epitopes can be identified empirically (e g using PEPSCAN [242,243] or similar methods), or they can be predicted (e g using the Jameson- Wolf antigenic index [244], matrix-based approaches [245], MAPITOPE [246], TEPITOPE [247,248], neural networks [249], OptiMer & EpiMer [250, 251], ADEPT [252], Tsites [253], hydrophilicity [254], antigenic index [255] or the methods disclosed in references 256-260, etc ) Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as "antigenic determinants"
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 word "substantially" does not exclude "completely" e g a composition which is "substantially free" from Y may be completely free from Y Where necessary, the word "substantially" may be omitted from the definition of the invention
The term "about" in relation to a numerical value x is optional and means, foi example, x+10%
Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing Thus components can be mixed m any order Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc
Antibodies will generally be specific for their target Thus they will have a higher affinity for the target than for an irrelevant control protein, such as bovine serum albumin
References to a percentage sequence identity between two ammo acid sequences means that, when aligned, that percentage of ammo 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 m the art, for example those descπbed in section 7 7 18 of ref 261 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 m ref 262
MODES FOR CARRYING OUT THE INVENTION
Construction ofRrgB chimeras
Two different pill have been identified in pneumococcus [2] PI-I and PI-2 Knockout studies showed that loss of PI-2 had little effect, but loss of PI-I reduced a strain's ability to colonise, and thus led to lower bacteremia and lung wash titres Thus blocking of PI-I has a better prospect of protecting against pneumococcal disease than blocking PI-2
PI-I RrgB protein has three different clades Fifteen different RrgB ammo acid sequences were found m 45 different strains and Figure 9 shows their relationship The wild-type sequences are >98% conserved withm each clade RrgB protein was found to elicit immune responses which are protective against homologous strains (mtra-clade), but which fail to protect against strains having RrgB from a different clade (mter-clade) Thus it was decided to combine multiple RrgB clades into a single composition, thereby increasing the spectrum of strain coverage SEQ ID NOs 1, 2 and 3 are the full-length encoded sequences for RrgB from strains TIGR4, Fmland6B- 12 and Taiwan2jF-15 To construct chimeras of these three proteins their N- and C-teπmm were truncated to give SEQ ID NOs 4, 5 and 6 Restriction enzymes Nhel BamHI and Xhol were used in this procedure To join these fragments to make chimeras linkers SEQ ID NOs 8 and 10 were used, made of either a GIy- Ser or Leu-Gly dipeptide followed by SEQ ID NO 7 These linkers provide convenient restriction sites for ligation of fragments The N-termmus of the chimeras was provided as Met- Ala- Ser, and the C-termmus was a Leu-Gly dipeptide followed by a hexa-His tag (SEQ ID NO 9) to facilitate purification
Six chimeras were constructed, referred to hereafter as follows
RTgB I-II-III = SEQ ID NO 11 RTgB I-III-II = SEQ ID NO 13
RTgB III-II-I = SEQ ID NO 15
RrgB III-I-II = SEQ ID NO 17
RTgB II-III-I = SEQ ID NO 19
RTgB II-I-III = SEQ ID NO 21 Except for the I-III-II chimera (SEQ ID NO 13) the expressed chimeras had a molecular weight of 205kDa, could be expressed m E cob in soluble form, and were puπfied from the soluble proteins For example, figure 3 shows a gel of the I-II-III chimera at 1 6mg/ml with 90% purity
Efficacy testing
Various model systems of pneumococcal disease were used for testing efficacy of the chimeras In a mouse model of intraperitoneal infection, antigens were administered mtrapeπtoneally and the challenge was intraperitoneal Six-week-old, specific-pathogen- free female BALB/c or CDl mice were immunized mtrapeπtoneally on days 0, 14, and 28 Immunizations were done using single recombinant proteins (20 μg/mouse) or with a combination of them (10 μg each/mouse), along with aluminium hydroxide or Freund's adjuvant Controls received identical courses of salme plus adjuvant Mice were then challenged mtrapeπtoneally with a lethal dose of TIGR4 (typical challenge dose ~lxlθ2 CFU/mouse), Fmland6B-12 (~2 xlO4 CFU/mouse) or 35B-SME15 (~lxlθ4 CFU/mouse) These three strains express RrgB clades I, II or III, respectively, and the TIGR4 strain is very virulent Efficacy of immunisation is tested by evaluating the effect of vaccination on bacteremia (at 5 and/or 24 hours post infection) and mortality (monitored for at least 10 days following bacteπal challenge)
