WO2008152448A2 - Streptococcus proteins, and their use in vaccination - Google Patents

Streptococcus proteins, and their use in vaccination Download PDF

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Publication number
WO2008152448A2
WO2008152448A2 PCT/IB2007/004637 IB2007004637W WO2008152448A2 WO 2008152448 A2 WO2008152448 A2 WO 2008152448A2 IB 2007004637 W IB2007004637 W IB 2007004637W WO 2008152448 A2 WO2008152448 A2 WO 2008152448A2
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WO
WIPO (PCT)
Prior art keywords
seq
amino acids
polypeptide
group
pho3
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PCT/IB2007/004637
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French (fr)
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WO2008152448A3 (en
Inventor
Steven Neville Chatfield
Mark Redfern
Helen Harries
Joanne Bedwell
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Emergent Product Development Uk Limited
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Application filed by Emergent Product Development Uk Limited filed Critical Emergent Product Development Uk Limited
Priority to EP07874553A priority Critical patent/EP2104512A2/en
Publication of WO2008152448A2 publication Critical patent/WO2008152448A2/en
Publication of WO2008152448A3 publication Critical patent/WO2008152448A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/09—Lichens
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • This invention relates to the identification of combinations of proteins, and their use as a vaccine, a process of making the vaccine, and a method for immunizing a human or animal against a condition related to Group B Streptococcus (GBS).
  • GBS Group B Streptococcus
  • GBS also known as Streptococcus agalactiae
  • Streptococcus agalactiae is the causative agent of various conditions.
  • GBS causes: Early onset neonatal infection.
  • This infection usually begins in utero or within five days of birth and causes severe septicaemia, meningitis, pneumonia, cellulitis, osteomyelitis, and septic arthritis in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.
  • Schuchat (1998) Clinic. Microbiol. Rev. 11(3): 497-513. Newborns with early-onset GBS disease acquire the organism intrapartum from their mothers, who are colonized with GBS in the genital track. See id. GBS colonizes the vagina of about 25% of young women, and approximately 1% of infants born via a vaginal birth to colonized mothers will become infected. See id. Late onset neonatal infection.
  • the late onset neonatal infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including pneumonia, cellulitis, osteomyelitis, septic arthritis, abscesses, endocarditis, epiglottitis, and endopthalmitis. Adult infections.
  • GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.
  • GBS is Gram positive coccus belonging to Lancei ⁇ eld group B.
  • serotypes based on their capsular polysaccharide structure (types Ia, Ib, Ia/c, II, III, IV, V, VI, VII, or VIII) identified to date. See Shet et al. (2004) Indian J. Med. Res. 120: 141-150.
  • types Ia, II, and III account for 85 percent of the isolates from infants. See id.
  • GBS serotypes Ia, III, and V (in a descending order) account for 78-87% of early onset invasive disease in newborn infants and parturient women.
  • the present invention is based on the identification of beneficial combinations of GBS' proteins which can be used to provide an immunogenic response, useful in therapy.
  • the composition provides an improved therapeutic effect compared to the single peptides.
  • the present invention is directed to a method of inducing an immune response against
  • Group B Streptococcus in an animal comprising administering to the animal in need thereof an effective amount of an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1-59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101- 181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5; (c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO
  • the present invention is directed to a method of inducing an immune response against Group B Streptococcus in an animal comprising administering to the animal in need thereof an effective amount of an isolated first polynucleotide encoding the first polypeptide described above and an isolated second polynucleotide encoding the second polypeptide described above.
  • the immune response induced by the first polynucleotide and the second polynucleotide prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal.
  • the first polynucleotide and the second polynucleotide may be located in one or more expression vectors. More than one expression vectors may be identical or different or a combination thereof.
  • Streptococcus in an animal comprises administering to the animal in need thereof an effective amount of an isolated first antibody or antigen binding fragment thereof raised against the first polypeptide described herein and an isolated second antibody or antigen binding fragment thereof raised against the second polypeptide described herein.
  • the immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal.
  • the method of inducing an immune response further comprises administering an isolated third polypeptide, an isolated third antibody or antigen-binding fragment thereof raised against an isolated third polypeptide, or an isolated third polynucleotide.
  • the third polynucleotide described herein may comprise a promoter operably linked to the first polynucleotide or the second polynucleotide.
  • the third polynucleotide may encode a third polypeptide.
  • the third polypeptide may be an antigenic or immunogenic polypeptide, e.g., a polypeptide derived from a bacterium, a virus, a fungus, or a protozoan.
  • the antigenic or immunogenic polypeptide may be derived from Group B Streptococcus.
  • the third polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1-59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101-181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5;(c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (a Phol-13
  • SEQ DD NO: 18 (g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ DD NO: 21; (ii) amino acids 1 -77 of SEQ DD NO: 21; (iii) amino acids 106- 225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ DD NO:21; (h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 24; (ii) amino acids 43-84 of SEQ DD NO: 24; (iii) amino acids 1-84 of SEQ DD NO: 24; (iv) amino acids 88-122 of SEQ DD NO: 24; and (v) amino acids 38-122 of SEQ DD NO: 24; (i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO:
  • the first polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 33; (ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ED NO: 33; and (iv) amino acids 24-192 of SEQ ED NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ED NO: 21; (iv) amino acids 272-360 of SEQ ED NO: 21; and (v) amino acids 106-360 of SEQ
  • the present invention includes a method comprising administering an isolated first polypeptide and an isolated second polypeptide, wherein the first polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ E) NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ E) NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 68, (ii) amino acids 1-138 of SEQ E) NO: 68; (iii) amino acids 285-363 of SEQ E) NO: 68; (iv) amino acids 210-363
  • the method of the present invention comprises administering an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 21; (ii) amino acids 1-77 of SEQ E) NO: 21; (iii) amino acids 106- 225 of SEQ E) NO:21; (iv) amino acids 272-360 of SEQ E) NO:21; and (v) amino acids 106-360 of SEQ E) NO:21; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 68, (ii) amino acids 1-138 of SEQ E) NO: 68; (iii) amino acids 285-363 of SEQ E) NO: 68; (i) SEQ E) NO:
  • the present invention includes a method of inducing an immune response comprising administering an isolated first polypeptide, an isolated second polypeptide, and an isolated third polypeptide, wherein the first polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 33; (ii) amino acids 113-192 of SEQ E) NO: 33; (iii) amino acids 71-192 of SEQ E) NO: 33; and (iv) amino acids 24-192 of SEQ E ) NO: 33, the second polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 21; (ii) amino acids 1-77 of SEQ E) NO: 21; (iii) amino acids 106-225 of SEQ E) NO
  • the method may further comprise administering one or more carriers or one or more adjuvants, e.g., Alum and a CPG oligonucleotide.
  • one or more carriers or one or more adjuvants e.g., Alum and a CPG oligonucleotide.
  • the method of the present invention induces an immune response when administered in an animal in need thereof.
  • the immune response may be a humoral immune response or a cell- mediated immune response or a combination thereof.
  • the first polypeptide, the first polynucleotide, or the first antibody or antigen binding fragment thereof may be administered prior to or simultaneously with the second polypeptide, second polynucleotide, or second antibody or antigen binding fragment thereof.
  • the second polypeptide, second polynucleotide, or second antibody or antigen binding fragment thereof may be administered prior to the first polypeptide, the first polynucleotide, or the first antibody or antigen binding fragment thereof.
  • the polypeptides, polynucleotides or antibody or antigen binding fragment thereof can be administered as one or multiple doses.
  • polypeptides, polynucleotides, or antibody or antigen-binding fragment thereof used in the present invention may be administered via intramuscular injection, subcutaneous injection, intra-dermal injection, intradural injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.
  • the present invention also includes a composition comprising an isolated first polypeptide and an isolated second polypeptide, a composition comprising an isolated first polynucleotide encoding the first polypeptide and an isolated second polynucleotide encoding the second polypeptide, and a composition comprising an isolated first antibody or antigen-binding fragment thereof specifically binds the first polypeptide and an isolated second antibody or antigen binding fragment thereof specifically binds the second polypeptide described herein.
  • composition comprising an isolated first polypeptide and an isolated second polypeptide, an isolated first antibody or antigen binding fragment thereof and an isolated second antibody or antigen binding fragment thereof, or an isolated first polynucleotide and an isolated second polynucleotide further comprises an isolated third polypeptide, an isolated third antibody or antigen-binding fragment thereof raised against a third polypeptide, or an isolated third polynucleotide, respectively.
  • the third polynucleotide may encode a third polypeptide.
  • the third polypeptide used in the present invention may be an antigenic or immunogenic polypeptide, e.g., a polypeptide derived from Group B Streptococcus.
  • the third polypeptide used in the present invention may be the GBS polypeptides, fragments, derivatives, or variants thereof.
  • the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO
  • the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444 of S
  • the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ DD NO:21 ; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-3
  • the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:
  • composition of the present invention may further comprise one or more polysaccharides, carriers, or adjuvants, e.g., Alum and a CpG polynucleotide, or a combination thereof.
  • polysaccharides, carriers, or adjuvants e.g., Alum and a CpG polynucleotide, or a combination thereof.
  • composition of the present invention when administered in an animal, induces an immune response such as a humoral immune response or a cell-mediated immune response.
  • a composition comprises two or more of polypeptides, wherein the polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • a reference amino acid sequence selected from the group consisting of Phol-13, Phol-14, Phol-5,
  • a composition may comprise two or more of polynucleotides, wherein the polynucleotide comprises a nucleic acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference nucleic acid sequence encoding an amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2- 15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3- 23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polynucleotide encodes a polypeptide recognizable by an antibody preparation that specifically binds to a polypeptide consisting
  • composition of the present invention may comprise two or more of vectors, wherein the vector encodes a polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2- 15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3- 23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • a reference amino acid sequence selected from group consisting of Phol-13, Phol-14
  • a composition may comprise two or more of host cells, wherein the host cell comprises a vector encoding a polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2- 10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3- 22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • a reference amino acid sequence selected from group consisting of Phol-13
  • a composition may comprise antibodies raised against two or more of polypeptides, wherein the polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 ( ⁇ hol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol
  • composition of the present invention further comprises a carrier, an , adjuvant, or a targeting molecule.
  • the present invention also includes a diagnostic reagent comprising the composition of the present invention described herein.
  • the present invention includes a method of detecting anti-Group B
  • Streptococcus antibodies in a test sample comprising the steps of (a) contacting the sample with the composition described herein to form Group B Streptococcus antigen: anti-Group B Streptococcus antibody immunocomplexes; and (b) detecting the presence of or measuring the amount of the immunocomplexes formed during step (a) or a diagnostic kit for detecting an antibody or antigen-binding fragment thereof to Group B Streptococcus, wherein the kit comprises the composition described elsewhere herein, a container means for contacting the polypeptide or composition with a test sample, and a reagent means for detecting or measuring Group B Streptococcus antigen: anti-Group B Streptococcus antibody immunocomplexes formed between the polypeptide and the antibody or antigen-binding fragment thereof.
  • BRIEF DESCRIPTION OF THE DRAWINGS BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1A depicts the Hopp/Woods antigenicity profile of the GBS polypeptides: IA:
  • Figure 2 depicts Opsonophagocytosis of Group B Streptococcus strain A909 opsonised with monkey sera raised against GBS vaccine comprising Pho 3-1 (SEQ ID NO: 33) and MSlO (NPGAP: SEQ ID NO: 68).
  • the present invention is directed to polypeptides and polynucleotides derived from GBS strains, composition comprising the polypeptides and polynucleotides, and methods of administering the composition to prevent or treat diseases related to Group B Streptococcus infection.
  • Organisms likely to contain the polypeptides include, but are not limited to, Enterococcus species ⁇ e.g., Enterococcus faecalis or Enterococcus faecium), Lactobacillus species ⁇ e.g.
  • Lactobacillus casei, Lactobacillus sakei, or Lactobacillus salivarius Lactococcus species ⁇ e.g., Lactococcus lactis), Leuconostoc species ⁇ e.g., Leuconostoc mesenteroides), Oenococcus species ⁇ e.g., Oenococcus oeni), Pediococcus species ⁇ e.g., Pediococcus pentosaceus), Staphylococcus species ⁇ e.g., Staphylococcus aureus or Staphylococcus epidermidis), or Streptococcus species ⁇ e.g., Streptococcus agalatiae, Streptococcus bovis, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumonia
  • Vaccines to each of these organisms may be developed in the same way as described for
  • a or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • nucleic acid or “nucleic acid fragment” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide or construct.
  • Two or more, nucleic acids of the present invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate (non-identical) polynucleotide constructs, e.g., on separate plasmids.
  • any nucleic acid or nucleic acid fragment may encode a single polypeptide, e.g.
  • a single antigen, cytokine, or regulatory polypeptide may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides.
  • a nucleic acid may encode a regulatory element such as a promoter or a transcription terminator, or may encode a specialized element or motif of a polypeptide or protein, such as a secretory signal peptide or a functional domain.
  • polynucleotide refers to an isolated molecule or construct, e.g., a virus genome (e.g., a non-infectious viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles as described in (Darquet, A-M et al, Gene Therapy 4:1341-1349 (1997)) comprising a polynucleotide.
  • virus genome e.g., a non-infectious viral genome
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • derivatives of pDNA e.g., minicircles as described in (Darquet, A-M et al, Gene Therapy 4:1341-1349 (1997) comprising a polynucleotide.
  • a polynucleotide may be provided in linear (e.g., mRNA), circular (e.g., plasmid), or branched form as well as double- stranded or single-stranded forms.
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and comprises any chain or chains of two or more amino acids.
  • terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of a “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • polypeptides which have undergone post-translational modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • GNS polypeptide encompasses full length polypeptide, serotypic, and other variants of full length polypeptide, fragments of full length polypeptide, serotypic or other variants of fragments of full length polypeptide, derivatives of full-length polypeptide, derivatives of fragments of full-length polypeptide, analogues of full-length polypeptide, analogues of fragments of full-length polypeptide, and chimeric and fusion polypeptide comprising full length polypeptide or one or more fragments of full length polypeptide.
  • fragment when referring to GBS polypeptides , of the present invention include any polypeptides which retain at least some of the, immunogenicity or antigenicity of the naturally-occurring proteins.
  • Fragments of GBS polypeptides of the present invention include proteolytic fragments, deletion fragments and, in particular, fragments which exhibit increased solubility during expression, purification, and/or administration to an animal. Fragments of GBS polypeptides further include proteolytic fragments or deletion fragments which exhibit reduced pathogenicity when delivered to a subject.
  • Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three- dimensional epitopes.
  • An “epitopic fragment” of a polypeptide antigen is a portion of the antigen that contains an epitope.
  • An “epitopic fragment” may, but need not, contain amino acid sequence in addition to one or more epitopes.
  • variant refers to a polypeptide that differs from the recited polypeptide due to amino acid substitutions, deletions, insertions, and/or modifications. Variants may occur naturally, such as a serotypic variant.
  • serotypic variant is intended polypeptides or polynucleotides that are present in a different GBS serotype. The serotypic variants are naturally occurring variants, but it can also be produced using art-known mutagenesis techniques. In the case of GBS, at least nine different serotypes, or "serotypic variants,” have been identified.
  • GBS serotypes include, but not limited to, serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VII, and VIII. There may be sequence variations between serotypes in some genes, for example, the genes encoding GBS Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS16, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, and PGK polypeptide.
  • Table 5 shows that an amino acid sequence of Phol-13 of GBS serotype III strain M732 have 98% homology to an amino acid sequence of the polypeptide found in GBS sero
  • Naturally occurring variants can also be found in related bacteria, e.g., Streptococcus agalatiae, Streptococcus bovis, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis, Streptococcus /nutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus suis, or Streptococcus thermophilus.
  • Polypeptides having homology to the GBS polypeptides can be found in these microorganisms.
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variant polypeptides differ from an identified sequence by substitution, deletion or addition of five amino acids or fewer.
  • Such variants may generally be identified by modifying a polypeptide sequence, and evaluating the antigenic properties of the ⁇ modified polypeptide using, for example, the representative procedures described herein.
  • Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • Polypeptide variants preferably exhibit at least about 60-70%, for example 75%, 80%,
  • variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.
  • Derivatives of GBS polypeptides of the present invention are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins.
  • An analog is another form of the GBS polypeptides of the present invention. An example is a proprotein which can be activated by cleavage of the proprotein to produce an active mature polypeptide.
  • Variants may also, or alternatively, contain other modifications, whereby, for example, a polypeptide may be conjugated or coupled, e.g., fused to a heterologous polypeptide, e.g., a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein.
  • the polypeptide may also be conjugated or produced coupled to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., 6-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated or coupled to an immunoglobulin Fc region.
  • the polypeptide may also be conjugated or coupled to a sequence that imparts or modulates the immune response to the polypeptide (e.g. a T-cell epitope, B-cell epitope, cytokine, chemokine, etc.) and/or enhances uptake and/or processing of the polypeptide by antigen presenting cells or other immune system cells.
  • the polypeptide may also be conjugated or coupled to other polypeptides/epitopes from GBS and/or from other bacteria and'or other viruses to generate a hybrid immunogenic protein that alone or in combination with various adjuvants can elicit protective immunity to other pathogenic organisms.
  • an "isolated" GBS polypeptide or a fragment, variant, or derivative thereof is intended a GBS polypeptide or protein that is not in its natural form. No particular level of purification is required.
  • an isolated GBS polypeptide can be removed from its native or natural environment.
  • Recombinantly produced GBS polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention, as are native or recombinant GBS polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • an isolated GBS polypeptide or protein can be provided as a live or inactivated expression vector expressing an isolated GBS polypeptide and can include those found in inactivated GBS vaccine compositions.
  • sequence identity refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences.
  • sequence identity is used herein interchangebly with the terms “sequence similarity” or “sequence homology.” When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position.
  • the percentage “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of "identical” positions.
  • the number of "identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity.” Percentage of "sequence identity” is determined by comparing two optimally aligned sequences over a comparison window (e.g., SEQ ID NO: 2 and a homologous polypeptide from another GBS isolate).
  • a comparison window e.g., SEQ ID NO: 2 and a homologous polypeptide from another GBS isolate.
  • the portion of a polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions termed gaps while the reference sequence (e.g. SEQ ID NO: 2) is kept constant.
  • An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical” positions between the reference and comparator sequences.
  • Sequence identity between two sequences can be determined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology Information as of September 1, 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. ScL USA 90(12):5873-5877, 1993).
  • BLASTN for nucleotide sequence comparison
  • BLASTP for polypeptide sequence comparison
  • polypeptide or “at least two different polynucleotide,” as used herein, refers to the polypeptides or polynucleotides comprising two or more polypeptides or polynucleotide, respectively, that are not identical.
  • first polypeptide and the second polypeptide have no homology or less than 100% homology or identity to each other.
  • at least two different is used herein interchangeably with “at least two,” “more than one,” “two or more,” or “a combination of two or more.”
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, for example a mammal, for example, a human.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an immune response in an animal, as determined by any method known in the art.
  • antigenic epitope as used herein, is defined as a portion of a protein to which an antibody or T-cell receptor can immunospecifically bind its antigen as determined by any method well known in the art. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Whereas all immunogenic epitopes are antigenic, antigenic epitopes need not be immunogenic.
  • a "coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, and the like, are outside the coding region.
  • compositions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio, hi some embodiments, the polypeptide, polynucleotides, compositions, and vaccines of the present invention are pharmaceutically acceptable.
  • an "effective amount” is that amount the administration of which to an individual, either in a single dose or as part of a series, is effective for treatment or prevention.
  • An amount is effective, for example, when its administration results in a reduced incidence of Group B Streptococcus infection relative to an untreated individual, as determined two weeks after challenge with infectious GBS. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated ⁇ e.g. human, nonhuman primate, primate, etc.), the responsive capacity of the individual's immune system, the degree of protection desired, the formulation of the vaccine, a professional assessment of the medical situation, and other relevant factors.
  • a single dose is from about 10 ⁇ g to 10 mg/kg body weight of purified polypeptide or an amount of a modified carrier organism or virus, or a fragment or remnant thereof, sufficient to provide a comparable quantity of the recombinantly expressed GBS polypeptides.
  • peptide vaccine or "subunit vaccine” refers to a composition comprising one or more polypeptides of the present invention, which when administered to an animal are useful in stimulating an immune response against GBS infection.
  • additive effect refers to the effect, e.g., raising an immune response, that is the sum of the effects of two or more polypeptides acting independently.
  • immunized sera raised against the Pho3-1 polypeptide and the MSlO polypeptide separately, opsonize GBS and result in about 10% GBS uptake and in about 15% GBS uptake, respectively, immunized sera raised against both the Pho3- 1 polypeptide and MSlO polypeptide together opsonize GBS and result in about 25% GBS uptake.
  • the term "synergistic effect” or “synergy” is used herein to denote the effect that is more than additive, i.e., the combined immunostimulatory effect is greater than the expected total stimulatory effect calculated on the basis of the sum of the effects resulting from two or more polypeptides acting independently.
  • the immunized sera raised against both the Pho3-1 polypeptide and MSlO polypeptide together in the above example may opsonize GBS and result in more than 25% GBS uptake.
  • subject is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, immunization, or therapy is desired.
  • Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals such as bears, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on.
  • the animal is a human subject.
  • mammals and comprises mammals and birds, as well as fish, reptiles, and amphibians.
  • the term animal also encompasses model animals, e.g., disease model animals.
  • the term animal includes valuable animals, either economically or otherwise, e.g., economically important breeding stock, racing animals, shqw animals, heirloom animals, rare or endangered animals, or companion animals.
  • the mammal can be a human subject, a food animal or a companion animal.
  • an "subject in need thereof refers to an individual for whom it is desirable to treat, i.e., to prevent, cure, retard, or reduce the severity of disease symptoms related to GBS infection, and/or result in no worsening of GBS disease over a specified period of time.
  • priming or “primary” and “boost” or “boosting” as used herein to refer to the initial and subsequent immunizations, respectively, i.e., in accordance with the definitions these terms normally have in immunology. However, in certain embodiments, e.g., where the priming component and boosting component are in a single formulation, initial and subsequent immunizations may not be necessary as both the “prime” and the “boost” compositions are administered simultaneously.
  • passive immunity refers to the immunity to an antigen developed by a host animal, the host animal being given antibodies produced by another animal, rather than producing its own antibodies to the antigen.
  • active immunity refers to the production of an antibody by a host animal as a result of the presence of the target antigen.
  • the present invention includes a combination of more than two polypeptides, e.g., a combination of an isolated first polypeptide and an isolated second polypeptide.
  • the polypeptides, used as an isolated first polypeptide or an isolated second polypeptide comprises an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence found in GBS serotype III strain M732, fragments, derivatives, or variants thereof.
  • Variants commonly occur with many genes of simple, unicellular organisms. Examples of variants are serotypic variants. Another example of variants can be found within a same strain. For example, even within a same strain, different isolates obtained from different patients and/or geographical locations can exhibit some variation.
  • GBS outer membrane proteins or polypeptides and polynucleotides encoding the polypeptides or proteins are disclosed in International Appl. No. PCT/GB99/04377, filed December 22, 1999 and published as WO0037646 and International Appl. No. PCT/GB02/01089, filed March 11, 2002 and published as WO 02072623, both incorporated herein by reference in their entireties. Additional GBS outer surface proteins or polypeptides and polynucleotides are also disclosed in International Appl. No. PCT/GB99/04376, filed on December 22, 1999 and published as WO 00/37490 incorporated herein by reference in its entirety.
  • Phol-13 polypeptide sequence (SEQ ID NO: 2) found in GBS strain M732 is publicly available and have Accession No. CAC09086 in Genbank.
  • polynucleotide sequence encoding the Phol-13 polypeptide found in GBS M732 is publicly available as Accession No. AX026644 in Genbank and is designated herein as SEQ ID NO: 1.
  • Serotypic variants of the Phol-13 polypeptide can be found in other GBS strains.
  • the corresponding polypeptide found in GBS strain A909 is publicly available, and the locus tag is designated as SAK l600 (SEQ ID NO: 3).
  • the Phol-14 polypeptide sequence (SEQ ID NO: 5) found in GBS strain M732 is publicly available and has Accession No. CAC09087 in Genbank. [0081] SEQ ID NO: 5
  • the phol-14 polynucleotide sequence (SEQ ID NO: 4) encoding the Phol-14 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026646 in Genbank.
  • the Phol-5 polypeptide sequence (SEQ ID NO: 8) found in GBS strain M732 is a transglycosylase SLT domain family protein.
  • the polypeptide sequence is publicly available and has Accession No. CAC09088 in Genbank.
  • SEQ ID NO: 7 The phol-5 polynucleotide sequence (SEQ ID NO: 7) encoding the Phol-5 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026648 in Genbank. [0089] SEQ ID NO: 7
  • a serotypic variant of the Phol-5 polypeptide found in GBS strain A909 is publicly available and has locus tagNo. SAK_1205 (SEQ ID NO: 9). [0091] SEQ DD NO: 9
  • the pho3-3 polynucleotide sequence (SEQ DD NO: 10) encoding the Pho3-3 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026650 in Genbank. [0095] SEQDDNO: 10
  • a serotypic variant of the Pho3-3 polypeptide in GBS strain A909 is publicly available and has locus tag no. SAK_1286 (SEQ DD NO: 12). [0097] SEQDDNO: 12
  • the pho2-10 polynucleotide sequence (SEQ ID NO: 13) encoding the Pho2-10 polypeptides found in GBS strain M732 is publicly available and has Accession No. AX026652 in Genbank.
  • a serotypic variant of the Pho2-10 polypeptide is found in GBS strain A909 and has locus tag no. SAK_1175 (SEQ ID NO: 16).
  • the pho2-15 polynucleotide sequence (SEQ ED NO: 17) encoding the Pho2-15 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026655 in Genbank.
  • a serotypic variant of the Pho2-15 polypeptide is found in GBS strain A909 and has locus tag no. SAKJ893 (SEQ E) NO: 19). [00111] SEQE)NO: 19
  • SEQ E The pho2-2 polynucleotide sequence (SEQ E) NO: 20) encoding the Pho2-2 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026657 in Genbank. [00115] SEQ E) NO: 20
  • a serotypic variant of the Pho2-2 polypeptide is found in GBS strain A909 and has locus tag no. SAK_O3O3 (SEQ ED NO: 22). [00117] SEQ ID NO: 22
  • the Pho3-14 polypeptide sequence (SEQ ID NO: 24) in GBS strain M732 is publicly available and has AccessionNo. CAC09094 in Genbank. [00119] SEQ ID NO: 24
  • the pho3-14 polynucleotide sequence (SEQ ID NO: 23) encoding the Pho3-14 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026659 in Genbank.
  • a serotypic variant of the Pho3-14 polypeptide is found in GBS strain A909 and has locus tag no. SAK_2026 (SEQ ID NO: 25). [00123] SEQ ID NO: 25
  • the pho3-17 polynucleotide sequence (SEQ ID NO: 26) encoding the Pho3-17 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026661 in Genbank.
  • a serotypic variant of the Pho3-17 polypeptide is found in GBS strain A909 and has locus tagno. SAK_0144 (SEQ E) NO: 28).
  • the pho3-18 polynucleotide sequence (SEQ E) NO: 29) encoding the Pho3-18 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026663 in Genbank.
  • a serotypic variant of the Pho3-18 polypeptide is found in GBS strain A909 and has locus tagno. SAK_1796 (SEQ ID NO: 31). [00135] SEQK)NO: 31
  • the Pho3-1 polypeptide sequence (SEQ ID NO: 33) in GBS strain M732 is publicly available and has Accession No. CAC09097 in Genbank. [00137] SEQ ID NO: 33
  • pho3-l (ME: P31) polynucleotide sequence (SEQ ID NO: 32) encoding the Pho3-1 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026665 ih Genbank.
  • a serotypic variant of the Pho3-1 is found in GBS strain A909 and has locus tag no.
  • Thepho3-21 (ME: P31) polynucleotide sequence (SEQ ID NO: 35) encoding the Pho3-21 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026667 in Genbank.
  • a serotypic variant of the Pho3-21 polypeptide is found in GBS strain A909 and has locus tagno. SAK l656 (SEQ ID NO: 37). [00147] SEQ ID NO: 37:
  • the pho3-22 polynucleotide sequence (SEQ ID NO: 38) encoding the Pho3-22 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026669 in Genbank.
  • a serotypic variant of the Pho3-22 polypeptide is found in GBS strain A909 and has locus tag no. SAK_0807 (SEQ ID NO: 40). [00153] SEQ ID NO: 40:
  • the Pho3-23 polypeptide sequence (SEQ ED NO: 42) in GBS strain M732 is publicly available and has locus tag No. CAC09100 in Genbank. [00155] SEQ ID NO: 42
  • the pho3-23 polynucleotide sequence (SEQ ID NO: 41) encoding the Pho3-23 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026671 in Genbank.
  • a serotypic variant of the Pho3-23 polypeptide is found in GBS strain A909 and has locus tag no. SAKJ281 (SEQ ID NO: 43). [00159] SEQ ID NO: 43:
  • SEQ ID NO: 45 The Pho3-24 polypeptide sequence found in GBS strain M732 is publicly available and has Accession No. CAC09101 in Genbank. [00161] SEQ ID NO: 45 1 mfmgipqyff ylilavlpiy iglffkkrfa lyeiifslsf ivmmltgstf nqlksllayv
  • the pho3-24 polynucleotide sequence (SEQ ID NO: 44) encoding the Pho3-24 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026673 in Genbank.
  • a serotypic variant of the Pho3-24 polypeptide is found in GBS strain A909 and has locus tag no. SAK_1811 (SEQ ID NO: 46). [00165] SEQ ID NO: 46:
  • the Pho3-29 polypeptide sequence (SEQ ID NO: 48) in GBS strain M732 is publicly available and has Accession No. CAC09102 in Genbank. [00167] SEQ ID NO: 48
  • the pho3-29 polynucleotide sequence (SEQ ID NO: 47) encoding the Pho3-29 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026675 in Genbank.
  • SEQ ID NO: 47 1 atgaataaaa taacgacatt atcaaccatc gccctgactt taatgctttg cgttggatgt 61 tctgccaata aagataatca aaaactaaa actgaggatc
  • a serotypic variant of the Pho3-29 polypeptide is found in GBS strain A909 and has locus tag no. SAK_0809 (SEQ ID NO: 49). [00171] SEQ ID NO: 49:
  • the pho3-50 polynucleotide sequence (SEQ ID NO: 50) encoding the Pho3-50 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026677 in Genbank.
  • a serotypic variant of the Pho3-50 polypeptide is found in GBS strain A909 and has locus tag no. SAK 0890 (SEQ ID NO: 52). [00177] SEQ ID NO: 52:
  • MS4 polypeptide sequence in GBS strain M732 is publicly available and has Accession No. CAC09078 in Genbank. [00179] SEQ ID NO: 54
  • MS4 polynucleotide sequence (SEQ ID NO: 53) encoding the MS4 polypeptide in
  • GBS strain M732 is publicly available and has AccessionNo. AX026599 in Genbank. [00181] SEQ ID NO: 53
  • a serotypic variant of the MS4 polypeptide is found in GBS strain A909 and has locus
  • MSIl polypeptide sequence (SEQ ID NO: 57) in GBS strain M732 is publicly available and has Accession No. CAC09079 in Genbank. [00185] SEQ ID NO: 57
  • MSl1 polynucleotide sequence (SEQ ID NO: 56) encoding the MSl1 polypeptide in
  • GBS strain M732 is publicly available andhas Accession No. AX026601 in Genbank. [00187] SEQ ID NO: 56
  • a serotypic variant ofthe MS4 polypeptide is found in GBS strain A909 and has locus tagno. SAK l788 (SEQ ID NO: 58). [00189] SEQ ID NO: 58:
  • MS16 polypeptide sequence I (SEQ ID NO: 60 ⁇ in GBS strain M732 is publicly available and has Accession No. CAC09080 in Genbank. [00191] SEQ ID NO: 60
  • MS16 polynucleotide sequence I (SEQ ID NO: 59) encoding the MS16 polypeptide in GBS strain M732 is publicly available andhas AccessionNo. AX026603 in Genbank. [00193] SEQ ID NO: 59
  • MS16 polypeptide sequence II (SEQ ID NO: 62) in GBS strain M732 is publicly available and has Accession No. CAC09081 in Genbank.
  • MS16 polynucleotide sequence II (SEQ ID NO: 61) encoding the MS16 polypeptide in GBS strain M732 is publicly available andhas Accession No. AX026605 in Genbank. [00197] SEQ ID NO: 61
  • a serotypic variant ofthe MS16 polypeptide is found in GBS strain A909 and has locus tag no. SAK_0475 (SEQ ID NO: 63). [00199] SEQ ID NO: 63:
  • MS14 polypeptide sequence (SEQ ID NO: 65) in GBS strain M732 is publicly available and has Accession No. CAC09082 in Genbank. [00201] SEQ ID NO: 65
  • MS14 polynucleotide sequence (SEQ ID NO: 64) encoding the MS14 polypeptide in
  • GBS strain M732 is publicly available and has Accession No. AX026607 in Genbank. [00203] SEQ ID NO: 64
  • a serotypic variant ofthe MS14 polypeptide is found in GBS strain A909 and has locus tag no. SAK_1267 (SEQ ID NO: 66). [00205] SEQ ID NO: 66:
  • MSlO polypeptide sequence (SEQ ID NO: 68) in GBS strain M732 is publicly available and has Accession No. CAC09083 in Genbank.
  • MSlO NP GAP; ME:NAP
  • SEQ ID NO: 67 The MSlO polypeptide in GBS strain M732 is publicly available and has Accession No. AX026609 in Genbank.
  • a serotypic variant of the MSlO polypeptide is found in GBS strain A909 and has locus tag no. SAK_0947 (SEQ ID NO: 69). [00211] SEQ ID NO: 69:
  • a combination of two or more polypeptides induces an immune response.
  • the immune response may have additive effect.
  • a combination of two or more polypeptides or polynucleotides may induce synergistic immune response.
  • Polypeptides, and fragments, derivatives, analogs, or variants thereof of the present invention can be antigenic and immunogenic fragment of the GBS polypeptides, which may also be used to prevent or treat, i.e., cure, ameliorate, lessen the severity of, or prevent or reduce contagion of infectious disease caused by GBS, or other species as disclosed herein.
  • antigens and its related term “antigenic” as used herein and in the claims refer to a substance that binds specifically to an antibody or T-cell receptor. In some embodiments the antigens are immunogenic.
  • immunogenic refers to an antigen capable of inducing an immune response directed against itself when administered to a subject.
  • Peptides or polypeptides e.g., immunogenic epitopes, capable of eliciting an immunogenic response are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins nor to the ammo or carboxyl terminals.
  • Polypeptides that are extremely hydrophobic, and those of six or fewer residues generally are ineffective at inducing antibodies, but may still bind antibodies raised against larger portions of the polypeptide; longer peptides, especially those containing proline residues, usually are effective (Sutcliffe, J. G., et al., Science 219:660-666 (1983)).
  • antigenic epitopes or antigenic fragments can contain a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 contained within the amino acid sequence of the polypeptides of the invention.
  • Antigenic as well as immunogenic epitopes may be linear, i.e., be comprised of contiguous amino acids in a polypeptide, or may be three dimensional, i.e., where an epitope is comprised of non-contiguous amino acids which come together due to the secondary or tertiary structure of the polypeptide, thereby forming an epitope.
  • a fragment comprising an antigenic and immunogenic polypeptide of the present invention can be predicted based on antigenicity of the GBS polypeptides described herein.
  • Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J.
  • linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 2.0 software program available from the Oxford Molecular Group.
  • This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, parker antigenicity method, Parker et al., Biochem. (1986), 25, 5425-5432; Protrusion Index antigenicity, Thornton et al., EMBO Journal (1986) 5: 409-413; and Welling antigenicity, FEBS Lett. (1985) 188:215-218.
  • Figure 1 shows the Hope/Woods antigenicity profile of the GBS polypeptides described herein.
  • Other techniques e.g., the Kyte-Doolittle technique, Kyte et al., J. MoI. Biol. (1982) 157:105-132, may be used to measure hydropathy of the GBS polypeptides.
  • an antigenic or immunogenic fragment of the Phol-13 polypeptide may be selected from the group consisting of amino acids 1- 21, 28-34, 51-56, 27-41, 55-93, 43-53, 94-116, 119-146, 119-146, 138-153, 154-180, 146-182, 159-166, and 168-175 of SEQ E) NO: 2.
  • an antigenic or immunogenic Phol-13 polypeptide fragment may be amino acids 21-59, 132-181, 101-181, or 1- 181 of SEQ ID NO: 2.
  • An antigenic or immunogenic polypeptide fragment of the Phol-14 polypeptide may be selected from the group consisting of amino acids 25-31, 19-31, or 1-45 of SEQ ID NO: 5.
  • An antigenic or immunogenic polypeptide fragment of the Phol-5 polypeptide may be selected from the group consisting of amino acids 1-14, 1-22, 43-115, 45-87, 127-220, 178-192, 145-157, and 145-163 or SEQ ID NO: 8.
  • an antigenic or immunogenic Phol-5 polypeptide fragment may be amino acids 43-115, 139-192, or 43-192 of SEQ ID NO: 8.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-3 polypeptide may be selected from the group consisting of amino acids 1-18, 18-42, 58-68, 47-53, 69-110, and 165-197 of SEQ ID NO: 11. In preferred embodiments, an antigenic or immunogenic Pho3-3 polypeptide fragment may be amino acids 1-88, 1-110, or 165-197 of SEQ ID NO: 11. [00221] An antigenic or immunogenic polypeptide fragment of the Pho2-10 polypeptide may be selected from the group consisting of amino acids 13-19, 37-61, 80-98, 1-24, 85-101, 121-133, 127-143, 130-157, 64-84 and 175-197 of SEQ ID NO: 14. In preferred embodiments, an antigenic or immunogenic Pho2-10 polypeptide fragment may be amino acids 1-59, 84-149, or 1- 149 of SEQ ID NO: 14 or SEQ ID NO: 15.
  • An antigenic or immunogenic polypeptide fragment of the Pho2-15 polypeptide may be amino acids 29-95, 1-95, 68-79 or 29-125 of SEQ DD NO: 18. In preferred embodiments, an antigenic or immunogenic Pho2-15 polypeptide fragment may be amino acids 29-95, 1-95, or 29- 125 of SEQ ID NO: 18.
  • An antigenic or immunogenic polypeptide fragment of the Pho2-2 polypeptide may be selected from the group consisting of amino acids 70-78, 106-147, 168-200, 273-290, and 301- 350 of SEQ ID NO:21.
  • an antigenic or immunogenic Pho2-2 polypeptide fragment may include amino acids 1-77, 106-225, 272-360, or 106-360 of SEQ ID NO:21.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-14 polypeptide may be selected from the group consisting of amino acids 1-36, 43-65, 65-91, and 91-122 of SEQ ID NO: 24.
  • an antigenic or immunogenic Pho3-14 polypeptide fragment may be amino acids 43-84, 1-84, 88-122, or 38-122 of SEQ BD NO: 24.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-17 polypeptide may be selected from the group consisting of amino acids 27-40, 29-62, 29-84, 101-122, 97-189, 110- 140, 131-149, 154-189, and 155-170 of SEQ ID NO: 27.
