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WO2011067758A2 - Fragments immunogènes et multimères pour protéines de streptococcus pneumoniae - Google Patents

Fragments immunogènes et multimères pour protéines de streptococcus pneumoniae Download PDF

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Publication number
WO2011067758A2
WO2011067758A2 PCT/IL2010/001009 IL2010001009W WO2011067758A2 WO 2011067758 A2 WO2011067758 A2 WO 2011067758A2 IL 2010001009 W IL2010001009 W IL 2010001009W WO 2011067758 A2 WO2011067758 A2 WO 2011067758A2
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WIPO (PCT)
Prior art keywords
polypeptide
pneumoniae
protein
seq
adjuvant
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PCT/IL2010/001009
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English (en)
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WO2011067758A3 (fr
Inventor
Yaffa Mizrachi-Nebenzahl
Michael Tal
Ron Dagan
Maxim Portnoi
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Protea Vaccine Technologies Ltd.
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Publication of WO2011067758A2 publication Critical patent/WO2011067758A2/fr
Publication of WO2011067758A3 publication Critical patent/WO2011067758A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins

Definitions

  • the present invention relates to the immunogenic polypeptide fragments derived from Streptococcus pneumoniae (S. pneumoniae) cell wall or cell membrane proteins and to their use in protection against infection with the bacteria.
  • the present invention relates to immunogenic polypeptide fragments and multimers derived from cell wall or cell membrane proteins of S. pneumoniae which exhibit age-dependent antigenicity.
  • Streptococcus pneumoniae belongs to the commensal flora of the human respiratory tract, but can also cause invasive infections such as meningitis and sepsis. Mortality due to pneumococcal infection remains high all over the world, augmented by a wide-spread antibiotic resistance in many pneumococcal strains (Dagan et al., Pneumococcal Infections, in: Feigin R, et al, eds. Textbook of Pediatric Infectious Diseases. 5 ed. Philadelphia: Saunders Co, 2004:1204-58).
  • the current polysaccharide- based vaccines (including polysaccharide conjugates) elicit a strain-specific protection in children and the elderly, who are the main targets for pneumococcal infections.
  • the available vaccines either do not elicit long lasting protection or are limited in strain coverage.
  • Development of new preventive interventions is hampered due to the incomplete understanding of pneumococcal pathogenesis.
  • S. pneumoniae is the leading cause of non-epidemic childhood meningitis in Africa and other regions of the developing world. Approximately, one million children die from pneumococcal inflicted diseases each year. Specifically, when considering deaths of children under five years of age worldwide, about 20% are from pneumococcal pneumonia. These high morbidity and mortality rates and the persistent emergence of antibiotic-resistant strains of S.
  • the optimal anti-pneumococcal vaccine should be safe, efficacious, wide-spectrum (covering most or all pneumococcal strains), affordable, and available in large quantities.
  • Vaccination with multivalent polysaccharide conjugate vaccines has been shown to be associated with serotype replacement, whereby non-vaccine serotype strains have elevated levels of carriage in populations with reduced incidence of vaccine serotype strains, which means that the effectiveness of conjugate vaccines is expected to diminish over time.
  • the mucosal epithelial surfaces with their tight junctions constitute the first line of defense that prevents the entry of pathogens and their products.
  • S. pneumoniae adhere to the nasopharyngeal mucosal cells (Tuomanen E. 1999, Curr. Opin. Microbiol., 2:35-9), causing carriage without an overt inflammatory response.
  • S. pneumoniae have to spread from the nasopharynx into the middle ear or the lungs or cross the mucosal epithelial cell layer and be deposited basally within the submucosa (Ring et al., J. Clin. Invest. 1998, 102:347-60).
  • Molecules involved in adhesion, spread and invasion of S. pneumoniae include capsular polysaccharides, cell-wall peptidoglycan and surface proteins (Jedrzejas MJ. Microbiol. Mol. Biol. Rev. 2001, 65, 187-207).
  • pneumoniae proteins demonstrate age- dependent antigenicity. All proteins tested to date elicit a protective immune response against the bacteria. These proteins are identified for use in vaccines especially in age groups (infants) which do not produce anti-S. pneumoniae antibodies following inoculation with polysaccharide-based vaccines or who do not mount significant antibody responses to these vaccines (elderly).