In a model of intravenous infection, antigens were administered mtrapeπtoneally and the challenge was intravenous Five-week-old CDl or BALB/c mice were immunized mtrapeπtoneally on days 0, 14, and 28 Immunizations were done using recombinant proteins individually (20 μg/mouse) or with a combination of them (10 μg each/mouse), along with Freund's adjuvant Controls received identical courses of salme plus adjuvant Mice were then challenged intravenously with a lethal dose of TIGR4 (typical challenge dose ~5xlO6 CFU/mouse), Fmland6B-12 (~2 xlO7 CFU/mouse) or 35B-SME15 (~5xlO7 CFU/mouse) Efficacy of vaccine candidates is tested by evaluating the effect of vaccination on bacteremia (at 48 hours post-mfection) and mortality (monitored for 10 days following bacteπal challenge or longer, depending on the infecting strain)
For example, CDl mice were immunised with the chimeras and then challenged with TIGR4 Figure 1 shows bacteremia after the challenge Geometric mean CFUs were as follows, together with a U-test comparison against the control group
Figure imgf000059_0001
Figure 2 shows mortality after the challenge Median survival times m days weie as follows
Figure imgf000059_0002
Figures 30 to 33 show the results of bacteremia and mortality assays for mice immunised mtrapeπtoneally with 20μg of the III-II-I chimera Figure 30 shows data for i v challenge with TIGR4, Figure 31 shows data for i p challenge with TIGR4, Figure 32 shows data for i v challenge with 35B-SME15 and Figure 33 shows data for i v challenge with 6B Finland 12
The following table summarises results obtained m two different models of challenge with three different strains which express, respectively, RrgB m clade I, II or III
Figure imgf000060_0001
+++ = P<0 01 against control, ++ = PO 05, + = P<0 1
Therefore the combination of different clades of RrgB allows for broader coverage against pneumococcal strains than single RrgB antigens
In further tests RrgB chimeras were adjuvanted with alum and tested for protection against TIGR4 intraperitoneal challenge Chimeras I-II-III and HI-II-I were highly protective against bacteremia, and the III-II-I chimera was also protective m terms of survival (Figure 7)
Further tests used intranasal challenge after intraperitoneal immunisation with one of four different chimeras (I-II-III, III-II-I, II-III-I, II-I-III) All chimeras showed efficacy or a trend to reduce bacteremia after intranasal TIGR4 challenge The II-III-I chimera gave good decrease of bacteremia and a non- significant trend of survival increase upon T4 challenge A PsaA control showed almost no efficacy, measured either by bacteremia or mortality, whereas the II-III-I chimera decreased bacteremia and increased survival Figure 13 shows results for an RrgB III-II-I Chimera m a 24hour bacteremia assay (Figure 13A) and a mortality assay (Figure 13B) m BalB/c mice, immunized mtrapeπtoneally with 20μg chimera (0-14-28 days) and challenged mtranasally with TIGR4 Antibodies against all five RrgB chimeras were also found to mediate in vitro killing of pneumococci in OPKA For instance, figure 8 shows results against the TIGR4 strain Figure 10 shows results against S pneumoniae serotype 6B m an OPKA assay (rabbits subcutaneously immunized with lOOμg of each chimera at days 0, 21 and 35), which shows that no difference m killing percentage is observed between the five chimeras and that the chimeras show killing that is comparable to the conjugate vaccine PCV7 Figure 1 1 shows that killing is specific and dependent on antibody concentration, showing that by increasing the dilution up to 1/131220, the percentage killing decreases m the tested chimera curves similarly to the positive control
Figure 12 shows a 48 hoar bacteremia (Figure 12A) and mortality (Figure 12B) assay using a III-II-I chimera (immunised i p and challenged i p with 35B-SME15) is comparable when using different chimera doses (2 μg and 20 μg) Figure 14 shows that RrgB IH-II-I chimera is protective using MF59 adjuvant in BalB/c mice, intraperitoneal immunisation with 20 μg chimera (0-14-28 days) and challenged mtransally
Figure 15 shows that RrgB HI-II-I chimera is protective upon subcutaneous immunization in BalB/c mice, immunized subcutaneously and challenged mtrapeπtoneally with TIGR4 (130CFU/mouse) Figure 15A shows a 24hour bacteremia assay and Figure 15B shows a mortality assay
Figure 16 shows that RrgB IH-II-I chimera elicits production of functional antibodies in a passive protection study, compared to a Normal Rabbit Serum (NRS) control, in a 24hour bacteremia assay
Figure 17 shows that antibodies are functional m OPA against strains of the three clades and Figure 18 shows that the OPA activity is specifically due to the antibodies against RrgB IH-II-I chimera Figure 19 shows that sible RrgB domains confer protection in vivo Specifically, the data show % survival of BalB/c mice immunised with the RrgB D 1 domain or the RrgB D4 domain (i p immunization 20μg, 0-14-28 days, i p challenge with TIGR4 lOOCFU)