  • an antigenic or immunogenic Pho3-17 polypeptide fragment may be amino acids 29-79, 101-189, or 29-189 of SEQ ID NO: 27.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-18 polypeptide may be selected from the group consisting of amino acids 23-45, 80-105, 105-142, 146-182, 190-217, 269-278, and 302-325 of SEQ ID NO: 30.
  • an antigenic or immunogenic Pho3-18 polypeptide fragment may be amino acids 79-182, 79-217, 23-217, or 23- 278 of SEQ ID NO: 30.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-1 polypeptide may be selected from the group consisting of amino acids 24-39, 71-99, 113-150, 150-192, and 113-192 of SEQ DD NO: 33.
  • an antigenic or immunogenic Pho3-1 polypeptide fragment may be amino acids 113-192, 71-192, or 24-192 of SEQ DD NO: 33.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-21 polypeptide may be selected from the group consisting of amino acids 25-40, 56-80, 98-107, 133-142, 153-172, and 181-202 of SEQ DD NO: 36. In preferred embodiments, an antigenic or immunogenic Pho3-21 polypeptide fragment may be amino acids 25-88, 132-202, 25-172, or 25-202 of SEQ DD NO: 36. [00229] An antigenic or immunogenic polypeptide fragment of the Pho3-22 polypeptide may be selected from the group consisting of amino acids 27-60, 60-94, and 27-94 of SEQ ID NO: 39. In preferred embodiments, an antigenic or immunogenic Pho3-22 polypeptide fragment may be amino acids 27-94 of SEQ ID NO: 39.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-23 polypeptide may be selected from the group consisting of amino acids 76-87, 111 -180, 111 -234, 72-234, or 40-335 of SEQ ID NO: 42.
  • an antigenic or immunogenic Pho3-23 polypeptide fragment may be amino acids 111-180, 111-234, 72-234, or 40-335 of SEQ ID NO: 42.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-24 polypeptide may be selected from the group consisting of amino acids 159-180, 260-277, 346-370, and 300-310 of SEQ ID NO: 45.
  • an antigenic or immunogenic Pho3-24 polypeptide fragment may be amino acids 98-180, 259-370, or 98-370 of SEQ ID NO: 45.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-29 polypeptide may be amino acids 21-33 of SEQ ID NO: 48.
  • An antigenic or immunogenic polypeptide fragment of the Pho3-50 polypeptide may be selected from the group consisting of amino acids 1-20, 28-37, 79-95, 102-112, 140-155, 192- 202, and 202-217 of SEQ ID NO: 51.
  • an antigenic or immunogenic Pho3-50 polypeptide fragment may be amino acids 78-154, 78-217, or 1-154 of SEQ ID NO: 51.
  • An antigenic or immunogenic polypeptide fragment of the MS4 polypeptide may be selected from the group consisting of amino acids 1-44, 51-63, 80-102, 102-117, 191 -205, 205- 225, 231-253, and 281-321 of SEQ ID NO: 54.
  • an antigenic or immunogenic MS4 polypeptide fragment may be amino acids 190-261, 190-321 or 1-118 of SEQ ID NO: 54.
  • An antigenic or immunogenic polypeptide fragment of the MS 11 polypeptide may be selected from the group consisting of amino acids 1-24, 24-35, 56-79, 112-140, 91-111, 182-202, 205-220, 240-270, 274-295, and 297-310 of SEQ ID NO: 57.
  • an antigenic or immunogenic MSI l polypeptide fragment may be amino acids 1-139, 184-311, or 184-398 of SEQ ID NO: 57.
  • An antigenic or immunogenic polypeptide fragment of the MS 16 polypeptide may be selected from the group consisting of amino acids 48-77, 101-116, 128-145, and 157-172 of SEQ ID NO: 60 and amino acids 1-30 and 75-105 of SEQ ID NO: 62.
  • an antigenic or immunogenic MS 16 polypeptide fragment may be amino acids 24-77, 101-172, or 24-172 of SEQ ID NO: 60 or SEQ ID NO: 62.
  • An antigenic or immunogenic polypeptide fragment of the MS 14 polypeptide may be selected from the group consisting of amino acids 1-18, 32-45, 141-154, 154-162, 162-180, and 241-268 of SEQ ID NO: 65.
  • an antigenic or immunogenic MS14 polypeptide fragment may be amino acids 1-95, 141-181, or 141-268 of SEQ ID NO: 65.
  • An antigenic or immunogenic polypeptide fragment of the MSlO polypeptide may be selected from the group consisting of amino acids 31-50, 78-89, 105-138, 212-220, 255-270, 285- 315, 330-360, 382-400, and 405-445 of SEQ ID NO: 68.
  • an antigenic or immunogenic MSlO polypeptide fragment may be amino acids 1-138, 285-363, 210-363, 210- 444, or 285-444 of SEQ ID NO: 68.
  • the beginning and ending residues of the antigenic or immunogenic polypeptide fragment may vary depending upon the computer modeling program used or the method used for determining the domains.
  • Isolated antigenic and immunogenic polypeptides of the present invention in addition to those encoded by polynucleotides of the invention, may be provided as a recombinant protein, a purified subunit, a viral vector expressing the protein, or may be provided in the form of an inactivated GBS vaccine, e.g., a live-attenuated virus vaccine, a heat-killed virus vaccine, etc.
  • an inactivated GBS vaccine e.g., a live-attenuated virus vaccine, a heat-killed virus vaccine, etc.
  • Production of the polypeptides of the present invention can be achieved by culturing the host cells, expressing the polynucleotides of the present invention, and recovering the polypeptides. Determining conditions for culturing the host cells and expressing the polynucleotide are generally specific to the host cell and the expression system and are within the knowledge • of one of skill in the art. Likewise, appropriate methods for recovering the polypeptide of interest are known to those in the art, and include, but are not limited to. chromatography, filtration, precipitation, or centrifugation.
  • polypeptides of the present invention are suitable for using as a vaccine composition as the polypeptides have low levels of sequence identity with human proteins.
  • the compositions comprising a combination of the polypeptides can elicit a strong immune response without producing an immune response against natural human polypeptides, thereby improving the safety profile of the combinations.
  • Table 3 shows the polypeptides of the invention and the levels of homology between the polypeptides and other polypeptides found in humans.
  • GBS polypeptides are known from different serotypes, different strains, and different species.
  • sequence identity between the GBS polypeptides and the corresponding polypeptide sequences from- different serotypes or strains or different species are publicly available as shown in Table 4.
  • Table 5 shows the polypeptide sequence identities between the GBS A909 and different species, serotypes, and strains.
  • Serotypic variation of GBS polypeptides is lower than the variation among species.
  • Serotypic variants are therefore expected to be immunologically cross-reactive.
  • the present invention thus includes GBS polypeptides found in other Streptococcus spp, e.g., Streptococcus agalactiae serotypes such as, but not limited to, Ia, Ib, Ia/c, II, III, IV, V, VI, VII, and VIII, or Streptococcus agalactiae, Streptococcus bovis, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus suis, or Streptococcus thermophilus or fragments thereof.
  • Streptococcus spp e.
  • the present invention is directed to an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1- 59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101-181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5;
  • the first polypeptide and the second polypeptide induces an immune response against GBS.
  • the immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal.
  • the first polypeptide and the second polypeptide further comprises an isolated third polypeptide.
  • the third polypeptide may be an antigenic or immunogenic polypeptide, e.g., a polypeptide derived from a bacterium, a virus, a fungus, or a protozoan.
  • the antigenic or immunogenic polypeptide may be derived from Group B Streptococcus.
  • the third polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ E) NO: 2; (ii) amino acids 1-59 of SEQ E) NO: 2; (iii) amino acids 132-181 of SEQ E ) NO: 2; (iv) amino acids 101-181 of SEQ E ) NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5;(c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ID NO:
  • the first polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ DD NO: 21; (iv) amino acids 272-360 of SEQ DD NO: 21
  • the first polypeptide comprises an amino acid sequence at least 80%, 85%,
  • the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444
  • the present invention includes a combination of an isolated first polypeptide, an isolated second polypeptide, and an isolated third polypeptide, wherein the first polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33, the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ ID
  • an antibody raised against the first and second polypeptides of the present invention may show cross reactivity against the corresponding polypeptides found in multiple GBS strains or serotypes.
  • the first and second polypeptide used in the present invention may further show cross protection against different GBS strains or serotypes.
  • a first polypeptide comprising SEQ DD NO: 33 and a second polypeptide comprising SEQ DD NO: 68 may show protection against GBS strain M732 as well as GBS strain GBS strain A909 (serotype Ia/c).
  • the present invention also includes a combination of two or more polypeptides wherein the first polypeptides and second polypeptides are from different GBS serotypes.
  • the present invention may include an isolated first polypeptide comprising SEQ DD NO: 33 (Pho3- 1) found in GBS M732 (serotype III) and a second polypeptide comprising SEQ ID NO: 69 (MS 10 corresponding polypeptide) found in GBS A909 (serotype Ia).
  • the first polypeptide and second polypeptide may therefore show protection against both GBS A909 and M732, when administered to a host in need thereof.
  • the first and second polypeptide may also show protection against GBS serotypes Ib, Ib/c, II, IV, V, VI, VII, and/or VIII. Likewise, other combinations of the GBS polypeptides from different serotypes are also possible.
  • the present invention is directed to at least two different GBS polypeptides, wherein the polypeptides further comprising a heterologous polypeptide.
  • the heterologous polypeptide can, in some embodiments, be fused to the GBS polypeptide.
  • the heterologous polypeptide can be translated from various heterologous nucleic acids.
  • heterologous polypeptides can be used, and can be selected from the group consisting of an N- or C-terminal peptide imparting stabilization, secretion, or simplified purification, i.e., His-tag, ubiquitin tag, NusA tag, chitin binding domain, ompT, ompA, pelB, DsbA, DsbC, c-myc, KSI, polyaspartic acid, (Ala-Trp-Trp-Pro)n, polyphenylalanine, polycysteine, polyarginine, B-tag, HSB-tag, green fluorescent protein (GFP), hemagglutinin influenza virus (HAI), calmodulin binding protein (CBP), galactose-binding protein, maltose binding protein (MBP), cellulose binding domains (CBD's), dihydrofolate reductase (DHFR), glutathione-S-transferase (GST), strepto
  • heterologous polypeptides can further include any pre- and/or pro- sequences that facilitate the transport, translocations, processing and/or purification of the GBS polypeptides from a host cell or any useful immunogenic sequence, including but not limited to sequences that encode a T-cell epitope of a microbial pathogen, or other immunogenic proteins and/or epitopes.
  • heterologous polypeptides may include other GBS proteins (either native proteins or variants, fragments, or derivatives thereof.)
  • the heterologous polypeptide may comprise one or more polypeptides from different GBS serotypes or strains. Accordingly, the at least two GBS polypeptides can induce an immune response against various serotypes of GBS.
  • the heterologous polypeptide may be from other organisms.
  • the polypeptide of the present invention can exist as a homopolymer, comprising multiple copies of the same polypeptide.
  • these polypeptides fused with a heterologous polypeptide can include a peptide linker sequence joining sequences that comprise two or more epitopes.
  • Suitable peptide linker sequences may be chosen based on their ability to adopt a flexible, extended conformation, or a secondary structure that could interact with joined epitopes, or based on their ability to increase overall solubility of the fusion polypeptide, or based on their lack of electrostatic or water-interaction effects that influence joined epitopes.
  • Peptide and polypeptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows: A (alanine); R (arginine); N (asparagine); D (aspartic acid); C (cysteine); Q (glutamine); E (glutamic acid); G (glycine); H (histidine); I (isoleucine); L (leucine); K (lysine); M (methionine); F (phenylalanine); P (proline); S (serine); T (threonine); W (tryptophan); Y (tyrosine); and V (valine).
  • the present invention also include an isolated first polynucleotide and an isolated second polynucleotide, wherein the first polynucleotide and the second polynucleotide encode the GBS polypeptides, fragments, derivatives, analogs, or variants thereof described herein.
  • Polynucleotides or nucleic acid sequences defined herein are represented by one-letter symbols for the bases as follows: A (adenine) C (cytosine) G (guanine) T (thymine) U (uracil) M (A or C) R (A or G) W (A or T/U); S(C or G); Y (C or T/U); K (G or T/U); V (A or C or G; not T/U) ; H (A or C or T/U; not G); D (A or G or T/U; not C); B (C or G or T/U; not A); N (A or C or G or T/U) or (unknown).
  • an isolated first polynucleotide used in the present invention encodes a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polynucleotide used in the present invention encodes a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ED NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225
  • an isolated first polynucleotide encodes a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 33;'(ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ED NO: 33; and (iv) amino acids 24-192 of SEQ ED NO: 33; and an isolated second polynucleotide encodes a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ
  • an isolated first polynucleotide encodes a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated second polynucleotide encodes a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii)
  • the present invention is directed to a polynucleotide further comprising a heterologous nucleic acid.
  • the heterologous nucleic acid can, in some embodiments, encode a heterologous polypeptide fused to the polypeptide.
  • the heterologous nucleic acid may comprise a promoter operably linked to the first, second, or third polynucleotide.
  • the heterologous nucleic acid may encode a third polypeptide.
  • the third polypeptide may be an antigenic or immunogenic polypeptide.
  • an antigenic or immunogenic polypeptide may be derived from a bacterium, a virus, a fungus, or a protozoan.
  • a heterologous nucleic acid may encode a polypeptide derived from Group B Streptococcus.
  • the polypeptide derived from GBS may be any GBS polypeptides, fragments, derivatives, or variants described herein.
  • GBS polypeptide variants are also included within the scope of the invention.
  • Modifications of nucleic acids encoding the GBS polypeptides can readily be accomplished by those skilled in the art, for example, by oligonucleotide-directed site-specific mutagenesis of a polynucleotide coding for a GBS polypeptide.
  • modified polypeptide can be encoded by a codon optimized nucleotide sequence.
  • Such modifications impart one or more amino acid substitutions, insertions, deletions, and/or modifications to expressed GBS polypeptides including fragments, variants, and derivatives.
  • modifications may enhance the immunogenicity of the GBS antigens, for example, by increasing cellular immune responses compared with unmodified polypeptides. Such modification may enhance solubility of the polypeptides. Alternatively, such modifications may have no effect.
  • the polynucleotide encoding the GBS polypeptide may be modified by introduction, deletion or modification of particular cleavage sites for proteolytic enzymes active in antigen presenting cells, to enhance immune responses to particular epitopes.
  • various nucleic acid coding regions will encode the same polypeptide due to the redundancy of the genetic code.
  • Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code” which shows which codons encode which amino acids is reproduced herein as Table 6. As a result, many amino acids are designated by more than one codon.
  • amino acids alanine and proline are coded for, by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet.
  • This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the polypeptides encoded by the DNA.
  • Codon preference or codon bias differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the "Codon Usage Database” available at www.kazusa.or.jp/codon/ (visited August 6, 2007), and these tables can be adapted in a number of ways. See Nakamura, Y., et al, "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
  • codon usage tables By utilizing the codon usage tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment comprising a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species.
  • the coding region is codon-optimized for expression in E.coli.
  • Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly.
  • various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and the "backtranslate" function in the GCG ⁇ Wisconsin Package, available from Accelrys, Inc., San Diego, CA. Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill.
  • Codon placement in a polynucleotide at an optimized frequency to encode a given polypeptide sequence can also be done in a directed manner.
  • a codon may be assigned to a particular amino acid so as to create or destroy a restriction enzyme cleavage site. Creation or destruction of restriction enzyme sites may facilitate DNA manipulation by assisting with cloning or forming identifying markers.
  • a codon may be assigned, to a particular amino acid so as to achieve a desired secondary structure of the polynucleotide.
  • an entire coding region encoding a polypeptide sequence, or fragment, variant, or derivative thereof can be codon optimized by any of the methods described herein or by other methods.
  • Various desired fragments, variants or derivatives may be designed, and each can then be codon-optimized individually.
  • partially codon-optimized coding regions of the present invention can be designed and constructed.
  • the invention includes a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide in which at least about 1%, 2%, 3,% 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codon positions have been codon-optimized for a given species.
  • the partially codon optimized coding regions contain a codon that is preferentially used in the genes of a desired species, e.g., E.coli, in place of a codon that is normally used in the native nucleic acid sequence.
  • the present invention further provides a vector or more than one vectors comprising one or more polynucleotides encoding the GBS polypeptides, fragments, derivatives, or variants thereof.
  • a vector which comprises at least two nucleic acid sequences encoding the GBS polypeptides or fragments, variants, derivatives, or analogs thereof described herein.
  • the at least two nucleic acid sequence may comprises an isolated first nucleic acid sequence and an isolated second nucleic acid sequence, which are located on one expression vector.
  • the present invention is further directed to at least two vectors, each of which comprises a nucleic acid fragment encoding a GBS polypeptide.
  • the at least two vectors can be identical or different or a combination thereof.
  • the nucleic acid fragment may be a fragment of a codon-optimized coding region operably encoding any GBS polypeptides described herein.
  • Additional GBS-derived coding or non-coding regions may also be included on the vector, e.g., a plasmid, or on a separate vector, and expressed, either using native GBS codons or codons optimized for expression in the host in which the polypeptide is being expressed.
  • a vector is delivered to a host, e.g., to a bacterial, plant or eukaryotic cell, or alternatively, in vivo to a tissue of the animal to be treated or immunized, the transcriptional unit will thus express the encoded gene product.
  • the level of expression of the gene product will depend to a significant extent on the strength of the associated promoter and the presence and activation of an associated enhancer element, as well as the optimization of the coding region.
  • vector refers to any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors may be DNA or RNA.
  • Vectors include, but are not limited to, plasmids, phage, phagemids, bacterial genomes, and virus genomes and virus-like particles.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis.
  • phage replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • plasmid refers to a circular, double-stranded construct made up of genetic material (i.e., nucleic acids), wherein the genetic material is extrachromosomal and replicates autonomously.
  • a polynucleotide of the present invention may be in a circular or linearized plasmid or vector, or other linear DNA which may also be non-infectious and nonintegrating (i.e., does not integrate into the genome of host cells).
  • a variety of host-expression vector systems may be utilized to express the polypeptides of the present invention.
  • expression vector refers to a vector that is capable of expressing the polypeptide of the present invention, i.e., the vector sequence contains the regulatory sequences required for polypeptide expression such as promoters, ribosome binding sites, etc.
  • expression refers to the biological production of a product encoded by a coding sequence. In most cases a DNA sequence, including the coding sequence, is transcribed to form a messenger-RNA (mRNA). The messenger-RNA is then translated to form a polypeptide product which has a relevant biological activity. Also, the process of expression may involve further processing steps to the RNA product of transcription, such as splicing to remove introns, and/or post-translational processing of a polypeptide product.
  • mRNA messenger-RNA
  • Vector-host systems include, but are not limited to, systems such as bacterial, mammalian, yeast, insect or plant cell systems. either in vivo, e.g., in an animal or in vitro, e.g., in mammalian cell cultures. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention as described above.
  • one aspect of the invention is directed to a host cell comprising a vector which contains a polynucleotide of the present invention.
  • the engineered host cell can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • transfect refers to any procedure whereby eukaryotic cells are induced to accept and incorporate into their genome isolated DNA, including but not limited to DNA in the form of a plasmid.
  • transform refers to any procedure whereby bacterial cells are induced to accept and incorporate into their genome isolated DNA, including but not limited to DNA in the form of a plasmid.
  • Bacterial host-expression vector systems include, but are not limited to, a prokaryote
  • PFENEXTM PFENEX TM (Dowpharma)
  • Streptomyces sp. or Staphylococcus sp.
  • PFENEXTM PFENEX TM (Dowpharma)
  • Streptomyces sp. or Staphylococcus sp.
  • PFENEXTM PFENEXTM
  • the PFENEXTM system is used.
  • the PFENEXTM expression system utilizes P. fluorescens biovar I, designated MBlOl, and compatible plasmids.
  • the plasmids used with P. fluorescens use the tac promoter system regulated by the Lad protein via IPTG induction.
  • the bacterial host can have an auxotrophic chromosomal deletion, e.g., pyrF, in which the deletion is complemented by the vector, to alleviate the need for antibiotic-resistance genes.
  • auxotrophic chromosomal deletion e.g., pyrF
  • a large number of suitable vectors are known to those of skill in the art, and are commercially available.
  • the following bacterial vectors are provided by way of example: pET, pET28, pBAD, pTrcHIS, pBR322, pQE70, pQE60, pQE-9 (Qiagen), phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, pPSIO, RSFlOlO, pRIT5 (Pharmacia); pCR (Invitrogen); pLex (Invitrogen), and pUC plasmid derivatives.
  • the expression vector comprises the plasmid pLex.
  • the pLex plasmid comprises a multiple cloning site that is tightly regulated by a tryptophan-inducible expression system utilizing the strong P L promoter from bacteriophage lambda, and the cl repressor protein. This pLex expression vector is especially useful for the expression of potentially toxic proteins in E. coli.
  • the lambda promoter provides high-level expression of recombinant proteins.
  • a GBS polypeptide antigen can be recombinantly expressed directly in a carrier organism, or virus, all or part of which is subsequently incorporated in a vaccine composition, as described in Eko, et al, Immunol., 173:3375-3382, 2004, which is expressly incorporated by reference herein.
  • GBS polypeptide vaccine antigens can be purified from infected-host cell culture.
  • a suitable expression vector contains regulatory sequences which can be operably joined to an inserted nucleotide sequence encoding a GBS polypeptide vaccine antigen.
  • regulatory sequences means nucleotide sequences which are necessary for or conducive to the transcription of an inserted sequence coding a GBS vaccine antigen by a host cell and/or which are necessary for or conducive to the translation by a host cell of the resulting transcript into the desired GBS polypeptide.
  • Regulatory sequences include, but are not limited to, 5 1 sequences such as operators, promoters and ribosome binding sequences, and 3' sequences such as polyadenylation signals. Regulatory sequences may also include enhancer sequences or upstream activator sequences.
  • bacterial vectors will include origins of replication and selectable markers, e.g., the ampicillin, tetracycline, kanamycin, resistance genes of E. coli, permitting transformation of the host cell and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters include, but are not limited to, the T7 promoter, lambda ( ⁇ ) promoter, T5 promoter, and lac promoter, or promoters derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others.
  • the polynucleotide of the invention is cloned downstream of the promoter, often in a polylinker region.
  • This plasmid is transformed into an appropriate bacterial strain, and DNA is prepared using standard techniques.
  • the orientation and DNA sequence of the polynucleotide as well as all other elements included in the vector, are confirmed using restriction mapping, DNA sequence analysis, and/or PCR analysis.
  • Bacterial cells harboring the correct plasmid can be stored as cell banks.
  • Examples of mammalian host-expression systems include cell lines capable of expressing a compatible vector, for example, the COS, C127, 3T3, CHO, HeLa and BHK cell lines.
  • suitable expression vectors include pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia), p75.6 (Valentis), pCEP (Invitrogen), pCEI (Epimmune), pZERO, P Trc99A, pUC19, pUC18, pKK223-3, pEXl, pCAL, pET, pSPUTK, pTrxFus, pFastBac, pThioHis, pTrcHis, and pLex, pET-17b, pET-1 Ia, pET-24a-d(+
  • mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • promoters may also be derived from viral sources, such as, e.g., human cytomegalovirus (CMV-EE promoter), herpes simplex virus type-1 (HSV TK promoter), the adenovirus late promoter; and the vaccinia virus 7.5K promoter, or can be derived from the genome of mammalian cells (e.g., metallothionein promoter).
  • Nucleic acid sequences derived from the SV40 splice and polyadenylation sites can be used to provide the required nontranscribed genetic elements.
  • a variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in animal cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • Other transcription control regions include those derived from animal genes such as actin, heat shock protein, bovine growth hormone and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells.
  • transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • lymphokine-inducible promoters e.g., promoters inducible by interferons or interleukins.
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, elements from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • Yeast host-expression systems include a yeast host (e.g., Saccharomyces, Pichia,
  • Suitable yeast expression vectors are known to those in the art and include, but are not limited to, e.g., pAL19, paR3, pBGl, pDBlet, pDB248X, pEA500, pFL20, pIRT2, pJK148, pON163, pSPl, pSP3, pUR19, pARTl, pCHY21 , REP41, pYZIN, pSLF104, pSLF172, pDS472, pSGP572, pSLF1072, REP41MH-N, pFA6a-kanMX6, pARTCM, and pALL.
  • insect host system and plant cell systems are also included.
  • the present invention is also directed to a combination of an isolated first antibody or antigen binding fragment thereof and an isolated second antibody or antigen binding fragment thereof, wherein both first and second antibodies and antigen binding fragments thereof are raised against the GBS polypeptides described herein.
  • antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, multivalent antibodies, multispecif ⁇ c antibodies, single chain antibodies, and fragments produced by a Fab expression library or epitope-binding fragments, e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), and disulfide-linked Fv fragments (sdFv).
  • Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • the antibodies and fragments thereof may be humanized antibodies, for example as described in EP-A-0239400, incorporated herein by reference in its entirety.
  • antibodies with fully human variable regions (or their fragments), for example, as described in US Patents Nos. 5,545,807 and 6,075,181 (both incorporated herein by reference in their entireties) may also be used.
  • Immunoglobulin or antibody molecules of the invention can be of any type ⁇ e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class or subclass ⁇ e.g., IgGl, IgG2a, IgG2b, IgG2c, and IgG3) of immunoglobulin molecule.
  • immunoglobulin or antibody molecules of the invention are IgG2a antibodies.
  • a selected mammal ⁇ e.g., mouse, rabbit, goat, horse, etc.
  • an immunogenic composition comprising the combination of polypeptides or peptides of the present invention.
  • various adjuvants described elsewhere herein, may be used to increase immunological response.
  • Monoclonal antibodies directed against epitopes obtainable from a peptide used in the present invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Antibodies both monoclonal and polyclonal, which are directed against epitopes obtainable from the polypeptides or peptides of the present invention, are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired.
  • the present invention includes a pharmaceutical, vaccine, or immunological composition comprising an isolated first polypeptide and an isolated second polypeptide, wherein the first polypeptide and second polypeptide are selected from the group consisting of the GBS polypeptides described herein.
  • a pharmaceutical, vaccine, or immunological composition which comprises an isolated first polynucleotide and an isolated second polynucleotide, wherein the first polynucleotide and the second polynucleotide are selected from the group consisting of the polynucleotides described herein.
  • the present invention includes an isolated first antibody or antigen binding fragment thereof and an isolated second antibody or antigen binding fragment thereof, wherein the first antibody or antigen binding fragment thereof and the second antibody or antigen binding fragment thereof are selected from the group consisting of the antibodies or antigen binding fragments thereof raised against the GBS polypeptides described herein.
  • the composition of the present invention includes an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68
  • the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210
  • the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and second polypeptide specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to the reference amino acid sequence.
  • the pharmaceutical, immunological, or vaccine composition of the present invention comprises an isolated first polynucleotide encoding the first polypeptide and an isolated second polynucleotide encoding the second polypeptide. Furthermore, the composition comprises an isolated first antibody or antigen-binding fragment thereof that specifically binds the first polypeptide described above and an isolated second antibody or antigen-binding fragment thereof that specifically binds the second polypeptide described above.
  • the present invention also includes at least two compositions, wherein a first composition comprises an isolated first polypeptide, polynucleotide, or antibody or antigen binding fragment thereof described herein and a second composition comprises an isolated second polypeptide, polynucleotide, or antibody or antigen binding fragment thereof described herein. Also included is a method of inducing an immune response comprising administering the at least two compositions in an animal in need thereof.
  • the composition comprising the first and second polypeptides further comprises an isolated third polypeptide; the composition comprising the first and second polynucleotides comprises an isolated third polynucleotide; and the composition comprising the first and second antibodies or antigen binding fragments thereof further comprises an isolated third antibody or antigen binding fragment thereof that specifically binds the third polypeptide.
  • the third polynucleotide may encode a third polypeptide.
  • the. third polypeptide may be an antigenic or immunogenic polypeptide. Such antigenic or immunogenic polypeptide may be derived from Group B Streptococcus.
  • the antigenic or immunogenic polypeptide may be the GBS polypeptides, fragments, derivatives, or variants described herein.
  • the composition can comprise any polypeptides disclosed in U.S. Patent Publication No. 2007/0036828, U.S. Patent Publication No. 2006/0275315, and U.S. Patent Publication No. 2006/0258849 (all incorporated herein by reference in their entireties).
  • composition of the present invention further comprising an isolated third polypeptide may therefore comprise an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24- 192 of SEQ ID NO: 33; an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of S
  • the composition of the present invention comprises two or more of isolated polypeptides, wherein each of said polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • a reference amino acid sequence selected from the group consisting of P
  • the composition comprises two or more of polynucleotides, wherein each of said polynucleotide comprises a nucleic acid sequence at least 80% identical to a reference nucleic acid sequence encoding an amino acid sequence selected from group consisting of Phol- 13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSl 1, MS14, MSlO(NP GAP, ME:NAP), PBP, pho.3-9, OCT, PGK, and NPGAP, wherein each of said polynucleotide encodes a polypeptide recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • each of said polynucleotide encodes
  • the composition comprises two or more of vectors, wherein each of said vector encodes a polypeptide comprising an amino acid sequence at least 80% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • the vector may comprise two or more polynucleotides encoding the two or more polypeptides.
  • the present invention includes a composition comprising isolated antibodies raised against two or more of polypeptides, wherein each of said polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein each of said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
  • a reference amino acid sequence selected from
  • compositions e.g, vaccine compositions, of the present invention can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington 's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., Mack Publishing Co., Easton, PA (1980), and Remington 's Pharmaceutical Sciences, 19th Edition, A.R. Gennaro, ed., Mack Publishing Co., Easton, PA (1995), both of which are incorporated herein by reference in their entireties.
  • the composition may be administered as an aqueous solution, it can also be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art.
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions e.g., vaccine, pharmaceutical, or immunological compositions
  • lipopeptides eg.,Vitiello, A. et al, J. Clin. Invest. 95:341, 1995
  • PEG poly(DL-lactide-co-glycolide)
  • concentration of polypeptides of the invention in the compositions of the invention can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • compositions in accordance with the invention comprise more than one polypeptide.
  • a composition e.g., a vaccine composition of the present invention can further include a polypeptide selected from the group consisting of, but not limited to, C5a peptidase, ⁇ -component of the C protein, LmbP, Sip, or LrrG as well as more than one GBS polypeptide described herein.
  • the present invention is also directed to a vaccine composition effective against GBS infection further comprising an adjuvant, carrier and/or excipient to the at least two isolated GBS polypeptides.
  • an adjuvant comprising an adjuvant, carrier and/or excipient to the at least two isolated GBS polypeptides.
  • the terms "adjuvant,” “carrier,” and “excipient” overlap to a significant extent.
  • a compound which acts as an "adjuvant” may also be a "carrier,” as well as an “excipient,” and certain other compounds normally thought of, e.g., as carriers, may also function as an adjuvant.
  • adjuvant refers to any material having the ability to (1) alter or increase the immune response to a particular antigen or (2) increase or aid an effect of a pharmacological agent.
  • immunogenic carrier refers to an isolated first polypeptide or fragment, variant, or derivative thereof which enhances the immunogenicity of an isolated second polypeptide or fragment, variant, or derivative thereof.
  • Carriers that can be used with compositions of the invention are well known in the art, and include, without limitation, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like.
  • aqueous carriers may be used, e.g. , water, buffered watery 0.8% saline, 0.3% glycine, hyaluronic acid and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the carrier is an immunogenic carrier.
  • an immunogenic carrier may be fused to or conjugated to a desired polypeptide or fragment thereof. See, e.g., European Patent No. EP 0385610 Bl, which is incorporated herein by reference in its entirety.
  • vaccines which contain immunogenic peptides as active ingredients, is known to one skilled in the art.
  • such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the peptide encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.
  • the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
  • the peptides of the invention may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with, the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic.
  • Salts formed with, the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • compositions of the present invention may be administered by direct injection.
  • the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • polypeptides, polynucleotides, or antibodies or antigen binding fragment thereof of the invention may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine before, after, or concurrently with the polypeptides, polynucleotides, or antibody or antigen binding fragment thereof.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine before, after, or concurrently with the polypeptides, polynucleotides, or antibody or antigen binding fragment thereof.
  • Potential adjuvants which may be screened for their ability to enhance the immune response according to the present invention include, but are not limited to: inert carriers, such as alum, bentonite, latex, and acrylic particles; pluronic block polymers, such as TITERMAX ® (block copolymer CRL-8941, squalene (a metabolizable oil) and a microparticulate silica stabilizer), depot formers, such as Freund's adjuvant, surface active materials, such as saponin, lysolecithin, retinal, Quil A, liposomes, and pluronic polymer formulations; macrophage stimulators, such as bacterial lipopolysaccharide; polycationic polymers such as chitosan; alternate pathway complement activators, such as insulin, zymosan, endotoxin, and levamisole; and non-ionic surfactants, such as poloxamers, poly(oxyethylene)-poly(oxypropylene) tri
  • TLR adjuvants include compounds that stimulate the TLRs ⁇ e.g., TLRl - TLRl 7), resulting in an increased immune system response to the vaccine composition of the present invention.
  • TLR adjuvants include, but are not limited to CpG (Coley Pharmaceutical Group Inc.) and MPL (Corixa).
  • CpG Coley Pharmaceutical Group Inc.
  • MPL Corixa
  • One example of a CpG oligonucleotide adjuvant is CpG7909, described in International Pat. Pub. No. WO 98/018810, US Patent Appl. Pub. No. 2002/016434 IA, US Pat. No. 6,727,230, and International Pat. Pub. No. WO98/32462, which are incorporated herein by reference in their entireties.
  • adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in- oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(l '-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBITM, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% s
  • adjuvants such as Amphigen (oil-in-water), Alhydrogel
  • aluminum hydroxide or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
  • the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (A12O3 basis).
  • the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 mg/ml, preferably 5 to 50 mg/ml, most preferably 15 mg/ml.
  • more than one adjuvant can be used in the methods and/or the composition.
  • a TLR adjuvant e.g., a CpG oligonucleotide, and Alum together can be formulated with a first polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof, and a second polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof in the methods and compositions of the present. invention.
  • Dosages of the adjuvants can vary according to the specific adjuvants.
  • dosage ranges can include: 10 ⁇ g/dose to 500 ⁇ g/dose, or 50 ⁇ g/dose to 200 ⁇ g/dose for CpG.
  • Dosage ranges can include: 2 ⁇ g/dose to 100 ⁇ g/dose, or 10 ⁇ g/dose to 30 ⁇ g/dose for MPL.
  • Dosage ranges can include: 10 ⁇ g/dose to 500 ⁇ g/dose, or 50 ⁇ g/dose to 100 ⁇ g/dose for aluminum hydroxide.
  • an adjuvant may be used with either the priming immunization, the booster immunization, or both.
  • the adjuvant is a cytokine.
  • Certain compositions of the present invention comprise one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines, or a polynucleotide encoding one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines.
  • cytokines include, but are not limited to granulocyte macrophage colony stimulating factor (GM- CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M- CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-I l), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 14 (IL-14), interleukin 15 (EL-15), interleukin 16 (IL-16), interleukin 17 (DL-17), interleukin 18 (IL-18), interferon alpha (IFN), interferon beta (IFN), interferon alpha
  • an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in disease symptoms.
  • an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen
  • an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion.
  • An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th2 response into a primarily cellular, or Thj response. Immune responses to a given antigen may be tested by various immunoassays well known to those of ordinary skill in the art, and/or described elsewhere herein.
  • the composition of the present invention may further comprise bacterial saccharides, such as the capsular saccharides from a GBS, Neisseria sp., Streptococcus pneumoniae sp. or Haemophilus influenzae type-b bacteria.
  • the saccharide antigen can be conjugated to a carrier molecule.
  • the polypeptides of the invention can also be administered via liposome carriers, which serve to target the polypeptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the polypeptide composition.
  • liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the polypeptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells (such as monoclonal antibodies which bind to the CD45 antigen or other costimulatory factor) or with other therapeutic or immunogenic compositions.
  • a molecule which binds to a receptor prevalent among lymphoid cells such as monoclonal antibodies which bind to the CD45 antigen or other costimulatory factor
  • liposomes either filled or decorated with a desired polypeptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the polypeptide compositions.
  • Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • a liposome suspension containing a polypeptide of the invention may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the polypeptide being delivered, and the stage of the disease being treated.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more polypeptides of the invention, often at a concentration of 25%-75%.
  • the immunogenic polypeptides can be supplied in finely divided form, optionally along with a surfactant and, propellant and/or a mucoadhesive, e.g., chitosan.
  • a surfactant e.g., a hydroxybenzoic acid
  • propellant e.g., benzoic acid
  • mucoadhesive e.g., chitosan.
  • Typical percentages of polypeptides are 0.01%-20% by weight, often l%-10%.
  • the surfactant must, of course, be pharmaceutically acceptable, and in some embodiments soluble in the propellant.
  • esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, in some embodiments 0.25-5% by weight.
  • the balance of the composition is ordinarily propellant, although an atomizer may be used in which no propellant is necessary and other percentages are adjusted accordingly. In.
  • the immunogenic polypeptides can be incorporated within an aerodynamically light particle, such as those particles desc ⁇ bed in U.S. Pat. No. 6,942,868 or U.S. Pat. Pub. No. 2005/0008633.
  • a carrier can also be included, e.g., lecithin for intranasal delivery.
  • Another embodiment of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the combination of polypeptides, or fragments thereof, adequate to produce antibody and/or T cell immune response to protect the animal from infection by Group B Streptococci.
  • the present invention is directed to a method to induce an immune response in a subject comprising: administering to the animal in need thereof an isolated first polypeptide, polynucleotide, antibody or antigen binding fragment thereof, vector, or host cell described herein and an isolated second polypeptides, polynucleotides, antibody or antigen binding fragment thereof, vectors, or host cells described herein.
  • the subject is an animal, e.g., a mammal, e.g., a primate, e.g., a human.
  • the invention is directed to a method of inducing an immune response, wherein the immune response treats, prevents, or ameliorates a disease or disorder related to GBS in a subject, e.g., a host animal, comprising administering an effective amount of an isolated first polypeptide, polynucleotide, antibody or antigen binding fragment thereof, vector, or host cell described herein and an isolated second polypeptide, polynucleotide, antibody or antigen binding fragment thereof, vector, or host cell described herein.
  • an animal can be treated with a composition comprising at least two different polypeptides, at least two different polynucleotides, vectors comprising the polynucleotides, or host cells comprising the vector prophylactically, e.g., as a prophylactic vaccine, to establish or enhance immunity to one or more GBS serotypes in a healthy animal prior to exposure to GBS or contraction of a GBS symptom, thus preventing the disease or reducing the severity of disease symptoms.
  • the composition may be used to induce an immune response or to treat an animal already exposed to GBS, or already suffering from GBS-related symptom to further stimulate the immune system of the animal, thus reducing or eliminating the symptoms associated with that exposure.
  • the composition of the present invention can be used to provide prophylactic immunity to a neonate.
  • treatment of an animal refers to the use of two or more compositions comprising a single GBS polypeptide, or a composition comprising two or more GBS polypeptides, polynucleotides, vectors, or host cells of the present invention to prevent, cure, retard, or reduce the severity of GBS symptoms in an animal, and/or result in no worsening of GBS symptoms over a specified period of time. It is not required that any composition, polypeptide, polynucleotide, a vector, or a host cell of the present invention provides total protection against GBS infection or totally cure or eliminate all GBS symptoms.