  • Multi-epitope vaccines against influenza virus are disclosed in WO 2009/016639.
  • Multi-epitope DNA vaccines are discussed in Subbramanian et al. (J. Virol. 2003, 77, 10113-10118). Multivalent minigene vaccines containing B-cell, CTL and Th epitopes from several pathogens are described in Ling-Ling and Whitton (J. Virol 1997, 71 2292- 2302).
  • the present invention provides immunogenic polypeptides and vaccines against S. pneumoniae.
  • the polypeptides of the present invention are specific fragments of S. pneumoniae antigens referred to herein as age-dependent proteins.
  • the antibody response to S. pneumoniae proteins increases with age in infants, and this increase correlates with decreased morbidity. It was previously shown, using sera longitudinally collected from healthy children exposed to bacterial colonization, that there is an age-dependent enhancement of the antibody response to certain S. pneumoniae surface protein antigens. This enhancement, with age, of antibody responses against a set of specific pneumococcal surface proteins is implicated in the development of natural immunity and was used to identify candidate protein antigens (herein "age dependent proteins”) for use in vaccine compositions against the bacteria.
  • age dependent proteins candidate protein antigens
  • polypeptides of the present invention possess reduced homology to human sequences compared to the intact protein, minimizing the risk of developing autoimmunity against the patient's own proteins. Furthermore, the polypeptides of the present invention have increased sequence identity to many different S. pneumoniae strains making them ideal for wide-spectrum vaccines against the bacteria.
  • immunogenic protein fragments can be produced recombinantly, as isolated polypeptides or polypeptide-multimers, or as part of a fusion protein, or synthetically by peptide synthesis, or by linking several identical and/or different synthetic polypeptide fragments.
  • Recombinant or synthetic production can be used, according to the present invention, to introduce specific mutations and/or variations in the peptide sequence for improving specific properties such as solubility and stability.
  • a fragment of an immunogenic protein may comprise several immunogenic epitopes but lack portions of the proteins which are not immunogenic or which confer undesired properties to the protein (e.g. toxicity, binding, cross reactivity to human sequences etc.)
  • polypeptides of the present invention can be used in vaccine compositions against S. pneumoniae alone, in mixture with other immunogenic peptides, protein fragments or proteins, as part of a chimeric protein which may be used as an adjuvant, or mixed or formulated with an external adjuvant.
  • the present invention provides a synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a S. pneumoniae protein selected from the group consisting of: phosphoglucomutase/phosphomannomutase family protein (Accession No. NP_346006, SEQ ID NO:l); elongation factor G/tetracycline resistance protein (tetO), (Accession No. NP_34481 1, SEQ ID NO:2); Aspartyl/glutamyl- tRNA amidotransferase subunit C (Accession No. NP_344960, SEQ ID NO:3); L-lactate dehydrogenase (Accession No.
  • a S. pneumoniae protein selected from the group consisting of: phosphoglucomutase/phosphomannomutase family protein (Accession No. NP_346006, SEQ ID NO:l); elongation factor G/tetracycline resistance protein (tetO), (Access
  • NP 345686 SEQ ID NO:4
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • NP_346439 SEQ ID NO:5
  • UDP-glucose 4- epimerase accesion No. NP_346261, SEQ ID NO:6
  • elongation factor Tu family protein accesion No. NP_358192, SEQ ID NO:7
  • Bifunctional GMP synthase/glutamine amidotransferase protein accesion No. NP_345899, SEQ ID NO:8
  • glutamate dehydrogenase accesion No.
  • NP_345769 SEQ ID NO:9; Elongation factor TS (Accession No. NP_346622, SEQ ID NO: 10); phosphoglycerate kinase (TIGR4) (Accession No. AAK74657, SEQ ID NO: 11); 30S ribosomal protein SI (Accession No. NP_345350, SEQ ID NO: 12); 6-phosphogluconate dehydrogenase (Accession No. NP_357929, SEQ ID NO: 13); aminopeptidase C (Accession No. NP_344819, SEQ ID NO: 14); carbamoyl-phosphate synthase (large subunit) (Accession No.