Figure 23 shows a 48 hour bacteremia (Figure 23A) and mortality (Figure 23B) assay using a IH-II-I chimera when combined with different combinations of further polypeptide antigens (20μg antigens with alum, immunised i p and challenged i v with 6B-Fmland 1 2E+08 CFU/mouse) In both (A) and (B) column 1 shows a combination of spiOO57, spr0096 and spr2021, column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA, column 3 shows RrgB IH-II-I chimera, column 4 shows RrgB IH-II-I chimera combined with spr0057, spr0096 and spr2021 , column 5 shows RrgB IH-II-I chimera combined with SP2216-1 , SP 1732-3 and PsaA, and column 6 shows an alum control These data show that the efficacy of a combination of SP2216-1 , SP1732-3 and PsaA is significantly increased when combined with the RrgB chimera
Figure 24 shows a 48 hour bacteremia (Figure 24A) and mortality (Figure 24B) assay using a HI-II-I chimera when combined with different combinations of further polypeptide antigens (20μg antigens with alum, immunised i p and challenged i v with 35B-SME15 5 2E+07 CFU/mouse) In both (A) and (B) column 1 shows a combination of spr0057, spr0096 and spr2021, column 2 shows a combination of SP2216-1, SP 1732-3 and PsaA, column 3 shows RrgB HI-II-I chimera, column 4 shows RrgB IH-II-I chimera combined with spr0057, spr0096 and spr2021 , column 5 shows RrgB IH-II-I chimera combined with SP2216-1, SPl 732-3 and PsaA, and column 6 shows an alum control These data show that the RrgB IH-II-I chimera, and the combinations of the RrgB IH-II-I chimera with other antigens, are all protective
Figure 25 shows (A) a 24 hour bacteremia assay and (B) mortality data in BALB/c mice using a IH-II-I chimera that contains a polyhistidine tag compared to a tag-less IH-II-I chimera and an alum control (i p immunisation, i p challenge with TIGR4 2 1E+02 CFU/mouse) These data show that both the his- tagged and tag-less chimeras significantly protect against TIGR4 both m terms of bacteremia and survival, with the tag-less chimera showing the most significant protection Figure 26 shows similar data i e a 24 hour bacteremia assay in BALB/c mice using a IH-II-I chimera that contains a polyhistidme tag compared to a tag-less IH-II-I chimera and an alum control, further compared to a combination of 5 sprOO57, spr0096 and spr2021 antigens, and a combination of the spr0057, spr0096 and spr2021 antigens with the tag-less IH-II-I chimera, (i p immunisation, i p challenge with TIGR4 1 6E+02 CFU/mouse) Figures 27 and 28 show data for i v challenge with 35B-SME15 (Figure 27) and 6BFmlandl2 (Figure 28), showing that tag-less IH-II-I chimera showed the same protective efficacy as his-tagged IH-II-I chimera against 35B-SME15 and 6BFmlandl2 i v challenge Similarly, Figure 29 shows that both tag- 0 less and his-tagged HI-II-I chimeras are protective against i v TIGR4 challenge
Figure 34 shows the results of 48 hour bacteremia and mortality assays for III-II-I chimera comparing a TIGR4 challenging strain over-expressmg pilus to a challenging strain that only expresses very low amounts of pilus These data show that protection is very good when the pilus is overexpressed and also when the pilus is only present at very low levels Figure 35 shows similar bacteremia data for both 5 III-II-I and H-I-III chimeras compaπng a 6BFmll2 challenging strain over-expressmg pilus (Figure 35A) to a 6BFmI 12 challenging strain under-expressing pilus (Figure 35B) The chimeras show significant protection against both the strain overexpressmg and the strain underexpressmg pilus
Antimicrobial Resistance
Figure 36 shows that pilus- 1 is more prevalent m pneumococcal strains that are iesistant to antiobioticsO (erythromycm-resistance, penicillin-resistance and multiple-drug- resistance) compared to strains that aie susceptible to antibiotics There is a significant association between pilus- 1 presence and antibiotic resistance An increase in the presence of pilus- 1 in antibiotic-resistant strains has also been observed in the multi-resistant PMEN strain collection (data not shown) These data suggest that immunising against pilus- 1 using an immunogenic composition including multiple RrgB clades will have the additional
15 advantage of protecting against pneumococci that are resistant to antibiotic treatment, for example erythromycin-resistant strains, penicillin-resistant strains and multiply-resistant strains
Monoclonal antibodies