  • an animal in need of therapeutic and/or preventative immunity refers to an animal which it is desirable to treat, i.e., to prevent, cure, retard, or reduce the severity of GBS symptoms, and/or result in no worsening of GBS symptoms over a specified period of time.
  • an antibody specifically reactive with a GBS organism is isolated from the serum of the host animal which has been administered a polypeptide or polynucleotide of the present invention.
  • the invention is directed to a method of providing passive immunity comprising administering the antibody specifically reactive with a GBS organism (which was isolated from the serum of a host animal) to an animal in need thereof.
  • the present invention is directed to a method of inducing an immune response comprising administering an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide is selected from the group consisting of the GBS polypeptides described herein.
  • the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to said reference amino acid sequence.
  • the method of inducing an immune response comprises administering an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106- 225 of SEQ ID NO: 21; (iv) amino acids 272-360 of SEQ ID NO: 21; and
  • the method comprises administering an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or ] 00% identical to a.
  • a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second poly
  • the method comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285- 363 of SEQ ID NO: 68; (iv) amino acids
  • the present invention includes a method of inducing immune response comprising further administering an isolated third polypeptide, polynucleotide, or antibody or antigen binding fragment thereof.
  • the third polypeptide, polynucleotide, and antibody or antigen binding fragment thereof are described elsewhere herein.
  • Treatment with the GBS polypeptides, polynucleotides, antibody or antigen binding fragments, or a pharmaceutical composition comprising the same can occur separately or in conjunction with other treatments, as appropriate.
  • compositions, polypeptides or polynucleotides are administered to a patient in an amount sufficient to elicit an effective CTL response to the GBS- derived polypeptide to cure or at least partially arrest symptoms and/or complications.
  • Amounts effective for this use will depend on, e.g., the polypeptide or polynucleotide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization for polypeptide vaccines is (that is for therapeutic or prophylactic administration) from about 1.0 ⁇ g to about 5000 ⁇ g of polypeptide, in some embodiments about 10 ⁇ g to about 30 ⁇ g, for a 70 kg patient, followed by boosting dosages of from about 1.0 ⁇ g to about 1000 ⁇ g, in some embodiments 10 ⁇ g to about 30 ⁇ g, of polypeptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood.
  • the dose range for the initial immunization is from about 1.0 ⁇ g to about 20,000 ⁇ g of polypeptide for a 70 kg patient, in some embodiments 2 ⁇ g -, 5 ⁇ g - 10 ⁇ g -, 15 ⁇ g -, 20 ⁇ g.-, 25 ⁇ g -, 30 ⁇ g -, 40 ⁇ g -, or 50 ⁇ g -2000 ⁇ g, followed by boosting dosages in the same dose range pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL (cytotoxic T lymphocytes) activity in the patient's blood.
  • CTL cytotoxic T lymphocytes
  • approximately 0.01 to 2000 ⁇ g, or in some embodiments 2 ⁇ g to 200 ⁇ g or 10 ⁇ g to 30 ⁇ g, of a polypeptide or polynucleotide of the present invention, or its fragment, derivative variant, or analog is administered to a host.
  • the amount of polynucleotide in the initial immunization depends upon a number of factors including, for example, the antigen being expressed, the expression vector being used, the age and weight of the subject, the precise condition requiring treatment and its severity, and the route of administration. Based on the above factors, determining the precise amount, number of doses, and timing of doses are within the ordinary skill in the art and will be readily determined by the attending physician or veterinarian. In some embodiments, doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g DNA or RNA per patient. [00349] In non-limiting embodiments of the invention, an effective amount of a composition of the invention produces an elevation of antibody titer to at least three times the antibody titer prior to administration.
  • polypeptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the polypeptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these polypeptide compositions.
  • administration should begin at the first sign of GBS infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
  • Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals.
  • the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection.
  • the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.
  • compositions of the present invention may be administered to any tissue of an animal, including, but not limited to, muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue, e.g , myocardium, endocardium, and pericardium, lymph tissue, blood tissue, bone tissue, pancreas tissue, kidney tissue, gall bladder tissue, stomach tissue, intestinal tissue, testicular tissue, ovarian tissue, uterine tissue, vaginal tissue, rectal tissue, nervous system tissue, eye tissue, glandular tissue, tongue tissue, and connective tissue, e.g., cartilage.
  • tissue of an animal including, but not limited to, muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue, e.g , myocardium, endocardium, and pericardium, lymph tissue, blood tissue, bone tissue, pancreas tissue, kidney tissue, gall bladder tissue, stomach tissue, intestinal
  • compositions of the present invention may be administered to any internal cavity of a vertebrate, including, but not limited to, the lungs, the mouth, the nasal cavity, the stomach, the peritoneal cavity, the intestine, any heart chamber, veins, arteries, capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity, the rectal cavity, joint cavities, ventricles in brain, spinal canal in spinal cord, the ocular cavities, the lumen of a duct of a salivary gland or a liver.
  • a vertebrate including, but not limited to, the lungs, the mouth, the nasal cavity, the stomach, the peritoneal cavity, the intestine, any heart chamber, veins, arteries, capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity, the rectal cavity, joint cavities, ventricles in brain, spinal canal in spinal cord, the ocular cavities, the lumen of a duct of a salivary gland or a liver.
  • compositions of the present invention When the compositions of the present invention is administered to the lumen of a duct of a salivary gland or a liver, the desired polypeptide is encoded in each of the salivary gland and the liver such that the polypeptide is delivered into the blood stream of the vertebrate from each of the salivary gland and the liver.
  • Certain modes for administration to secretory organs of a gastrointestinal system using the salivary gland, liver and pancreas to release a desired polypeptide into the bloodstream are disclosed in U.S. Patent Nos. 5,837,693 and 6,004,944, both of which are incorporated herein by reference in their entireties.
  • the compositions are administered to muscle, either skeletal muscle or cardiac muscle, or lung tissue.
  • compositions of the present invention are delivered to an animal by methods described herein, thereby achieving an effective immune response, and/or an effective therapeutic or preventative immune response.
  • an effective immune response and/or an effective therapeutic or preventative immune response.
  • Any mode of administration can be used so long as the mode results in the delivery and/or expression of the desired polypeptide in the desired tissue, in an amount sufficient to generate an immune response to GBS, and/or to generate a prophylactically or therapeutically effective immune response to GBS, in an animal in need of such response.
  • compositions of the present invention can be administered by injection, parenterally, for example intravenously, intramuscularly or subcutaneously.
  • parenteral administration the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions of the present invention can be administered by mucosal delivery, transdermal delivery, subcutaneous injection, intravenous injection, oral administration, pulmonary administration, intramuscular " (i.m.) administration, or via intradural injection.
  • Transdermal delivery includes, but not limited to intradermal (e.g., into the dermis or epidermis), transdermal (e.g., percutaneous) and transmucosal administration (i.e., into or through skin or mucosal tissue).
  • Intracavity administration includes, but not limited to administration into oral, vaginal, rectal, nasal, peritoneal, or intestinal cavities as well as, intrathecal (i.e., into spinal canal), intraventricular (i.e., into the brain ventricles or the heart ventricles), intraatrial (i.e., into the heart atrium) and sub arachnoid (i.e., into the sub arachnoid spaces of the brain) administration.
  • intrathecal i.e., into spinal canal
  • intraventricular i.e., into the brain ventricles or the heart ventricles
  • intraatrial i.e., into the heart atrium
  • sub arachnoid i.e., into the sub arachnoid spaces of the brain
  • the compositions, polypeptides, polynucleotides, or antibody or antigen binding fragment thereof or any of the present invention may also be administered by a transdermal patch, sublingual wafer, or automjection device.
  • Any mode of administration can be used so long as the mode results in the delivery and/or expression of the desired polypeptide in the desired tissue, in an amount sufficient to generate an immune response against GBS, and/or to generate a prophylactically or therapeutically effective immune response against GBS, in an animal in need of such response.
  • Administration means of the present invention include needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., "gene guns” or pneumatic "needleless” injectors) Med-E-Jet (Vahlsing, H., et al, J. Immunol.
  • gelfoam sponge depots other commercially available depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, use of polynucleotide coated suture (Qin, Y., et al, Life Sciences 65, 2193-2203 (1999)) or topical applications during surgery. Certain modes of administration are intramuscular needle-based injection and pulmonary application via catheter infusion.
  • the immune system of the host responds to the vaccine by producing large amounts of HTLs (helper T lymphocytes) and/or CTLs (cytotoxic T lymphocytes) specific for the desired antigen. Consequently, the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection.
  • HTLs helper T lymphocytes
  • CTLs cytotoxic T lymphocytes
  • polypeptides or polynucleotides of the present invention stimulate a cell-mediated immune response sufficient for protection of an animal against GBS infection. In other embodiments, polypeptides or polynucleotides of the present invention stimulate both a humoral and a cell-mediated response, the combination of which is sufficient for protection of an animal against GBS protection.
  • components that induce T cell responses are combined with components that induce antibody responses to the target antigen of interest.
  • vaccine compositions of the invention are combined with polypeptides or polynucleotides which induce or facilitate neutralizing antibody responses to the target antigen of interest.
  • One embodiment of such a composition comprises a class I epitope in accordance with the invention, along with a PADRE ® (Epimmune, San Diego, CA) molecule (described, for example, in U.S. Patent Number 5,736,142, which is incorporated herein by reference in its entirety).
  • the polynucleotides of the present invention can be incorporated into the cells of the animal in vivo, and an antigenic amount of the Streptococcus agalactiae-de ⁇ ved polypeptide, or fragments, variants, or derivatives thereof, is produced in vivo.
  • an antigenic amount of the Streptococcus agalactiae-de ⁇ ved polypeptide, or fragments, variants, or derivatives thereof is produced in vivo.
  • the Streptococcus agalactiae-de ⁇ ved polypeptide is expressed in the animal in an amount sufficient to elicit an immune response.
  • Such an immune response might be used, for example, to generate antibodies to GBS for use in diagnostic assays or as laboratory reagents.
  • the present invention further provides a method for generating, enhancing, or modulating a protective and/or therapeutic immune response against GBS in an animal, comprising administering to the animal in need of therapeutic and/or preventative immunity one or more of the compositions, at least two polypeptides, polynucleotides, vectors, host cells, or composition thereof described herein.
  • the polypeptides, polynucleotides, antibody or antigen binding fragments, or compositions of the present invention can be administered to an animal at any time during the lifecycle of the animal to which it is being administered.
  • the composition can be given shortly after birth.
  • administration can occur while other vaccines are being administered, e.g., at birth, 2 months, 4 months, 6 months, 9 months, at 1 year, at 5 years, or at the onset of puberty.
  • administration can occur when the human become sexually active.
  • polypeptides, polynucleotides, antibody or antigen binding fragments thereof, or compositions of the invention can be used in any desired immunization or administration regimen; e.g., in a single administration or alternatively as part of periodic vaccinations such as annual vaccinations, or as in a prime-boost regime wherein the polypeptide or polynucleotide of the present invention is administered either before or after the administration of the same or of a different polypeptide or polynucleotide.
  • one or more compositions of, the present invention can be utilized in a "prime boost" regimen.
  • An example of a "prime boost” regimen may be found in Yang, Z. et al. J. Virol. 77:799-803 (2002), which is incorporated herein by reference in its entirety.
  • one or more polypeptide, polynucleotide, or antibody compositions of the present invention are delivered to an animal, thereby priming the immune response of the animal to a GBS polypeptide of the invention, and then a second immunogenic composition is utilized as a boost vaccination.
  • compositions of the present invention are used to prime immunity, and then a second immunogenic composition, e.g., a recombinant viral vaccine or vaccines, e.g., a recombinant vaccine virus, e.g., a recombinant MVA vaccinia virus, a different polynucleotide vaccine, or one or more purified subunit of the GBS polypeptides or fragments, variants or derivatives thereof is used to boost the anti-GBS immune response.
  • a second immunogenic composition e.g., a recombinant viral vaccine or vaccines, e.g., a recombinant vaccine virus, e.g., a recombinant MVA vaccinia virus, a different polynucleotide vaccine, or one or more purified subunit of the GBS polypeptides or fragments, variants or derivatives thereof is used to boost the anti-GBS immune response.
  • a priming composition and a boosting composition are combined in a single composition or single formulation.
  • a single composition may comprise an isolated GBS polypeptide or a fragment, variant, or derivative thereof as the priming component and a polynucleotide encoding a GBS polypeptide as the boosting component.
  • the compositions may be contained in a single vial where the priming component and boosting component are mixed together.
  • the polynucleotide component may provide a boost to the isolated polypeptide component.
  • compositions comprising both a priming component and a boosting component are referred to herein as "combinatorial vaccine compositions" or “single formulation heterologous prime-boost vaccine compositions.”
  • the priming composition may be administered before the boosting composition, or even after the boosting composition, if the boosting composition is expected to take longer to act.
  • the priming composition may be administered simultaneously with the boosting composition, but in separate formulations where the priming component and the boosting component are separated.
  • At least two GBS polypeptides, polynucleotides, antibody or antigen binding fragments thereof, vectors, or host cells or compositions comprising the same can be administered separately or at the same time.
  • the first polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof may be administered prior to or after the second polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof.
  • the first polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof is administered simultaneously with the second polypeptide, polynucleotide, or antibody or antigen- binding fragment thereof.
  • At least two GBS polypeptides, polynucleotides, antibody or antigen binding fragments thereof, vectors, or host cells or compositions comprising the same may be administered in one or multiple doses.
  • the administering may be performed in at least one dose, at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses or more.
  • at least three doses may be administered at a first month, at a second month, and at a third month.
  • at least three doses may be at a first month, at a second month, and at a fourth month.
  • At least three doses may be administered at a first month, at a second month, and at a sixth month.
  • the methods may further comprise administration of a fourth dose.
  • the fourth dose may be administered at least once between twelfth and sixtieth month after the administration of the first dose.
  • Pho3-1 (ME:P31), one of the selected proteins, was administered in 47 healthy adult volunteers.
  • the purpose of this trial was to evaluate the safety and immunogenicity of this protein.
  • Pho3-1 (ME:P31) was adjuvanted with alum and tested at four different strengths, with two doses given 28 days apart.
  • Pho3-1 (ME:P31) was immunogenic at all doses tested, the immune response rate was 83% at the lowest dose tested and 100% at the highest dose tested.
  • Pho3-1 (ME:P31) was well tolerated at all dose levels tested, with no serious adverse events reported.
  • the opsonophagocytosis assay will demonstrate opsonisation of GBS with immune sera raised against the proteins and their subsequent uptake by a phagocytic cell line. It is well known to those skilled in the art that this up-take is a precursor to the killing of GBS by the phagocyte.
  • the opsonophagocytosis activity is expressed as a percentage of HL-60 cells (phagocytes) which have taken up fluorescence labeled GBS bacteria.
  • MSlO polypeptide (NPGAP; SEQ ID NO: 68).
  • the sera (individually and in combination) were obtained to opsonise a GBS type Ia/c strain, A909, and measured for the increase in phagocytic uptake mediated by the immune sera compared with the preimmune sera. Antisera against both these candidates could individually mediate a phagocytic uptake. It was also shown that combining equal volumes of the two sera at dilutions >l:100 results in an additive opsonophagocytic effect (See figure 2).

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Abstract

The present invention is directed to providing a vaccine to enhance the immune response of an animal in need of protection against a Group B Streptococcus (GBS) infection. The present invention is also directed toward a composition comprising two or more of polypeptides, wherein said polypeptide comprises an amino acid sequence at least 80% identical to the reference amino acid sequence selected from the group consisting of Pho1-13, Pho1-14, Pho1-5, Pho3-3 (pho1-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3- 29, Pho3-50, MS4, MS11, MS16, MS14, MS10(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, a fragment, variant, analog, or derivative thereof, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence. In some embodiments, the present invention is directed to a method of inducing an immune response in a host comprising administering to said host an effective amount of the composition of the present invention.

Description

PROTEINS3 AND THEIR USE
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This invention relates to the identification of combinations of proteins, and their use as a vaccine, a process of making the vaccine, and a method for immunizing a human or animal against a condition related to Group B Streptococcus (GBS).
[0002] GBS, also known as Streptococcus agalactiae, is the causative agent of various conditions. In particular, GBS causes: Early onset neonatal infection.
[0003] This infection usually begins in utero or within five days of birth and causes severe septicaemia, meningitis, pneumonia, cellulitis, osteomyelitis, and septic arthritis in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate. See Schuchat (1998) Clinic. Microbiol. Rev. 11(3): 497-513. Newborns with early-onset GBS disease acquire the organism intrapartum from their mothers, who are colonized with GBS in the genital track. See id. GBS colonizes the vagina of about 25% of young women, and approximately 1% of infants born via a vaginal birth to colonized mothers will become infected. See id. Late onset neonatal infection.
[0004] The late onset neonatal infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including pneumonia, cellulitis, osteomyelitis, septic arthritis, abscesses, endocarditis, epiglottitis, and endopthalmitis. Adult infections.
[0005] These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterized by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis. Urinary tract infections. [0006] GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections. Veterinary infections.
[0007] GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.
[0008] GBS is Gram positive coccus belonging to Lanceiϊeld group B. There are at least nine antigenically distinct serotypes based on their capsular polysaccharide structure (types Ia, Ib, Ia/c, II, III, IV, V, VI, VII, or VIII) identified to date. See Shet et al. (2004) Indian J. Med. Res. 120: 141-150. In the US and Western Europe, types Ia, II, and III account for 85 percent of the isolates from infants. See id. In the US, GBS serotypes Ia, III, and V (in a descending order) account for 78-87% of early onset invasive disease in newborn infants and parturient women. See Zaleznik et al. (2000) Clin Infect Dis 30: 276-81; See also Harrison et al. (1998) J Infect Dis 177: 998- 1002. Late-onset GBS disease in infants 7-90 days of age is dominated by serotype III, followed by serotypes Ia and V. See Harrison et al. (1998) J Infect Dis 111: 998-1002. However, the types of GBS most prevalent in India are types III, II and Ib, and the types of GBS prevalent in Japan are types VI and VII.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention is based on the identification of beneficial combinations of GBS' proteins which can be used to provide an immunogenic response, useful in therapy. The composition provides an improved therapeutic effect compared to the single peptides.
[0010] The present invention is directed to a method of inducing an immune response against
Group B Streptococcus in an animal comprising administering to the animal in need thereof an effective amount of an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1-59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101- 181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5; (c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ID NO: 8; (iii) amino acids 139-192 of SEQ ID NO: 8; and (iv) amino acids 43-192 of SEQ ID NO: 8; (d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (ii) amino acids 1-88 of SEQ ID NO: 11; (iii) amino acids 1-110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11; (e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ TD NO: 14; (ii) amino acids 1-59 of SEQ ED NO: 14; (iii) amino acids 84-149 of SEQ DD NO: 14; and (iv) amino acids 1-149 of SEQ DD NO: 14; and (v) SEQ DD NO: 15; (f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 18; (ii) amino acids 29-95 of SEQ DD NO: 18; (iii) amino acids 1-95 of SEQ DD NO: 18; and (iv) amino acids 29-125 of SEQ DD NO: 18; (g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ DD NO:21; (h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 24; (ii) amino acids 43-84 of SEQ DD NO: 24; (iii) amino acids 1-84 of SEQ ID NO: 24; (iv) amino acids 88-122 of SEQ DD NO: 24; and (v) amino acids 38-122 of SEQ DD NO: 24; (i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 27; (ii) amino acids 29-79 of SEQ DD NO: 27; (iii) amino acids 101-189 of SEQ DD NO: 27; and (iv) amino acids 29-189 of SEQ DD NO: 27; (j) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 30; (ii) amino acids 79-182 of SEQ DD NO: 30; (iii) amino acids 79-217 of SEQ DD NO: 30; (iv) amino acids 23-217 of SEQ DD NO: 30; and (v) amino acids 23-278 of SEQ DD NO: 30; (k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; (1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 36; (ii) amino acids 25-88 of SEQ DD NO: 36; (iii) amino acids 132-202 of SEQ DD NO:,36; (iv) amino acids 25-172 of SEQ DD NO: 36; and (v) amino acids 25-202 of SEQ DD NO: 36; (m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 39 and (ii) amino acids 27-94 of SEQ DD NO: 39; (n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 42; (ii) amino acids 111-180 of SEQ DD NO: 42; (iii) amino acids 111-234 of SEQ DD NO: 42; (iv) amino acids 72-234 of SEQ DD NO: 42; and (v) amino acids 40-335 of SEQ DD NO: 42; (o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 45; (ii) amino acids 98-180 of SEQ DD NO: 45; (iii) amino acids 259-370 of SEQ DD NO: 45; and (iv) amino acids 98-370 of SEQ DD NO: 45; (p) a Pho3-29 polypeptide, which is SEQ DD NO: 48; (q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 51; (ii) amino acids 78-154 of SEQ DD NO: 51; (iii) amino acids 78-217 of SEQ DD NO: 51; and (iv) amino acids 1-154 of SEQ DD NO: 51; (r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 54; (ii) amino acids 190-261 of SEQ DD NO: 54; (iii) amino acids 190-321 of SEQ DD NO: 54; and (iv) amino acids 1- 118 of SEQ DD NO: 54; (s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 57; (ii) amino acids 1-139 of SEQ DD NO: 57; (iii) amino acids 184-311 of SEQ ID NO: 57; and (iv) amino acids 184-398 of SEQ DD NO: 57; (t) an MS16 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 60, (ii) amino acids 24-77 of SEQ ID NO: 60; (iii) amino acids 101-172 of SEQ ID NO: 60; (iv) amino acids 24-172 of SEQ ID NO: 60; and (v) SEQ ID NO: 62; (u) an MS 14 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 65; (ii) amino acids 1-95 of SEQ ID NO: 65; (iii) amino acids 141-181 of SEQ ID NO: 65; and (iv) amino acids 141-268 of SEQ ID NO: 65; and (v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68, wherein the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to the reference amino acid sequence. In one aspect, the immune response induced by the first polypeptide and the second polypeptide prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal.
[0011] In one embodiment, the present invention is directed to a method of inducing an immune response against Group B Streptococcus in an animal comprising administering to the animal in need thereof an effective amount of an isolated first polynucleotide encoding the first polypeptide described above and an isolated second polynucleotide encoding the second polypeptide described above. In certain aspects, the immune response induced by the first polynucleotide and the second polynucleotide prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal. The first polynucleotide and the second polynucleotide may be located in one or more expression vectors. More than one expression vectors may be identical or different or a combination thereof.
[0012] In other embodiments, a method of inducing an immune response against Group B
Streptococcus in an animal comprises administering to the animal in need thereof an effective amount of an isolated first antibody or antigen binding fragment thereof raised against the first polypeptide described herein and an isolated second antibody or antigen binding fragment thereof raised against the second polypeptide described herein. In certain aspects, the immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal.
[0013] In certain embodiments, the method of inducing an immune response further comprises administering an isolated third polypeptide, an isolated third antibody or antigen-binding fragment thereof raised against an isolated third polypeptide, or an isolated third polynucleotide. The third polynucleotide described herein may comprise a promoter operably linked to the first polynucleotide or the second polynucleotide. In another aspect, the third polynucleotide may encode a third polypeptide. The third polypeptide may be an antigenic or immunogenic polypeptide, e.g., a polypeptide derived from a bacterium, a virus, a fungus, or a protozoan. For example, the antigenic or immunogenic polypeptide may be derived from Group B Streptococcus. In further aspects, the third polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1-59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101-181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5;(c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ID NO: 8; (iii) amino acids 139-192 of SEQ ID NO: 8; and (iv) amino acids 43-192 of SEQ ID NO: 8; (d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (ii) amino acids 1-88 of SEQ ID NO: 11; (iii) amino acids 1-110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11; (e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 14; (ii) amino acids 1-59 of SEQ ID NO: 14; (iii) amino acids 84- 149 of SEQ DD NO: 14; and (iv) amino acids 1-149 of SEQ DD NO: 14; and (v) SEQ DD NO: 15; (f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 18; (ii) amino acids 29-95 of SEQ DD NO: 18; (iii) amino acids 1-95 of SEQ DD NO: 18; and (iv) amino acids 29-125 of. SEQ DD NO: 18; (g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ DD NO: 21; (ii) amino acids 1 -77 of SEQ DD NO: 21; (iii) amino acids 106- 225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ DD NO:21; (h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 24; (ii) amino acids 43-84 of SEQ DD NO: 24; (iii) amino acids 1-84 of SEQ DD NO: 24; (iv) amino acids 88-122 of SEQ DD NO: 24; and (v) amino acids 38-122 of SEQ DD NO: 24; (i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 27; (ii) amino acids 29-79 of SEQ DD NO: 27; (iii) amino acids 101-189 of SEQ DD NO: 27; and (iv) amino acids 29-189 of SEQ DD NO: 27; (j) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 30; (ii) amino acids 79-182 of SEQ DD NO: 30; (iii) amino acids 79-217 of SEQ DD NO: 30; (iv) amino acids 23-217 of SEQ DD NO: 30; (v) amino acids 23- 278 of SEQ DD NO: 30; (k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; (1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 36; (ii) amino acids 25-88 of SEQ DD NO: 36; (iii) amino acids 132-202 of SEQ DD NO: 36; (iv) amino acids 25-172 of SEQ DD NO: 36; and (v) amino acids 25-202 of SEQ DD NO: 36; (m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 39 and (ii) amino acids 27-94 of SEQ DD NO: 39; (n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 42; (ii) amino acids 111-180 of SEQ ID NO: 42; (iii) amino acids 111-234 of SEQ ID NO: 42; (iv) amino acids 72-234 of SEQ ID NO: 42; and (v) amino acids 40-335 of SEQ ED NO: 42; (o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 45; (ii) amino acids 98-180 of SEQ ID NO: 45; (iii) amino acids 259-370 of SEQ ID NO: 45; and (iv) amino acids 98- 370 of SEQ ID NO: 45; (p) a Pho3-29 polypeptide, which is SEQ ID NO: 48; (q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 51; (ii) amino acids 78-154 of SEQ ID NO: 51; (iii) amino acids 78-217 of SEQ ID NO: 51; and (iv) amino acids 1- 154 of SEQ ED NO: 51; (r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 54; (ii) amino acids 190-261 of SEQ ED NO: 54; (iii) amino acids 190-321 of SEQ ID NO: 54; and (iv) amino acids 1-118 of SEQ ED NO: 54; (s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 57; (ii) amino acids 1-139 of SEQ ED NO: 57; (iii) amino acids 184-311 of SEQ ED NO: 57; and (iv) amino acids 184-398 of SEQ ED NO: 57; (t) an MS16 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 60, (ii) amino acids 24-77 of SEQ ED NO: 60; (iii) amino acids 101-172 of SEQ ED NO: 60; (iv) amino acids 24-172 of SEQ ED NO: 60; and (v) SEQ ED NO: 62; (u) an MS 14 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 65; (ii) amino acids 1-95 of SEQ ID NO: 65; (iii) amino acids 141 -181 of SEQ ED NO: 65; and (iv) amino acids 141-268 of SEQ ED NO: 65; and (v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 68, (ii) amino acids 1-138 of SEQ ED NO: 68; (iii) amino acids 285-363 of SEQ ED NO: 68; (iv) amino acids 210-363 of SEQ ED NO: 68; (v) amino acids 210-444 of SEQ ED NO: 68; and (vi) amino acids 285-444 of SEQ ED NO: 68, wherein the third polypeptide is different from, or not identical to, the first polypeptide and the second polypeptide and specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to the reference amino acid sequence.
[0014] In accordance with one embodiment of the present invention, the first polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 33; (ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ED NO: 33; and (iv) amino acids 24-192 of SEQ ED NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ED NO: 21; (iv) amino acids 272-360 of SEQ ED NO: 21; and (v) amino acids 106-360 of SEQ ED NO: 21.
[0015] Also, the present invention includes a method comprising administering an isolated first polypeptide and an isolated second polypeptide, wherein the first polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ E) NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ E) NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 68, (ii) amino acids 1-138 of SEQ E) NO: 68; (iii) amino acids 285-363 of SEQ E) NO: 68; (iv) amino acids 210-363 of SEQ E) NO: 68; (v) amino acids 210-444 of SEQ E) NO: 68; and (vi) amino acids 285-444 of SEQ E) NO: 68.
[0016] In further embodiments, the method of the present invention comprises administering an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 21; (ii) amino acids 1-77 of SEQ E) NO: 21; (iii) amino acids 106- 225 of SEQ E) NO:21; (iv) amino acids 272-360 of SEQ E) NO:21; and (v) amino acids 106-360 of SEQ E) NO:21; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 68, (ii) amino acids 1-138 of SEQ E) NO: 68; (iii) amino acids 285-363 of SEQ E) NO: 68; (iv) amino acids 210-363 of SEQ E) NO: 68; (v) amino acids 210-444 of SEQ E) NO: 68; and (vi) amino acids 285-444 of SEQ E) NO: 68.
[0017] Furthermore, the present invention includes a method of inducing an immune response comprising administering an isolated first polypeptide, an isolated second polypeptide, and an isolated third polypeptide, wherein the first polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 33; (ii) amino acids 113-192 of SEQ E) NO: 33; (iii) amino acids 71-192 of SEQ E) NO: 33; and (iv) amino acids 24-192 of SEQ E) NO: 33, the second polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 21; (ii) amino acids 1-77 of SEQ E) NO: 21; (iii) amino acids 106-225 of SEQ E) NO:21; (iv) amino acids 272-360 of SEQ E) NO:21; and (v) amino acids 106-360 of SEQ E) NO:21, and the third polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ E) NO: 68, (ii) amino acids 1-138 of SEQ E) NO: 68; (iii) amino acids 285-363 of SEQ E) NO: 68; (iv) amino acids 210-363 of SEQ E) NO: 68; (v) amino acids 210-444 of SEQ E) NO: 68; and (vi) amino acids 285-444 of SEQ E) NO: 68. [0018] In one aspect, the method of the present invention further comprises administering a polysaccharide, which may be derived from Group B Streptococcus.
[0019] In another aspect, the method may further comprise administering one or more carriers or one or more adjuvants, e.g., Alum and a CPG oligonucleotide.
[0020] The method of the present invention induces an immune response when administered in an animal in need thereof. The immune response may be a humoral immune response or a cell- mediated immune response or a combination thereof.
[0021] In the present invention, the first polypeptide, the first polynucleotide, or the first antibody or antigen binding fragment thereof may be administered prior to or simultaneously with the second polypeptide, second polynucleotide, or second antibody or antigen binding fragment thereof. In another aspect, the second polypeptide, second polynucleotide, or second antibody or antigen binding fragment thereof may be administered prior to the first polypeptide, the first polynucleotide, or the first antibody or antigen binding fragment thereof. The polypeptides, polynucleotides or antibody or antigen binding fragment thereof can be administered as one or multiple doses.
[0022] The polypeptides, polynucleotides, or antibody or antigen-binding fragment thereof used in the present invention may be administered via intramuscular injection, subcutaneous injection, intra-dermal injection, intradural injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.
[0023] In one aspect, the present invention also includes a composition comprising an isolated first polypeptide and an isolated second polypeptide, a composition comprising an isolated first polynucleotide encoding the first polypeptide and an isolated second polynucleotide encoding the second polypeptide, and a composition comprising an isolated first antibody or antigen-binding fragment thereof specifically binds the first polypeptide and an isolated second antibody or antigen binding fragment thereof specifically binds the second polypeptide described herein.
[0024] In further aspects, the composition comprising an isolated first polypeptide and an isolated second polypeptide, an isolated first antibody or antigen binding fragment thereof and an isolated second antibody or antigen binding fragment thereof, or an isolated first polynucleotide and an isolated second polynucleotide further comprises an isolated third polypeptide, an isolated third antibody or antigen-binding fragment thereof raised against a third polypeptide, or an isolated third polynucleotide, respectively. The third polynucleotide may encode a third polypeptide. The third polypeptide used in the present invention may be an antigenic or immunogenic polypeptide, e.g., a polypeptide derived from Group B Streptococcus. In certain embodiments, the third polypeptide used in the present invention may be the GBS polypeptides, fragments, derivatives, or variants thereof. [0025] In one embodiment of this invention, the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ ID NO:21.
[0026] In another embodiment, the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444 of SEQ DD NO: 68; and (vi) amino acids 285-444 of SEQ DD NO: 68.
[0027] In certain embodiments, the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ DD NO:21 ; and an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444 of SEQ DD NO: 68; and (vi) amino acids 285-444 of SEQ DD NO: 68.
[0028] hi other embodiments, the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; an isolated second polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated third polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ED NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210- 444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ED NO: 68.
[0029] Furthermore, the composition of the present invention may further comprise one or more polysaccharides, carriers, or adjuvants, e.g., Alum and a CpG polynucleotide, or a combination thereof.
[0030] The composition of the present invention, when administered in an animal, induces an immune response such as a humoral immune response or a cell-mediated immune response.
[0031] In certain embodiments, a composition comprises two or more of polypeptides, wherein the polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from the group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[0032] Also, a composition may comprise two or more of polynucleotides, wherein the polynucleotide comprises a nucleic acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference nucleic acid sequence encoding an amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2- 15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3- 23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polynucleotide encodes a polypeptide recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[0033] Moreover, a composition of the present invention may comprise two or more of vectors, wherein the vector encodes a polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2- 15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3- 23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[0034] In another embodiment, a composition may comprise two or more of host cells, wherein the host cell comprises a vector encoding a polypeptide comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2- 10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3- 22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[0035] In other embodiments, a composition may comprise antibodies raised against two or more of polypeptides, wherein the polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (ρhol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein the polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[0036] In another aspect, the composition of the present invention further comprises a carrier, an , adjuvant, or a targeting molecule.
[0037] The present invention also includes a diagnostic reagent comprising the composition of the present invention described herein.
[0038] In one aspect, the present invention includes a method of detecting anti-Group B
Streptococcus antibodies in a test sample comprising the steps of (a) contacting the sample with the composition described herein to form Group B Streptococcus antigen: anti-Group B Streptococcus antibody immunocomplexes; and (b) detecting the presence of or measuring the amount of the immunocomplexes formed during step (a) or a diagnostic kit for detecting an antibody or antigen-binding fragment thereof to Group B Streptococcus, wherein the kit comprises the composition described elsewhere herein, a container means for contacting the polypeptide or composition with a test sample, and a reagent means for detecting or measuring Group B Streptococcus antigen: anti-Group B Streptococcus antibody immunocomplexes formed between the polypeptide and the antibody or antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure IA-W: depicts the Hopp/Woods antigenicity profile of the GBS polypeptides: IA:
Phol-13; IB: Phol-14; 1C: Phol-5; ID: Pho3-3; lE-a: Pho2-10; lE-b: Pho2-10 II; IF: Pho2-15: IG: Pho2-2; IH: Pho3-14; II: Pho3-17; U: Pho3-18; IK: Pho3-1; IL: Pho3-21; IM: Pho3-22: IN: Pho3-23; 10: Pho3-24; IP: Pho3-29; IQ: Pho3-50; IR: MS4; IS: MSI l; IT: MS16; IU: MS16II; IV: MS14; and IW: MSlO.
[0040] Figure 2: depicts Opsonophagocytosis of Group B Streptococcus strain A909 opsonised with monkey sera raised against GBS vaccine comprising Pho 3-1 (SEQ ID NO: 33) and MSlO (NPGAP: SEQ ID NO: 68).
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention incorporates by reference International Appl. No.
PCT/GB99/04377, filed on December 22, 1999 and published as WO 00/37646, International Appl. No. PCT/GB99/04376, filed on December 22, 1999 and published as WO 00/37490, and International Appl. No. PCT/GB02/01089, filed on March 11, 2002 and published as WO 02/07623 in their entireties.
[0042] The present invention is directed to polypeptides and polynucleotides derived from GBS strains, composition comprising the polypeptides and polynucleotides, and methods of administering the composition to prevent or treat diseases related to Group B Streptococcus infection. Organisms likely to contain the polypeptides include, but are not limited to, Enterococcus species {e.g., Enterococcus faecalis or Enterococcus faecium), Lactobacillus species {e.g. , Lactobacillus casei, Lactobacillus sakei, or Lactobacillus salivarius), Lactococcus species {e.g., Lactococcus lactis), Leuconostoc species {e.g., Leuconostoc mesenteroides), Oenococcus species {e.g., Oenococcus oeni), Pediococcus species {e.g., Pediococcus pentosaceus), Staphylococcus species {e.g., Staphylococcus aureus or Staphylococcus epidermidis), or Streptococcus species {e.g., Streptococcus agalatiae, Streptococcus bovis, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus suis, or Streptococcus thermophilus).
[0043] Vaccines to each of these organisms may be developed in the same way as described for
GBS.
[0044] Sequence identifiers are listed in Table 1 as follows. TABLEl
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Definitions
[0045] The term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide," is understood to represent one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
[0046] The term "nucleic acid" or "nucleic acid fragment" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide or construct. Two or more, nucleic acids of the present invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate (non-identical) polynucleotide constructs, e.g., on separate plasmids. Furthermore, any nucleic acid or nucleic acid fragment may encode a single polypeptide, e.g. , a single antigen, cytokine, or regulatory polypeptide, or may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides. In addition, a nucleic acid may encode a regulatory element such as a promoter or a transcription terminator, or may encode a specialized element or motif of a polypeptide or protein, such as a secretory signal peptide or a functional domain.
[0047] The term "polynucleotide," "polynucleotides," "nucleotide," or "nucleotides" or any variations thereof is intended to encompass a single nucleic acid or nucleic acid fragment as well as plural nucleic acids or nucleic acid fragments, and refers to an isolated molecule or construct, e.g., a virus genome (e.g., a non-infectious viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles as described in (Darquet, A-M et al, Gene Therapy 4:1341-1349 (1997)) comprising a polynucleotide. A polynucleotide may be provided in linear (e.g., mRNA), circular (e.g., plasmid), or branched form as well as double- stranded or single-stranded forms. A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
[0048] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and comprises any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term further includes polypeptides which have undergone post-translational modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
[0049] The term "GBS polypeptide," as used herein, encompasses full length polypeptide, serotypic, and other variants of full length polypeptide, fragments of full length polypeptide, serotypic or other variants of fragments of full length polypeptide, derivatives of full-length polypeptide, derivatives of fragments of full-length polypeptide, analogues of full-length polypeptide, analogues of fragments of full-length polypeptide, and chimeric and fusion polypeptide comprising full length polypeptide or one or more fragments of full length polypeptide.
[0050] The terms "fragment," "analog," "derivative," or "variant" when referring to GBS polypeptides , of the present invention include any polypeptides which retain at least some of the, immunogenicity or antigenicity of the naturally-occurring proteins. Fragments of GBS polypeptides of the present invention include proteolytic fragments, deletion fragments and, in particular, fragments which exhibit increased solubility during expression, purification, and/or administration to an animal. Fragments of GBS polypeptides further include proteolytic fragments or deletion fragments which exhibit reduced pathogenicity when delivered to a subject. Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three- dimensional epitopes.
[0051] An "epitopic fragment" of a polypeptide antigen is a portion of the antigen that contains an epitope. An "epitopic fragment" may, but need not, contain amino acid sequence in addition to one or more epitopes.