  • NP_345739, SEQ ID NO: 15 PTS system, mannose-specific IIAB components (Accession No. NP_344822, SEQ ID NO: 16); 30S ribosomal protein S2 (Accession No. NP 346623, SEQ ID NO: 17); dihydroorotate dehydrogenase IB (Accession No. NP_358460, SEQ ID NO: 18); aspartate carbamoyltransferase catalytic subunit (Accession No. NP_345741, SEQ ID NO: 19); elongation factor Tu (Accession No.
  • NP_345941, SEQ ID NO:20 Pneumococcal surface immunogenic protein A (PsipA) (Accession No. NP_344634, SEQ ID NO:21); phosphoglycerate kinase (R6) (Accession No. NP_358035, SEQ ID NO:22); ABC transporter substrate-binding protein (Accession No. NP_344690, SEQ ID NO:23); endopeptidase O (Accession No. NP_346087, SEQ ID NO:24); Pneumococcal surface immunogenic protein C (PsipC) (Accession No. NP_345081, SEQ ID NO:25), and variants and analogs thereof.
  • PsipA Pneumococcal surface immunogenic protein A
  • R6 phosphoglycerate kinase
  • R6 phosphoglycerate kinase
  • R6 phosphoglycerate kinase
  • the synthetic or recombinant polypeptide of 51 - 250 amino acids is selected from the group consisting of SEQ ID NOS: 26-75.
  • the polypeptide consists of 101-250 amino acids. According to other embodiments the polypeptide consists of 51- 100 amino acids.
  • a polypeptide according to the present invention consists of a sequence selected from SEQ ID NO:26-75. According to some embodiments, the polypeptides of the present invention share less than 30% sequence identity with the sequence of the homologous human proteins. According to other embodiments, the polypeptides according to the invention share less than 10%) sequence identity with such human proteins. According to yet another embodiment, when aligning the sequence of a polypeptide according to the invention with the corresponding sequence of a human protein, no more than nine contiguous amino acid residues are identical between the two sequences.
  • Variants of the peptides of the present invention include substitution of one amino acid residue per maximum of each contiguous sequence of nine amino acid residues in a peptide sequence, namely, peptides having about 90% or more identity are included within the scope of the present invention. According to other embodiments, sequences having at least 97% identity to the peptides of the present invention are provided.
  • the present invention provides a synthetic or recombinant polypeptide comprising at least one polypeptide fragment of 51-250 amino acids, derived from the sequence of an S. pneumoniae protein associated with an age-dependent immune response, wherein the peptide sequence of 51-250 amino acids is selected from the group consisting of:
  • polypeptide is a separate embodiment of the invention.
  • the present invention provides a synthetic or recombinant peptide of 51-100 amino acids selected from the group of SEQ ID NOS: 26, 27, 28, 29, 30, 31, 36, 37, 39, 40, 43, 44, 45, 48, 49, 52, 53, 56, 57, 59, 60, 61, 64, 65, 66, 67, 69, 70, 71, 72, and 75.
  • the present invention provides a synthetic or recombinant peptide of 101-250 amino acids selected from the group of SEQ ID NOS: 32, 33, 34, 35, 38, 41, 42, 46, 47, 50, 51, 54, 58, 62, 63, 68, 73, and 74.
  • the present invention provides, according to some specific embodiments, a synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a Streptococcus pneumoniae (S. pneumoniae) cell wall or cell membrane protein associated with an age-dependent immune response, wherein the cell wall or cell membrane protein associated with an age-dependent immune response is selected from the group consisting of SEQ ID NO:l to SEQ ID NO:25 and the synthetic or recombinant polypeptide is selected from SEQ ID NO: 26 to SEQ ID NO:75, and variants and analogs having at least about 90% sequence identity, or at least about 97% identity to said synthetic or recombinant polypeptide.
  • S. pneumoniae Streptococcus pneumoniae
  • the present invention provides a synthetic or recombinant polypeptide (herein denoted "multimer") comprising a plurality of S. pneumoniae derived polypeptide fragments.
  • the multimer may contain a plurality of repeats not necessarily adjacent, of a specific fragment, a plurality of different fragments, from same or different protein, a plurality of repeats of a plurality of fragments, or a combination of any of these options.
  • a plurality according to the present invention means that at least two copies of & pneumoniae derived polypeptide or polypeptides fragment or fragments are present in a single polypeptide-based multimer construct.
  • the synthetic or recombinant multimer comprises a sequence selected from SEQ ID NOs: 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94.