Monoclonal antibodies were raised against the RrgB from TIGR4 Four mAbs were studied m more detail (named 23B8/B6, 23F8/10, 23E1/A9 and 30A8/A8) 23B8/B6 and 23F8/10 bound to the full- »0 length RrgB from TIGR4, to the Dl domain fragment, and also to a D1-D2-D3 fragment, but not to a D4 fragment Conveisely, 23E1/A9 bound to the full-length protein and so the D4 domain fragment but not to a D1-D2-D3 fragment, or a D4 fragment 30A8/A8 bound to the full-length RrgB protein but not to any of the domain fragments The mAbs did not bind to RrgB protein from Finland68- 12 or 23F strains, but they did bind to all five chimeras which were expressed The binding results are shown in figure 5 and confirm that the RrgB retains epitopes in its hybnd form
As shown in figure 4A, each of the four tested anti-TIGR4 mAbs was able to reduce bacteremia in a passive protection test, with the best results coming from 23F8/10 Each of the four tested anti-TIGR4 mAbs also guaranteed a significant (p<0 01 for all MAbs except 23B8/B6, P=O 021) survival increase m a mortality assay (Figure 4B)
To determine the epitope recognized by each of the four protective MAbs, the different RrgB domains were cloned, as single domains (Dl, D2, D3, D4) or as multi-domain fragments (Dl-3, D2-4, D3-4), expressed in E CoIi as His-tagged polypeptides and successfully purified in a soluble form by affinity chiomatography on His-trap high performance columns (GE Healthcare) The recombinant proteins were then probed m western blot analysis against the MAbs by using FL RrgB clade 1 and BSA as positive and negative controls respectively
The results, as shown in Figure 20, showed that monoclonal antibodies have a different and specific reactivity on the recombinant proteins Both mAb 23F8/10 and mAb 23 B8/B6 were able to specifically recognize the N-termmal domain Dl, the mAb 23 E1/A9 recognized the C-terminal D4, while 30A8/A8, was able to detect only D2-4, suggesting the recognition of a conformational epitope between D2 and D4 These data were then subsequently confirmed by ELISA (data not shown)
Monoclonal antibodies were also raised against the RrgB from Finland68- 12 Two particular mAbs (2A5/29, 3A5/19) bound to the full-length RrgB from Fmland6B-12, but not to the RrgB protein from TIGR4 or 23F strains The mAbs also bound to all five chimeras which were expressed The binding results are shown m figure 6
Epitope Mapping of Protective mAb 23F8/10
To map the region on the Dl domain containing the protective epitope recognized by mAb 23 F8/10 mass spectrometry analysis, Western Blot detection and limited proteolysis of the recombinant proteins were used m combination This approach can be summarized in four mam steps (i) enzymatic or chemical partial cleavage of the protein, (n) definition of sequence coverage of the generated fragments by MS analysis after their separation by SDS-PAGE, (in) western blot analysis of the generated fragments, (iv) comparison of positive and negative bands in western blot m order to localize the epitope The first step was to obtain from the full length RrgB a significant number of polypeptides showing a well resolved pattern after separation on SDS-PAGE The protease selected for these expenments was trypsin, which cleaves proteins at the C-termmal side of arginme (R) and lysine (K) residues 20 μg of full length RrgB was digested and the products of the digestion were separated with SDS-PAGE (5 μg of the full length protein, and 12 μg of the product of digestion) As noted above, and as shown m Figure 20, the monoclonal antibody 23F8/10 recognized both the full length recombinant RrgB and the RrgB D 1 , as well as a high number of polypeptides deπved from the cleavage of the full length protein with trypsin The identification of both the positive and negative bands m the western blot analysis (with respect to the same Coomassie stained sample) was important for the epitope identification The western blot with monoclonal antibody 23F8/C10 is shown as Figure 21 About 20 Coomassie-stamed proteolitic fragments, comprising both western blot (immunoblotted with MAb 23F8/C10) positive (green arrows) and negative (red arrows) bands, were excised from the gel and in situ digested with trypsin O/N and analyzed by MALDI-TOF/TOF mass spectrometry, in order to define the sequence coverage for each of them The sequence coverage obtained for each analyzed fragment was defined between the most "N-termmal" and the most "C-termmal" tryptic peptides identified m the PMF spectra ( peptide mass fingerprints ) A schematic sequence coverage of the electrophoretic pattern of the trypsin products denved from full length RrgB, m association with western blot results, was prepared This analysis suggested that the 23F8/10 epitope is between ammo acid 32 and ammo acid 141 of full length RrgB