[0052] The term "variant," as used herein, refers to a polypeptide that differs from the recited polypeptide due to amino acid substitutions, deletions, insertions, and/or modifications. Variants may occur naturally, such as a serotypic variant. The term "serotypic variant" is intended polypeptides or polynucleotides that are present in a different GBS serotype. The serotypic variants are naturally occurring variants, but it can also be produced using art-known mutagenesis techniques. In the case of GBS, at least nine different serotypes, or "serotypic variants," have been identified. GBS serotypes include, but not limited to, serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VII, and VIII. There may be sequence variations between serotypes in some genes, for example, the genes encoding GBS Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS16, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, and PGK polypeptide. Table 5, as a non-limiting example, shows that an amino acid sequence of Phol-13 of GBS serotype III strain M732 have 98% homology to an amino acid sequence of the polypeptide found in GBS serotype Ia strain A909.
[0053] Naturally occurring variants can also be found in related bacteria, e.g., Streptococcus agalatiae, Streptococcus bovis, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis, Streptococcus /nutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus suis, or Streptococcus thermophilus. Polypeptides having homology to the GBS polypeptides can be found in these microorganisms.
[0054] Non-naturally occurring variants may be produced using art-known mutagenesis techniques. In a preferred embodiment, variant polypeptides differ from an identified sequence by substitution, deletion or addition of five amino acids or fewer. Such variants may generally be identified by modifying a polypeptide sequence, and evaluating the antigenic properties of the ■modified polypeptide using, for example, the representative procedures described herein.
[0055] As a non-limiting example, conservative substitutions may be made as shown in Table 2.
Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
TABLE 2
Figure imgf000018_0001
[0056] Polypeptide variants preferably exhibit at least about 60-70%, for example 75%, 80%,
85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with identified polypeptides. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of GBS polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. An analog is another form of the GBS polypeptides of the present invention. An example is a proprotein which can be activated by cleavage of the proprotein to produce an active mature polypeptide.
[0057] Variants may also, or alternatively, contain other modifications, whereby, for example, a polypeptide may be conjugated or coupled, e.g., fused to a heterologous polypeptide, e.g., a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein. The polypeptide may also be conjugated or produced coupled to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., 6-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated or coupled to an immunoglobulin Fc region. The polypeptide may also be conjugated or coupled to a sequence that imparts or modulates the immune response to the polypeptide (e.g. a T-cell epitope, B-cell epitope, cytokine, chemokine, etc.) and/or enhances uptake and/or processing of the polypeptide by antigen presenting cells or other immune system cells. The polypeptide may also be conjugated or coupled to other polypeptides/epitopes from GBS and/or from other bacteria and'or other viruses to generate a hybrid immunogenic protein that alone or in combination with various adjuvants can elicit protective immunity to other pathogenic organisms.
[0058] By an "isolated" GBS polypeptide or a fragment, variant, or derivative thereof is intended a GBS polypeptide or protein that is not in its natural form. No particular level of purification is required. For example, an isolated GBS polypeptide can be removed from its native or natural environment. Recombinantly produced GBS polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention, as are native or recombinant GBS polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Li addition, an isolated GBS polypeptide or protein can be provided as a live or inactivated expression vector expressing an isolated GBS polypeptide and can include those found in inactivated GBS vaccine compositions.
[0059] The term "sequence identity" as used herein refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. The term "sequence identity" is used herein interchangebly with the terms "sequence similarity" or "sequence homology." When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be "identical" at that position. The percentage "sequence identity" is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity." Percentage of "sequence identity" is determined by comparing two optimally aligned sequences over a comparison window (e.g., SEQ ID NO: 2 and a homologous polypeptide from another GBS isolate). In order to optimally align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions termed gaps while the reference sequence (e.g. SEQ ID NO: 2) is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical" positions between the reference and comparator sequences. Percentage "sequence identity" between two sequences can be determined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology Information as of September 1, 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. ScL USA 90(12):5873-5877, 1993). When utilizing "BLAST 2 Sequences," parameters that were default parameters as of September 1, 2004, can be used for word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expect value (10) and any other required parameter including but not limited to matrix option. Other publicly, available computer based methods for determining identity include the FASTA (Atschul et ah, J. Molec. Biol., 1990; 215:403-410), the BLASTX program available from NCBI, and the Gap program from Genetics Computer Group, Madison WI.
[0060] The term "at least two different polypeptide" or "at least two different polynucleotide," as used herein, refers to the polypeptides or polynucleotides comprising two or more polypeptides or polynucleotide, respectively, that are not identical. For example, the first polypeptide and the second polypeptide have no homology or less than 100% homology or identity to each other. The term "at least two different" is used herein interchangeably with "at least two," "more than one," "two or more," or "a combination of two or more."
[0061] The term "epitopes," as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, for example a mammal, for example, a human. An "immunogenic epitope," as used herein, is defined as a portion of a protein that elicits an immune response in an animal, as determined by any method known in the art. The term "antigenic epitope," as used herein, is defined as a portion of a protein to which an antibody or T-cell receptor can immunospecifically bind its antigen as determined by any method well known in the art. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Whereas all immunogenic epitopes are antigenic, antigenic epitopes need not be immunogenic.
[0062] As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, and the like, are outside the coding region.
[0063] The term "pharmaceutically acceptable" refers to compositions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio, hi some embodiments, the polypeptide, polynucleotides, compositions, and vaccines of the present invention are pharmaceutically acceptable.
[0064] An "effective amount" is that amount the administration of which to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. An amount is effective, for example, when its administration results in a reduced incidence of Group B Streptococcus infection relative to an untreated individual, as determined two weeks after challenge with infectious GBS. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated {e.g. human, nonhuman primate, primate, etc.), the responsive capacity of the individual's immune system, the degree of protection desired, the formulation of the vaccine, a professional assessment of the medical situation, and other relevant factors. It is expected that the effective amount will fall in a relatively broad range that can be determined through routine trials. Typically a single dose is from about 10 μg to 10 mg/kg body weight of purified polypeptide or an amount of a modified carrier organism or virus, or a fragment or remnant thereof, sufficient to provide a comparable quantity of the recombinantly expressed GBS polypeptides. The term "peptide vaccine" or "subunit vaccine" refers to a composition comprising one or more polypeptides of the present invention, which when administered to an animal are useful in stimulating an immune response against GBS infection.
[0065] The term "additive effect" as used herein refers to the effect, e.g., raising an immune response, that is the sum of the effects of two or more polypeptides acting independently. For example, if in an opsonophagocytosis assay immunized sera raised against the Pho3-1 polypeptide and the MSlO polypeptide, separately, opsonize GBS and result in about 10% GBS uptake and in about 15% GBS uptake, respectively, immunized sera raised against both the Pho3- 1 polypeptide and MSlO polypeptide together opsonize GBS and result in about 25% GBS uptake. [0066] The term "synergistic effect" or "synergy" is used herein to denote the effect that is more than additive, i.e., the combined immunostimulatory effect is greater than the expected total stimulatory effect calculated on the basis of the sum of the effects resulting from two or more polypeptides acting independently. For example, the immunized sera raised against both the Pho3-1 polypeptide and MSlO polypeptide together in the above example may opsonize GBS and result in more than 25% GBS uptake.
[0067] The term "subject" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, immunization, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals such as bears, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the animal is a human subject.
[0068] The term "animal" is intended to encompass a singular "animal" as well as plural
"animals" and comprises mammals and birds, as well as fish, reptiles, and amphibians. The term animal also encompasses model animals, e.g., disease model animals. In some embodiments, the term animal includes valuable animals, either economically or otherwise, e.g., economically important breeding stock, racing animals, shqw animals, heirloom animals, rare or endangered animals, or companion animals. In particular, the mammal can be a human subject, a food animal or a companion animal.
[0069] As used herein, an "subject in need thereof refers to an individual for whom it is desirable to treat, i.e., to prevent, cure, retard, or reduce the severity of disease symptoms related to GBS infection, and/or result in no worsening of GBS disease over a specified period of time.
[0070] The terms "priming" or "primary" and "boost" or "boosting" as used herein to refer to the initial and subsequent immunizations, respectively, i.e., in accordance with the definitions these terms normally have in immunology. However, in certain embodiments, e.g., where the priming component and boosting component are in a single formulation, initial and subsequent immunizations may not be necessary as both the "prime" and the "boost" compositions are administered simultaneously.
[0071] The term "passive immunity" refers to the immunity to an antigen developed by a host animal, the host animal being given antibodies produced by another animal, rather than producing its own antibodies to the antigen. The term "active immunity" refers to the production of an antibody by a host animal as a result of the presence of the target antigen.
Polypeptides [0072] The present invention includes a combination of more than two polypeptides, e.g., a combination of an isolated first polypeptide and an isolated second polypeptide. The polypeptides, used as an isolated first polypeptide or an isolated second polypeptide comprises an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence found in GBS serotype III strain M732, fragments, derivatives, or variants thereof. Variants commonly occur with many genes of simple, unicellular organisms. Examples of variants are serotypic variants. Another example of variants can be found within a same strain. For example, even within a same strain, different isolates obtained from different patients and/or geographical locations can exhibit some variation.
[0073] GBS outer membrane proteins or polypeptides and polynucleotides encoding the polypeptides or proteins are disclosed in International Appl. No. PCT/GB99/04377, filed December 22, 1999 and published as WO0037646 and International Appl. No. PCT/GB02/01089, filed March 11, 2002 and published as WO 02072623, both incorporated herein by reference in their entireties. Additional GBS outer surface proteins or polypeptides and polynucleotides are also disclosed in International Appl. No. PCT/GB99/04376, filed on December 22, 1999 and published as WO 00/37490 incorporated herein by reference in its entirety.
[0074] The Phol-13 polypeptide sequence (SEQ ID NO: 2) found in GBS strain M732 is publicly available and have Accession No. CAC09086 in Genbank.
[0075] SEQ ID NO: 2
1 mipwieqts rgersydiys rllkdri iml tgqvednman siiaqllf ld aqdntkdiyl
61 yvntpggsvs aglaivdtmn fiksdvqtiv mgmaasmgti iassgakgkr fmlpnaeymi
121 hqpmggtggg tqqsdmaiaa ehllktrhtl ekiladnsgq siekvhddae rdrwmsaqet
181 ldygfideim anne
[0076] The polynucleotide sequence encoding the Phol-13 polypeptide found in GBS M732 is publicly available as Accession No. AX026644 in Genbank and is designated herein as SEQ ID NO: 1.
[0077] SEQ ID NO: 1
1 atgatcccag tagtaatcga acaaacaagt cgtggtgaac gttcttatga tatttactca
61 cgtcttttaa aagatcgtat tattatgttg acaggccaag ttgaggataa tatggccaat
121 agtatcattg cacagttatt gtttctcgat gcacaagata atacaaagga tatttacctt
181 tatgtcaata caccaggtgg ttcagtatcg gctggacttg ctattgtgga caccatgaac
241 ttcattaaat cggacgtaca gacgattgtt atggggatgg ctgcttcgat gggaaccatt
301 attgcttcaa gtggtgctaa aggaaaacgt tttatgttac cgaatgcaga atatatgatc
361 caccaaccaa tgggcggaac aggcggaggt acacagcaat ctgatatggc tatcgctgct
421 gagcatcttt taaaaacgcg tcatacttta gaaaaaatct tagctgataa ttctggtcaa
481 tctattgaaa aagtccatga tgatgcagag cgtgatcgtt ggatgagtgc tcaagaaaca
541 cttgattatg gctttattga tgaaatcatg gctaataatg aataagg
[0078] Serotypic variants of the Phol-13 polypeptide can be found in other GBS strains. For example, the corresponding polypeptide found in GBS strain A909 is publicly available, and the locus tag is designated as SAK l600 (SEQ ID NO: 3).
[0079] SEQ ID NO: 3 MI PWIEQTSRGERSYDIYSRLLKDRI IMLTGQVEDNLVIANSI IAQLLFLDAQDNTKDI YL YVNTPGGSVSAGLAIVDTMNFIKSDVQTIVMGMAASMGTI IASSGAKGKRFMLPNAEYMI HQPMGGTGGGTQQSDMAIAAEHLLKTRHTLEKILADNSGQSIEKVHDDAERDRWMSAQET LDYGFIDAIMENNNLQ
[0080] The Phol-14 polypeptide sequence (SEQ ID NO: 5) found in GBS strain M732 is publicly available and has Accession No. CAC09087 in Genbank. [0081] SEQ ID NO: 5
1 mraysgplsv flprfkacdi ivnvrrtiml fkekipglil cfiiaipswl lglylpliga 61 pvfailigii vg
[0082] The phol-14 polynucleotide sequence (SEQ ID NO: 4) encoding the Phol-14 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026646 in Genbank.
[0083] SEQ ID NO: 4
1 atcagagcat attctggtcc tctttcggtt ttcctgccac gttttaaagc ttgtgatata
61 atagtcaatg tgaggaggac tatcatgtta tttaaggaaa aaattcctgg actaatatta
121 tgctttatta ttgctatacc atcttggttg cttgggcttt atctcccttt aataggagca
181 ccagtctttg ctatcttgat tggaataatt gttggatc
[0084] A serotypic variant ofthe Phol-14 polypeptide found in GBS strain A909 and has locus tagNo. SAK_1212 in Genbank (SEQ ID NO: 6). [0085] SEQ ID NO: 6:
MLFKEKIPGLILCFIIAIPSWI1LGLYLPLIGAPVFAILIGIIVGSFYQNRQLFNKGIAFTSKYILQTAWL. LGFGLNLMQVMKVGISSLPIIIMTISISLIIAYVLQKLFKLDKTIATLIGVGSSICGGSAIAATAPVINAK DDEVAQAISVIFLFNILAALIFPTLGNFIGLSDHGFALFAGTAVNDTSSVTATATAWDAINHSNTLGGATI VKLTRTLAIIPITIVLSIYHMKQTQKEQSVSVTKIFPKFVLYFILASLLTTIVASLGFSLRIFEPLKVLSK FFIVMAMGAIGINTNVSKLIKTGGKSILLGAACWLGIIIVSLTMQAILGTW
[0086] The Phol-5 polypeptide sequence (SEQ ID NO: 8) found in GBS strain M732 is a transglycosylase SLT domain family protein. The polypeptide sequence is publicly available and has Accession No. CAC09088 in Genbank.
[0087] SEQ ID NO: 8
1 mnkrrklskl nvkkqhlayg aitlvalfsc ilavtvifks sqvtteslsk adkvrvakks
61 kmtkatsksk vedvkqapkp sqasneapks ssqsteansq qqvtaseeta veqawteip
121 llpvrhnnly avtetpynpa qppdqvarye qwkycqavgs aaaaqmaaat gvpqstwehi
181 iaresngnpn vanasgasgl fqtmpgwgst atvqdqviql lkliraqgls agyq
[0088] The phol-5 polynucleotide sequence (SEQ ID NO: 7) encoding the Phol-5 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026648 in Genbank. [0089] SEQ ID NO: 7
1 atgaataaaa gaagaaaatt atcaaaattg aatgtaaaaa arcaacattt agcttatgga
61 gctatcactt tagtagccct tttttcatgt attttggctg taacggtcat ctttaaaagt
121 tcacaagtta ctactgaatc tttgtcaaaa gcagataaag ttcgcgtagc caaaaaatca
181 aaaatgacta aggcgacatc taaatcaaaa gtagaagatg taaaacaggc tccaaaacct
241 tctcaggcat ctaatgaagc cccaaaatca agttctcaat ctacagaagc taattctcag
301 caacaagtta ctgcgagtga agagacggct gtagaacaag cagttgtaac agaaataccc
361 ctgctaccag tcaggcacaa caacctttat gctgttactg agacacctta caaccctgct
421 caaccaccag accaagtggc caggtatgag caatggaaat actgccaggc ggtcggatct 481 gctgctgcag cacaaatggc tgctgcaaca ggagtccctc agtctacttg ggaacatatt
541 attgcccgtg aatcaaatgg taatcctaat gttgctaatg cctcaggagc ttcaggactt
601 ttccaaacga tgccaggttg gggttcaaca gctacagttc aggatcaagt aattcagcta
661 ttaaagctta ttcgtgctca agggttatca gctgggtacc agtga
[0090] A serotypic variant of the Phol-5 polypeptide found in GBS strain A909 is publicly available and has locus tagNo. SAK_1205 (SEQ ID NO: 9). [0091] SEQ DD NO: 9
MNVKKQHLAYGAITLVALFSCILAVTVIFKSSQVTTESLSKADKVRVAKKSKMTKATSKSKVEDVKQAPKP SQASNEAPKSSSQSTEANSQQQVTASEEAAVEQAWTENTPATSQAQQAYAVTETTYRPAQHQTSGQVLSN GNTAGAIGSAAAAQMAAATGVPQSTWEHIIARESNGNPNVANASGASGLFQTMPGWGSTATVQNQVNSAIK AYRAQGLSAWGY
[0092] The Pho3-3 polypeptide sequence (SEQ ID NO: 11) found in GBS strain M732 is publicly available and have Accession No. CAC09089 in Genbank. [0093] SEQIDNO: 11
1 mtepffdkel tcrpieaipe llefditvrg dnrgwfkenf qkekmiplgf pesffeadkl
61 qnnisftkkn tlrglhaepw dkyvsiadeg rvigtwvdlr egdsfgnvyq tiidaskgif
121 vprgvangfq vlsdkaayty lvndywalel kpkyafvnya dpnlgiqwen leeaevsead
181 knhpllkdvk plkkedl
[0094] The pho3-3 polynucleotide sequence (SEQ DD NO: 10) encoding the Pho3-3 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026650 in Genbank. [0095] SEQDDNO: 10
1 atgactgaac cattttttga taaagaatta acttgtcgcc caattgaagc cattcctgaa
61 ttgttggaat tcgatattac cgttcgtgga gacaaccgtg gatggttcaa agagaacttt
121 caaaaagaaa aaatgatacc gcttggtttc ccagaaagct tcttcgaggc agacaaacta
181 caaaataata tttcgtttac aaaaaaaaat actttgcgag gtctccatgc agagccttgg
241 gataaatatg tttcgatcgc tgatgaagga cgtgtgatcg gtacttgggt tgacctccgt
301 gaaggtgaca gttttggtaa cgtttaccaa acgattatcg atgcctcaaa aggtattttt
361 gttccacgcg gcgttgctaa tggtttccaa gttctttcag ataaagcagc ttatacttat
421 ctcgttaacg attattgggc acttgaactc aaaccaaaat atgctttcgt taactatgca
481 gatccaaatc taggcattca gtgggaaaat ctwgaagaag cagaagtctc agaagcagat
541 aagaatcacc cacttctcaa agatgtaaaa cctttgaaga aggaagattt gtaa
[0096] A serotypic variant of the Pho3-3 polypeptide in GBS strain A909 is publicly available and has locus tag no. SAK_1286 (SEQ DD NO: 12). [0097] SEQDDNO: 12
MTEQFFDKELTCRPIEAIPGLLEFDIPVRGDNRGWFKENFQKEKMIPLGFPESFFEADKLQNNISFNKKNT LRGLHAEPWDKYVSIADEGRVIGTWVDLREGDSFGNVYQTIIDASKGIFVPRGVANGFQVLSDKAAYTYLV NDYWALELKPKYAFVNYADPNLGIQWENLEEAEVSEADKNHPLLKDVKPLKKEDL
[0098] The Pho2-10 polypeptide sequence I (SEQ ID NO: 14) in GBS strain M732 is publicly available and has Accession No. CAC09090 in Genbank. [0099] SEQ DD NO: 14
1 yyligglaem qhvnhssfdk askagfiial givygdigts plytmqslve nqggissvte
61 sfilgsisli iwtltlitti kyvlvalkad nhheggifsl ytlvrkmtpw livpavigga
121 tllsdgaltp avtvlqplri kwpslqhis rirvcyfatl lftvtfaiqg lervllella
181 imlyglpfgl [00100] The Pho2-10 polypeptide sequence II (SEQ ID NO: 15) in GBS strain M732 is publicly available and has Accession No. CAC09091 in Genbank. [00101] SEQIDNO: 15
1 dlfswrqtga ealysdlghv grgnihvswp fvkvaiilsy cgqgawilan knagnelnpf 61 fasipsqftm hwilatlaa iiasqalis
[00102] The pho2-10 polynucleotide sequence (SEQ ID NO: 13) encoding the Pho2-10 polypeptides found in GBS strain M732 is publicly available and has Accession No. AX026652 in Genbank.
[00103] SEQIDNO: 13
1 tattatttaa tcggagggtt ggcagaaatg caacatgtca atcattcttc ttttgataaa
61 gcatcaaaag caggatttat tattgcttta ggcattgttt atggagatat tggtacaagc
121 ccactctata cgatgcaatc attggttgaa aaccaaggtg gtatttctag tgtcacagaa
181 tcgtttatct taggttctat atctttaatc atatggacct tgacacttat tacaactatc
241 aagtatgtgc ttgtagcttt aaaggcggat aatcaccacg aaggtggtat tttttcttta
301 tatacccttg ttagaaaaat gacaccttgg ttaattgttc cggctgttat tggaggtgca
361 accttgttgt cagatggagc tttgacgcca gctgtaaccg tacttcagcc gttaaggatt
421 aaagtagttc ctagtttgca gcatatttcc agaatcagag tatgttattt tgcgaccttg
481 ttatttactg ttacttttgc catccaaggt ttggaacggg tgttattgga attattggcc
541 attatgttat atggtttgcc ttttggttta ncggtctcct tatagttttg cccatccaga
601 agttttcaag cattaatcca tactacggtt tgaaattgtt atttagtcca gagaatcata
661 aaggtatttt tattttagga tctattttcc tggcgacaaa cgggagcaga agcactatac
721 tctgacttag gtcatgttgg gcgtggaaat atacatgttt catggccgtt cgttaaggtt
781 gccattatac tttcttattg tgggcaaggg gcatggattt tagctaataa gaacgcagga
841 aatgaattga atcccttttt tgctagtatt ccttcgcaat ttacaatgca tgtcgttatt
901 ttagctactt tggcagctat catcgcttca caggcactga tttctggatc aatttacctt
961 aagttctgag ctatgcgact aaaaatattc ccacaatttc gttcaactta tcctgttgac
1021 aatattgggt caaacctaca tacctggtat taattggttc ttatttgcca ttacaacctc
1081 tattggtttg ctttttaaga cttcagcgca catggaagca gcatatggat tagcgataac
1141 aattacgatg ctaatgacaa ctattttact gtctttcttt ttaattcaaa aaggagtaaa
1201 gagaggtttt agctatt
[00104] A serotypic variant of the Pho2-10 polypeptide is found in GBS strain A909 and has locus tag no. SAK_1175 (SEQ ID NO: 16).
[00105] SEQIDNO: 16
MQHVNHSSFDKASKAGFIIALGIVYGDIGTSPLYTMQSLVENQGGISSVTESFILGSISLIIWTLTLITTI KYVLVALKADNHHEGGIFSLYTLVRKMTPWLIVPAVIGGATLLSDGALTPAVTVTSAVEGLKWPSLQHIF QNQSNVIFATLFILLLLFAIQRFGTGVIGKLFGPIMFIWFAFLGISGFLNSFAHPEVFKAINPYYGLKLLF SPENHKGIFILGSIFLATTGAEALYSDLGHVGRGNIHVSWPFVKVAIILSYCGQGAWILANKNAGNELNPF FASIPSQFTMHWILATLAAIIASQALISGSFTLVSEAMRLKIFPQFRSTYPGDNIGQTYIPVINWFLFAI TTSIVLLFKTSAHMEAAYGLAITITMLMTTILLSFFLIQKGVKRGLVLLMMIFFGILEGIFFLASAVKFMH GGYVWIIAVAIIFIMTIWYKGSKIVSRYVKLLDLKDYIGQLDKLRHDHRYPIYHTNWYLTNRMEGDMID KSIMYSILDKRPKKAQVYWFVNIKVTDEPYTAEYKVDMMGTDFIVKVELYLGFKMRQTVSRYLRTIVEELL ESGRLPKQGKTYSVRPDSKVGDFRFIVLDERFSSSQNLKPGERFVMLMKSSIKHWTATPIRWFGLQFSEVT TEWPLIFTANRGLPIKEKIELTTTGD
[00106] The Pho2-15 polypeptide sequence (SEQ ID NO: 18) in GBS strain M732 is publicly available and has Accession No. CAC09092 in Genbank. [00107] SEQIDNO: 18
1 mqvflnivnk ffdpvihmgs gwmlivmtg lamifgvkfs kaleggikla ialtgigaii 61 giltgafses lqafvkntgi nlsiidvgwa platitwgsp ytlyflliml ivnivmivmk 121 kkrip [00108] The pho2-15 polynucleotide sequence (SEQ ED NO: 17) encoding the Pho2-15 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026655 in Genbank.
[00109] SEQIDNO: 17
1 atgcaggtat ttttaaatat tgtcaataaa ttctttgatc cagttattca tatgggttcg
61 ggagttgtga tgctaattgt catgacaggt ttagccatga tatttggagt gaagttttct
121 aaagcacttg aaggtggtat taagttagct attgctctta cgggtattgg tgctattatt
181 ggtattttaa ctggtgcttt ttccgaatca cttcaagctt ttgttaaaaa tacaggaatc
241 aatctaagca ttattgacgt tggttgggct ccattagcaa ctattacatg gggatcacca
301 tatacgcttt acttcttatt aatcatgctt attgtcaata ttgttatgat tgttatgaaa
361 aaaaaacgga taccttag
[00110] A serotypic variant of the Pho2-15 polypeptide is found in GBS strain A909 and has locus tag no. SAKJ893 (SEQ E) NO: 19). [00111] SEQE)NO: 19
MQVFLNIVNKFFDPIIHMGSGWMLIVMTGLAMIFGVKFSKALEGGIKLAIALTGIGAIIGILTGAFSESL QAFVKNTGINLSIIDVGWAPLATITWGSPYTLYFLLIMLIVNIVMIVMKKTDTLDVDIFDIWHLSITGLLI
FDKVFPGLDKYDFDAAKLNKAIGFWGSKFFIGMILGLVIGIMGNPVFSFAALGGWFSLGFTAGACLELFSL IGSWFIAAVEPLSQGITNFANGKMHGRRFNIGLDWPFIAGRAEIWACANILAPIMLVEAILLSKVGNGILP LAGIIAMGVTPALLWTRGRLIRMITFGTLLLPLFLLSGTMIAPFATELAKKVGAFPAGARAGSLITHSTL EGPMEKIFGYVIGKATTGQLSAIITLIIFATAYLGLFMWYAKQMKRRNAEYAANQK
[00112] The Pho2-2 polypeptide sequence (SEQ E) NO: 21) in GBS strain M732 is publicly available and has Accession No. CAC09093 in Genbank. [00113] SEQ E) NO: 21
1 MGQEPIIEYQ NINKVYGENV AVEDINLKIY PGDFVCFIGT SGSGKTTLMR MVNHMLKPTN
61 GTLLFKGKDI STINPIELRR RIGYVIQNIG LMPHMTIYEN IVLVPKLLKW SEEAKRAKAR
121 ELIKLVELPE EYLDRYPSEL SGGQQQRIGV IRALAADQDI ILMDEPFGAL DPITREGIQD
181 LVKSLQEEMG KTIILVTHDM DEALKLATKI IVMDNGKMVQ EGTPNDLLHH PATSFVEQMI
241 GEERLLHAQA DITPVKQIML NNPVSITAEK TLTEAITLMR QKRVDSLLVT DNGKLIGFID
301 LESLSSKYKK DRLVSDILKH TDFYVMEDDL LRNTAERILK RGLKYAPWD HENNXKGIVT
361 RASLVDMLYD IIWGDTETED Q
[00114] The pho2-2 polynucleotide sequence (SEQ E) NO: 20) encoding the Pho2-2 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026657 in Genbank. [00115] SEQ E) NO: 20
1 ATGGGCCAAG AACCTATCAT CGAATATCAA AATATCAATA AAGTGTATGG GGAAAATGTT
61 GCGGTTGAAG ATATTAACCT TAAAATTTAC CCTGGTGATT TCGTTTGTTT CATCGGTACG
121 AGTGGATCAG GTAAAACAAC ATTAATGCGT ATGGTTAACC ATATGTTAAA ACCAACAAAT
181 GGTACTCTAT TATTTAAGGG AAAAGATATT TCTACTATTA ACCCCATTGA ATTAAGACGC
241 AGAATTGGAT ATGTTATCCA AAACATTGGT TTAATGCCTC ATATGACCAT TTACGAAAAT
301 ATAGTTCTTG TACCAAAATT ATTGAAATGG TCAGAAGAAG CTAAAAGAGC TAAAGCAAGG
361 GAACTTATTA AATTAGTTGA ATTACCCGAA GAATATTTGG ATCGCTACCC TAGTGAGTTG
421 TCTGGCGGTC AGCAACAACG TATCGGTGTC ATTCGCGCTC TTGCAGCAGA CCAAGATATT
481 ATTTTAATGG ATGAGCCTTT TGGAGCTCTG GATCCTATTA CTAGAGAAGG TATTCAAGAC
541 TTAGTCAAGT CTCTTCAGGA AGAAATGGGG AAAACTATCA TCTTAGTTAC TCATGATATG
601 GATGAAGCCC TCAAGTTAGC AACAAAAATT ATTGTTATGG ACAATGGTAA AATGGTCCAA
661 GAAGGGACAC CCAATGATCT CTTACATCAT CCTGCTACCA GTTTCGTTGA ACAAATGATT
721 GGGGAAGAGC GTCTTCTTCA TGCGCAGGCT GATATTACCC CTGTTAAACA GATAATGTTA
781 AATAATCCTG TTTCAATAAC TGCTGAAAAA ACACTAACTG AAGCTATTAC ACTAATGCGC
841 CAAAAACGCG TTGACTCACT TCTAGTAACC GATAACGGTA AATTAATTGG TTTTATTGAC
901 TTAGAATCTC TAAGCAGTAA ATATAAGAAA GAYCGACTTG TTTCTGATAT CTTAAAACAT 961 ACTGATTTTT ATGTTATGGA AGACGACTTA CTTAGAAATA CTGCTGAGCG TATTTTAAAA 1021 CGTGGTTTAA AATACGCTCC AGTTGTTGAC CATGAGAATA ACYTAAAGGG CATTGTTACT 1081 CGTGCATCCC TAGTTGATAT GTTATACGAT ATTATTTGGG GCGATACTGA AACGGAGGAT 1141 CAATAA
[00116] A serotypic variant of the Pho2-2 polypeptide is found in GBS strain A909 and has locus tag no. SAK_O3O3 (SEQ ED NO: 22). [00117] SEQ ID NO: 22
MGQEPIIEYQNINKVYGENVAVEDINLKIYPGDFVCFIGTSGSGKTTLMRMVNHMLKPTNGTLLFKGKDIS TINPIELRRRIGYVIQNIGLMPHMTIYENIVLVPKLLKWSEEAKRAKARELIKLVELPEEYLDRYPSELSG GQQQRIGVIRALAADQDIILMDEPFGALDPITREGIQDLVKSLQEEMGKTIILVTHDMDEALKLATKIIVM DNGKMVQEGTPNDLLHHPATSFVEQMIGEERLLHAQADITPVKQIMLNNPVSITAEKTLTEAITLMRQKRV
KGIVTRASLVDMLYDIIWGDTETEDQ
[00118] The Pho3-14 polypeptide sequence (SEQ ID NO: 24) in GBS strain M732 is publicly available and has AccessionNo. CAC09094 in Genbank. [00119] SEQ ID NO: 24
1 mpysdvfatg gflyyvtial syllgssiwl fivqfiayyv sgiyfyklvy yvaqseivsi 61 gmtlifyimn ivlgfggmyp iqwalpfmli slwflikfcv dnivdeafif ygilaafslf 121 id
[00120] The pho3-14 polynucleotide sequence (SEQ ID NO: 23) encoding the Pho3-14 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026659 in Genbank.
[00121] SEQ ID NO: 23
1 atcccttata gtgatgtttt tgctacagga ggatttttat actatgtaac gattgctcta
61 agttaccttt tagggtctag tatctggtta tttattgtac agtttattgc ttactatgta
121 tctggaattt atttttataa attagtttat tatgtggcac aaagtgaaat tgtctcgata
181 ggcatgacgt tgattttcta tataatgaat attgtcttag gattcggtgg tatgtaccca
241 atacagtggg cattaccttt tatgctcatt tcgctatggt ttttaattaa attttgtgtc
301 gataatatcg ttgatgaagc atttatattt tatggtattt tagcagcatt ctcactattt
361 atagatc
[00122] A serotypic variant of the Pho3-14 polypeptide is found in GBS strain A909 and has locus tag no. SAK_2026 (SEQ ID NO: 25). [00123] SEQ ID NO: 25
MSKRTAIRMKKSSRFSILLYSVLSTLLAIANPLLTYFANGLQTQNLYTGLMMTKGQIPYSDVFATGGFLYY VTIALSYLLGSSIWLLIVQFIAYYVSGIYFYKLVYYVAQSEIVSIGMTLIFYIMNIVLGFGGMYPIQWALP FMLISLWFLIKFCVDNIVDEAFIFYGILAAFSLFIDPQTLIFWLCSFVLLTATNIKQKQSLRGFYQFLCW FGMILIAYTVGYFMFNLQIISSYIDKAIFYPFTYFARTNHSFLLSLAIQIWLLGSGCLFGLWDFIQNRKK ASYQIGLNFIACIFIIYAIMAIFSRDFNLYHFLPALPFGLLLTSNKITILYQKVIDRRSHRRQYFSGKSLI VDLFVKKTYYLPLLLVSLSIGLLVYNTYQNVTLSKERRDISHYLTTKIDRDGKIYVWDKVASIYSQTRLKS ASQFVLPHINTAQKNNEKILKDELLQHGAKYFILNKNEKLPNELKSDIKKHYQEVPLSNITHFVLYRFK
[00124] The Pho3-17 polypeptide sequence (SEQ ID NO: 27) in GBS strain M732 is publicly available and has Accession No. CAC09095 in Genbank. [00125] SEQ ID NO: 27
1 mrkrfsllnf iwtfiffff ilfpllnhkg kvdansrqsv tytkeefiqk ivpdaqdlgk
61 sygirpsfii aqaaldsdfg ekysysiiic wllaepgtps itlndsstgk kqekqfthyk
121 swkysmddyl ahiksgatgk kdsytimvsv knpktlvqkl qdsgfdndkk yakkmteiid 181 lydltrydk
[00126] The pho3-17 polynucleotide sequence (SEQ ID NO: 26) encoding the Pho3-17 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026661 in Genbank.
[00127] SEQ E) NO: 26
1 atgaggaaac gtttttcctt gctaaatttt attgttgtta cttttatttt ctttttcttt 61 attctttttc cgcttttaaa ccataaggga aaagtagatg ctaattctag gcagagtgtt 121 acctacacca aagaagaatt tatacaaaaa attgtgccag atgcgcaaga tctaggaaag 181 tcgtacggta ttcgtccttc atttattatt gcacaggcgg ctttggattc tgatttcgga 241 gagaaatata gctatagtat cataatctgt tggttgcttg cagaaccagg aacgccctca 301 attaccttaa atgatagtag tacaggaaaa aaacaggaaa agcaatttac tcattataaa 361 tcttggaagt attcaatgga tgattacctt gctcatataa aatctggagc gacaggcaaa 421 aaagattcat atactataat ggtgtctgtt aaaaatccaa aaactttagt gcaaaaatta 481 caagatagtg gttttgataa tgacaaaaag tacgctaaaa aaatgacgga aatcattgat 541 ttgtatgatt taacaagata tgataagtga
[00128] A serotypic variant of the Pho3-17 polypeptide is found in GBS strain A909 and has locus tagno. SAK_0144 (SEQ E) NO: 28).
[00129] SEQ E) NO: 28
MRKRFSLLNFIWTFIFFFFILFPLLNHKGKVDANSRQSVTYTKEEFIQKIVPDAQDLGKSYGIRPSFI IA QAALDSDFGEKILANKYHNLFGLLAEPGTPSITLNDSSTGKKQEKQFTHYKSWKYSMYDYLAHIKSGATGK KDSYT IMVSVKNPKTLVQKLQDSGFDNDKKYAKKMTE I I DLYDLTRYDK
[00130] The Pho3-18 polypeptide sequence (SEQ E) NO: 30) in GBS strain M732 is publicly available and has Accession No. CAC09096 in Genbank. [00131] SEQ E) NO: 30
1 mlviiliivl asltvtiisy pkmteltksv ekqlednadn lsdqltyqie vaqkdqiyvt 61 nqlnrmqqei isrlpicvqn ksaltesrdr sdkrlelins nlsqsvqkmq dsmknawikc 121 akllrkswkk rykrvanffe tvsrqlesvn qglgrwklcq dvgtteqsls ntktrgilge 181 lqlgqiiedi mtvsqyeref ptvsgsserv eyaikylemv reiisicllt lsfsrrllpi 241 grclwnwvtr fkwnsirnly wasirkfakd innkylnppe ttnfgimflp teglysewr 301 natffdslrr deniwagps tlsay
[00132] The pho3-18 polynucleotide sequence (SEQ E) NO: 29) encoding the Pho3-18 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026663 in Genbank.
[00133] SEQ E) NO: 29
1 atgcttgtca tcattttgat cattgtacta gctagtctga cagtgacgat aatttcttac 61 ccaaaaatga cggaattaac aaagtccgtt gaaaaacaac ttgaagataa tgctgataat 121 ctatcagacc aactgacata tcagatagaa gtggcgcaaa aagatcaaat ctacgtgact 181 aatcagctaa accgtatgca acaggaaatt atcagtcgct taccgatatg cgtacagaat 241 aaatcagcat taacggagag tcgagatcga tcagacaaac gcttggaatt gattaactcc 301 aatttatctc agtcagttca gaaaatgcaa gattcaatga aaaacgcttg gatcaaatgc 361 gccaaactgt tgaggaaaag ctggaaaaaa cgctacaaac gcgttgcaaa cttctttgaa 421 actgtatcgc gtcaactaga gagcgtcaat caaggtctgg gtagatggaa actgtgccaa 481 gatgttggta ccactgaaca aagtctgtca aatactaaga caaggggaat attaggggag 541 ttacaactcg gtcaaattat agaagatatt atgacagtta gtcaatatga gagagaattt 601 cctacggtgt ctggctcttc tgagcgtgtt gaatatgcta ttaaatacct ggaaatggtc 661 agggagatta tatctatttg cctattgact ctaagtttct ctagaagatt attaccgatt
721 gggagatgct tatggaattg ggtgaccagg ttcaaatgga actctattcg taatctttac
781 tgggcaagta ttcgtaaatt tgcaaaagat ataaacaata agtacttaaa tcctcctgaa
841 acgacaaatt ttggtatcat gttcttacca actgaagggc tctattctga agtggtaaga
901 aatgcaacat tctttgatag tctaagacgt gacgaaaata ttgtagtagc tggaccgtca
961 accttatctg cttactaa
[00134] A serotypic variant of the Pho3-18 polypeptide is found in GBS strain A909 and has locus tagno. SAK_1796 (SEQ ID NO: 31). [00135] SEQK)NO: 31
MLVIILIIALASLTVTIISYQKMTELTKSVEKQLEDNADNLSDQLTYQIEAAQKDQILTLTNQLNRMQQEI YQLLTDMRTELNQHLTESRDRSDKRLELINSNLSQSVQKMQDSNEKRLDQMRQTVEEKLEKTLQTRLQTSF ETVSRQLESVNQGLGEMKTVAQDVGTLNKVLSNTKTRGILGELQLGQIIEDIMTVSQYEREFPTVSGSSER VEYAIKLPGNGQGDYIYLPIDSKFPLEDYYRLEDAYELGDKVQIELYRKSLLASIRKFAKDINNKYLNPPE TTNFGIMFLPTEGLYSEWRNATFFDSLRRDENIWAGPSTLSALLNSLSVGFKTLNIQKNANDISKILGN VKVEFGKFGGMLSKAQKQLNTASKSIDSLLTTRTNAIIRALNTVEEHQDQATTSLLNLPITEEEEINEN
[00136] The Pho3-1 polypeptide sequence (SEQ ID NO: 33) in GBS strain M732 is publicly available and has Accession No. CAC09097 in Genbank. [00137] SEQ ID NO: 33
1 mrkevtpeml nynkypgpqf ihfenivksd diefqlvine ksafdvtvfg qrfseillky
61 dfivgdwgne qlrlrgfykd astirknsri srledyikey cnfgcayfvl enpnprdikf
121 dderphkrrk srsksqssks qtrnnrsqsn anahftskkr kdtkrrqerh ikeeqdkemt
181 sakqhllfvr kn
[00138] The pho3-l (ME: P31) polynucleotide sequence (SEQ ID NO: 32) encoding the Pho3-1 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026665 ih Genbank.