  • a synthetic or recombinant polypeptide multimer consisting of a sequence selected from SEQ ID NOs: 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94 is provided.
  • a multimer according to some embodiments is produced as part of a fusion protein comprising a carrier sequence which may, according to some embodiments, serve as an adjuvant.
  • the adjuvant provides a scaffold for better expression of the polypeptides.
  • the adjuvant provides T-helper epitopes to the expressed polypeptides.
  • the adjuvant or formulation has properties of a delivery system.
  • the fusion protein comprises detoxified pneumolysin or a fragment thereof.
  • the fusion protein comprises heat shock protein 60 (hsp60) or a fragment thereof
  • the present invention comprises a multimer comprising multiple copies of a plurality of different S. pneumoniae derived fragments, providing a high -density vaccine.
  • the multimer can be produced recombinantly, as an isolated polypeptide or as a fusion protein, or synthetically by linking a plurality of synthetic polypeptide fragments, or can be mixed or formulated with an external adjuvant and/or delivery system.
  • the present invention provides a synthetic or recombinant multimer comprising multiple copies of a plurality of S. pneumoniae derived fragments arranged in an alternating sequential polymeric structure ( ⁇ 2 ⁇ 3 ⁇ ..) ⁇ or in a block copolymer structure (Xi) n (X2)n(3 ⁇ 4)n—(Xm)n-
  • a synthetic or recombinant multimer according to the present invention is selected according to a specific embodiment from the group consisting of: i. B(XiZX 2 Z ...X m ) n B; and ii. B(Xi) n Z(X 2 ) n Z...(X m ) n B;
  • B is an optional sequence of 1-4 amino acid residues; n is at each occurrence independently an integer of 2-4; m is an integer of 2-4; each of X ⁇ , X 2 ...X m is an immunogenic S. pneumoniae derived fragment consisting of 51-250 amino acid residues; Z at each occurrence is a bond or a spacer of 1-20 amino acid residues, and wherein the maximal number of amino acid residues in the multimer is about 1000.
  • the spacer Z is selected from the group consisting of: Ala, Ala-Ala, Ala-Ala-Ala, Gly, Gly-Gly, Gly-Gly-Gly, Pro, Ser and Lys.
  • At least one amino acid of the spacer induces a specific conformation on a segment of the polypeptide (e.g. one or more proline residue).
  • the spacer comprises a cleavable sequence.
  • the cleavable spacer is cleaved by intracellular enzymes.
  • the cleavable spacer comprises a proteaseOspecific cleavable sequence.
  • at least one fragment or multimer of the present invention is produced as part of a fusion protein comprising a carrier sequence, namely the polypeptide sequences are inserted within a sequence of a carrier polypeptide or are fused to a free amino group or a free carboxy group of a carrier protein sequence, which according to certain embodiments is a S. pneumoniae protein or fragment.
  • the carrier protein sequence serves as an adjuvant.
  • the carrier polypeptide is selected from the group consisting of: detoxified pneumolysin, hsp60 or a fragment thereof.
  • the present invention provides, according to another aspect, isolated polynucleotide sequences encoding a polypeptide according to any one of SEQ ID NOS: 26-75.
  • the isolated polynucleotide sequence encoding a multimer according to the present invention comprises a sequence selected from SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93 and 95.
  • the present invention provides isolated polynucleotide sequences encoding a chimeric or fusion polypeptide comprising at least one peptide of SEQ ID NOS: 26-75.
  • the present invention provides vaccine compositions for immunization of a subject against S. pneumoniae comprising at least one synthetic or recombinant polypeptide of 51-250 amino acids derived from an age-dependent S. pneumoniae cell wall or cell membrane protein.
  • a vaccine composition according to the present invention further comprises at least one additional S. pneumoniae peptide, polypeptide or protein sequence.
  • the vaccine composition further comprises an adjuvant. According to other embodiments, the vaccine does not contain an adjuvant.
  • the vaccine composition further comprises a delivery system. According to other embodiments, the vaccine does not contain a delivery system.
  • compositions include, but are not limited to water-in-oil emulsion, lipid emulsion, and liposomes.
  • the adjuvant is selected from the group consisting of: CCS/C ® , Montanide ® , alum, muramyl dipeptide, Gelvac ® , chitin microparticles, chitosan, cholera toxin subunit B, labile toxin, AS21V, AS02V, Intralipid ® , and Lipofundin ® .