The same strategy was then used on the N-terminal domain Dl, in order to narrow the region containing the epitope recognized by MAb 23F8/C10 20 μg of Dl was digested and the products of the digestion were separated with SDS-PAGE (5 μg of the full length protem, and 12 μg of the product of digestion) Unlike full length digested RrgB, m this experiment the monoclonal antibody 23F8/10 recognized only full length Dl and some of the polypeptides deπved from trypsin Dl digestion Afterwards, both positive and negative bands were taken into consideration for further analysis About 10 Coomassie-stamed peptide fragments, comprising both positive and negative bands, were excised from the gel and in situ digested with trypsin O/N and analyzed by MALDI-TOF/TOF mass spectrometry, in order to define the sequence coverage for each of them The sequence coverage obtained for each analyzed fiagment was defined between the most "N-termmal" and the most "C-termmal" tryptic peptides identified in the PMF spectra ( peptide mas v fingerprints ) The sequence coverage of the electrophoretic band of the trypsin products deπved from RrgB D 1 domain, as previously established m association with the results of the western blot, suggested that the region recognised by MAb 23F8/10 containing the protective epitope is from ammo acid residue 55 to ammo acid residue 89 of RrgB The Dl ammo acid sequence (for which no structural data are yet available) was modeled onto the domain 1 crystal structure of the S pyogenes pilus backbone SpyO128 (overall homology about 27%) The residues that the data suggest are the epitope (aa 55-89) were mapped onto the model (Figure 22A) In a 3D reconstruction of the electron density map of the pilus, obtained performing a rigid body fitting of the RrgB D 1 -4 structure, this epitope is shown to be surface exposed (Figure 22 B & 22C) Rr gB chimeras as carrier proteins
In addition to acting as vaccine components, the RrgB chimeras are suitable for use as earner proteins m sacchaπde- carrier conjugates The I-II-III and III-II-I chimeras were conjugated to a saccharide immunogen and IgG responses (GMT) against the saccharide were then measured by ELISA Results were compared to a number of other pneumococcal proteins, and also to Nl 9 and CRM 197 as positive controls Results from study VFVII were as follows
Figure imgf000065_0001
It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spiπt of the invention
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Claims

1 An immunogenic composition composing at least two of
(a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 1 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO
1 ,
(b) a second polypeptide, comprising a second ammo acid sequence, where the second ammo acid sequence composes an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (n) consisting of a fiagment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
(c) a third polypeptide, composing a third ammo acid sequence, where the third ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (ii) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO
3 2 A polypeptide composing at least two of
(a) a first ammo acid sequence comprising an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 1 and/or (n) consisting of a fiagment of at least 7 contiguous ammo acids from SEQ ID NO 1 ,
(b) a second ammo acid sequence comprising an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (u) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
(c) a third ammo acid sequence composing an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 3 3 A polypeptide composing ammo acid sequence
A-{-X-L-}n-B wherein each X is an ammo acid sequence of first polypeptide, second polypeptide or third polypeptide as defined m claim 1 , L is an optional linker ammo acid sequence, A is an optional N terminal ammo acid sequence, B is an optional C terminal ammo acid sequence, n is an integer of 2 or more
4 The polypeptide of claim 2 or claim 3, composing an ammo acid sequence selected from the group consisting of SEQ ID NOs 1 1, 13, 15, 17, 19 and 21
5 A bacterium which expresses at least two of (a) a first polypeptide comprising a first ammo acid sequence, where the first ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 1 and/or (π) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 1, (b) a second polypeptide, comprising a second ammo acid sequence, where the second ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID NO 2 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO 2, and/or
(c) a third polypeptide, comprising a third ammo acid sequence, where the third ammo acid sequence comprises an ammo acid sequence (i) having at least 90% sequence identity to SEQ ID
NO 3 and/or (n) consisting of a fragment of at least 7 contiguous ammo acids from SEQ ID NO
3 A polypeptide comprising ammo acid sequence SEQ ID NO 83
PCT/IB2010/052445 2009-06-01 2010-06-01 COMBINATIONS OF PNEUMOCOCCAL RrgB CLADES WO2010140119A1 (en)

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