[00139] SEQ ID NO: 32
1 atgcgaaaag aagtgacacc agagatgctt aactataata agtatcctgg cccacagttt
61 attcactttg aaaatatcgt taaaagtgat gatattgaat ttcaacttgt tattaatgaa
121 aaatcagctt ttgatgtgac tgtctttgga caacgttttt ctgagatttt attaaaatat
181 gattttatcg ttggcgattg gggtaacgag cagttgaggc taagaggctt ttacaaagat
241 gctagtacga ttagaaaaaa tagccggatt tcacgtttag aagattatat taaagagtat
301 tgtaactttg gttgtgctta ttttgtgttg gagaatccaa atcctagaga tattaaattt
361 gatgatgaaa gacctcataa gcgtcgtaag tcaagatcca aatcacaatc atcaaagtca
421 caaactagaa ataatcgttc ccagtcaaat gccaatgctc attttacaag taaaaagcgt
481 aaagacacaa aacgccgtca agaacgtcat attaaagaag agcaagataa ggaaatgacc
541 tctgcaaagc agcatttgtt attcgtaaga aaaaattaa
[00140] A serotypic variant of the Pho3-1 is found in GBS strain A909 and has locus tag no.
SAK_0558 (SEQ ID NO: 34). [00141] SEQ ID NO: 34:
MRKEVTPEMLNYNKYPGPQFIHFENIVKSDDIEFQLVINEKSAFDVTVFGQRFSEILLKYDFIVGDWGNEQ LRLRGFYKDASTIRKNSRISRLEDYIKEYCNFGCAYFVLENPNPRDIKFDDERPHKRRKSRSKSQSSKSQT RNNRSQSNANAHFTSKKRKDTKRRQERHIKEEQDKEMTSAKQHFVIRKKK
[00142] The Pho3-21 polypeptide sequence (SEQ ID NO: 36) in GBS strain M732 is publicly available and has Accession No. CAC09098 in Genbank. [00143] SEQ ID NO: 36
1 mtikkvlsvt giilvtvasl aacsskshtt ktgkkevnfa tvgttapf sy vkdgkltgfd 61 ievakavfkg sdnykvtfkk tewssvftgi dsgkfqmggn nisyssersq kylfsypigs 121 tpsvlavpkn snikayndis ghktqwqgt ttakqlenfn kehqknpvtl kytnenitqi 181 ltnlsdgkad fklltdqllt 11
[00144] Thepho3-21 (ME: P31) polynucleotide sequence (SEQ ID NO: 35) encoding the Pho3-21 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026667 in Genbank.
[00145] SEQ ID NO: 35
1 atgacaataa aaaaagtgtt aagtgtaaca ggaattattt tagtgacagt agcgtctcta
61 gctgcttgta gctcaaaatc tcatactact aagacgggca aaaaagaagt taattttgca
121 actgttggaa caacggcacc tttttcttat gtgaaggatg ggaaactgac tggctttgat
181 attgaagtag ccaaagctgt ttttaaaggt tcagataact ataaagtcac ttttaaaaaa
241 acagaatggt catcggtatt taccggcatt gattcaggaa agtttcaaat gggtggaaat
301 aatatttctt attcatcaga gagatctcaa aaatayttat tttcataccc aataggctct
361 actccttcag ttttagcagt tcctaagaat agtaatatca aagcttataa tgatattagt
421 ggtcataaaa cacaggttgt ccaaggaacg acaactgcca agcaattaga aaatttcaat
481 aaagagcatc agaaaaatcc tgttactcta aaatatacta atgaaaatat tacacagatt
541 ctaacgaatt tgagtgatgg aaaagctgat tttaaacttt tgacggacca actgttaacg
601 ctattataa
[00146] A serotypic variant of the Pho3-21 polypeptide is found in GBS strain A909 and has locus tagno. SAK l656 (SEQ ID NO: 37). [00147] SEQ ID NO: 37:
MTIKKVLSVTGIILVTVASLAACSSKSHTTKTGKKEVNFATVGTTAPFSYVKDGKLTGFDIEVAKAVFKGS DNYKVTFKKTEWSSVFTGIDSGKFQMGGNNISYSSERSQKYLFSYPIGSTPSVLAVPKNSNIKAYNDISGH KTQWQGTTTAKQLENFNKEHQKNPVTLKYI1NENITQILTNLSDGKADFKLFDCFPTVNAIIKNQGLTNLKT
IPLTMRDQPYIYFIFGQDQKDLQKYVNNRLKQLRKDGTLSKIAKEYLGGDYVPNEKDLVTPKEK
[00148] The Pho3-22 polypeptide sequence (SEQ ID NO: 39) in GBS strain M732 is publicly, available and has Accession No. CAC09099 in Genbank. [00149] SEQ ID NO: 39
1 mknitklstv alslllctac aasntstskt qshhpkqtkl tdkqkeepkn keaadqemhp 61 qgavdltkyk akpvkdygkk idvgdgkkmn iyetgqgkip ivfipgqaei rhamliri
[00150] The pho3-22 polynucleotide sequence (SEQ ID NO: 38) encoding the Pho3-22 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026669 in Genbank.
[00151] SEQ ID NO: 38
1 atgaagaata taacaaagct atcaactgtt gctttaagcc tactactttg tacggcgtgt
61 gctgcatcaa acacgtctac atctaaaaca cagtctcatc atcctaaaca aactaaactc
121 acagataagc aaaaagaaga acccaaaaac aaagaagctg ctgatcaaga gatgcatccc
181 caaggcgctg ttgatttgac aaaatataag gcaaaaccgg tcaaagatta tggaaaaaaa
241 atcgatgttg gtgatggcaa gaaaatgaac atttatgaaa ctggtcaggg aaaaattcca
301 attgttttta ttcctggtca agctgagatt cgccacgcta tgcttataag aatttaa
[00152] A serotypic variant of the Pho3-22 polypeptide is found in GBS strain A909 and has locus tag no. SAK_0807 (SEQ ID NO: 40). [00153] SEQ ID NO: 40:
MKNITKLSTVALSLLLCTACAASNTSTSKTQSHHPKQTKLTDKQKEEPKNKEAADQEMHPQGAVDLTKYKA KPVKDYGKKIDVGDGKKMNIYETGQGKIPIVFIPGQAEISPRYAYKNLIERLSKKYKIYTVEPLGYGLSDI PTKPRTLENITKEIHTGLNKIGVKNFYLAAHSLGGMYSLNYAKNYPEEVRGFIGMDTSTPWMEGEQKTKYD PESAKWAMKLPDVDDKTNEQYLSIAKKINGNKSQRDEDERLGDNLGKLKNVKFPKGMPTKYFLADQSVEGI KLRQADFKGLKNWDQQHIDLSEDPKDASVEILKGSHNIFQTQYEHMAKAIDGFIQSHQK
[00154] The Pho3-23 polypeptide sequence (SEQ ED NO: 42) in GBS strain M732 is publicly available and has locus tag No. CAC09100 in Genbank. [00155] SEQ ID NO: 42
1 mnestirkef kiwfkwiln nqavialmit flvfltifif tkisfmfkpv fdflavlilp 61 lvisgllyyl lkpmvtflek rgikrvtail svftiiilll iwamssfipm msnqlrhfme 121 dlpsyvnkvq metssfidhn pwlksykgei ssmlsnissq avsyaekfsk nvldwagnla 181 stvarvtvat imapfilfyl lrdsrnmkng flmvlptklr qpadrilrem nsqmsgyvqg 241 qiivaitvgv ifsimysiig lrygvtlgii agvlnmvpyl gsfvaqipvf ilalvagpvm 301 wkvaivfvi eqtlegrfvs plvlgnklsi hpitimfill tsgamfgvwg vflsipiyas 361 ikwvkelfd wykavsglyt idwteerse evknve
[00156] The pho3-23 polynucleotide sequence (SEQ ID NO: 41) encoding the Pho3-23 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026671 in Genbank.
[00157] SEQ ID NO: 41
1 gtgaatgaat cgaccatcag aaaagaattt aaaatagttg tttttaaatg gatcttaaat
61 aatcaagcag ttattgctct catgattacc tttttggtat ttttaacgat ttttattttt
121 accaaaatct cttttatgtt taaacctgtg tttgattttc ttgctgtgct gatattgccg
181 cttgtaattt ctggcttgct ttattaccta ttaaaaccta tggttacatt tttagagaag
241 cggggaatta agcgtgtaac agcgatatta tcagttttta ctattataat ccttctgtta
301 atttgggcaa tgtctagttt tattcccatg atgagtaatc aattacgcca ttttatggaa
361 gatctccctt catatgtgaa taaagtgcaa atggaaacaa gttcgtttat agatcacaac
421 ccttggttaa aatcttataa aggggaaata tcgagcatgt tatctaatat cagtagccaa
481 gcggtctctt atgctgaaaa attttcaaag aatgttttag attgggcagg aaatttagct
541 agtacagttg cacgtgtgac agtagcaaca atcatggctc cctttatttt gttttatctt
601 ttaagagata gtcgcaacat gaagaatggt ttcttaatgg ttttaccaac caaactacgc
661 caaccagctg atcgtatttt gcgagaaatg aatagtcaaa tgtcaggata tgtgcaagga
721 caaatcattg ttgctattac tgttggtgtt attttttcaa taatgtatag tattataggc
781 cttagatatg gcgtgacatt agggattatt gccggtgtgt taaatatggt tccctatttg
841 ggaagttttg tcgcccaaat tccagtgttt atcttagcgc ttgtcgcagg acctgttatg
901 gttgttaaag ttgcgattgt ttttgttatt gagcaaactc tagagggacg ctttgtctca
961 cctttggttt taggtaataa acttagcatt catccaatta caattatgtt tattttatta
1021 acctctggag cgatgtttgg tgtttgggga gtattcctca gtattccgat ttatgcatct
1081 atcaaagttg ttgttaaaga attgtttgat tggtacaaag ctgtcagtgg gctatataca
1141 atagatgttg ttactgaaga aagaagtgaa gaagttaaaa atgttgaata g
[00158] A serotypic variant of the Pho3-23 polypeptide is found in GBS strain A909 and has locus tag no. SAKJ281 (SEQ ID NO: 43). [00159] SEQ ID NO: 43:
MNRPSEKEFKNSLFFKWILNNQAVIALMITFLVFLTIFIFTKISFMFKPVFDFLAVLILPLVISGLLYYLL KPMVTFLEKRGIKRVTAILSVFTI I ILLLIWAMSSFIPMMSNQLRHFMEDLPSYVNKVQMETSSFIDHNPW LKSYKGEISSMLSNISSQAVSYAEKFSKNVLDWAGNLAS TVARVTVATIMAPFILFYLLRDSRNM KNGFLM VLPTKLRQPTDRILREMNSQMSGYVQGQI IVAITVGVIFSIMYSIIGLRYGVTLGI IAGVLNMVPYLGSFV AQIPVFTLALVAGPVMWKVAIVFVIEQTLEGRFVSPLVLGNKLSIHPITIMFILLTSGAMFGVWGVFLSI PIYASIKVWKELFDWYKAVSGLYTVDWTEERSEEVKNVE
[00160] The Pho3-24 polypeptide sequence (SEQ ID NO: 45) found in GBS strain M732 is publicly available and has Accession No. CAC09101 in Genbank. [00161] SEQ ID NO: 45 1 mfmgipqyff ylilavlpiy iglffkkrfa lyeiifslsf ivmmltgstf nqlksllayv
61 vgqsllvfiy kayrkrfnht lvfyvtvcls ifplflvkli paisedghqs lfgflgisyl
121 tfravamiie mrdgvlkeft lweflrfllf fptfssgpid rfkrfnedyi nipdrnelld
181 mlgqaihylm lgflykfila yifgslimpp lkelaleqgg vfnwptlgvm yafgfdlffd
241 fagytmfala isnlmgiksp infdkpfksr dlkefwnrwh mslsfwfrdf vfmrlvkllv
301 knkvfknrnv tssvayiinm llmgfwhglt wyyiayglfh giglvindaw vrkkknxnke
361 rrlakkpllp enkwtyalgv fitfnwmfs flifsgfldl lwfpqphnk
[00162] The pho3-24 polynucleotide sequence (SEQ ID NO: 44) encoding the Pho3-24 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026673 in Genbank.
[00163] SEQ IDNO: 44
1 atgtttatgg gaatcccaca atatttcttc taccttatct tagctgtcct accaatttac
61 atcggcttat tctttaagaa gcgttttgcc ttatatgaga ttatttttag tctaagtttt
121 attgtaatga tgttgactgg tagtactttt aatcaattga agtcactatt ggcatacgtt
181 gtcggacagt ctctgctagt ttttatctat aaagcttacc ggaaacgatt taatcatact
241 ttggtctttt atgtaacggt ttgtttatct atttttccgc tatttttggt aaaattaatt
301 ccagctatat ctgaggatgg gcatcagtca ctttttgggt ttctaggaat ttcttacctt
361 acttttagag ctgttgctat gattattgaa atgagagacg gtgtcttgaa agaatttact
421 ttatgggaat tcttaagatt tttactcttc tttccaactt tctcaagtgg accaattgat
481 cgttttaaac gattcaatga ggattacatt aatatcccag atcgaaacga actcctagat
541 atgttaggtc aagcgattca ttatttgatg ttaggttttc tctataagtt tattttagcc
601 tatatttttg gaagtctgat tatgcctcct ctaaaagaat tagcgctaga acagggtggt
661 gtgtttaatt ggccaacact tggggttatg tatgcctttg gttttgattt gttctttgat
721 tttgcaggtt acacaatgtt tgcgttggct atttctaacc taatggggat taagtctccg
781 attaactttg acaaaccttt caaatcacgc gacctaaaag aattttggaa tagatggcat
841 atgagccttt ctttctggtt tagagacttt gttttcatga ggcttgttaa gcttttagtt
901 aaaaataaag tttttaaaaa ccgtaatgtt acttcaagtg tagcttatat tatcaatatα
961 cttcttatgg gattctggca tgggttaact tggtactata tagcctatgg tctctttcat
1021 gggattggcc tagttattaa tgacgcttgg gtacgtaaga agaaaaatay taataaagaa
1081 agaagattgg ctaaaaaacc acttttacca gaaaacaaat ggacttatgc tttgggtgtc
1141 ttcatcacct ttaatgtagt tatgttttct. ttcttgattt tttcaggatt tttagatctt
1201 ttgtggttcc cacaaccgca taacaaataa
[00164] A serotypic variant of the Pho3-24 polypeptide is found in GBS strain A909 and has locus tag no. SAK_1811 (SEQ ID NO: 46). [00165] SEQ ID NO: 46:
MIQAFLEKLPHLDVYGNPQYFFYLILAVLPIYIGLFFKKRFALYEIIFSLSFIVMMLTGSTFNQLKSLLAY WGQSLLVFIYKAYRKRFNHTLVFYVTVCLSIFPLFLVKLIPAISEDGHQSLFGFLGISYLTFRAVAMIIE MRDGVLKEFTLWEFLRFLLFFPTFSSGPIDRFKRFNEDYINIPDRNELLDMLGQAIHYLMLGFLYKFILAY IFGSLIMPPLKELALEQGGVFNWPTLGVMYAFGFDLFFDFAGYTMFALAISNLMGIKSPINFDKPFKSRDL KEFWNRWHMSLSFWFRDFVFMRLVKLLVKNKVFKNRNVTSSVAYIINMLLMGFWHGLTWYYIAYGLFHGIG LVINDAWVRKKKNINKERRLAKKPLLPENKWTYALGVFITFNWMFSFLIFSGFLDLLWFPQPHNK
[00166] The Pho3-29 polypeptide sequence (SEQ ID NO: 48) in GBS strain M732 is publicly available and has Accession No. CAC09102 in Genbank. [00167] SEQ ID NO: 48
1 mnkittlsti altlmlcvgc sankdnqktk ted
[00168] The pho3-29 polynucleotide sequence (SEQ ID NO: 47) encoding the Pho3-29 polypeptide found in GBS strain M732 is publicly available and has Accession No. AX026675 in Genbank.
[00169] SEQ ID NO: 47 1 atgaataaaa taacgacatt atcaaccatc gccctgactt taatgctttg cgttggatgt 61 tctgccaata aagataatca aaaaactaaa actgaggatc
[00170] A serotypic variant of the Pho3-29 polypeptide is found in GBS strain A909 and has locus tag no. SAK_0809 (SEQ ID NO: 49). [00171] SEQ ID NO: 49:
MNKITTLSTIALTLMLCVGCSANKDNQKTKTEDPKQENVTANPKEDKATAAKRMKLTKELEKYNKGTESFI SNKQYQKYIKTPIKEYGQKVKVKGKKMNVYTVGEGKVPIVFIPGQGTVTAKHQYHNLISNLSKTHKWVVE PFGSGLSDVIDQPRNLANITSDIHEALQKVGITGKYVIASHSIGGVYALKYISTYPKEVLGLIGLDTSTPG MEGGKQVDFAAPVLKELPKIPKVSDDINAQFFAIGHKILNNSNMKEEAKNSSNMINESANYKIPKGIPAMY LLARESDLGLQLRSLDIPIKGSWQEQHKELSEDPKEVSIHTLDGTHQIYDTNLKEVIDYTNQFMKTFEK
[00172] The Pho3-50 polypeptide sequence (SEQ ID NO: 51) in GBS strain M732 is publicly available and has Accession No. CAC09383 in Genbank. [00173] SEQ ID NO: 51
1 drgyqeamak lrktygeygl gvstgldlpe segyvpgkys lgttlmesfg qydaytpmql
61 gqyistiann gnrlaphws diyegndsnk faqlvrsitp ktlnkiaisd qelaiiqegf
121 ynwnsgsgy atgtsmrgnv ttisgktgta etfakningq tvstynlnai aydtnrkiav
181 avmyphvttd ttkshqlvar dmidqyisqs qdnkrgh
[00174] The pho3-50 polynucleotide sequence (SEQ ID NO: 50) encoding the Pho3-50 polypeptide in GBS strain M732 is publicly available and has Accession No. AX026677 in Genbank.
[00175] SEQ ID NO: 50
1 gatcgaggct atcaagaago aatggctaaa ctaaggaaaa cttacggcga atatggttta
61 ggggtttcta caggattaga tttacctgaa tcagaaggtc atgtacctgg aaaatacagc
121 ttaggaacaa ctctaatgga atcgttcggt cagtatgatg cctatacacc aatgcaactt
181 ggtcagtata tctcaactat tgcgaataat gggaatcgtt tagcacctca cgtggtttca
241 gatatctatg aagggaatga ttctaataag ttcgctcaat tggttcgttc aatcactcct
301 aaaacactaa ataagatagc tatctcagat caagagttag ccattattca agaaggtttt
361 tataacgttg tcaatagtgg aagtggctat gcaactggta cgtcaatgag ggggaatgtg
421 acaaccatta gyggtaaaac tggtaccgct gaaacatttg ctaaaaatat aaatggacaa
481 acagtttcta cctacaactt aaacgctatt gcctacgata ctaatcgtaa aatagcagta
541 gcggtaatgt atccgcatgt tacaactgat acaacaaaat cccatcaatt agttgcacga
601 gatatgattg atcaatatat ttcacagtca caggacaata agagaggaca ttga
[00176] A serotypic variant of the Pho3-50 polypeptide is found in GBS strain A909 and has locus tag no. SAK 0890 (SEQ ID NO: 52). [00177] SEQ ID NO: 52:
MLNRKKRYRLTVKKQNASIPRRLNLLFFIIVLLFTVLILRLEQMQIGQQSFYMKKLTALTSYTVKESKARG QIFDAKGWLVENDERPTVAFSRGNNISSQSIKELANKLSHYITLTEVASSDRAKRDYYLADKANYKKWE SLPDSKRYDKFGNHLAESTVYANAVAAVPVSAINYSEDELKWALFNQMSATPTFGSVKLSTGELSDDQIK KLDADKKELLGISVTSNWHRRKKGTSLSDILGTISTEKAGLPREEVKKYLKKGYSLNDRVGTSYLEKQYED DLQGIRQIRKVVWKKGKWSDNITQEGKSGRNLKLTIDLNYQNKVESILKQYYGSELSSGRASFSEGMYA VAIEPSTGKVLAMAGLKNDHGNLVDDSLGTIAKNFTPGSWKGATLSSGWENKVLRGNEVLYDQEIANIRS WFTRGLTPISAAQALEYSSNTYMVQVALRLMGQDYNTGDALTDRGYQEAMAKLRKTYGEYGLGVSTGLDLP ESEGYVPGKYSLGTTLMESFGQYDAYTPMQLGQYISTIANNGNRLAPHWSDIYEGNDSNKFAQLVRSITP KTLNKIAISDQELAIIQEGFYNWNSGSGYATGTSMRGNVTTISGKTGTAETFAKNVNGQTVSTYNLNAIA YDTNRKIAVAVMYPHVTTDTTKSHQLVARDMIDQYISQFTGQ
[00178] The MS4 polypeptide sequence (SEQ ID NO: 54) in GBS strain M732 is publicly available and has Accession No. CAC09078 in Genbank. [00179] SEQ ID NO: 54
1 mtqvfqgrsf laekdfsree feylidfsah lkdlkkrgvp hhylegknia llfektstrt
61 raafttaaid lgahpeylga ndiqlgkkes tedtakvlgr mfdgiefrgf sqrmveelae
121 fsgvpvwngl tdewhptqml adyltikenf gklegitlvy cgdgrnnvan sllvagtlmg
181 vnvhifspke lfpaeeivkl aegyakesga hvlvtdnvde avkgadvfyt dvwvsmgeed
241 kfkervellq pyqvnmelik kanndnlifl hclpafhdtn tvygkdvaek fgvkemevtd
301 evfrskyarh fdqaenrmht ikavmaatlg nlfipkv
[00180] The MS4 polynucleotide sequence (SEQ ID NO: 53) encoding the MS4 polypeptide in
GBS strain M732 is publicly available and has AccessionNo. AX026599 in Genbank. [00181] SEQ ID NO: 53
1 atgacacaag tatttcaagg acgtagtttc ttagcagaaa aagatttttc tcgtgaggaa
61 tttgaatatc ttattgattt ttcagctcat ttaaaagacc ttaaaaaacg tggtgttcct
121 catcattatc ttgaaggtaa aaatattgct ctcttatttg aaaaaacatc tactcgtact
181 cgcgcagcct ttacaactgc agcaattgac ctaggcgctc atccggaata ccttggtgca
241 aatgatattc aacttggtaa aaaagaatca acagaagata ctgctaaggt tttaggacgt
301 atgtttgatg gtattgaatt ccgtggtttt agccaaagaa tggttgaaga gcttgctgaa
361 ttttctggag tacctgtctg gaatggttta acagatgaat ggcatccaac acaaatgcta
421 gctgactacc ttactatcaa agaaaacttc ggtaaacttg aaggtattac tcttgtttac
481 tgtggtgacg gacgtaacaa tgttgccaac tcgcttttag tggctgggac tttgatgggg
541 gtcaatgtac acatcttttc tccaaaagaa cttttyccwg ctgaagagat tgttaaattg
601 gctgaaggat atgccaaaga atctggggct cacgttctcg ttactgataa tgtagacgaa
661 gctgtaaagg gagcagacgt cttttacact gatgtctggg tatcgatggg agaagaagat
721 aagttcaaag aacgcgttga acttcttcaa ccatatcaag taaacatgga actgattaaa
781 aaagctaata atgataatct tatcttctta cactgcttac ctgcattcca tgatacaaat
841 accgtttatg gcaaagacgt cgctgaaaaa tttggggtca aggaaatgga agttactgat
901 gaagtcttcc gtagcaaata tgctcgtcat ttcgaccaag ctgaaaatcg tatgcacact
961 attaaagctg taatggctgc aacccttgga aatcttttca ttccaaaagt ttaa
[00182] A serotypic variant of the MS4 polypeptide is found in GBS strain A909 and has locus
.tegno. SAK_2123 (SEQ ID NO: 55). [00183] SEQ ID NO: 55:
MTQVFQGRSFLAEKDFSREEFEYLIDFSAHLKDLKKRGVPHHYLEGKNIALLFEKTSTRTRAAFTTAAIDL GAHPEYLGANDIQLGKKESTEDTAKVLGRMFDGIEFRGFSQRMVEELAEFSGVPVWNGLTDEWHPTQMLAD
VTDNVDEAVKGADVFYTDVWVSMGEEDKFKERVELLQPYQVNMDLIKKANNDNLIFLHCLPAFHDTNTVYG KDVAEKFGVKEMEVTDEVFRSKYARHFDQAENRMHTIKAVMAATLGNLFIPKV
[00184] The MSIl polypeptide sequence (SEQ ID NO: 57) in GBS strain M732 is publicly available and has Accession No. CAC09079 in Genbank. [00185] SEQ ID NO: 57
1 makltvkdvd lkvkkvlvrv dfnvplkdgv itndnritaa lptikyiieq ggrailfshl
61 grvkeeadke gkslapvaad laaklgqdw fpgvtrgakl eeainaledg qvllventrf
121 edvdgkkesk ndeelgkywa slgdgifvnd afgtahraha snvgisanve kavagfllen
181 eiayiqeave tperpfvail ggskvsdkig vienllekad kvligggmty tfykaqgiei
241 gnslveedkl dvakdlleks ngklilpvds keanafagyt evrdtegeav segflgldig
301 pksiakfdea ltgaktwwn gpmgvfenpd fqagtigvmd aivkqpgvks iigggdsaaa
361 ainlgradkf swistgggas mellegkvlp glaaltek
[00186] The MSl1 polynucleotide sequence (SEQ ID NO: 56) encoding the MSl1 polypeptide in
GBS strain M732 is publicly available andhas Accession No. AX026601 in Genbank. [00187] SEQ ID NO: 56
1 atggctaaat tgactgttaa agacgttgat ttgaaggtaa aaaaagtcct cgttcgtgtt 61 gactttaatg tgcctttgaa agacggcgtt atcactaacg acaaccgtat cactgcggct 121 cttccaacaa tcaagtatat catcgaacaa ggtggtcgtg ctatcctctt ctctcacctt 181 ggacgtgtta aagaagaagc tgacaaagaa ggaaaatcac ttgcaccggt agctgctgat 241 ttagctgcta aacttggtca agatgttgta ttcccaggtg ttactcgtgg tgcaaaatta 301 gaagaagcaa tcaatgcttt ggaagatgga caagttcttt tggttgaaaa cactcgtttt 361 gaagatgttg acggtaagaa agaatctaag aatgacgaag aacttggtaa atactgggct 421 tcacttggag atggaatctt cgttaacgat gcatttggta cagcacaccg tgctcatgca 481 tcaaacgtag gtatttcagc aaacgttgaa aaagctgtag ctggtttcct tcttgaaaac 541 gaaattgctt acatccaaga agcagttgaa actccagaac gcccattcgt agctattctt 601 ggtggctcaa aagtttctga taagattggt gttatcgaaa accttcttga aaaagctgat 661 aaagttctta tcggtggtgg tatgacttac acattctaca aagctcaagg tatcgaaatc 721 ggtaactcac ttgtagaaga agacaaattg gatgttgcta aagacctcct tgaaaaatca 781 aacggtaaat tgatcttgcc agttgactca aaagaagcaa acgcatttgc tggttatact 841 gaagttcgcg acactgaagg tgaagcagtt tcagaagggt tccttggtct tgacatcggt 901 cctaaatcaa tcgctaaatt tgatgaagca cttactggtg ctaaaacagt tgtatggaac 961 ggacctatgg gtgtctttga aaaccctgac ttccaagctg gtacaatcgg tgtaatggac 1021 gctatcgtta aacaaccagg cgttaaatca atcatcggtg gtggtgattc agcagcagct 1081 gctatcaacc ttggtcgtgc tgacaaattc tcatggatct ctactggtgg tggagcaagc 1141 atggaattgc tcgaaggtaa agtattacca ggtttggcag cattgactga aaaataa
[00188] A serotypic variant ofthe MS4 polypeptide is found in GBS strain A909 and has locus tagno. SAK l788 (SEQ ID NO: 58). [00189] SEQ ID NO: 58:
MAKLTVKDVDLKGKKVLVRVDFNVPLKDGVITNDNRITAALPTIKYIIEQGGRAILFSHLGRVKEEADKEG KSLAPVAADLAAKLGQDWFPGVTRGAKLEEAINALEDGQVLLVENTRFEDVDGKKESKNDEELGKYWASL GDGIFVNDAFGTAHRAHASNVGISANVEKAVAGFLLENEIAYIQEAVETPERPFVAILGGSKVSDKIGVIE NLLEKADKVLIGGGMTYTFYKAQGIEIGNSLVEEDKLDVAKDLLEKSNGKLILPVDSKEANAFAGYTEVRD TEGEAVSEGFLGLDIGPKSIAKFDEALTGAKTWWNGPMGVFENPDFQAGTIGVMDAIVKQPGVKSIIGGG DSAAAAINLGRADKFSWISTGGGASMELLEGKVLPGLAALTEK
[00190J The MS16 polypeptide sequence I (SEQ ID NO: 60} in GBS strain M732 is publicly available and has Accession No. CAC09080 in Genbank. [00191] SEQ ID NO: 60
1 mthitfdlfk vlgqfvgehe ldylppqvsa adaflrqgtg pgsdflgwme ppenydkeef 61 sriqkaaeki ksdsevlwi giggsylgak aaidflnnhf anlqtaeerk apqilyagns 121 isstyladlv eyvqdkefsv nvisksgttt epaiafrvfk ellvkkyrsr rs
[00192] The MS16 polynucleotide sequence I (SEQ ID NO: 59) encoding the MS16 polypeptide in GBS strain M732 is publicly available andhas AccessionNo. AX026603 in Genbank. [00193] SEQ ID NO: 59
1 atgacacata ttacatttga cttattcaaa gtcttgggtc aatttgtagg cgaacacgag
61 ttagactacc taccaccaca agtaagtgca gcagatgctt tccttcgtca agggactggt
121 cctggatcag attttctcgg atggatggaa cctccagaaa actatgacaa agaagaattt
181 tctcgcattc aaaaagccgc tgaaaagatt aaatcagata gcgaagtact cgtggttatt
241 ggtattggtg gttcgtacct tggtgcaaaa gcagcaattg actttttgaa taatcatttt
301 gctaatttgc aaaccgcaga agaacgtaaa gcgcctcaga ttctttatgc tggaaattct
361 atttcatcta cttaccttgc cgatttagtt gaatacgtcc aagataaaga attctcagta
421 aatgtcattt caaaatcagg tacaacaact gaaccagcga ttgctttccg tgtatttaaa
481 gaacttctag ttaaaaagta ccggtcaaga agaagc
[00194] The MS16 polypeptide sequence II (SEQ ID NO: 62) in GBS strain M732 is publicly available and has Accession No. CAC09081 in Genbank.
[00195] SEQ ID NO: 62
1 mnrrfrwswl ssrkdvdfvn kkatdgvlla htdggvpnmf vtlptqdayt lgytiyffel 61 aiglsgylns vnpfdqpgve aykrnmfafg kpgfeelsae lnarl
[00196] The MS16 polynucleotide sequence II (SEQ ID NO: 61) encoding the MS16 polypeptide in GBS strain M732 is publicly available andhas Accession No. AX026605 in Genbank. [00197] SEQ ID NO: 61
1 attaaccgaa gatttagatg gtcttggtta tcttcaagaa aagatgtaga ttttgttaat
61 aaaaaagcaa cagatggtgt gcttcttgct catacagatg gtggggttcc aaatatgttt
121 gtaacgcttc ctacacaaga cgcttacact cttggttaca ctatttactt ctttgagtta
181 gcaattggcc tttcaggtta tcttaactca gtaaatccat ttgatcaacc gggggtagaa
241 gcatataaac gtaatatgtt cgcatttggt aaacctggat tcgaagagct tagcgctgaa
301 ttgaatgcac gtctttaa
[00198] A serotypic variant ofthe MS16 polypeptide is found in GBS strain A909 and has locus tag no. SAK_0475 (SEQ ID NO: 63). [00199] SEQ ID NO: 63:
MTHITFDYSKVLGQFVGEHELDYLQPQVSAADAFLRQGTGPGSDFLGWMDLPENYDKEEFSRIQKAAEKIK SDSEVLWIGIGGSYLGAKAAIDFLNNHFANLQTAEERKAPQILYAGNSISSTYLADLVEYVQDKEFSVNV ISKSGTTTEPAIAFRVFKELLVKKYGQEEANKRIYATTDKVKGAVKVEADANNWETFWPDNVGGRFSVLT AVGLLPIAASGADITALMEGANAARKDLSSDKISENIAYQYAAVRNVLYRKGYITEILANYEPSLQYFGEW WKQLAGESEGKDQKGIYPTSANFSTDLHSLGQFIQEGYRNLFETWRVEKPRKNVTIPELTEDLDGLGYLQ GKDVDFVNKKATDGVLLAHTDGGVPNMFVTLPTQDAYTLGYTIYFFELAIGLSGYLNSVNPFDQPGVEAYK RNMFALLGKPGFEELSAELNARL
[00200] The MS14 polypeptide sequence (SEQ ID NO: 65) in GBS strain M732 is publicly available and has Accession No. CAC09082 in Genbank. [00201] SEQ ID NO: 65
1 mtllekinet rdflqakgvt apefglilgs glgelaeeie npiwdyadi' pnwgqstwg
61 hagkfsvwdl sgrkvlalqg rfhfyegntm ewtfpyrim ralachsvlv tnaaggigyg
121 pgtlmlikdh inmigtnpli genleefgpr fpdmsdayta tyrqkahqia endikleegv
181 ylgvsgptye tpaeirafqt mgaqavgmst vpevivaahs glkvlgisai tnlaagfqse
241 lnheewevt qrikedfkgl gkslvael
[00202] The MS14 polynucleotide sequence (SEQ ID NO: 64) encoding the MS14 polypeptide in
GBS strain M732 is publicly available and has Accession No. AX026607 in Genbank. [00203] SEQ ID NO: 64
1 atgacattat tagaaaaaat taatgagact agagactttt tgcaagcaaa aggcgtcaca
61 gcaccagaat ttggycttat tttaggctct ggtttaggag aattggctga agaaatcgaa
121 aatcctattg ttgtggatta tgcagacatc ccmaattggg gacagtcaac agtagttggt
181 catgctggaa aatttagtgt atgggattta tcaggccgta aggtattagc gcttcaaggt
241 cgttttcatt tttaygaagg waatacaatg gaagtcgtta ctttcccagt acgtatcatg
301 agagcattgg cttgccacag tgtgcttgtg actaatgcag cgggtgggat tggatacgga
361 ccaggaactt taatgctgat caaagaccac atcaatatga ttgggactaa ccctctcata
421 ggtgagaacc ttgaagaatt tggaccacgt ttcccagaca tgtcggatgc ttayacagca
481 acatatcgac aaaaagctca ccaaattgct gaaaacgata tcaaactcga agaaggtgtg
541 tacttgggtg tatcaggacc cacttatgaa acacctgcag aaattcgtgc attccaaaca
601 atgggcgcac aagcggtagg tatgtccacg gttccagagg tgatcgttgc agctcactca
661 gggcttaaag tgttaggaat ttcagcaatt actaaccttg ccgctggctt ccaatcagag
721 ctcaatcatg aggaggtcgt tgaagttact cagcgtatta aagaagattt caagggatta
781 ggtaaatcat tagttgctga actc
[00204] A serotypic variant ofthe MS14 polypeptide is found in GBS strain A909 and has locus tag no. SAK_1267 (SEQ ID NO: 66). [00205] SEQ ID NO: 66:
MTLLEKINETRDFLQAKGVTAPEFGLILGSGLGELAEEIENPIWDYADI PNWGQSTWGHAGKLVYGDLS GRKVLALQGRFHFYEGNTMEWTFPVRIMRALACHSVLVTNAAGGIGYGPGTLMLIKDHINMIGTNPLIGE NLEEFGPRFPDMSDAYTATYRQKAHQIAEKQNIKLEEGVYLGVSGPTYETPAEIRAFQTMGAQAVGMSTVP EVIVAAHSGLKVLGISAITNFAAGFQSELNHEEWEVTQRIKEDFKGLVKSLVAEL
[00206] The MSlO polypeptide sequence (SEQ ID NO: 68) in GBS strain M732 is publicly available and has Accession No. CAC09083 in Genbank.
[00207] SEQ ID NO: 68
1 mtkeyqnyvn gewkssvnqi eilspiddss lgfvpamtre evdhamkagr ealpawaalt
61 vyeraqylhk aadiierdke eiatvlakei skaynasvte wrtadliry aaeegirlst
121 sadeggkmda stghklavir rqpvgivlai apynypvnls gskiapalig gnwmfkppt
181 qgsvsglvla kafaeaglpa gvfntitgrg seigdyiveh eevnfinftg stpvgqrigk
241 lagmrpimle lggkdagivl adadldnaak qivagaydys gqrctaikrv lweevadel
301 aekisenvak lsvgdpfdna tvtpviddns adfieslwd arqkgakeln efkrdgrllt
361 pglfdhvtld mklaweepfg pilpiirvkd aeeavaiank sdfglqssvf trdfqkafdi
421 anklevgtvh innktgrgpd nfpflglkgs gagvqgirys ieamtnvksi vldmk
[00208] The MSlO (NP GAP; ME:NAP) polynucleotide sequence (SEQ ID NO: 67) encoding the MSlO polypeptide in GBS strain M732 is publicly available and has Accession No. AX026609 in Genbank.