  • the adjuvant CCS/C ® is included in the vaccine formulation.
  • the vaccine is formulated for intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal and transdermal delivery. In some embodiments, the vaccine is formulated for intramuscular administration. In other embodiments, the vaccine is formulated for oral administration. In yet other embodiments, the vaccine is formulated for intranasal administration. According to some embodiments, formulations for various of these routes of delivery contain delivery systems such as liposomes, ISCOMs, or other macromolecular carriers.
  • the present invention provides according to a further aspect a method for inducing an immune response and conferring protection against S. pneumoniae in a subject, comprising administering a vaccine composition comprising at least one synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a cell-wall or cell-membrane protein of S. pneumoniae associated with age-dependent immune response, and variants and analogs thereof.
  • the composition comprises a multimer or a fusion polypeptide comprising at least one synthetic or recombinant polypeptide, variant or analog of 51-250 amino acids derived from the sequence of a cell-wall or cell-membrane protein of S. pneumoniae associated with age-dependent immune response.
  • the route of administration of the vaccine is selected from intramuscular, oral, intranasal, intraperitoneal, subcutaneous, topical, intradermal, and transdermal delivery. According to preferred embodiments the vaccine is administered by intramuscular, intranasal or oral routes.
  • composition comprising at least one synthetic or recombinant S. pneumoniae derived polypeptide of 51-250 amino acids, and variants, analogs, multimers and fusion polypeptides thereof in protection against an S. pneumoniae infection in a subject is provided.
  • a peptide or polypeptide derived from the sequence of an age-dependent cell-wall or cell-membrane protein of S. pneumoniae, and variants and analogs thereof, for preparation of a vaccine composition for immunization against S. pneumoniae is also within the scope of the present invention. Further aspects provide use of an isolated polynucleotide according to the invention for production of a polypeptide of 51-250 amino acids, and variants, analogs, multimers and fusion polypeptides thereof, and for vaccination against an S. pneumoniae infection in a subject.
  • FIG. 1 Colonization of CBA/N xid mice following immunization with PS20 emulsified with CFA as adjuvant.
  • the antigens were emulsified with CFA as an adjuvant in the first immunization and with IFA in the two following immunizations.
  • Mice were subsequently challenged intranasally (IN) with S. pneumoniae serotype 3 strain WU2 (7.5* 10 5 CFU per mouse).
  • mice Forty eight hours later, mice were sacrificed and nasopharynx (A) and right lobe-lung (B) of each mouse were homogenized and plated on blood agar for bacterial enumeration. Asterisks represent significant values (p value ⁇ 0.05) compared to control mice immunized with adjuvant only, as indicated by student t-test.
  • FIG. 1 Colonization of CBA/N xid mice following immunization with PS20 formulated in CCS/C as adjuvant.
  • Mice were also subcutaneously immunized with l( ⁇ g non-lectin protein extract of S. pneumonia serotype 3 strain WU2 cell wall fraction emulsified with CFA as a positive control.
  • the CFA emulsified group was emulsified with CFA as an adjuvant in the first immunization and with IFA in the two following immunizations. Mice were subsequently challenged IN with 5".
  • mice Forty eight hours later, mice were sacrificed and nasopharynx (A) and right lobe- lung (B) of each mouse were homogenized and plated on blood agar for bacterial enumeration. Asterisks represent significant values (p value ⁇ 0.05) compared to control mice immunized with adjuvant only, as indicated by student t-test.
  • FIG. 3 Survival rate of CBA N xid mice following immunization with PS19 with CFA as adjuvant.
  • Mice were subsequently challenged IN with a lethal dose of S. pneumoniae serotype 3 strain WU2 (3* 10 6 CFU per mouse). Survival rate were monitored daily in the following 7 days. Mice that survived for more than 7 days were marked as alive.
  • FIG. 4 Survival rate of CBA/N xid mice following immunization with PS19 with CCS/C as adjuvant.
  • Mice were subsequently challenged IN with a lethal dose of S. pneumoniae serotype 3 strain WU2 (3 * 10 6 CFU per mouse). Survival rates were monitored daily in the following 7 days. Mice that survived for more than 7 days were marked as alive.
  • FIG. 5 Colonization of CBA/N xid mice following immunization with PS19 formulated in CCS/C as adjuvant.
  • Mice were subsequently challenged IN with S. pneumoniae serotype 3 strain WU2 (1.9* 10 6 CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx (A) and right lobe-lung (B) of each mouse were homogenized and plated on blood agar for bacterial enumeration.
  • Asterisks represent significant values (p value ⁇ 0.05) compared to control mice immunized with adjuvant only, as indicated by student t-test.
  • Novel therapeutic strategies are necessary to counter the prevalence of antibiotic- resistant pneumococci and the limitations of currently available vaccines.
  • Future discovery of therapeutic modalities requires a better understanding of the dynamic interplay between pathogen and host, which leads either to S. pneumoniae clearance or to carriage and disease development. It is suspected that inappropriate or altered immune responses underlie the switch from benign carriage to clinical disease. It has been observed in infants that the antibody response and antibody levels to S. pneumoniae increase with age and correlates negatively with morbidity.
  • the development of a universal vaccine against S. pneumoniae will prevent replacement carriage and disease development, caused by serotypes not included in the conjugate vaccine observed following immunization with the polysaccharide conjugate vaccine. Furthermore, such a vaccine may be used in subjects previously immunized with the polysaccharide vaccine.
  • a cell wall fraction was extracted from S. pneumoniae.
  • the proteins (around 150) in the cell wall fraction were screened by 2D-immunoblotting using sera obtained longitudinally from children attending day care centers and sera from healthy adult volunteers.
  • membrane extracts were resolved by 2D-PAGE and screened with sera obtained longitudinally from children attending day care centers and sera from healthy adult. Thirty eight proteins exhibited age-dependent antigenicity and are therefore denoted "age-dependent”.
  • the sequences of the age-dependent proteins were determined and the proteins were identified (for example SEQ ID NOS 1-25).
  • the polypeptides of the present invention have the advantage of reduced homology or no to human sequences. If a microbial antigen has significant sequence homology to a human protein, then use of such an antigen in a vaccine would entail the risk of eliciting autoimmune responses directed against the particular human protein - an unacceptable outcome. Therefore, it is very important to remove any such sequences - homologous between the microbial antigen and the human protein - from the antigen in order that it would have utility as a vaccine.
  • antigen presentation means the expression of antigen on the surface of a cell in association with major histocompatibility complex class I or class II molecules (MHC-I or MHC-II) of animals or with the HLA-I and HLA-II of humans.
  • MHC-I or MHC-II major histocompatibility complex class I or class II molecules
  • immunogenicity or “immunogenic” relates to the ability of a substance to stimulate or elicit an immune response. Immunogenicity is measured by determining the ability to produce antibodies specific for the substance. The presence of antibodies is detected by methods known in the art, for example using ELISA.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules.
  • a "chimeric protein/polypeptide” or a “fusion protein/polypeptide” are used interchangeably and refer to an immunogenic peptide or peptides operatively linked to a polypeptide or protein.
  • a “multimer” refers to a construct comprising at least two covalently linked, immunogenic protein fragments or analogs thereof according to the invention.
  • the at least two protein fragments or analogs thereof may be identical or different, and the multimer may include at least one sequence of a carrier protein or a protein fragment which is optional functionalized as an adjuvant.
  • polypeptides of the present invention may be synthesized chemically using methods known in the art for synthesis of peptides and polypeptides. These methods generally rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis.
  • polypeptide indicates a sequence of amino acids linked by peptide bonds.
  • a polypeptide is generally a peptide of about 51 or more amino acids.
  • Polypeptide analogs and peptidomimetics are also included within the scope of the invention as well as salts and esters of the polypeptides of the invention.
  • a polypeptide analog according to the present invention may optionally comprise at least one non-natural amino acid and/or at least one blocking group at either the C terminus or N terminus.
  • Salts of the polypeptides of the invention are physiologically acceptable organic and inorganic salts. The design of appropriate "analogs" may be computer assisted.
  • peptidomimetic means that a polypeptide sequence according to the invention is modified in such a way that it includes at least one non-peptidic bond such as, for example, urea, carbamate, sulfonamide, hydrazine, or any other covalent bond.
  • non-peptidic bond such as, for example, urea, carbamate, sulfonamide, hydrazine, or any other covalent bond.
  • the design of appropriate "peptidomimetic" may be computer assisted.