[00209] SEQ ID NO: 67 i ttgacaaaag aatatcaaaa ttatgtcaat ggcgaatgga aatcatctgt taatcagatt
61 gagattttgt caccaattga tgattcttca ttgggattcg tgccagcgat gactcgagaa
121 gaagttgatc atgctatgaa agcgggtcgt gaggctttac cagcttgggc tgctttaaca
181 gtatatgaac gtgcacaata ccttcataaa gccgcagaca ttattgaacg tgataaagaa
241 gaaattgcta ctgttttagc aaaagaaatt tctaaagctt acaatgcttc agtaaccgag
301 gttgtaagga cagctgatct tattcgttat gcagcagaag aaggaattcg tttatcaact
361 tcagctgacg aaggtggaaa aatggatgct tcaacaggtc ataagttggc tgttattcgt
421 cgtcaaccag taggtatcgt tttagcaatc gcaccttata attaccctgt taacctctca
481 ggatcaaaaa ttgcgccagc tctaattggt ggaaacgttg tgatgtttaa accaccaaca
541 caaggttcag tctcaggact tgttttagca aaagcttttg cagaagcagg tcttccagca
601 ggtgtcttta atactattac aggacgcggt tctgagattg gagattacat tgttgagcat
661 gaagaagtta attttattaa ctttacagga tcaacgccag ttggacaacg tattggtaag
721 ttggcaggaa tgcgtccaat tatgcttgag ttgggcggta aggatgcagg tatcgtctta
781 gctgatgctg accttgataa cgctgctaaa caaatcgttg caggtgctta tgattactct
841 ggacaacgct gtacggcaat taagcgtgtg cttgtcgttg aagaagttgc wgatgaattg
901 gcagaaaaaa tatctgaaaa tgtagcaaaa ttatcagtag gtgatccatt tgataatgca
961 acggtgacac cggttattga tgataattca gctgacttta ttgaaagctt agtagtagat
1021 gcacgtcaaa aaggtgcgaa agaattgaat gaatttaaac gtgatggtcg tctattaact
1081 ccaggattgt ttgatcatgt tactttagat atgaaactag cttgggaaga gccttttgga
1141 ccaattctcc caattattcg tgtcaaggat gcagaagaag ctgttgctat tgccaacaaa
1201 tctgattttg gattacaatc atcagtcttt acacgtgatt tccaaaaagc atttgatata
1261 gcaaataaac ttgaagttgg tacagttcac attaacaata agactggacg tggtccwgat
1321 aatttcccat tcttaggact caaaggatct ggtgcaggtg ttcaaggtat cagatattca
1381 attgaagcaa tgacaaatgt aaaatcgatt gttctcgata tgaaatag
[00210] A serotypic variant of the MSlO polypeptide is found in GBS strain A909 and has locus tag no. SAK_0947 (SEQ ID NO: 69). [00211] SEQ ID NO: 69:
MTKEYQNYVNGEWKSSVNQIEILSPIDDSSLGFVPAMTREEVDHAMKAGREALPAWAALTVYERAQYLHKA ADI IERDKEEIATVLAKEISKAYNASVTEWRTADLIRYAAEEGIRLSTSADEGGKMDASTGHKLAVIRRQ PVGIVLAIAPYNYPVNLSGSKIAPALIGGNWMFKPPTQGSVSGLVLAKAFAEAGLPAGVFNTITGRGSEI GDYIVEHEEVNFINFTGSTPVGKRIGKLAGMRPIMLELGGKDAGWLAD ADLDNAAKQIVAGAYDYSGQRC TAIKRVL WEEVADELAEKISENVAKLSVGDPFDNATVTPVIDDNSADFIESLWD ARQKGAKELNEFKRD GRLLTPGLFDHVTLDMKLAWEEPFGPILPIIRVKDAEEAVAIANKSDFGLQSSVFTRDFQKAFDIANKLEV GTVHINNKTGRGPDNFPFLGLKGSGAGVQGIRYSIEAMTNVKSIVFDMK
[00212] In one embodiment, a combination of two or more polypeptides induces an immune response. In one aspect, the immune response may have additive effect. In another embodiment, a combination of two or more polypeptides or polynucleotides may induce synergistic immune response.
Fragments
[00213] Polypeptides, and fragments, derivatives, analogs, or variants thereof of the present invention can be antigenic and immunogenic fragment of the GBS polypeptides, which may also be used to prevent or treat, i.e., cure, ameliorate, lessen the severity of, or prevent or reduce contagion of infectious disease caused by GBS, or other species as disclosed herein. The term "antigens" and its related term "antigenic" as used herein and in the claims refer to a substance that binds specifically to an antibody or T-cell receptor. In some embodiments the antigens are immunogenic. The term "immunogen," as used herein, refers to an antigen capable of inducing an immune response directed against itself when administered to a subject.
[00214] Peptides or polypeptides, e.g., immunogenic epitopes, capable of eliciting an immunogenic response are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins nor to the ammo or carboxyl terminals. Polypeptides that are extremely hydrophobic, and those of six or fewer residues generally are ineffective at inducing antibodies, but may still bind antibodies raised against larger portions of the polypeptide; longer peptides, especially those containing proline residues, usually are effective (Sutcliffe, J. G., et al., Science 219:660-666 (1983)).
[00215] In certain aspects of the present invention, antigenic epitopes or antigenic fragments can contain a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 contained within the amino acid sequence of the polypeptides of the invention. Antigenic as well as immunogenic epitopes may be linear, i.e., be comprised of contiguous amino acids in a polypeptide, or may be three dimensional, i.e., where an epitope is comprised of non-contiguous amino acids which come together due to the secondary or tertiary structure of the polypeptide, thereby forming an epitope.
[00216] A fragment comprising an antigenic and immunogenic polypeptide of the present invention can be predicted based on antigenicity of the GBS polypeptides described herein. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. ScL USA 81 :3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g. , x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 2.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, parker antigenicity method, Parker et al., Biochem. (1986), 25, 5425-5432; Protrusion Index antigenicity, Thornton et al., EMBO Journal (1986) 5: 409-413; and Welling antigenicity, FEBS Lett. (1985) 188:215-218. For example, Figure 1 shows the Hope/Woods antigenicity profile of the GBS polypeptides described herein. Other techniques, e.g., the Kyte-Doolittle technique, Kyte et al., J. MoI. Biol. (1982) 157:105-132, may be used to measure hydropathy of the GBS polypeptides.
[002J.7] According to the Hopp/Wood . antigenicity method, an antigenic or immunogenic fragment of the Phol-13 polypeptide may be selected from the group consisting of amino acids 1- 21, 28-34, 51-56, 27-41, 55-93, 43-53, 94-116, 119-146, 119-146, 138-153, 154-180, 146-182, 159-166, and 168-175 of SEQ E) NO: 2. In preferred embodiments, an antigenic or immunogenic Phol-13 polypeptide fragment may be amino acids 21-59, 132-181, 101-181, or 1- 181 of SEQ ID NO: 2.
[00218] An antigenic or immunogenic polypeptide fragment of the Phol-14 polypeptide may be selected from the group consisting of amino acids 25-31, 19-31, or 1-45 of SEQ ID NO: 5.
[00219] An antigenic or immunogenic polypeptide fragment of the Phol-5 polypeptide may be selected from the group consisting of amino acids 1-14, 1-22, 43-115, 45-87, 127-220, 178-192, 145-157, and 145-163 or SEQ ID NO: 8. In preferred embodiments, an antigenic or immunogenic Phol-5 polypeptide fragment may be amino acids 43-115, 139-192, or 43-192 of SEQ ID NO: 8.
[00220] An antigenic or immunogenic polypeptide fragment of the Pho3-3 polypeptide may be selected from the group consisting of amino acids 1-18, 18-42, 58-68, 47-53, 69-110, and 165-197 of SEQ ID NO: 11. In preferred embodiments, an antigenic or immunogenic Pho3-3 polypeptide fragment may be amino acids 1-88, 1-110, or 165-197 of SEQ ID NO: 11. [00221] An antigenic or immunogenic polypeptide fragment of the Pho2-10 polypeptide may be selected from the group consisting of amino acids 13-19, 37-61, 80-98, 1-24, 85-101, 121-133, 127-143, 130-157, 64-84 and 175-197 of SEQ ID NO: 14. In preferred embodiments, an antigenic or immunogenic Pho2-10 polypeptide fragment may be amino acids 1-59, 84-149, or 1- 149 of SEQ ID NO: 14 or SEQ ID NO: 15.
[00222] An antigenic or immunogenic polypeptide fragment of the Pho2-15 polypeptide may be amino acids 29-95, 1-95, 68-79 or 29-125 of SEQ DD NO: 18. In preferred embodiments, an antigenic or immunogenic Pho2-15 polypeptide fragment may be amino acids 29-95, 1-95, or 29- 125 of SEQ ID NO: 18.
[00223] An antigenic or immunogenic polypeptide fragment of the Pho2-2 polypeptide may be selected from the group consisting of amino acids 70-78, 106-147, 168-200, 273-290, and 301- 350 of SEQ ID NO:21. In preferred embodiments, an antigenic or immunogenic Pho2-2 polypeptide fragment may include amino acids 1-77, 106-225, 272-360, or 106-360 of SEQ ID NO:21.
[00224] An antigenic or immunogenic polypeptide fragment of the Pho3-14 polypeptide may be selected from the group consisting of amino acids 1-36, 43-65, 65-91, and 91-122 of SEQ ID NO: 24. In preferred embodiments, an antigenic or immunogenic Pho3-14 polypeptide fragment may be amino acids 43-84, 1-84, 88-122, or 38-122 of SEQ BD NO: 24.
[00225] An antigenic or immunogenic polypeptide fragment of the Pho3-17 polypeptide may be selected from the group consisting of amino acids 27-40, 29-62, 29-84, 101-122, 97-189, 110- 140, 131-149, 154-189, and 155-170 of SEQ ID NO: 27. In preferred embodiments, an antigenic or immunogenic Pho3-17 polypeptide fragment may be amino acids 29-79, 101-189, or 29-189 of SEQ ID NO: 27.
[00226] An antigenic or immunogenic polypeptide fragment of the Pho3-18 polypeptide may be selected from the group consisting of amino acids 23-45, 80-105, 105-142, 146-182, 190-217, 269-278, and 302-325 of SEQ ID NO: 30. In preferred embodiments, an antigenic or immunogenic Pho3-18 polypeptide fragment may be amino acids 79-182, 79-217, 23-217, or 23- 278 of SEQ ID NO: 30.
[00227] An antigenic or immunogenic polypeptide fragment of the Pho3-1 polypeptide may be selected from the group consisting of amino acids 24-39, 71-99, 113-150, 150-192, and 113-192 of SEQ DD NO: 33. In preferred embodiments, an antigenic or immunogenic Pho3-1 polypeptide fragment may be amino acids 113-192, 71-192, or 24-192 of SEQ DD NO: 33.
[00228] An antigenic or immunogenic polypeptide fragment of the Pho3-21 polypeptide may be selected from the group consisting of amino acids 25-40, 56-80, 98-107, 133-142, 153-172, and 181-202 of SEQ DD NO: 36. In preferred embodiments, an antigenic or immunogenic Pho3-21 polypeptide fragment may be amino acids 25-88, 132-202, 25-172, or 25-202 of SEQ DD NO: 36. [00229] An antigenic or immunogenic polypeptide fragment of the Pho3-22 polypeptide may be selected from the group consisting of amino acids 27-60, 60-94, and 27-94 of SEQ ID NO: 39. In preferred embodiments, an antigenic or immunogenic Pho3-22 polypeptide fragment may be amino acids 27-94 of SEQ ID NO: 39.
[00230] An antigenic or immunogenic polypeptide fragment of the Pho3-23 polypeptide may be selected from the group consisting of amino acids 76-87, 111 -180, 111 -234, 72-234, or 40-335 of SEQ ID NO: 42. In preferred embodiments, an antigenic or immunogenic Pho3-23 polypeptide fragment may be amino acids 111-180, 111-234, 72-234, or 40-335 of SEQ ID NO: 42.
[00231] An antigenic or immunogenic polypeptide fragment of the Pho3-24 polypeptide may be selected from the group consisting of amino acids 159-180, 260-277, 346-370, and 300-310 of SEQ ID NO: 45. In preferred embodiments, an antigenic or immunogenic Pho3-24 polypeptide fragment may be amino acids 98-180, 259-370, or 98-370 of SEQ ID NO: 45.
[00232] An antigenic or immunogenic polypeptide fragment of the Pho3-29 polypeptide may be amino acids 21-33 of SEQ ID NO: 48.
[00233] An antigenic or immunogenic polypeptide fragment of the Pho3-50 polypeptide may be selected from the group consisting of amino acids 1-20, 28-37, 79-95, 102-112, 140-155, 192- 202, and 202-217 of SEQ ID NO: 51. In preferred embodiments, an antigenic or immunogenic Pho3-50 polypeptide fragment may be amino acids 78-154, 78-217, or 1-154 of SEQ ID NO: 51.
[00234] An antigenic or immunogenic polypeptide fragment of the MS4 polypeptide may be selected from the group consisting of amino acids 1-44, 51-63, 80-102, 102-117, 191 -205, 205- 225, 231-253, and 281-321 of SEQ ID NO: 54. In preferred embodiments, an antigenic or immunogenic MS4 polypeptide fragment may be amino acids 190-261, 190-321 or 1-118 of SEQ ID NO: 54.
[00235] An antigenic or immunogenic polypeptide fragment of the MS 11 polypeptide may be selected from the group consisting of amino acids 1-24, 24-35, 56-79, 112-140, 91-111, 182-202, 205-220, 240-270, 274-295, and 297-310 of SEQ ID NO: 57. In preferred embodiments, an antigenic or immunogenic MSI l polypeptide fragment may be amino acids 1-139, 184-311, or 184-398 of SEQ ID NO: 57.
[00236] An antigenic or immunogenic polypeptide fragment of the MS 16 polypeptide may be selected from the group consisting of amino acids 48-77, 101-116, 128-145, and 157-172 of SEQ ID NO: 60 and amino acids 1-30 and 75-105 of SEQ ID NO: 62. In preferred embodiments, an antigenic or immunogenic MS 16 polypeptide fragment may be amino acids 24-77, 101-172, or 24-172 of SEQ ID NO: 60 or SEQ ID NO: 62.
[00237] An antigenic or immunogenic polypeptide fragment of the MS 14 polypeptide may be selected from the group consisting of amino acids 1-18, 32-45, 141-154, 154-162, 162-180, and 241-268 of SEQ ID NO: 65. In preferred embodiments, an antigenic or immunogenic MS14 polypeptide fragment may be amino acids 1-95, 141-181, or 141-268 of SEQ ID NO: 65.
[00238] An antigenic or immunogenic polypeptide fragment of the MSlO polypeptide may be selected from the group consisting of amino acids 31-50, 78-89, 105-138, 212-220, 255-270, 285- 315, 330-360, 382-400, and 405-445 of SEQ ID NO: 68. In preferred embodiments, an antigenic or immunogenic MSlO polypeptide fragment may be amino acids 1-138, 285-363, 210-363, 210- 444, or 285-444 of SEQ ID NO: 68.
[00239] As one of skill in the art will appreciate, the beginning and ending residues of the antigenic or immunogenic polypeptide fragment may vary depending upon the computer modeling program used or the method used for determining the domains.
[00240] Isolated antigenic and immunogenic polypeptides of the present invention in addition to those encoded by polynucleotides of the invention, may be provided as a recombinant protein, a purified subunit, a viral vector expressing the protein, or may be provided in the form of an inactivated GBS vaccine, e.g., a live-attenuated virus vaccine, a heat-killed virus vaccine, etc.
[00241] Production of the polypeptides of the present invention can be achieved by culturing the host cells, expressing the polynucleotides of the present invention, and recovering the polypeptides. Determining conditions for culturing the host cells and expressing the polynucleotide are generally specific to the host cell and the expression system and are within the knowledge • of one of skill in the art. Likewise, appropriate methods for recovering the polypeptide of interest are known to those in the art, and include, but are not limited to. chromatography, filtration, precipitation, or centrifugation.
[00242] In addition, the polypeptides of the present invention are suitable for using as a vaccine composition as the polypeptides have low levels of sequence identity with human proteins. The compositions comprising a combination of the polypeptides can elicit a strong immune response without producing an immune response against natural human polypeptides, thereby improving the safety profile of the combinations. Table 3 shows the polypeptides of the invention and the levels of homology between the polypeptides and other polypeptides found in humans.
TABLE 3
Blast search of the human sequences in the "nr" database using Pho 1-13 protein sequence
Accession Description of amino Length of E-value Number of Identity Similarity Number acid sequence aa subjects in between between query sequence of query that query and and subject (%) subject matched query subject (%) endopeptidase CIp
NP_006003 277 aa 8e-50 90/190 47 72 precursor
(In total = 1 BLAST Hit)
Blast search of the human sequences in the "nr" database using Pho 1-14 protein sequence
Accession Description of amino Length of E-value Number of Identity Similarity Number acid sequence aa subjects in between between query
Figure imgf000044_0001
Blast search of the human sequences in the "nr" database using Pho 3-3 (Pho 1-7) protein sequence
Figure imgf000044_0002
Figure imgf000044_0003
BAB84904
FUOO 149 protein 792 aa 0 .64 26/100 26 43 unnamed protein
BAD18683 959 aa 0 .68 21/97 21 36 product
The e-values of other BLAST Hits are greater than 1.0 and are not considered to be significant, therefore they were not included in the table (In total = 50 BLAST Hits).
Figure imgf000045_0001
Blast search of the human sequences in the "nr" database using Pho 2-15 protein sequence
Accession Description of Length of E-value Number of Identity similarity
Number amino acid aa subjects in between between query sequence sequence of query that query and and subject (%) subject matched query subject (%)
No significant similarity found.
Blast search of the human sequences in the "nr" database using phO3-21 protein sequence
Accession Description of Length of E-value Number of Identity Similarity
Number amino acid aa subjects in between between query sequence sequence of query that query and and subject (%) subject matched query subject (%)
No significant similarity found.
Blast search of the human se uences in the "nr" database usin Pho 3-50 rotein se uence
Figure imgf000045_0002
Blast search of the human se uences in the "nr"* database usin Pho 3-23 rotein se uence
Figure imgf000045_0003
Figure imgf000046_0001
Blast search of the human se uences in the "nr"* database usin Pho 3-29 rotein se uence
Figure imgf000046_0002
Blast search of the human se uences in the "nr"* database usin Pho 3-24 rotein se uence
Figure imgf000046_0003
Figure imgf000047_0001
Figure imgf000047_0002
Blast search of the human sequences in the "nr"* database using Pho3-22 protein sequence
Figure imgf000048_0001
Figure imgf000049_0001
Blast search of the human sequences in the "nr" database using Pho 3-18 protein sequence
Figure imgf000049_0002
Figure imgf000050_0001
Blast search of the human sequences in the "nr" database using Pho 3-17 (Pho 3-9) protein sequence
Figure imgf000050_0002
Blast search of the human sequences in the "nr" database using 03AA42 (ME-P31) protein sequence
Figure imgf000050_0003
Blast search of the human sequences in the "nr"* database using phO2-2 (ME-P22) protein sequence:
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000053_0002
Blast search of the human sequences in the "nr" database usin MSIl rotein se uence
Figure imgf000053_0003
Blast search of the human se uences in the "nr" database usin MS16 rotein se uence
Figure imgf000053_0004
Figure imgf000054_0001
Blast search of the human sequences in the "nr" database usin MS14 rotein sequence
Figure imgf000054_0002
* purine nucleoside phosphorylase
Blast search of the human sequences in the "nr" database using MSlO (03AC12) protein sequence
Figure imgf000054_0003
Figure imgf000055_0001
Variants
[00243] The sequences of GBS polypeptides are known from different serotypes, different strains, and different species. The sequence identity between the GBS polypeptides and the corresponding polypeptide sequences from- different serotypes or strains or different species are publicly available as shown in Table 4.
TABLE 4
Figure imgf000055_0002
Figure imgf000056_0001
Gene identifiers in sequenced GBS strains
[00244] Table 5 shows the polypeptide sequence identities between the GBS A909 and different species, serotypes, and strains.
TABLE 5: GBS CANDIDATES. HOMOLOGY WITH OTHER BACTERIAL PROTEINS.
Figure imgf000056_0002
Figure imgf000056_0003
Conservation phol-5
Figure imgf000056_0004
Figure imgf000057_0001
Figure imgf000057_0002
Figure imgf000057_0003
Figure imgf000057_0004
Figure imgf000058_0001
Figure imgf000058_0002
Figure imgf000058_0003
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000059_0003
Figure imgf000059_0004
Figure imgf000060_0001
Figure imgf000060_0002
Figure imgf000060_0003
Figure imgf000060_0004
Figure imgf000061_0002
Figure imgf000061_0003
Figure imgf000061_0001
Figure imgf000061_0005
Figure imgf000062_0001
MS16
NB. at contains 2 artial se uences N and C terminus
Figure imgf000062_0002
[00245] Serotypic variation of GBS polypeptides is lower than the variation among species.
Serotypic variants are therefore expected to be immunologically cross-reactive. The present invention thus includes GBS polypeptides found in other Streptococcus spp, e.g., Streptococcus agalactiae serotypes such as, but not limited to, Ia, Ib, Ia/c, II, III, IV, V, VI, VII, and VIII, or Streptococcus agalactiae, Streptococcus bovis, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus suis, or Streptococcus thermophilus or fragments thereof. These proteins can be obtained using the methods described above or available to a person of ordinary skill in the art. Therefore, the present invention is directed to an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1- 59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101-181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5; (c)a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ BD NO: 8; (iii) amino acids 139-192 of SEQ ID NO: 8; and (iv) amino acids 43-192 of SEQ ID NO: 8; (d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (ii) amino acids 1-88 of SEQ ID NO: 11; (iii) amino acids 1- 110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11; (e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 14; (ii) amino acids 1-59 of SEQ ID NO: 14; (iii) amino acids 84-149 of SEQ ID NO: 14; and (iv) amino acids 1-149 of SEQ ID NO: 14; and (v) SEQ ID NO: 15; (f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 18; (ii) amino acids 29-95 of SEQ ID NO: 18; (iii) amino acids 1-95 of SEQ ID NO: 18; and (iv) ammo acids 29-125 of SEQ ID NO: 18; (g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 21 ; (ii) amino acids 1-77 of SEQ TD NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; (h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 24; (ii) amino acids 43-84 of SEQ ID NO: 24; (iii) amino acids 1-84 of SEQ ID NO: 24; (iv) amino acids 88-122 of SEQ ID NO: 24; and (v) amino acids 38-122 of SEQ ID NO: 24; (i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 27; (ii) amino acids 29-79 of SEQ ID NO: 27; (iii) amino acids 101-189 of SEQ ID NO: 27; and (iv) amino acids 29-189 of SEQ ID NO: 27; 0) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 30; (ii) amino acids 79-182 of SEQ ID NO: 30; (iii) amino acids 79-217 of SEQ ID NO: 30; (iv) amino acids 23-217 of SEQ ID NO: 30; and (v) amino acids 23-278 of SEQ ID NO: 30; (k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ED NO: 33; (1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 36; (ii) amino acids 25-88 of SEQ ID NO: 36; (iii) amino acids 132-202 of SEQ ID NO: 36; (iv) amino acids 25-172 of SEQ ID NO: 36; and (v) amino acids 25-202 of SEQ ID NO: 36; (m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 39 and (ii) amino acids 27-94 of SEQ ID NO: 39; (n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 42; (ii) amino acids 111-180 of SEQ E) NO: 42; (iii) amino acids 111-234 of SEQ ID NO: 42; (iv) amino acids 72-234 of SEQ ID NO: 42; and (v) amino acids 40-335 of SEQ ID NO: 42; (o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 45; (ii) amino acids 98-180 of SEQ ID NO: 45; (iii) amino acids 259-370 of SEQ E) NO: 45; and (iv) amino acids 98-370 of SEQ E) NO: 45; (p) a Pho3-29 polypeptide, which is SEQ E) NO: 48; (q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 51; (ii) amino acids 78-154 of SEQ E) NO: 51; (iii) amino acids 78-217 of SEQ E) NO: 51; and (iv) amino acids 1-154 of SEQ E) NO: 51; (r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ E) NO: 54; (ii) amino acids 190-261 of SEQ E) NO: 54; (iii) amino acids 190-321 of SEQ E) NO: 54; and (iv) amino acids 1- 118 of SEQ E) NO: 54; (s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ E) NO: 57; (ii) amino acids 1-139 of SEQ E) NO: 57; (iii) amino acids 184-311 of SEQ E) NO: 57; and (iv) amino acids 184-398 of SEQ E) NO: 57; (t) an MS16 polypeptide, which is selected from the group consisting of: (i) SEQ E) NO: 60, (ii) amino acids 24-77 of SEQ E) NO: 60; (iii) amino acids 101-172of SEQ E) NO: 60; (iv) amino acids 24-172 of SEQ E) NO: 60; and (v) SEQ E) NO: 62; (u) an MS 14 polypeptide, which is selected from the group consisting of: (i) SEQ E) NO: 65; (ii) amino acids 1-95 of SEQ E) NO: 65; (iii) amino acids 141-181 of SEQ E) NO: 65; and (iv) amino acids 141-268 of SEQ E) NO: 65; or (v) an MSl 0 polypeptide, which is selected from the group consisting of: (i) SEQ E) NO: 68, (ii) amino acids 1-138 of SEQ E) NO: 68; (iii) amino acids 285-363 of SEQ E) NO: 68; (iv) amino acids 210-363 of SEQ E) NO: 68; (v) amino acids 210-444 of SEQ E) NO: 68; and (vi) amino acids 285-444 of SEQ E) NO: 68, wherein the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to the reference amino acid sequence. In one aspect, the first polypeptide and the second polypeptide induces an immune response against GBS. In further aspects, the immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in the animal. In certain embodiments, the first polypeptide and the second polypeptide further comprises an isolated third polypeptide. The third polypeptide may be an antigenic or immunogenic polypeptide, e.g., a polypeptide derived from a bacterium, a virus, a fungus, or a protozoan. For example, the antigenic or immunogenic polypeptide may be derived from Group B Streptococcus. In further aspects, the third polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ E) NO: 2; (ii) amino acids 1-59 of SEQ E) NO: 2; (iii) amino acids 132-181 of SEQ E) NO: 2; (iv) amino acids 101-181 of SEQ E) NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5;(c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ID NO: 8; (iii) amino acids 139-192 of SEQ DD NO: 8; and (iv) amino acids 43-192 of SEQ ID NO: 8; (d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (ii) amino acids 1-88 of SEQ ED NO: 11; (iii) amino acids 1-110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11; (e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 14; (ii) amino acids 1-59 of SEQ DD NO: 14; (iii) amino acids 84-149 of SEQ DD NO: 14; and (iv) amino acids 1-149 of SEQ DD NO: 14; and (v) SEQ DD NO: 15; (f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 18; (ii) amino acids 29-95 of SEQ DD NO: 18; (iii) amino acids 1-95 of SEQ ID NO: 18; and (iv) amino acids 29-125 of SEQ DD NO: 18; (g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ DD NO:21; (h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 24; (ii) amino acids 43-84 of SEQ DD NO: 24; (iii) amino acids 1- 84 of SEQ JD NO: 24; (iv) amino acids 88-122 of SEQ JD NO: 24; and (v) amino acids 38-122 of SEQ DD NO: 24: (i) a Pho3-17 polypeptide, which is' selected from the group consisting of: (i) SEQ DD NO: 27; (ii) amino acids 29-79 of SEQ DD NO: 27; (iii) amino acids 101-189 of SEQ DD NO: 27; and (iv) amino acids 29-189 of SEQ JD NO: 27; (j) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 30; (ii) amino acids 79-182 of SEQ DD NO: 30; (iii) amino acids 79-217 of SEQ DD NO: 30; (iv) amino acids 23-217 of SEQ DD NO: 30; and (v) amino acids 23-278 of SEQ DD NO: 30; (k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; (1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 36; (ii) amino acids 25-88 of SEQ D) NO: 36; (iii) amino acids 132-202 of SEQ JD NO: 36; (iv) amino acids 25-172 of SEQ JD NO: 36; and (v) amino acids 25-202 of SEQ DD NO: 36; (m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 39 and (ii) amino acids 27-94 of SEQ DD NO: 39; (n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 42; (ii) amino acids 111-180 of SEQ DD NO: 42; (iii) amino acids 111-234 of SEQ JD NO: 42; (iv) amino acids 72-234 of SEQ DD NO: 42; and (v) amino acids 40-335 of SEQ DD NO: 42; (o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 45; (ii) amino acids 98-180 of SEQ DD NO: 45; (iii) amino acids 259-370 of SEQ DD NO: 45; and (iv) amino acids 98-370 of SEQ DD NO: 45; (p) a Pho3-29 polypeptide, which is SEQ DD NO: 48; (q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 51; (ii) amino acids 78-154 of SEQ DD NO: 51; (iii) amino acids 78-217 of SEQ ID NO: 51; and (iv) amino acids 1-154 of SEQ ID NO: 51; (r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 54; (ii) amino acids 190-261 of SEQ DD NO: 54; (iii) amino acids 190-321 of SEQ DD NO: 54; and (iv) amino acids 1-118 of SEQ DD NO: 54; (s) an MSI l polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 57; (ii) amino acids 1-139 of SEQ DD NO: 57; (iii) amino acids 184-311 of SEQ DD NO: 57; and (iv) amino acids 184- 398 of SEQ DD NO: 57; (t) an MS 16 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 60, (ii) amino acids 24-77 of SEQ DD NO: 60; (iii) amino acids 101-172of SEQ DD NO: 60; (iv) amino acids 24-172 of SEQ DD NO: 60; and (v) SEQ DD NO: 62; (u) an MS14 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 65; (ii) amino acids 1-95 of SEQ DD NO: 65; (iii) amino acids 141-181 of SEQ DD NO: 65; and (iv) amino acids 141- 268 of SEQ DD NO: 65; or (v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444 of SEQ DD NO: 68; and (vi) amino acids 285-444 of SEQ DD NO: 68, wherein the third polypeptide is different from, or not identical to, the first and second polypeptides and specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to the reference amino acid sequence.
[00248] In accordance with one embodiment of the present invention, the first polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ DD NO: 21; (iv) amino acids 272-360 of SEQ DD NO: 21; and (v) amino acids 106-360 of SEQ DD NO: 21.
[00249] Alternatively, the first polypeptide comprises an amino acid sequence at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33; and the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210- 444 of SEQ DD NO: 68; and (vi) amino acids 285-444 of SEQ DD NO: 68. [00250] In certain embodiments, the first polypeptide comprises an amino acid sequence at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ED NO:21; and the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444 of SEQ DD NO: 68; and (vi) amino acids 285-444 of SEQ DD NO: 68.
[00251] Furthermore, the present invention includes a combination of an isolated first polypeptide, an isolated second polypeptide, and an isolated third polypeptide, wherein the first polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33, the second polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ DD NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21: (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ ID NO:21, and the third polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ DD NO: 68, (ii) amino acids 1-138 of SEQ DD NO: 68; (iii) amino acids 285-363 of SEQ DD NO: 68; (iv) amino acids 210-363 of SEQ DD NO: 68; (v) amino acids 210-444 of SEQ DD NO: 68; and (vi) amino acids 285-444 of SEQ DD NO: 68.
[00252] Surprisingly, an antibody raised against the first and second polypeptides of the present invention may show cross reactivity against the corresponding polypeptides found in multiple GBS strains or serotypes. The first and second polypeptide used in the present invention may further show cross protection against different GBS strains or serotypes. For example, a first polypeptide comprising SEQ DD NO: 33 and a second polypeptide comprising SEQ DD NO: 68 may show protection against GBS strain M732 as well as GBS strain GBS strain A909 (serotype Ia/c).
[00253] The present invention also includes a combination of two or more polypeptides wherein the first polypeptides and second polypeptides are from different GBS serotypes. For example, the present invention may include an isolated first polypeptide comprising SEQ DD NO: 33 (Pho3- 1) found in GBS M732 (serotype III) and a second polypeptide comprising SEQ ID NO: 69 (MS 10 corresponding polypeptide) found in GBS A909 (serotype Ia). The first polypeptide and second polypeptide may therefore show protection against both GBS A909 and M732, when administered to a host in need thereof. The first and second polypeptide may also show protection against GBS serotypes Ib, Ib/c, II, IV, V, VI, VII, and/or VIII. Likewise, other combinations of the GBS polypeptides from different serotypes are also possible.
[00254] In some embodiments, the present invention is directed to at least two different GBS polypeptides, wherein the polypeptides further comprising a heterologous polypeptide. The heterologous polypeptide can, in some embodiments, be fused to the GBS polypeptide. The heterologous polypeptide can be translated from various heterologous nucleic acids. Various heterologous polypeptides can be used, and can be selected from the group consisting of an N- or C-terminal peptide imparting stabilization, secretion, or simplified purification, i.e., His-tag, ubiquitin tag, NusA tag, chitin binding domain, ompT, ompA, pelB, DsbA, DsbC, c-myc, KSI, polyaspartic acid, (Ala-Trp-Trp-Pro)n, polyphenylalanine, polycysteine, polyarginine, B-tag, HSB-tag, green fluorescent protein (GFP), hemagglutinin influenza virus (HAI), calmodulin binding protein (CBP), galactose-binding protein, maltose binding protein (MBP), cellulose binding domains (CBD's), dihydrofolate reductase (DHFR), glutathione-S-transferase (GST), streptococcal protein G, staphylococcal protein A, T7genelO, avidin/streptavidin/Strep-tag, trpE, chloramphenicol acetyltransferase, lacZ (β-Galactosidase), His-patch thioredoxin, thioredoxin, FLAG™ peptide (Sigma-Aldrich), S-tag, and T7-tag. See e.g., Stevens, R.C., Structure, 8:R177- Rl 85 (2000). The heterologous polypeptides can further include any pre- and/or pro- sequences that facilitate the transport, translocations, processing and/or purification of the GBS polypeptides from a host cell or any useful immunogenic sequence, including but not limited to sequences that encode a T-cell epitope of a microbial pathogen, or other immunogenic proteins and/or epitopes.
[00255] Other suitable heterologous polypeptides may include other GBS proteins (either native proteins or variants, fragments, or derivatives thereof.) In one embodiment, the heterologous polypeptide may comprise one or more polypeptides from different GBS serotypes or strains. Accordingly, the at least two GBS polypeptides can induce an immune response against various serotypes of GBS. In another embodiment, the heterologous polypeptide may be from other organisms. In other embodiments, the polypeptide of the present invention can exist as a homopolymer, comprising multiple copies of the same polypeptide.
[00256] Optionally, these polypeptides fused with a heterologous polypeptide can include a peptide linker sequence joining sequences that comprise two or more epitopes. Suitable peptide linker sequences may be chosen based on their ability to adopt a flexible, extended conformation, or a secondary structure that could interact with joined epitopes, or based on their ability to increase overall solubility of the fusion polypeptide, or based on their lack of electrostatic or water-interaction effects that influence joined epitopes. [00257] Peptide and polypeptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows: A (alanine); R (arginine); N (asparagine); D (aspartic acid); C (cysteine); Q (glutamine); E (glutamic acid); G (glycine); H (histidine); I (isoleucine); L (leucine); K (lysine); M (methionine); F (phenylalanine); P (proline); S (serine); T (threonine); W (tryptophan); Y (tyrosine); and V (valine).
Polynucleotides
[00258] The present invention also include an isolated first polynucleotide and an isolated second polynucleotide, wherein the first polynucleotide and the second polynucleotide encode the GBS polypeptides, fragments, derivatives, analogs, or variants thereof described herein.
[00259] Polynucleotides or nucleic acid sequences defined herein are represented by one-letter symbols for the bases as follows: A (adenine) C (cytosine) G (guanine) T (thymine) U (uracil) M (A or C) R (A or G) W (A or T/U); S(C or G); Y (C or T/U); K (G or T/U); V (A or C or G; not T/U) ; H (A or C or T/U; not G); D (A or G or T/U; not C); B (C or G or T/U; not A); N (A or C or G or T/U) or (unknown).
[00260] In one embodiment, an isolated first polynucleotide used in the present invention encodes a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polynucleotide used in the present invention encodes a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ED NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ED NO: 21; (iv) amino acids 272-360 of SEQ ED NO: 21; and (v) amino acids 106-360 of SEQ ED NO: 21, wherein the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting essentially of an amino acid sequence identical to the reference amino acid sequence.
[00261] In another embodiment, an isolated first polynucleotide encodes a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ED NO: 33;'(ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ED NO: 33; and (iv) amino acids 24-192 of SEQ ED NO: 33; and an isolated second polynucleotide encodes a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68, wherein the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting essentially of an amino acid sequence identical to the reference amino acid sequence..
[00262] In other embodiments, an isolated first polynucleotide encodes a first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated second polynucleotide encodes a second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68, wherein the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting essentially of an amino acid sequence identical to the reference amino acid sequence.
[00263] In some embodiments, the present invention is directed to a polynucleotide further comprising a heterologous nucleic acid. The heterologous nucleic acid can, in some embodiments, encode a heterologous polypeptide fused to the polypeptide. The heterologous nucleic acid may comprise a promoter operably linked to the first, second, or third polynucleotide. In other aspects, the heterologous nucleic acid may encode a third polypeptide. The third polypeptide may be an antigenic or immunogenic polypeptide. For example, an antigenic or immunogenic polypeptide may be derived from a bacterium, a virus, a fungus, or a protozoan. Therefore, a heterologous nucleic acid may encode a polypeptide derived from Group B Streptococcus. For example, the polypeptide derived from GBS may be any GBS polypeptides, fragments, derivatives, or variants described herein.
Codon Optimization
[00264] Also included within the scope of the invention are genetically engineered polynucleotides encoding the GBS polypeptide variants. Modifications of nucleic acids encoding the GBS polypeptides can readily be accomplished by those skilled in the art, for example, by oligonucleotide-directed site-specific mutagenesis of a polynucleotide coding for a GBS polypeptide. Such modified polypeptide can be encoded by a codon optimized nucleotide sequence. Such modifications impart one or more amino acid substitutions, insertions, deletions, and/or modifications to expressed GBS polypeptides including fragments, variants, and derivatives. Such modifications may enhance the immunogenicity of the GBS antigens, for example, by increasing cellular immune responses compared with unmodified polypeptides. Such modification may enhance solubility of the polypeptides. Alternatively, such modifications may have no effect. For example, the polynucleotide encoding the GBS polypeptide may be modified by introduction, deletion or modification of particular cleavage sites for proteolytic enzymes active in antigen presenting cells, to enhance immune responses to particular epitopes. As appreciated by one of ordinary skill in the art, various nucleic acid coding regions will encode the same polypeptide due to the redundancy of the genetic code. Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code" which shows which codons encode which amino acids is reproduced herein as Table 6. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for, by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the polypeptides encoded by the DNA.
TABLE 6: The Standard Genetic Code
Figure imgf000072_0001
[00266] It is to be appreciated that any polynucleotide that encodes a polypeptide in accordance with the invention falls within the scope of this invention, irregardless of the codons used.
[00267] Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
[00268] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www.kazusa.or.jp/codon/ (visited August 6, 2007), and these tables can be adapted in a number of ways. See Nakamura, Y., et al, "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). [00269] By utilizing the codon usage tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment comprising a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species. For example, in some embodiments of the present invention, the coding region is codon-optimized for expression in E.coli.
[00270] Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and the "backtranslate" function in the GCG~Wisconsin Package, available from Accelrys, Inc., San Diego, CA. Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill.
[00271] Codon placement in a polynucleotide at an optimized frequency to encode a given polypeptide sequence can also be done in a directed manner. For example, a codon may be assigned to a particular amino acid so as to create or destroy a restriction enzyme cleavage site. Creation or destruction of restriction enzyme sites may facilitate DNA manipulation by assisting with cloning or forming identifying markers. Alternatively, a codon may be assigned, to a particular amino acid so as to achieve a desired secondary structure of the polynucleotide.