  • Salts and esters of the polypeptides of the invention are encompassed within the scope of the invention.
  • Salts of the polypeptides of the invention are physiologically acceptable organic and inorganic salts.
  • Functional derivatives of the polypeptides of the invention covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the polypeptide and do not confer toxic properties on compositions containing it.
  • These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.
  • acyl moieties e.g., alkanoyl or carbocyclic aroyl groups
  • O-acyl derivatives of free hydroxyl group for example that of seryl or threonyl residues
  • amino acid refers to compounds which have an amino group and a carboxylic acid group, preferably in a 1,2- 1,3-, or 1,4- substitution pattern on a carbon backbone.
  • oc-Amino acids are most preferred, and include the 20 natural amino acids (which are L-amino acids except for glycine) which are found in proteins, the corresponding D-amino acids, the corresponding N-methyl amino acids, side chain modified amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy-proline, 5 -hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, ⁇ -cyanolanine), and synthetically derived -amino acids, such as amino- isobutyric acid, norleucine, norvaline, homocysteine and homoserine.
  • ⁇ -Alanine and ⁇ - amino butyric acid are examples of 1,3 and 1,4-amino acids, respectively, and many others are well known to the art.
  • Statine-like isosteres a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOH
  • hydroxyethylene isosteres a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOHCH 2
  • reduced amide isosteres a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CH 2 NH linkage
  • thioamide isosteres are also useful residues for this invention.
  • amino acids used in this invention are those that are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention.
  • Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used.
  • Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention, as long as antigenicity is preserved in the substituted peptide.
  • Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged.
  • substitutions may enhance oral bioavailability, penetration into the central nervous system, targeting to specific cell populations and the like.
  • substitutions may enhance oral bioavailability, penetration into the central nervous system, targeting to specific cell populations and the like.
  • One of skill in the art will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • polypeptides of the present invention can be prepared by expression from an expression vector per se or as a chimeric protein.
  • the methods to produce a chimeric or recombinant protein comprising one or more peptides derived from age-dependent proteins of S. pneumoniae are known to those with skill in the art.
  • a nucleic acid sequence encoding one or more polypeptide comprising at least one such peptide can be inserted into an expression vector for preparation of a polynucleotide construct for propagation and expression in host cells.
  • expression vector and "recombinant expression vector” as used herein refers to a DNA molecule, for example a plasmid or virus, containing a desired and appropriate nucleic acid sequences necessary for the expression of the recombinant polypeptides for expression in a particular host cell.
  • operably linked refers to a functional linkage of at least two sequences. Operably linked includes linkage between a promoter and a second sequence, for example, a nucleic acid of the present invention, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • the regulatory regions necessary for transcription of the polypeptides can be provided by the expression vector.
  • the precise nature of the regulatory regions needed for gene expression may vary among vectors and host cells.
  • a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence.
  • Regulatory regions may include those 5' non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
  • a translation initiation codon (ATG) may also be provided.
  • linkers or adapters providing the appropriate compatible restriction sites are added during synthesis of the nucleic acids.
  • a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.
  • An alternative method to PCR is the use of synthetic gene.
  • the method allows production of an artificial gene which comprises an optimized sequence of nucleotides to be expressed in cells of a desired species (for example, E. coli).
  • a desired species for example, E. coli.
  • Redesigning a gene offers a means to improve gene expression in many cases. Rewriting the open reading frame is possible because of the redundancy of the genetic code. Thus it is possible to change up to about one-third of the nucleotides in an open reading frame and still produce the same protein.
  • optimization methods such as replacing rarely used codons with more common codons can result in dramatic effects. Further optimizations such as removing RNA secondary structures can also be included.
  • Computer programs are available to perform these and other simultaneous optimizations.
  • a well optimized gene can dramatically improve protein expression. Because of the large number of nucleotide changes made to the original DNA sequence, the only practical way
  • An expression construct comprising a polypeptide sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of polypeptide per se or as a recombinant fusion protein.
  • the expression vectors that may be used include but are not limited to plasmids, cosmids, phage, phagemids or modified viruses.
  • such expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the desired gene sequence, and one or more selection markers.
  • the recombinant polynucleotide construct comprising the expression vector and a polypeptide according to the invention should then be transferred into a host cell where it can replicate (for example a bacterial cell), and then be transfected and expressed in an appropriate prokaryotic or eukaryotic host cell.