[00272] In certain embodiments, an entire coding region encoding a polypeptide sequence, or fragment, variant, or derivative thereof can be codon optimized by any of the methods described herein or by other methods. Various desired fragments, variants or derivatives may be designed, and each can then be codon-optimized individually. In addition, partially codon-optimized coding regions of the present invention can be designed and constructed. For example, the invention includes a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide in which at least about 1%, 2%, 3,% 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codon positions have been codon-optimized for a given species. The partially codon optimized coding regions contain a codon that is preferentially used in the genes of a desired species, e.g., E.coli, in place of a codon that is normally used in the native nucleic acid sequence.
Vectors and Expression Systems
[00273] The present invention further provides a vector or more than one vectors comprising one or more polynucleotides encoding the GBS polypeptides, fragments, derivatives, or variants thereof. [00274] In accordance with one aspect of the present invention, provided is a vector, which comprises at least two nucleic acid sequences encoding the GBS polypeptides or fragments, variants, derivatives, or analogs thereof described herein. The at least two nucleic acid sequence may comprises an isolated first nucleic acid sequence and an isolated second nucleic acid sequence, which are located on one expression vector.
[00275] In other embodiments, the present invention is further directed to at least two vectors, each of which comprises a nucleic acid fragment encoding a GBS polypeptide. The at least two vectors can be identical or different or a combination thereof. The nucleic acid fragment may be a fragment of a codon-optimized coding region operably encoding any GBS polypeptides described herein.
[00276] Additional GBS-derived coding or non-coding regions may also be included on the vector, e.g., a plasmid, or on a separate vector, and expressed, either using native GBS codons or codons optimized for expression in the host in which the polypeptide is being expressed. When such a vector is delivered to a host, e.g., to a bacterial, plant or eukaryotic cell, or alternatively, in vivo to a tissue of the animal to be treated or immunized, the transcriptional unit will thus express the encoded gene product. The level of expression of the gene product will depend to a significant extent on the strength of the associated promoter and the presence and activation of an associated enhancer element, as well as the optimization of the coding region.
[00277] The term "vector," as used herein, refers to any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors may be DNA or RNA. Vectors include, but are not limited to, plasmids, phage, phagemids, bacterial genomes, and virus genomes and virus-like particles. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
[00278] Any of a wide variety of suitable cloning vectors are known in the art and commercially available which can be used with appropriate hosts. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, cosmid, etc. As used herein, the term "plasmid" refers to a circular, double-stranded construct made up of genetic material (i.e., nucleic acids), wherein the genetic material is extrachromosomal and replicates autonomously. A polynucleotide of the present invention may be in a circular or linearized plasmid or vector, or other linear DNA which may also be non-infectious and nonintegrating (i.e., does not integrate into the genome of host cells). Procedures for inserting a nucleotide sequence into an expression vector, and transforming or transfecting into an appropriate host cell and cultivating under conditions suitable for expression are generally known in the art, as described generally in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989).
[00279] A variety of host-expression vector systems may be utilized to express the polypeptides of the present invention. The term "expression vector" refers to a vector that is capable of expressing the polypeptide of the present invention, i.e., the vector sequence contains the regulatory sequences required for polypeptide expression such as promoters, ribosome binding sites, etc. The term "expression" refers to the biological production of a product encoded by a coding sequence. In most cases a DNA sequence, including the coding sequence, is transcribed to form a messenger-RNA (mRNA). The messenger-RNA is then translated to form a polypeptide product which has a relevant biological activity. Also, the process of expression may involve further processing steps to the RNA product of transcription, such as splicing to remove introns, and/or post-translational processing of a polypeptide product.
[00280] Vector-host systems include, but are not limited to, systems such as bacterial, mammalian, yeast, insect or plant cell systems. either in vivo, e.g., in an animal or in vitro, e.g., in mammalian cell cultures. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
[00281] Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention as described above. Thus, one aspect of the invention is directed to a host cell comprising a vector which contains a polynucleotide of the present invention. The engineered host cell can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The term "transfect," as used herein, refers to any procedure whereby eukaryotic cells are induced to accept and incorporate into their genome isolated DNA, including but not limited to DNA in the form of a plasmid. The term "transform," as used herein, refers to any procedure whereby bacterial cells are induced to accept and incorporate into their genome isolated DNA, including but not limited to DNA in the form of a plasmid.
[00282] Bacterial host-expression vector systems include, but are not limited to, a prokaryote
(e.g., E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, e.g., P. aeruginosa or P. fluorescens (e.g. PFENEX ™ (Dowpharma)), Streptomyces sp., or Staphylococcus sp.) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing polypeptide coding regions of the present invention. In some embodiments, the PFENEX™ system is used. The PFENEX™ expression system utilizes P. fluorescens biovar I, designated MBlOl, and compatible plasmids. In some embodiments, the plasmids used with P. fluorescens use the tac promoter system regulated by the Lad protein via IPTG induction. Li some embodiments, the bacterial host can have an auxotrophic chromosomal deletion, e.g., pyrF, in which the deletion is complemented by the vector, to alleviate the need for antibiotic-resistance genes. A large number of suitable vectors are known to those of skill in the art, and are commercially available. The following bacterial vectors are provided by way of example: pET, pET28, pBAD, pTrcHIS, pBR322, pQE70, pQE60, pQE-9 (Qiagen), phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pBR322, pPSIO, RSFlOlO, pRIT5 (Pharmacia); pCR (Invitrogen); pLex (Invitrogen), and pUC plasmid derivatives. Another example of a suitable vector for the present invention is Vaccinia vectors, e.g., Modified Vaccinia Ankara (MVA) Vector. However, any other plasmid or vector can be used as long as it is replicable and viable in the host. In some embodiments, the expression vector comprises the plasmid pLex. The pLex plasmid comprises a multiple cloning site that is tightly regulated by a tryptophan-inducible expression system utilizing the strong PL promoter from bacteriophage lambda, and the cl repressor protein. This pLex expression vector is especially useful for the expression of potentially toxic proteins in E. coli. In addition, the lambda promoter provides high-level expression of recombinant proteins. Alternately, a GBS polypeptide antigen can be recombinantly expressed directly in a carrier organism, or virus, all or part of which is subsequently incorporated in a vaccine composition, as described in Eko, et al, Immunol., 173:3375-3382, 2004, which is expressly incorporated by reference herein. Alternatively, GBS polypeptide vaccine antigens can be purified from infected-host cell culture.
[00283] A suitable expression vector contains regulatory sequences which can be operably joined to an inserted nucleotide sequence encoding a GBS polypeptide vaccine antigen. As used herein, the term "regulatory sequences" means nucleotide sequences which are necessary for or conducive to the transcription of an inserted sequence coding a GBS vaccine antigen by a host cell and/or which are necessary for or conducive to the translation by a host cell of the resulting transcript into the desired GBS polypeptide. Regulatory sequences include, but are not limited to, 51 sequences such as operators, promoters and ribosome binding sequences, and 3' sequences such as polyadenylation signals. Regulatory sequences may also include enhancer sequences or upstream activator sequences.
[00284] Generally, bacterial vectors will include origins of replication and selectable markers, e.g., the ampicillin, tetracycline, kanamycin, resistance genes of E. coli, permitting transformation of the host cell and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters include, but are not limited to, the T7 promoter, lambda (λ) promoter, T5 promoter, and lac promoter, or promoters derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others.
[00285] Once an expression vector is selected, the polynucleotide of the invention is cloned downstream of the promoter, often in a polylinker region. This plasmid is transformed into an appropriate bacterial strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the polynucleotide as well as all other elements included in the vector, are confirmed using restriction mapping, DNA sequence analysis, and/or PCR analysis. Bacterial cells harboring the correct plasmid can be stored as cell banks.
[00286] Examples of mammalian host-expression systems include cell lines capable of expressing a compatible vector, for example, the COS, C127, 3T3, CHO, HeLa and BHK cell lines. Examples of suitable expression vectors include pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia), p75.6 (Valentis), pCEP (Invitrogen), pCEI (Epimmune), pZERO, PTrc99A, pUC19, pUC18, pKK223-3, pEXl, pCAL, pET, pSPUTK, pTrxFus, pFastBac, pThioHis, pTrcHis, and pLex, pET-17b, pET-1 Ia, pET-24a-d(+) and pET-9a pK233 (or any of the tac family of plasmids), pT7, lambda pSKF, and pET-28(a)+, vectors useful in yeast cells', including YIp, YRp1 YCP, YEp and YLp plasmids as well as viral genomes from which to construct viral vectors such as Simian virus 40 (SV40), bovine papilloma virus, pox virus such as vaccinia virus, e.g., W MVA, and parvovirus, including adeno-associated virus, retrovirus, herpesvirus, adenovirus, retroviral, e.g., murine leukemia virus and lentiviruses (e.g., human immunodeficiency virus), alphavirus, and picornavirus. References citing methods for the in vivo introduction of non-infectious virus genomes to animal tissues are well known to those of ordinary skill in the art. Any of a variety of methods known in the art can be used to insert a nucleotide sequence coding for a GBS polypeptide vaccine antigen into a suitable expression vector.
[00287] Generally, mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. Such promoters may also be derived from viral sources, such as, e.g., human cytomegalovirus (CMV-EE promoter), herpes simplex virus type-1 (HSV TK promoter), the adenovirus late promoter; and the vaccinia virus 7.5K promoter, or can be derived from the genome of mammalian cells (e.g., metallothionein promoter). Nucleic acid sequences derived from the SV40 splice and polyadenylation sites can be used to provide the required nontranscribed genetic elements. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in animal cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from animal genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, elements from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
[00288] Yeast host-expression systems include a yeast host (e.g., Saccharomyces, Pichia,
Hansenula, Kluyyeromyces, Schizosaccharomyces, Schwanniomyces and Yarrowia) transformed with recombinant yeast expression vectors containing polypeptide coding sequences, employing suitable vectors and control sequences. Suitable yeast expression vectors are known to those in the art and include, but are not limited to, e.g., pAL19, paR3, pBGl, pDBlet, pDB248X, pEA500, pFL20, pIRT2, pJK148, pON163, pSPl, pSP3, pUR19, pARTl, pCHY21 , REP41, pYZIN, pSLF104, pSLF172, pDS472, pSGP572, pSLF1072, REP41MH-N, pFA6a-kanMX6, pARTCM, and pALL.
[00289] Also included are insect host system and plant cell systems.
[00290] One of skill in the art will recognize that some of the above listed vectors are capable of replicating and expressing polypeptides in more than one type of host, e.g., the pOG44 plasmid can replicate and express polypeptides in both prokaryotic and eukaryotic cells.
Antibody or Antigen binding fragment thereof
[00291] The present invention is also directed to a combination of an isolated first antibody or antigen binding fragment thereof and an isolated second antibody or antigen binding fragment thereof, wherein both first and second antibodies and antigen binding fragments thereof are raised against the GBS polypeptides described herein.
[00292] For the purposes of this invention, antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, multivalent antibodies, multispecifϊc antibodies, single chain antibodies, and fragments produced by a Fab expression library or epitope-binding fragments, e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), and disulfide-linked Fv fragments (sdFv). Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. The antibodies and fragments thereof may be humanized antibodies, for example as described in EP-A-0239400, incorporated herein by reference in its entirety. Furthermore, antibodies with fully human variable regions (or their fragments), for example, as described in US Patents Nos. 5,545,807 and 6,075,181 (both incorporated herein by reference in their entireties) may also be used. Neutralizing antibodies, i.e., those which inhibit biological activity of the substance amino acid sequences, are especially preferred for diagnostics and therapeutics. Antibodies may be produced by standard techniques, such as by immunization or by using a phage display library. Immunoglobulin or antibody molecules of the invention can be of any type {e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class or subclass {e.g., IgGl, IgG2a, IgG2b, IgG2c, and IgG3) of immunoglobulin molecule. In a particular embodiment, immunoglobulin or antibody molecules of the invention are IgG2a antibodies.
[00293] For example, if polyclonal antibodies are desired, a selected mammal {e.g., mouse, rabbit, goat, horse, etc.) may be immunized with an immunogenic composition comprising the combination of polypeptides or peptides of the present invention. Depending on the host species, various adjuvants, described elsewhere herein, may be used to increase immunological response.
[00294] Monoclonal antibodies directed against epitopes obtainable from a peptide used in the present invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known.
[00295] In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452- 454). Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,779) can be adapted to produce the substance specific single chain antibodies.
[00296] Antibodies, both monoclonal and polyclonal, which are directed against epitopes obtainable from the polypeptides or peptides of the present invention, are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired.
Compositions
[00297] The present invention includes a pharmaceutical, vaccine, or immunological composition comprising an isolated first polypeptide and an isolated second polypeptide, wherein the first polypeptide and second polypeptide are selected from the group consisting of the GBS polypeptides described herein.
[00298] Also included is a pharmaceutical, vaccine, or immunological composition, which comprises an isolated first polynucleotide and an isolated second polynucleotide, wherein the first polynucleotide and the second polynucleotide are selected from the group consisting of the polynucleotides described herein.
[00299] Moreover, the present invention includes an isolated first antibody or antigen binding fragment thereof and an isolated second antibody or antigen binding fragment thereof, wherein the first antibody or antigen binding fragment thereof and the second antibody or antigen binding fragment thereof are selected from the group consisting of the antibodies or antigen binding fragments thereof raised against the GBS polypeptides described herein.
[00300] In one aspect, the composition of the present invention includes an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical, to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
[00301] In another aspect, the composition comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ED NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210- 444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
[00302] In other embodiments, the first polypeptide and the second polypeptide are not identical and each of the first polypeptide and second polypeptide specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to the reference amino acid sequence. [00303] The pharmaceutical, immunological, or vaccine composition of the present invention comprises an isolated first polynucleotide encoding the first polypeptide and an isolated second polynucleotide encoding the second polypeptide. Furthermore, the composition comprises an isolated first antibody or antigen-binding fragment thereof that specifically binds the first polypeptide described above and an isolated second antibody or antigen-binding fragment thereof that specifically binds the second polypeptide described above.
[00304] The present invention also includes at least two compositions, wherein a first composition comprises an isolated first polypeptide, polynucleotide, or antibody or antigen binding fragment thereof described herein and a second composition comprises an isolated second polypeptide, polynucleotide, or antibody or antigen binding fragment thereof described herein. Also included is a method of inducing an immune response comprising administering the at least two compositions in an animal in need thereof.
[00305] In one embodiment, the composition comprising the first and second polypeptides further comprises an isolated third polypeptide; the composition comprising the first and second polynucleotides comprises an isolated third polynucleotide; and the composition comprising the first and second antibodies or antigen binding fragments thereof further comprises an isolated third antibody or antigen binding fragment thereof that specifically binds the third polypeptide. The third polynucleotide may encode a third polypeptide. In one embodiment, the. third polypeptide may be an antigenic or immunogenic polypeptide. Such antigenic or immunogenic polypeptide may be derived from Group B Streptococcus. For example, the antigenic or immunogenic polypeptide may be the GBS polypeptides, fragments, derivatives, or variants described herein. In addition, the composition can comprise any polypeptides disclosed in U.S. Patent Publication No. 2007/0036828, U.S. Patent Publication No. 2006/0275315, and U.S. Patent Publication No. 2006/0258849 (all incorporated herein by reference in their entireties).
[00306] For example, the composition of the present invention further comprising an isolated third polypeptide may therefore comprise an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24- 192 of SEQ ID NO: 33; an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated third polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ED NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
[00307] In certain aspects, the composition of the present invention comprises two or more of isolated polypeptides, wherein each of said polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[00308] Alternatively, the composition comprises two or more of polynucleotides, wherein each of said polynucleotide comprises a nucleic acid sequence at least 80% identical to a reference nucleic acid sequence encoding an amino acid sequence selected from group consisting of Phol- 13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSl 1, MS14, MSlO(NP GAP, ME:NAP), PBP, pho.3-9, OCT, PGK, and NPGAP, wherein each of said polynucleotide encodes a polypeptide recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[00309] In further embodiments, the composition comprises two or more of vectors, wherein each of said vector encodes a polypeptide comprising an amino acid sequence at least 80% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence. In an alternative embodiment, the vector may comprise two or more polynucleotides encoding the two or more polypeptides. The composition may further comprise two or more of host cells, wherein each of said host cell comprises the vector or vectors described above.
[00310] In addition, the present invention includes a composition comprising isolated antibodies raised against two or more of polypeptides, wherein each of said polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein each of said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
[00311] Compositions, e.g, vaccine compositions, of the present invention can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington 's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., Mack Publishing Co., Easton, PA (1980), and Remington 's Pharmaceutical Sciences, 19th Edition, A.R. Gennaro, ed., Mack Publishing Co., Easton, PA (1995), both of which are incorporated herein by reference in their entireties. Although the composition may be administered as an aqueous solution, it can also be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art. In addition, the composition may contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.
[00312] Compositions, e.g., vaccine, pharmaceutical, or immunological compositions, may include, for example, lipopeptides (eg.,Vitiello, A. et al, J. Clin. Invest. 95:341, 1995), polypeptides encapsulated e.g., in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, el al., Molec. Immunol. 28:287-294, 1991: Alonso el al, Vaccine 12:299-306, 1994: Jones et al., Vaccine 13:675-681, 1995); polypeptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu, et al., Clin Exp Immunol. 113:235-243, 1998); multiple antigen peptide systems (MAPs) (see e.g., Tarn, J. P., Proc. Natl. Acad. ScL U.S.A. 85:5409-5413, 1988; Tarn, J.P., J. Immunol. Methods 196:17-32, 1996); particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al, Sem. Hematol. 30:16, 1993; FaIo, L. D., Jr. et al, Nature Med. 7:649, 1995); adjuvants (e.g., incomplete Freund's adjuvant) (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al, Vaccine 11:293, 1993); or liposomes (Reddy, R. et al, J. Immunol. 148:1585, 1992; Rock, K. L., Immunol Today 17:131, 1996).
[00313] The concentration of polypeptides of the invention in the compositions of the invention can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
[00314] Furthermore, compositions in accordance with the invention comprise more than one polypeptide. In some embodiments, a composition, e.g., a vaccine composition of the present invention can further include a polypeptide selected from the group consisting of, but not limited to, C5a peptidase, β-component of the C protein, LmbP, Sip, or LrrG as well as more than one GBS polypeptide described herein.
[00315] The present invention is also directed to a vaccine composition effective against GBS infection further comprising an adjuvant, carrier and/or excipient to the at least two isolated GBS polypeptides. As the person of ordinary skill in the art would appreciate, the terms "adjuvant," "carrier," and "excipient" overlap to a significant extent. For example, a compound which acts as an "adjuvant" may also be a "carrier," as well as an "excipient," and certain other compounds normally thought of, e.g., as carriers, may also function as an adjuvant. The term "adjuvant" refers to any material having the ability to (1) alter or increase the immune response to a particular antigen or (2) increase or aid an effect of a pharmacological agent. Any compound which may increase the expression, antigenicity or immunogenicity of the polypeptide is a potential adjuvant. The term "immunogenic carrier" as used herein refers to an isolated first polypeptide or fragment, variant, or derivative thereof which enhances the immunogenicity of an isolated second polypeptide or fragment, variant, or derivative thereof.
[00316] Carriers that can be used with compositions of the invention are well known in the art, and include, without limitation, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. A variety of aqueous carriers may be used, e.g. , water, buffered watery 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the carrier is an immunogenic carrier. Suitably, an immunogenic carrier may be fused to or conjugated to a desired polypeptide or fragment thereof. See, e.g., European Patent No. EP 0385610 Bl, which is incorporated herein by reference in its entirety.
[00317] The preparation of vaccines which contain immunogenic peptides as active ingredients, is known to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
[00318] The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
[00319] The peptides of the invention may be formulated into the vaccine as neutral or salt forms.
Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with, the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
[00320] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
[00321] The pharmaceutical and vaccine compositions of the present invention may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Typically, each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
[00322] The polypeptides, polynucleotides, or antibodies or antigen binding fragment thereof of the invention may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
[00323] In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine before, after, or concurrently with the polypeptides, polynucleotides, or antibody or antigen binding fragment thereof.
[00324] Potential adjuvants which may be screened for their ability to enhance the immune response according to the present invention include, but are not limited to: inert carriers, such as alum, bentonite, latex, and acrylic particles; pluronic block polymers, such as TITERMAX® (block copolymer CRL-8941, squalene (a metabolizable oil) and a microparticulate silica stabilizer), depot formers, such as Freund's adjuvant, surface active materials, such as saponin, lysolecithin, retinal, Quil A, liposomes, and pluronic polymer formulations; macrophage stimulators, such as bacterial lipopolysaccharide; polycationic polymers such as chitosan; alternate pathway complement activators, such as insulin, zymosan, endotoxin, and levamisole; and non-ionic surfactants, such as poloxamers, poly(oxyethylene)-poly(oxypropylene) tri-block copolymers, cytokines and growth factors; bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts such as aluminum hydroxide; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viruses and virally-derived materials, poisons, venoms, imidazoquiniline compounds, poloxamers, mLT, and cationic lipids. International Patent Application No. PCT/US95/09005 incorporated herein by reference in its entirety describes use of a mutated form of heat labile toxin of enterotoxigenic E. coli ("mLT") as- an adjuvant. U.S. Pat. No. 5,057,540. incorporated herein by reference, in its entirety, describes the adjuvant, Qs21. In some embodiments., the adjuvant is a toll-like receptor (TLR) stimulating adjuvant. See e.g., Science 372:184-187 (2006). TLR adjuvants include compounds that stimulate the TLRs {e.g., TLRl - TLRl 7), resulting in an increased immune system response to the vaccine composition of the present invention. TLR adjuvants include, but are not limited to CpG (Coley Pharmaceutical Group Inc.) and MPL (Corixa). One example of a CpG oligonucleotide adjuvant is CpG7909, described in International Pat. Pub. No. WO 98/018810, US Patent Appl. Pub. No. 2002/016434 IA, US Pat. No. 6,727,230, and International Pat. Pub. No. WO98/32462, which are incorporated herein by reference in their entireties.
[00325] Further examples of adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in- oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(l '-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI™, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN™ 80 emulsion, MF59™, SAF, monophospholipid A (MPL), Detox™, QS21, Stimulon™, a polyoxyetihylene ether, a polyoxyethylene ester, and aluminium salts or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan) or a combination of two or more of the adjuvants.
[00326] In certain embodiments, adjuvants such as Amphigen (oil-in-water), Alhydrogel
(aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
[00327] The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (A12O3 basis). Conveniently, the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 mg/ml, preferably 5 to 50 mg/ml, most preferably 15 mg/ml.
[00328] In one aspect of the invention, more than one adjuvant can be used in the methods and/or the composition. For example a TLR adjuvant, e.g., a CpG oligonucleotide, and Alum together can be formulated with a first polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof, and a second polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof in the methods and compositions of the present. invention.
[00329] Dosages of the adjuvants can vary according to the specific adjuvants. For example, in some aspects, dosage ranges can include: 10 μg/dose to 500 μg/dose, or 50 μg/dose to 200 μg/dose for CpG. Dosage ranges can include: 2 μg/dose to 100 μg/dose, or 10 μg/dose to 30 μg/dose for MPL. Dosage ranges can include: 10 μg/dose to 500 μg/dose, or 50 μg/dose to 100 μg/dose for aluminum hydroxide. In a prime-boost regimen, as described elsewhere herein, an adjuvant may be used with either the priming immunization, the booster immunization, or both.
[00330] In certain adjuvant compositions, the adjuvant is a cytokine. Certain compositions of the present invention comprise one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines, or a polynucleotide encoding one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines. Examples of cytokines include, but are not limited to granulocyte macrophage colony stimulating factor (GM- CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M- CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-I l), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 14 (IL-14), interleukin 15 (EL-15), interleukin 16 (IL-16), interleukin 17 (DL-17), interleukin 18 (IL-18), interferon alpha (IFN), interferon beta (IFN), interferon gamma (IFN), interferon omega (IFN), interferon tau (IFN), interferon gamma inducing factor I (IGIF), transforming growth factor beta (TGF), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MDP-I alpha and MIP-I beta), Leishmania elongation initiating factor (LEIF), and Flt-3 ligand.
[00331] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th2 response into a primarily cellular, or Thj response. Immune responses to a given antigen may be tested by various immunoassays well known to those of ordinary skill in the art, and/or described elsewhere herein.
[00332] In other embodiments, the composition of the present invention may further comprise bacterial saccharides, such as the capsular saccharides from a GBS, Neisseria sp., Streptococcus pneumoniae sp. or Haemophilus influenzae type-b bacteria. In some embodiments, the saccharide antigen can be conjugated to a carrier molecule.
[00333] The polypeptides of the invention can also be administered via liposome carriers, which serve to target the polypeptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the polypeptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the polypeptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells (such as monoclonal antibodies which bind to the CD45 antigen or other costimulatory factor) or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired polypeptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the polypeptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. A liposome suspension containing a polypeptide of the invention may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the polypeptide being delivered, and the stage of the disease being treated.
[00334] For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more polypeptides of the invention, often at a concentration of 25%-75%.
[00335] For aerosol or mucosal administration, the immunogenic polypeptides can be supplied in finely divided form, optionally along with a surfactant and, propellant and/or a mucoadhesive, e.g., chitosan. Typical percentages of polypeptides are 0.01%-20% by weight, often l%-10%. The surfactant must, of course, be pharmaceutically acceptable, and in some embodiments soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, in some embodiments 0.25-5% by weight. The balance of the composition is ordinarily propellant, although an atomizer may be used in which no propellant is necessary and other percentages are adjusted accordingly. In. some embodiments, the immunogenic polypeptides can be incorporated within an aerodynamically light particle, such as those particles descπbed in U.S. Pat. No. 6,942,868 or U.S. Pat. Pub. No. 2005/0008633. A carrier can also be included, e.g., lecithin for intranasal delivery.
Methods of Treatment/Prevention and Regimens
[00336] Another embodiment of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the combination of polypeptides, or fragments thereof, adequate to produce antibody and/or T cell immune response to protect the animal from infection by Group B Streptococci.
[00337] In one aspect, the present invention is directed to a method to induce an immune response in a subject comprising: administering to the animal in need thereof an isolated first polypeptide, polynucleotide, antibody or antigen binding fragment thereof, vector, or host cell described herein and an isolated second polypeptides, polynucleotides, antibody or antigen binding fragment thereof, vectors, or host cells described herein. In certain embodiments, the subject is an animal, e.g., a mammal, e.g., a primate, e.g., a human. [00338] In some embodiments, the invention is directed to a method of inducing an immune response, wherein the immune response treats, prevents, or ameliorates a disease or disorder related to GBS in a subject, e.g., a host animal, comprising administering an effective amount of an isolated first polypeptide, polynucleotide, antibody or antigen binding fragment thereof, vector, or host cell described herein and an isolated second polypeptide, polynucleotide, antibody or antigen binding fragment thereof, vector, or host cell described herein.
[00339] In some embodiments, an animal can be treated with a composition comprising at least two different polypeptides, at least two different polynucleotides, vectors comprising the polynucleotides, or host cells comprising the vector prophylactically, e.g., as a prophylactic vaccine, to establish or enhance immunity to one or more GBS serotypes in a healthy animal prior to exposure to GBS or contraction of a GBS symptom, thus preventing the disease or reducing the severity of disease symptoms. The composition may be used to induce an immune response or to treat an animal already exposed to GBS, or already suffering from GBS-related symptom to further stimulate the immune system of the animal, thus reducing or eliminating the symptoms associated with that exposure. In some embodiments, the composition of the present invention can be used to provide prophylactic immunity to a neonate. As defined herein, the term "treatment of an animal" refers to the use of two or more compositions comprising a single GBS polypeptide, or a composition comprising two or more GBS polypeptides, polynucleotides, vectors, or host cells of the present invention to prevent, cure, retard, or reduce the severity of GBS symptoms in an animal, and/or result in no worsening of GBS symptoms over a specified period of time. It is not required that any composition, polypeptide, polynucleotide, a vector, or a host cell of the present invention provides total protection against GBS infection or totally cure or eliminate all GBS symptoms. As used herein, "an animal in need of therapeutic and/or preventative immunity" refers to an animal which it is desirable to treat, i.e., to prevent, cure, retard, or reduce the severity of GBS symptoms, and/or result in no worsening of GBS symptoms over a specified period of time.
[00340] In some embodiments, an antibody specifically reactive with a GBS organism is isolated from the serum of the host animal which has been administered a polypeptide or polynucleotide of the present invention. In some embodiments, the invention is directed to a method of providing passive immunity comprising administering the antibody specifically reactive with a GBS organism (which was isolated from the serum of a host animal) to an animal in need thereof.
[00341] The present invention is directed to a method of inducing an immune response comprising administering an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide is selected from the group consisting of the GBS polypeptides described herein. The first polypeptide and the second polypeptide are not identical and each of the first polypeptide and the second polypeptide specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to said reference amino acid sequence.
[00342] For example, the method of inducing an immune response comprises administering an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106- 225 of SEQ ID NO: 21; (iv) amino acids 272-360 of SEQ ID NO: 21; and (v) amino acids 106- 360 of SEQ ID NO: 21.
[00343] In some embodiments, the method comprises administering an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or ] 00% identical to a. reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ BD NO: 68; (iii) amino acids 285 -363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
[00344] In other embodiments, the method comprises an isolated first polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and an isolated second polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference amino acid sequence selected from the group consisting of (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285- 363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
[00345] Moreover, the present invention includes a method of inducing immune response comprising further administering an isolated third polypeptide, polynucleotide, or antibody or antigen binding fragment thereof. The third polypeptide, polynucleotide, and antibody or antigen binding fragment thereof are described elsewhere herein. [00346] Treatment with the GBS polypeptides, polynucleotides, antibody or antigen binding fragments, or a pharmaceutical composition comprising the same can occur separately or in conjunction with other treatments, as appropriate.
[00347] In therapeutic applications, compositions, polypeptides or polynucleotides are administered to a patient in an amount sufficient to elicit an effective CTL response to the GBS- derived polypeptide to cure or at least partially arrest symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose" or "unit dose." Amounts effective for this use will depend on, e.g., the polypeptide or polynucleotide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization for polypeptide vaccines is (that is for therapeutic or prophylactic administration) from about 1.0 μg to about 5000 μg of polypeptide, in some embodiments about 10 μg to about 30 μg, for a 70 kg patient, followed by boosting dosages of from about 1.0 μg to about 1000 μg, in some embodiments 10 μg to about 30 μg, of polypeptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood. In alternative embodiments, generally for humans the dose range for the initial immunization (that is for therapeutic or prophylactic administration) is from about 1.0 μg to about 20,000 μg of polypeptide for a 70 kg patient, in some embodiments 2 μg -, 5 μg - 10 μg -, 15 μg -, 20 μg.-, 25 μg -, 30 μg -, 40 μg -, or 50 μg -2000 μg, followed by boosting dosages in the same dose range pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL (cytotoxic T lymphocytes) activity in the patient's blood. In a specific, non-limiting embodiment of the invention, approximately 0.01 to 2000 μg, or in some embodiments 2 μg to 200 μg or 10 μg to 30 μg, of a polypeptide or polynucleotide of the present invention, or its fragment, derivative variant, or analog is administered to a host.
[00348] In embodiments where DNA vaccine administration is used, the amount of polynucleotide in the initial immunization (that is for therapeutic or prophylactic administration) depends upon a number of factors including, for example, the antigen being expressed, the expression vector being used, the age and weight of the subject, the precise condition requiring treatment and its severity, and the route of administration. Based on the above factors, determining the precise amount, number of doses, and timing of doses are within the ordinary skill in the art and will be readily determined by the attending physician or veterinarian. In some embodiments, doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA or RNA per patient. [00349] In non-limiting embodiments of the invention, an effective amount of a composition of the invention produces an elevation of antibody titer to at least three times the antibody titer prior to administration.
[00350] It must be kept in mind that the polypeptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the polypeptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these polypeptide compositions.
[00351] For therapeutic use, administration should begin at the first sign of GBS infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
[00352] Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing chronic infection the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection. Where the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.
[00353] More specifically, the compositions of the present invention may be administered to any tissue of an animal, including, but not limited to, muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue, e.g , myocardium, endocardium, and pericardium, lymph tissue, blood tissue, bone tissue, pancreas tissue, kidney tissue, gall bladder tissue, stomach tissue, intestinal tissue, testicular tissue, ovarian tissue, uterine tissue, vaginal tissue, rectal tissue, nervous system tissue, eye tissue, glandular tissue, tongue tissue, and connective tissue, e.g., cartilage.
[00354] Furthermore, the compositions of the present invention may be administered to any internal cavity of a vertebrate, including, but not limited to, the lungs, the mouth, the nasal cavity, the stomach, the peritoneal cavity, the intestine, any heart chamber, veins, arteries, capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity, the rectal cavity, joint cavities, ventricles in brain, spinal canal in spinal cord, the ocular cavities, the lumen of a duct of a salivary gland or a liver. When the compositions of the present invention is administered to the lumen of a duct of a salivary gland or a liver, the desired polypeptide is encoded in each of the salivary gland and the liver such that the polypeptide is delivered into the blood stream of the vertebrate from each of the salivary gland and the liver. Certain modes for administration to secretory organs of a gastrointestinal system using the salivary gland, liver and pancreas to release a desired polypeptide into the bloodstream are disclosed in U.S. Patent Nos. 5,837,693 and 6,004,944, both of which are incorporated herein by reference in their entireties. [00355] In one embodiment, the compositions are administered to muscle, either skeletal muscle or cardiac muscle, or lung tissue. Specific, but non-limiting modes for administration to lung tissue are disclosed in Wheeler, C.J., et al, Proc. Natl. Acad. ScL USA 93:11454-11459 (1996), which is incorporated herein by reference in its entirety.
[00356] In certain embodiments, one or more compositions of the present invention are delivered to an animal by methods described herein, thereby achieving an effective immune response, and/or an effective therapeutic or preventative immune response. There may be different composition/formulation requirements dependent on the different delivery systems. Any mode of administration can be used so long as the mode results in the delivery and/or expression of the desired polypeptide in the desired tissue, in an amount sufficient to generate an immune response to GBS, and/or to generate a prophylactically or therapeutically effective immune response to GBS, in an animal in need of such response.
[00357] Where appropriate, the pharmaceutical compositions can be administered by injection, parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. According to the disclosed methods, compositions of the present invention can be administered by mucosal delivery, transdermal delivery, subcutaneous injection, intravenous injection, oral administration, pulmonary administration, intramuscular " (i.m.) administration, or via intradural injection. Other suitable routes of administration include, but not limited to intratracheal, transdermal, intraocular, intranasal, inhalation, intracavity, intraductal (e.g., into the pancreas) and intraparenchymal (i.e., into any tissue) administration. Transdermal delivery includes, but not limited to intradermal (e.g., into the dermis or epidermis), transdermal (e.g., percutaneous) and transmucosal administration (i.e., into or through skin or mucosal tissue). Intracavity administration includes, but not limited to administration into oral, vaginal, rectal, nasal, peritoneal, or intestinal cavities as well as, intrathecal (i.e., into spinal canal), intraventricular (i.e., into the brain ventricles or the heart ventricles), intraatrial (i.e., into the heart atrium) and sub arachnoid (i.e., into the sub arachnoid spaces of the brain) administration. The compositions, polypeptides, polynucleotides, or antibody or antigen binding fragment thereof or any of the present invention may also be administered by a transdermal patch, sublingual wafer, or automjection device.
[00358] Any mode of administration can be used so long as the mode results in the delivery and/or expression of the desired polypeptide in the desired tissue, in an amount sufficient to generate an immune response against GBS, and/or to generate a prophylactically or therapeutically effective immune response against GBS, in an animal in need of such response. Administration means of the present invention include needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., "gene guns" or pneumatic "needleless" injectors) Med-E-Jet (Vahlsing, H., et al, J. Immunol. Methods 171,11-22 (1994)), Pigjet (Schrijver, R., et al, Vaccine 15, 1908-1916 (1997)), Biojector (Davis, H., et al, Vaccine 12, 1503-1509 (1994); Gramzinski, R., et al, MoI Med. 4, 109-118 (1998)), AdvantaJet (Linmayer, I., et al, Diabetes Care 9:294-297 (1986)), Medi-jector (Martins, J., and Roedl, E. J. Occup. Med. 27:821-824 (1979)), gelfoam sponge depots, other commercially available depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, use of polynucleotide coated suture (Qin, Y., et al, Life Sciences 65, 2193-2203 (1999)) or topical applications during surgery. Certain modes of administration are intramuscular needle-based injection and pulmonary application via catheter infusion. Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
[00359] Upon immunization with a polypeptide or polynucleotide composition in accordance with the invention, the immune system of the host responds to the vaccine by producing large amounts of HTLs (helper T lymphocytes) and/or CTLs (cytotoxic T lymphocytes) specific for the desired antigen. Consequently, the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection.
[00360] In some embodiments, polypeptides or polynucleotides of the present invention stimulate a cell-mediated immune response sufficient for protection of an animal against GBS infection. In other embodiments, polypeptides or polynucleotides of the present invention stimulate both a humoral and a cell-mediated response, the combination of which is sufficient for protection of an animal against GBS protection.
[00361] In certain embodiments, components that induce T cell responses are combined with components that induce antibody responses to the target antigen of interest. Thus, in certain embodiments of the invention, vaccine compositions of the invention are combined with polypeptides or polynucleotides which induce or facilitate neutralizing antibody responses to the target antigen of interest. One embodiment of such a composition comprises a class I epitope in accordance with the invention, along with a PADRE® (Epimmune, San Diego, CA) molecule (described, for example, in U.S. Patent Number 5,736,142, which is incorporated herein by reference in its entirety).
[00362] The polynucleotides of the present invention, or vectors containing the polynucleotides of the present invention, can be incorporated into the cells of the animal in vivo, and an antigenic amount of the Streptococcus agalactiae-deήved polypeptide, or fragments, variants, or derivatives thereof, is produced in vivo. Upon administration of the composition according to this method, the Streptococcus agalactiae-deύved polypeptide is expressed in the animal in an amount sufficient to elicit an immune response. Such an immune response might be used, for example, to generate antibodies to GBS for use in diagnostic assays or as laboratory reagents. [00363] The present invention further provides a method for generating, enhancing, or modulating a protective and/or therapeutic immune response against GBS in an animal, comprising administering to the animal in need of therapeutic and/or preventative immunity one or more of the compositions, at least two polypeptides, polynucleotides, vectors, host cells, or composition thereof described herein.
[00364] The polypeptides, polynucleotides, antibody or antigen binding fragments, or compositions of the present invention can be administered to an animal at any time during the lifecycle of the animal to which it is being administered. For example, the composition can be given shortly after birth. In humans, administration can occur while other vaccines are being administered, e.g., at birth, 2 months, 4 months, 6 months, 9 months, at 1 year, at 5 years, or at the onset of puberty. In some embodiments, administration can occur when the human become sexually active.
[00365] Furthermore, the polypeptides, polynucleotides, antibody or antigen binding fragments thereof, or compositions of the invention can be used in any desired immunization or administration regimen; e.g., in a single administration or alternatively as part of periodic vaccinations such as annual vaccinations, or as in a prime-boost regime wherein the polypeptide or polynucleotide of the present invention is administered either before or after the administration of the same or of a different polypeptide or polynucleotide.
[00366] Recent studies have indicated that a prime-boost protocol is often a suitable method of
■administering vaccines. In a prime-boost protocol, one or more compositions of, the present invention can be utilized in a "prime boost" regimen. An example of a "prime boost" regimen may be found in Yang, Z. et al. J. Virol. 77:799-803 (2002), which is incorporated herein by reference in its entirety. In a non-limiting example, one or more polypeptide, polynucleotide, or antibody compositions of the present invention are delivered to an animal, thereby priming the immune response of the animal to a GBS polypeptide of the invention, and then a second immunogenic composition is utilized as a boost vaccination. One or more compositions of the present invention are used to prime immunity, and then a second immunogenic composition, e.g., a recombinant viral vaccine or vaccines, e.g., a recombinant vaccine virus, e.g., a recombinant MVA vaccinia virus, a different polynucleotide vaccine, or one or more purified subunit of the GBS polypeptides or fragments, variants or derivatives thereof is used to boost the anti-GBS immune response.
[00367] In another non-limiting example, a priming composition and a boosting composition are combined in a single composition or single formulation. For example, a single composition may comprise an isolated GBS polypeptide or a fragment, variant, or derivative thereof as the priming component and a polynucleotide encoding a GBS polypeptide as the boosting component. In this embodiment, the compositions may be contained in a single vial where the priming component and boosting component are mixed together. In general, because the peak levels of expression of polypeptide from the polynucleotide does not occur until later (e.g., 7-10 days) after administration, the polynucleotide component may provide a boost to the isolated polypeptide component. Compositions comprising both a priming component and a boosting component are referred to herein as "combinatorial vaccine compositions" or "single formulation heterologous prime-boost vaccine compositions." In addition, the priming composition may be administered before the boosting composition, or even after the boosting composition, if the boosting composition is expected to take longer to act.
[00368] In another embodiment, the priming composition may be administered simultaneously with the boosting composition, but in separate formulations where the priming component and the boosting component are separated.
[00369] In one aspect, at least two GBS polypeptides, polynucleotides, antibody or antigen binding fragments thereof, vectors, or host cells or compositions comprising the same can be administered separately or at the same time. For example, the first polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof may be administered prior to or after the second polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof. In another aspect, the first polypeptide, polynucleotide, or antibody or antigen-binding fragment thereof is administered simultaneously with the second polypeptide, polynucleotide, or antibody or antigen- binding fragment thereof.
[00370] Furthermore, at least two GBS polypeptides, polynucleotides, antibody or antigen binding fragments thereof, vectors, or host cells or compositions comprising the same may be administered in one or multiple doses. The administering may be performed in at least one dose, at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses or more. For example, at least three doses may be administered at a first month, at a second month, and at a third month. Or, at least three doses may be at a first month, at a second month, and at a fourth month. Alternatively, at least three doses may be administered at a first month, at a second month, and at a sixth month. The methods may further comprise administration of a fourth dose. For example, the fourth dose may be administered at least once between twelfth and sixtieth month after the administration of the first dose.
[00371] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et ah, ed., Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et ah, ed., Cold Springs Harbor Laboratory, New York (1992), DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis, M. J. Gait ed., (1984); Mullis et al U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, VoIs. 154 and 155 (Wu et al eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-TV, D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).
[00372] General principles of antibody engineering are set forth in Antibody Engineering, 2nd edition, C.A.K. Borrebaeck, Ed., Oxford Univ. Press (1995). General principles of protein engineering are set forth in Protein Engineering, A Practical Approach, Rickwood, D., et al, Eds., IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principles of antibodies are set forth in: Nisonoff, A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, MA (1984); and Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall, New York, NY (1984). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al (eds) , Basic and Clinical -Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).
[00373] Standard reference works setting forth general principles of immunology include Current
Protocols in Immunology, John Wiley & Sons, New York; Klein, J., Immunology: The Science of Self-Nonself Discrimination, John Wiley & Sons, New York (1982); Kennett, R., et al, eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A., "Monoclonal Antibody Technology" in Burden, R., et al, eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology 4th ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D., Immunology, 6th ed. London: Mosby (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular Immunology, Ed. 5, Elsevier Health Sciences Division (2005); Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer, Cold Spring Harbor Press (2003).
EXAMPLES
Example 1
[00374] A composition comprising Pho3-1 (ME:P31), one of the selected proteins, was administered in 47 healthy adult volunteers. The purpose of this trial was to evaluate the safety and immunogenicity of this protein. In this trial, Pho3-1 (ME:P31) was adjuvanted with alum and tested at four different strengths, with two doses given 28 days apart. Pho3-1 (ME:P31) was immunogenic at all doses tested, the immune response rate was 83% at the lowest dose tested and 100% at the highest dose tested. In addition, Pho3-1 (ME:P31) was well tolerated at all dose levels tested, with no serious adverse events reported.
Example 2:
Opsonophagocytosis of GBS strain A909 opsonised with monkey sera raised against the Pho 3-1 polypeptide and MSlO (NPGAP) polypeptide in GBS strain M732.
[00375] The opsonophagocytosis assay will demonstrate opsonisation of GBS with immune sera raised against the proteins and their subsequent uptake by a phagocytic cell line. It is well known to those skilled in the art that this up-take is a precursor to the killing of GBS by the phagocyte. The opsonophagocytosis activity is expressed as a percentage of HL-60 cells (phagocytes) which have taken up fluorescence labeled GBS bacteria.
[00376] Rhesus monkeys were immunized with the Pho3-1 polypeptide (SEQ ID NO: 33) and the
MSlO polypeptide (NPGAP; SEQ ID NO: 68). The sera (individually and in combination) were obtained to opsonise a GBS type Ia/c strain, A909, and measured for the increase in phagocytic uptake mediated by the immune sera compared with the preimmune sera. Antisera against both these candidates could individually mediate a phagocytic uptake. It was also shown that combining equal volumes of the two sera at dilutions >l:100 results in an additive opsonophagocytic effect (See figure 2).

Claims

We claim:
1. A method of inducing an immune response against Group B Streptococcus in an animal comprising administering to said animal in need thereof an effective amount of an isolated first polypeptide and an isolated second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1-59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101- 181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2;
(b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5;
(c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ID NO: 8; (iii) amino acids 139-192 of SEQ ID NO: 8; and (iv) amino acids 43-192 of SEQ ID NO: 8;
(d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (ii) amino acids 1-88 of SEQ ID NO: 11; (iii) amino acids 1-110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11;
(e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 14; (ii) amino acids 1-59 of SEQ ID NO: 14; (iii) amino acids 84-149 of SEQ ID NO: 14; and (iv) amino acids 1-149 of SEQ ID NO: 14;
(f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 18; (ii) amino acids 29-95 of SEQ ID NO: 18; (iii) amino acids 1-95 of SEQ ID NO: 18; and (iv) amino acids 29-125 of SEQ ID NO: 18;
(g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21;
(h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 24; (ii) amino acids 43-84 of SEQ ID NO: 24; (iii) amino acids 1-84 of SEQ ID NO: 24; (iv) amino acids 88-122 of SEQ ID NO: 24; and (v) amino acids 38-122 of SEQ ID NO: 24; (i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 27; (ii) amino acids 29-79 of SEQ ED NO: 27; (iii) amino acids 101-189 of SEQ ED NO: 27; and (iv) amino acids 29-189 of SEQ ED NO: 27;
(j) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 30; (ii) amino acids 79-182 of SEQ ED NO: 30; (iii) amino acids 79-217 of SEQ ED NO: 30; (iv) amino acids 23-217 of SEQ ED NO: 30; (v) amino acids 23-278 of SEQ ED NO: 30;
(k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 33; (ii) amino acids 113-192 of SEQ ED NO: 33; (iii) amino acids 71-192 of SEQ ED NO: 33; and (iv) amino acids 24-192 of SEQ ED NO: 33;
(1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 36; (ii) amino acids 25-88 of SEQ ED NO: 36; (iii) amino acids 132-202 of SEQ ED NO: 36; (iv) amino acids 25-172 of SEQ ED NO: 36; and (v) amino acids 25-202 of SEQ ED NO: 36;
(m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 39 and (ii) amino acids 27-94 of SEQ ED NO: 39;
(n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 42; (ii) amino acids 111-180 of SEQ ED NO: 42; (iii) amino acids 111-234 of SEQ ED NO: 42; (iv) amino acids 72-234 of SEQ ED NO: 42; and (v) amino acids 40-335 of SEQ ED NO: 42;
(o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 45; (ii) amino acids 98-180 of SEQ ED NO: 45; (iii) amino acids 259-370 of SEQ ED NO: 45; and (iv) amino acids 98-370 of SEQ ID NO: 45;
(p) a Pho3-29 polypeptide, which is SEQ ED NO: 48;
(q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 51; (ii) amino acids 78-154 of SEQ ED NO: 51; (iii) amino acids 78-217 of SEQ ED NO: 51; and (iv) amino acids 1-154 of SEQ ED NO: 51;
(r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 54; (ii) amino acids 190-261 of SEQ ED NO: 54; (iii) amino acids 190-321 of SEQ ED NO: 54; and (iv) amino acids 1 - 118 of SEQ ED NO: 54;
(s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 57; (ii) amino acids 1-139 of SEQ ID NO: 57; (iii) amino acids 184-311 of SEQ ED NO: 57; and (iv) amino acids 184-398 of SEQ ED NO: 57; (t) an MS 16 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 60, (ii) amino acids 24-77 of SEQ ID NO: 60; (iii) amino acids 101-172of SEQ ID NO: 60; (iv) amino acids 24-172 of SEQ ID NO: 60; and (v) SEQ ID NO: 62;
(u) an MS 14 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 65; (ii) amino acids 1-95 of SEQ ID NO: 65; (iii) amino acids 141-181 of SEQ ID NO: 65; and (iv) amino acids 141-268 of SEQ ID NO: 65; or
(v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68,
wherein said first polypeptide and said second polypeptide are not identical and each of said first polypeptide and said second polypeptide specifically bind to an antibody raised against a polypeptide consisting of an amino acid sequence identical to said reference amino acid sequence.
2. The method of claim 1, wherein said immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in said animal.
3. A method of inducing an immune response against Group B Streptococcus in an animal comprising administering to said animal in need thereof an effective amount of an isolated first polynucleotide encoding said first polypeptide of claim 1 and an isolated second polynucleotide encoding said second polypeptide of claim 1.
4. The method of claim 3, wherein said immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in said animal.
5. A method of inducing an immune response against Group B Streptococcus in an animal comprising administering to said animal in need thereof an effective amount of an isolated first antibody or antigen binding fragment thereof raised against said first polypeptide of claim 1 and an isolated second antibody or antigen binding fragment thereof raised against said second polypeptide of claim 1.
6. The method of claim 5, wherein said immune response prevents, treats, or ameliorates a condition or disorder associated with Group B Streptococcal infection in said animal.
7. The method of claim 3 or 4, wherein said first polynucleotide and said second polynucleotide are located in one or more expression vectors.
8. The method of claim 7, wherein said first polynucleotide and said second polynucleotide are located in one expression vector.
9. The method of claim 7, wherein said first polynucleotide and said second polynucleotide are located in more than one expression vectors.
10. The method of claim 9, wherein said more than one expression vectors are identical or different or a combination thereof.
11. The method of claim 1 or 2, which further comprises administering an isolated third polypeptide.
12. The method of claim 5 or 6, which further comprises administering an isolated third antibody or antigen-bindmg fragment thereof raised against a third polypeptide.
13. The method of claim 3 or 4, which further comprises administering an isolated third polynucleotide.
14. The method of claim 13, wherein said third polynucleotide comprises a promoter operably linked to said first polynucleotide or said second polynucleotide.
15. The method of claim 13, wherein said third polynucleotide encodes a third polypeptide.
16. The method of any one of claim 11, 12, or 15, wherein said third polypeptide is an antigenic or immunogenic polypeptide.
17. The method of claim 16, wherein said antigenic or immunogenic polypeptide is deπved from a bacterium, a virus, a fungus, or a protozoan.
18. The method of claim 16 or 17, wherein said antigenic or immunogenic polypeptide is derived from Group B Streptococcus.
19. The method of any one of claim 11, 12, or 15-18, wherein said third polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (n) amino acids 1-59 of SEQ ID NO: 2; (in) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101- 181 of SEQ ID NO: 2; and (v) ammo acids 1-181 of SEQ ID NO: 2; (b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ED NO: 5 and (π) amino acids 1-45 of SEQ ID NO: 5;
(c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ED NO: 8; (ii) amino acids 43-115 of SEQ ID NO: 8; (in) ammo acids 139-192 of SEQ ID NO: 8; and (iv) amino acids 43-192 of SEQ DD NO: 8;
(d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (n) amino acids 1-88 of SEQ ID NO: 11; (in) amino acids 1-110 of SEQ ED NO: 11; and (iv) ammo acids 165-197 of SEQ ED NO: 11;
(e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 14; (n) amino acids 1-59 of SEQ ED NO: 14; (in) amino acids 84-149 of SEQ ED NO: 14; and (iv) amino acids 1-149 of SEQ ED NO: 14;
(f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 18; (ii) amino acids 29-95 of SEQ ED NO: 18; (in) ammo acids 1-95 of SEQ ID NO: 18; and (iv) ammo acids 29-125 of SEQ ED NO: 18;
(g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ ED NO: 21; (n) ammo acids 1-77 of SEQ ED NO: 21; (in) amino acids 106-225 of SEQ ED NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) ammo acids 106-360 of SEQ ED NO:21;
(h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 24; (n) amino acids 43-84 of SEQ ED NO: 24; (in) ammo acids 1-84 of SEQ ED NO: 24; (iv) ammo acids 88-122 of SEQ ED NO: 24; and (v) amino acids 38-122 of SEQ ED NO: 24;
(i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 27; (n) amino acids 29-79 of SEQ ED NO: 27; (in) ammo acids 101-189 of SEQ ED NO: 27; and (iv) amino acids 29-189 of SEQ ID NO: 27;
(j) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 30; (n) ammo acids 79-182 of SEQ ED NO: 30; (in) ammo acids 79-217 of SEQ ED NO: 30; (iv) amino acids 23-217 of SEQ ED NO: 30; (v) amino acids 23-278 of SEQ ED NO: 30;
(k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 33; (n) ammo acids 113-192 of SEQ ID NO: 33; (in) amino acids 71-192 of SEQ ED NO: 33; and (iv) ammo acids 24-192 of SEQ ED NO: 33; (1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 36; (ii) amino acids 25-88 of SEQ ID NO: 36; (iii) amino acids 132-202 of SEQ ID NO: 36; (iv) amino acids 25-172 of SEQ ID NO: 36; and (v) amino acids 25-202 of SEQ ED NO: 36;
(m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 39 and (ii) amino acids 27-94 of SEQ ID NO: 39;
(n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 42; (ii) amino acids 111-180 of SEQ ID NO: 42; (iii) amino acids 111-234 of SEQ ID NO: 42; (iv) amino acids 72-234 of SEQ ID NO: 42; and (v) amino acids 40-335 of SEQ ID NO: 42;
(o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 45; (ii) amino acids 98-180 of SEQ ID NO: 45; (iii) amino acids 259-370 of SEQ ID NO: 45; and (iv) amino acids 98-370 of SEQ ID NO: 45;
(p) a Pho3-29 polypeptide, which is SEQ ID NO: 48;
(q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 51; (ii) amino acids 78-154 of SEQ ID NO: 51; (iii) amino acids 78-217 of SEQ ID NO: 51; and (iv) amino acids 1-154 of SEQ ID NO: 51;
(r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 54; (ii) amino acids 190-261 of SEQ ED NO: 54; (iii) amino acids 190-321 of SEQ ED NO: 54; and (iv) amino acids 1-118 of SEQ ED NO: 54;
(s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 57; (ii) amino acids 1-139 of SEQ ID NO: 57; (iii) amino acids 184-311 of SEQ ED NO: 57; and (iv) amino acids 184-398 of SEQ ED NO: 57;
(t) an MS 16 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 60, (ii) amino acids 24-77 of SEQ ED NO: 60; (iii) amino acids 101-172of SEQ ED NO: 60; (iv) amino acids 24-172 of SEQ ED NO: 60; and (v) SEQ ED NO: 62;
(u) an MS 14 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 65; (ii) amino acids 1-95 of SEQ ED NO: 65; (iii) amino acids 141-181 of SEQ ED NO: 65; and (iv) amino acids 141-268 of SEQ ED NO: 65; or
(v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 68, (ii) amino acids 1-138 of SEQ ED NO: 68; (iii) amino acids 285-363 of SEQ ED NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) ammo acids 285-444 of SEQ ID NO: 68,
wherein the third polypeptide is not identical to the first polypeptide and the second polypeptide and specifically binds to an antibody raised against a polypeptide consisting of an amino acid sequence identical to said reference amino acid sequence.
20. The method of any one of claim 1-19, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 33; (ii) ammo acids 113-192 of SEQ ID NO: 33; (in) ammo acids 71-192 of SEQ ED NO: 33; and (iv) ammo acids 24-192 of SEQ ID NO: 33; and
said second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 21; (n) ammo acids 1-77 of SEQ ID NO: 21; (in) amino acids 106-225 of SEQ ID NO: 21; (iv) amino acids 272-360 of SEQ ID NO: 21; and (v) amino acids 106-360 of SEQ ID NO: 21.
21. The method of any one of claim 1-19, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference ammo acid sequence selected from the group consisting of:
(i) SEQ ID NO: 33; (π) ammo acids 113-192 of SEQ ID NO: 33; (in) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and
said second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 68, (n) amino acids 1-138 of SEQ ID NO: 68; (in) ammo acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) ammo acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
22. The method of any one of claim 1-19, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference ammo acid sequence selected from the group consisting of: (i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21;. and
said second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
23. The method of any one of claim 11, 12, or 15-19, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33,
said second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21, and
said third polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
24. The method of any one of claim 1-23, wherein said amino acid sequence is 90% identical to the reference amino acid sequence.
25. The method of any one of claim 1-24, wherein said amino acid sequence is 95% identical to the reference amino acid sequence.
26. The method of any one of claim 1-25, wherein said amino acid sequence is identical to the reference amino acid sequence.
27. The method of any one of claim 1-26, further comprising administering a polysaccharide.
28. The method of claim 27, wherein said polysaccharide is derived from Group B Streptococcus.
29. The method of any one of claim 1-28, further comprising administering a carrier.
30. The method of any one of claim 1-29, further comprising administering an adjuvant.
31. The method of claim 30, wherein said adjuvant is selected from the group consisting of mLT, MF59™, SAF, Ribi™ adjuvant system, monophospholipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), Detox™, QS21, Stimulon™, complete Freund's adjuvant, incomplete Freund's adjuvant, cytokines, interferons, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), monophosphoryl lipid A (MPL), 3-O-deacylated MPL, a CpG oligonucleotide, a saponin, a polyoxyetihylene ether, a polyoxyethylene ester, and aluminium salts, and a combination of more than one adjuvants.
32. The method of claim 31, wherein said adjuvant is Alum and a CPG oligonucleotide.
33. The method of any one of claim 1-32, comprising a hormone, a vitamin, a polyalkylene, a lipid, a liposome, a lipoprotein, or a polycationic agent or a combination thereof.
34. The method of any one of claim 1-33, wherein said immune response is a humoral immune response or a cell-mediated immune response.
35. The method of any one of claim 1-34, wherein said immune response is a humoral immune response.
36. The method of any one of claim 1-35, wherein said immune response is an antibody response.
37. The method of any one of claim 1-36, wherein the animal is a mammal.
38. The method of claim 37, wherein said mammal is a human.
39. The method of claim 1 or 2, wherein said first polypeptide is administered prior to said second polypeptide.
40. The method of claim 1 or 2, wherein said first polypeptide is administered simultaneously with said second polypeptide.
41. The method of claim 1 or 2, wherein said second polypeptide is administered prior to said first polypeptide.
42. The method of claim 3 or 4, wherein said first polynucleotide is administered prior to said second polynucleotide.
43. The method of claim 3 or 4, wherein said first polynucleotide is administered simultaneously with said second polynucleotide.
44. The method of claim 3 or 4, wherein said second polynucleotide is administered prior to said first polynucleotide.
45. The method of claim 5 or 6, wherein said first antibody or fragment thereof is administered prior to said second antibody or fragment thereof.
46. The method of claim 5 or 6, wherein said first antibody or fragment thereof is administered simultaneously with said second antibody or fragment thereof.
47. The method of claim 5 or 6, wherein said second antibody or fragment thereof is administered prior to said first antibody or fragment thereof.
48. The method of any one of claim 1-47, wherein said administering is performed in one or multiple doses.
49. The method of any one of claim 1-48, wherein said administering is performed via intramuscular injection, subcutaneous injection, intra-dermal injection, intradural injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.
50. A composition comprising an isolated first polypeptide and an isolated second polypeptide, wherein each of said first polypeptide and said second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence of:
(a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) amino acids 1-59 of SEQ ID NO: 2; (iii) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101- 181 of SEQ ID NO: 2; and (v) amino acids 1-181 of SEQ ID NO: 2;
(b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) amino acids 1-45 of SEQ ID NO: 5; (c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ED NO: 8; (iii) amino acids 139-192 of SEQ ID NO: 8; and (iv) amino acids 43-192 of SEQ ID NO: 8;
(d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (ii) amino acids 1-88 of SEQ ID NO: 11; (iii) amino acids 1-110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11;
(e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 14; (ii) amino acids 1-59 of SEQ ID NO: 14; (iii) amino acids 84-149 of SEQ DD NO: 14; and (iv) amino acids 1-149 of SEQ DD NO: 14;
(f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 18; (ii) amino acids 29-95 of SEQ DD NO: 18; (iii) amino acids 1-95 of SEQ DD NO: 18; and (iv) amino acids 29-125 of SEQ DD NO: 18;
(g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ DD NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ DD NO:21; (iv) amino acids 272-360 of SEQ DD NO:21; and (v) amino acids 106-360 of SEQ DD NO:21;
(h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 24; (ii) amino acids 43-84 of SEQ DD NO: 24; (iii) amino acids 1-84 of SEQ DD NO: 24; (iv) amino acids 88-122 of SEQ DD NO: 24; and (v) amino acids 38-122 of SEQ DD NO: 24;
(i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 27; (ii) amino acids 29-79 of SEQ DD NO: 27; (iii) amino acids 101-189 of SEQ DD NO: 27; and (iv) amino acids 29-189 of SEQ DD NO: 27;
(j) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 30; (ii) amino acids 79-182 of SEQ DD NO: 30; (iii) amino acids 79-217 of SEQ DD NO: 30; (iv) amino acids 23-217 of SEQ DD NO: 30; (v) amino acids 23-278 of SEQ DD NO: 30;
(k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 33; (ii) amino acids 113-192 of SEQ DD NO: 33; (iii) amino acids 71-192 of SEQ DD NO: 33; and (iv) amino acids 24-192 of SEQ DD NO: 33;
(1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ DD NO: 36; (ii) amino acids 25-88 of SEQ DD NO: 36; (iii) amino acids 132-202 of SEQ DD NO: 36; (iv) amino acids 25-172 of SEQ DD NO: 36; and (v) amino acids 25-202 of SEQ DD NO: 36; (m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 39 and (ii) amino acids 27-94 of SEQ ED NO: 39;
(n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 42; (ii) amino acids 111-180 of SEQ ID NO: 42; (iii) amino acids 111-234 of SEQ ID NO: 42; (iv) amino acids 72-234 of SEQ ED NO: 42; and (v) amino acids 40-335 of SEQ ID NO: 42;
(o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 45; (ii) amino acids 98-180 of SEQ ID NO: 45; (iii) amino acids 259-370 of SEQ ID NO: 45; and (iv) amino acids 98-370 of SEQ ED NO: 45;
(p) a Pho3-29 polypeptide, which is SEQ ED NO: 48;
(q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 51; (ii) amino acids 78-154 of SEQ ED NO: 51; (iii) amino acids 78-217 of SEQ ED NO: 51; and (iv) amino acids 1-154 of SEQ ED NO: 51;
(r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 54; (ii) amino acids 190-261 of SEQ ED NO: 54; (iii) amino acids 190-321 of SEQ ED NO: 54; and (iv) amino acids l-118 of SEQ ED NO: 54;
(s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 57; (ii) amino acids 1-139 of SEQ ED NO: 57; (iii) amino acids 184-311 of SEQ ED NO: 57; and (iv) amino acids 184-398 of SEQ ED NO: 57;
(t) an MS 16 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 60, (ii) amino acids 24-77 of SEQ ED NO: 60; (iii) amino acids 101-172of SEQ ED NO: 60; (iv) amino acids 24-172 of SEQ ED NO: 60; and (v) SEQ ED NO: 62;
(u) an MS14 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 65; (ii) amino acids 1-95 of SEQ ED NO: 65; (iii) amino acids 141-181 of SEQ ED NO: 65; and (iv) amino acids 141-268 of SEQ ED NO: 65; or
(v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 68, (ii) amino acids 1-138 of SEQ ED NO: 68; (iii) amino acids 285-363 of SEQ ED NO: 68; (iv) amino acids 210-363 of SEQ ED NO: 68; (v) amino acids 210-444 of SEQ ED NO: 68; and (vi) amino acids 285-444 of SEQ ED NO: 68, wherein said first polypeptide and said second polypeptide are not identical and each of said first polypeptide and second polypeptide specifically bind to an antibody raised against a polypeptide consisting of an amino acid sequence identical to said reference amino acid sequence.
51. A composition comprising an isolated first polynucleotide which encodes said first polypeptide of claim 50 and an isolated second polynucleotide which encodes said second polypeptide of claim 50.
52. A composition comprising an isolated first antibody or antigen binding fragment thereof raised against said first polypeptide of claim 50 and an isolated second antibody or antigen binding fragment thereof raised against said second polypeptide of claim 50.
53. The composition of claim 50, further comprising an isolated third polypeptide.
54. The composition of claim 52, further comprising an isolated third antibody or antigen- bindmg fragment thereof raised against a third polypeptide.
55. The composition of claim 51, further comprising an isolated third polynucleotide.
56. The composition of claim 55, wherein said third polynucleotide encodes a third polypeptide.
57. The composition of any of claim 53, 54, or 56, wherein said third polypeptide is an antigenic or immunogenic polypeptide.
58. The composition of claim 57, wherein said antigenic or immunogenic polypeptide is derived from Group B Streptococcus.
59. The composition of claim 58, wherein said third polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(a) a Phol-13 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 2; (ii) ammo acids 1-59 of SEQ E) NO: 2; (in) amino acids 132-181 of SEQ ID NO: 2; (iv) amino acids 101- 181 of SEQ ID NO: 2; and (v) ammo acids 1-181 of SEQ ID NO: 2;
(b) a Phol-14 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 5 and (ii) ammo acids 1-45 of SEQ ID NO: 5; (c) a Phol-5 polypeptide, which is selected from the group consisting of (i) SEQ ID NO: 8; (ii) amino acids 43-115 of SEQ ID NO: 8; (in) amino acids 139-192 of SEQ ID NO: 8; and (iv) ammo acids 43-192 of SEQ ID NO: 8;
(d) a Pho3-3 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 11; (π) amino acids 1-88 of SEQ ID NO: 11; (in) amino acids 1-110 of SEQ ID NO: 11; and (iv) amino acids 165-197 of SEQ ID NO: 11;
(e) a Pho2-10 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 14; (π) ammo acids 1-59 of SEQ ID NO: 14; (in) ammo acids 84-149 of SEQ ID NO: 14; and (iv) amino acids l-149 of SEQ ID NO: 14;
(f) a Pho2-15 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 18; (ii) ammo acids 29-95 of SEQ ED NO: 18; (in) amino acids 1-95 of SEQ ED NO: 18; and (iv) amino acids 29-125 of SEQ ED NO: 18;
(g) a Pho2-2 polypeptide, which is selected from the group consisting of (i) SEQ ED NO: 21; (n) amino acids 1-77 of SEQ ED NO: 21; (in) ammo acids 106-225 of SEQ ED NO:21; (iv) ammo acids 272-360 of SEQ ID NO:21; and (v) ammo acids 106-360 of SEQ ED NO:21;
(h) a Pho3-14 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 24; (π) ammo acids 43-84 of SEQ ED NO: 24; (in) amino acids 1-84 of SEQ ED NO: 24; (iv) ammo acids 88-122 of SEQ ED NO: 24; and (v) ammo acids 38-122 of SEQ ED NO: 24;
(i) a Pho3-17 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 27; (n) amino acids 29-79 of SEQ ED NO: 27; (in) amino acids 101-189 of SEQ ED NO: 27; and (iv) amino acids 29-189 of SEQ ID NO: 27;
(]) a Pho3-18 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 30; (n) amino acids 79-182 of SEQ ED NO: 30; (in) ammo acids 79-217 of SEQ ED NO: 30; (iv) amino acids 23-217 of SEQ ED NO: 30; (v) ammo acids 23-278 of SEQ ED NO: 30;
(k) a Pho3-1 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 33; (n) amino acids 113-192 of SEQ ID NO: 33; (in) ammo acids 71-192 of SEQ ED NO: 33; and (iv) ammo acids 24-192 of SEQ ED NO: 33;
(1) a Pho3-21 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 36; (n) ammo acids 25-88 of SEQ ED NO: 36; (in) ammo acids 132-202 of SEQ ED NO: 36; (iv) amino acids 25-172 of SEQ ED NO: 36; and (v) ammo acids 25-202 of SEQ ED NO: 36; (m) a Pho3-22 polypeptide, which is selected from the group consisting of: (i) SEQ ID NO: 39 and (ii) amino acids 27-94 of SEQ ID NO: 39;
(n) a Pho3-23 polypeptide, which is selected from the group consisting of: (i) SEQ ED NO: 42; (ii) amino acids 111-180 of SEQ ID NO: 42; (iii) amino acids 111-234 of SEQ ED NO: 42; (iv) amino acids 72-234 of SEQ BD NO: 42; and (v) amino acids 40-335 of SEQ BD NO: 42;
(o) a Pho3-24 polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 45; (ii) amino acids 98-180 of SEQ BD NO: 45; (iii) amino acids 259-370 of SEQ BD NO: 45; and (iv) amino acids 98-370 of SEQ BD NO: 45;
(p) a Pho3-29 polypeptide, which is SEQ BD NO: 48;
(q) a Pho3-50 polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 51; (ii) amino acids 78-154 of SEQ BD NO: 51; (iii) amino acids 78-217 of SEQ BD NO: 51; and (iv) amino acids 1 - 154 of SEQ BD NO : 51 ;
(r) an MS4 polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 54; (ii) amino acids 190-261 of SEQ BD NO: 54; (iii) amino acids 190-321 of SEQ BD NO: 54; and (iv) amino acids 1 - 118 of SEQ BD NO: 54;
(s) an MSl 1 polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 57; (ii) amino acids 1-139 of SEQ BD NO: 57; (iii) amino acids 184-311 of SEQ BD NO: 57; and (iv) amino acids 184-398 of SEQ BD NO: 57;
(t) an MS 16 polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 60, (ii) amino acids 24-77 of SEQ BD NO: 60; (iii) amino acids 101-172of SEQ BD NO: 60; (iv) amino acids 24-172 of SEQ BD NO: 60; and (v) SEQ BD NO: 62;
(u) an MS14 polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 65; (ii) amino acids 1-95 of SEQ BD NO: 65; (iii) amino acids 141-181 of SEQ BD NO: 65; and (iv) amino acids 141-268 of SEQ BD NO: 65; or
(v) an MSlO polypeptide, which is selected from the group consisting of: (i) SEQ BD NO: 68, (ii) amino acids 1-138 of SEQ BD NO: 68; (iii) amino acids 285-363 of SEQ BD NO: 68; (iv) amino acids 210-363 of SEQ BD NO: 68; (v) amino acids 210-444 of SEQ BD NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68, wherein said third polypeptide is not identical to the first polypeptide and the second polypeptide and specifically binds to an antibody raised against a polypeptide consisting an amino acid sequence identical to said reference amino acid sequence.
60. The composition of any one of claim 50-59, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and
a second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 21; (ii) amino acids 70-78 of SEQ ID NO: 21; (iii) amino acids 106-147 of SEQ ID NO:21; (iv) amino acids 168-200 of SEQ ID NO:21; (v) amino acids 273-290 of SEQ ID NO:21; (vi) amino acids 301-350 of SEQ ID NO:21; and (vii) a combination of two or more of (i) to (vi).
61. The composition of any one of claim 50-59, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33; and
a second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
62. The composition of any one of claim 50-59, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and a second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
63. The composition of claim 59, wherein said first polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 33; (ii) amino acids 113-192 of SEQ ID NO: 33; (iii) amino acids 71-192 of SEQ ID NO: 33; and (iv) amino acids 24-192 of SEQ ID NO: 33;
a second polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 21; (ii) amino acids 1-77 of SEQ ID NO: 21; (iii) amino acids 106-225 of SEQ ID NO:21; (iv) amino acids 272-360 of SEQ ID NO:21; and (v) amino acids 106-360 of SEQ ID NO:21; and
a third polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 68, (ii) amino acids 1-138 of SEQ ID NO: 68; (iii) amino acids 285-363 of SEQ ID NO: 68; (iv) amino acids 210-363 of SEQ ID NO: 68; (v) amino acids 210-444 of SEQ ID NO: 68; and (vi) amino acids 285-444 of SEQ ID NO: 68.
64. The composition of any one of claim 50-63, wherein said amino acid sequence is 90% identical to the reference amino acid sequence.
65. The composition of any one- of claim 50-64, wherein said amino acid sequence is 95% identical to the reference amino acid sequence.
66. The composition of any one of claim 50-65, wherein said amino acid sequence is identical to the reference amino acid sequence.
67. The composition of any one of claim 50-66, further comprising a polysaccharide.
68. The composition of claim 67, wherein said polysaccharide is derived from Group B Streptococcus.
69. The composition of any one of claim 50-68, further comprising a carrier.
70. The composition of any one of claim 50-69, further comprising an adjuvant.
71. The composition of claim 70, wherein said adjuvant is selected from the group consisting of mLT, MF59™, SAF, Ribi™ adjuvant system, monophospholipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), Detox™, QS21, Stimulon™, complete Freund's adjuvant, incomplete Freund's adjuvant, cytokines, interferons, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), monophosphoryl lipid A (MPL), 3-O-deacylated MPL, a CpG oligonucleotide, a saponin, a polyoxyetihylene ether, a polyoxyethylene ester, and aluminium salts, and a combination of more than one adjuvants.
72. The composition of claim 71, wherein said adjuvant is Alum and a CpG polynucleotide.
73. The composition of any one of claim 50-72, further comprising a hormone, a vitamin, a polyakylene, a lipid, a liposome, a lipoprotein, or a polycationic agent or a combination thereof.
74. The composition of any one of claim 50-73, which induces an immune response when administered in an animal.
75. The composition of claim 74, wherein said immune response is a humoral immune response or a cell-mediated immune response.
76. The composition of claim 75, wherein said immune response is a humoral immune response.
77. The composition of claim 76, wherein said immune response is an antibody response.
78. A composition comprising two or more of isolated polypeptides, wherein said polypeptide comprises an amino acid sequence at least 80% identical to a reference amino acid sequence selected from the group consisting of Pho 1-13, Pho 1-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
79. The composition of claim 78, wherein said amino acid sequence is identical with the reference amino acid.
80. A composition comprising two or more of polynucleotides, wherein said polynucleotide comprises a nucleic acid sequence at least 80% identical to a reference nucleic acid sequence encoding an amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polynucleotide encodes a polypeptide recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
81. The composition of claim 80, wherein said nucleic acid sequence is identical with the reference nucleic acid.
82. A composition comprising two or more of vectors, wherein said vector encodes a polypeptide comprising an amino acid sequence at least 80% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3- 29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
83. A composition comprising two or more of host cells, wherein said host cell comprises a vector encoding a polypeptide comprising an ammo acid sequence at least 80% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
84. A composition comprising isolated antibodies raised against two or more of polypeptides, wherein said polypeptide comprises an ammo acid sequence at least 80% identical to a reference amino acid sequence selected from group consisting of Phol-13, Phol-14, Phol-5, Pho3-3 (phol-7), Pho2-10, Pho2-15, Pho2-2 (ME:P22), Pho3-14, Pho3-17, Pho3-18, Pho3-1 (ME:P31), Pho3-21, Pho3-22, Pho3-23, Pho3-24, Pho3-29, Pho3-50, MS4, MSI l, MS14, MSlO(NP GAP, ME:NAP), PBP, pho 3-9, OCT, PGK, and NPGAP, wherein said polypeptide is recognizable by an antibody preparation that specifically binds to a polypeptide consisting of the reference amino acid sequence.
85. The composition of any one of claim 78-84, further comprising a earner.
86. The composition of any one of claim 78-85, further comprising an adjuvant.
87. The composition of any one of claim 78-86, further comprising a targeting molecule.
88. The composition of claim 78, which comprises two polypeptides, wherein said polypeptide comprises an amino acid sequence at least 80% identical to the reference amino acid sequence of Pho2-2 (ME:P22) or Pho3-1.
89. The composition of claim 78, which comprises two polypeptides, wherein said polypeptide comprises an amino acid sequence at least 80% identical to the reference amino acid sequence of Pho2-2 (ME:P22) and MSlO (NP GAP, ME:NAP).
90. The composition of claim 78, which comprises two polypeptides, wherein said polypeptide comprises an amino acid sequence at least 80% identical to the reference amino acid sequence of Pho3-1 (ME:P31), MSlO(NP GAP, ME:NAP).
91. The composition of claim 78, which comprises three polypeptides, wherein said polypeptide comprises an amino acid sequence at least 80% identical to the reference amino acid sequence of Pho2-2 (ME:P22), Pho3-1, or MSlO (NP GAP, ME:NAP).
92. The composition of any one of claim 78-91, which induces a humoral immune response (HIR) or cell mediated immune response (CMI) against said polypeptide when administered to a mammal or a bird.
93. The composition of any one of claim 78-91, which induces both a humoral immune response (HIR) and cell mediated immune response (CMI) against said polypeptide when administered to a mammal or a bird.
94. A diagnostic reagent comprising the composition of any one of claim 78-93.
95. A method of detecting anti -Group B Streptococcus antibodies in a test sample comprising the steps of
(a) contacting said sample with the composition of any one of claim 78-93 to form Group B Streptococcus antigen: anti-Group B Streptococcus antibody immunocomplexes; and
(b) detecting the presence of or measuring the amount of said immunocomplexes formed during step (a).
96. A diagnostic kit for detecting an antibody or antigen-binding fragment thereof to Group B Streptococcus, wherein said kit comprises the composition of any one of claim 78-93, a container means for contacting said polypeptide or composition with a test sample, and a reagent means for detecting or measuring Group B Streptococcus antigen: anti-Group B Streptocuccus antibody immunocomplexes formed between said polypeptide and said antibody or antigen-binding fragment thereof.
97. A method of producing an immune response in a host comprising administering to said host an effective amount of the composition of any one of claim 78-93.
98. The method of claim 97, wherein said immune response is a humoral immune response.
99. The method of claim 98, wherein said immune response is a cell-mediated immune response.
100. The method of any one of claim 97-99, wherein the host is a mammal.
101. A method for the treatment or prevention of a condition associated with Group B Streptococcal infection in a host comprising administering to said host an effective amount of the composition of any one of claim 78-93.
102. The method of claim 101, wherein the infection is a focal infection.
103. The method of claim 101, wherein the infection is a urinary tract infection.
104. The method of any one of claim 97-103 wherein the host is a mammal.
PCT/IB2007/004637 2006-12-21 2007-12-21 Streptococcus proteins, and their use in vaccination WO2008152448A2 (en)

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