  • a host cell where it can replicate (for example a bacterial cell), and then be transfected and expressed in an appropriate prokaryotic or eukaryotic host cell.
  • the expression vector is used with a compatible prokaryotic or eukaryotic host cell which may be derived from bacteria, yeast, insects, mammals and humans.
  • the polypeptide or multimer can be separated from undesired components by a number of protein purification methods.
  • One such method uses a polyhistidine tag on the recombinant protein.
  • a polyhistidine-tag consists in at least six histidine (His) residues added to a recombinant protein, often at the N- or C-terminus.
  • Polyhistidine-tags are often used for affinity purification of polyhistidine- tagged recombinant proteins that are expressed in E. coli or other prokaryotic expression systems.
  • the bacterial cells are harvested by centrifugation and the resulting cell pellet can be lysed by physical means or with detergents or enzymes such as lysozyme.
  • the crude lysate contains at this stage the recombinant protein among several other proteins derived from the bacteria and are incubated with affinity media such as NTA-agarose, HisPur resin or Talon resin.
  • affinity media such as NTA-agarose, HisPur resin or Talon resin.
  • These affinity media contain bound metal ions, either nickel or cobalt to which the polyhistidine-tag binds with micromolar affinity.
  • the resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion.
  • the washing efficiency can be improved by the addition of 20 mM imidazole, and proteins are then usually eluted with 150-300 mM imidazole.
  • the polyhistidine tag may be subsequently removed using restriction enzymes, endoproteases or exoproteases. Kits for the purification of histidine-tagged proteins can be purchased for example from Qiagen.
  • inclusion bodies are aggregates of protein that may form when a recombinant polypeptide is expressed in a prokaryote. While the cDNA may properly code for a translatable mRNA, the protein that results may not fold correctly or completely, or the hydrophobicity of the sequence may cause the recombinant polypeptide to become relatively insoluble.
  • Inclusion bodies are easily purified by methods well known in the art. Various procedures for the purification of inclusion bodies are known in the art. In some embodiments the inclusion bodies are recovered from bacterial lysates by centrifugation and are washed with detergents and chelating agents to remove as much bacterial protein as possible from the aggregated recombinant protein.
  • the washed inclusion bodies are dissolved in denaturing agents and the released protein is then refolded by gradual removal of the denaturing reagents by dilution or dialysis (as described for example in Molecular cloning: a laboratory manual, 3rd edition, Sambrook, J. and Russell, D. W., 2001 ; CSHL Press).
  • proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column.
  • proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis.
  • proteins may be separated by polarity/hydrophobicity via high pressure liquid chromatography or reversed-phase chromatography.
  • proteins are often purified on a small scale by using 20- PAGE and are then analyzed by peptide mass fingerprinting to establish the protein identity. The purified protein is tracked by its molecular mass or other methods known in the art.
  • the amount of the specific protein has to be compared to the amount of total protein.
  • the latter can be determined by the Bradford or Lowry total protein assay or by absorbance of light at 280 nm; however some reagents used during the purification process may interfere with accurate quantification.
  • imidazole commonly used for purification of polyhistidine-tagged recombinant proteins
  • BCA bicinchoninic acid
  • SPR Surface Plasmon Resonance
  • SPR can detect binding of label free molecules on the surface of a chip. If the desired protein is an antibody, binding can be translated directly to the activity of the protein. One can express the active concentration of the protein as the percent of the total protein. SPR can be a powerful method for quickly determining protein activity and overall yield.
  • the vaccines of the present invention comprise at least one immunogenic polypeptide derived from S. pneumoniae age-dependent proteins, and optionally, an adjuvant and/or delivery system.
  • Formulation can contain one or more of a variety of additives, such as adjuvant, delivery system excipient, stabilizers, buffers, or preservatives.
  • the vaccine can be formulated for administration in one of many different modes.
  • the vaccine is formulated for parenteral administration, for example intramuscular administration.
  • the vaccine is formulated for oral administration.
  • the vaccine is formulated for intradermal administration. Needles specifically designed to deposit the vaccine intradermally are known in the art, as disclosed for example in US 6,843,781 and US 7,250,036 among others. According to other embodiments, administration is performed with a needleless injector.
  • the vaccine is formulated for intranasal administration.
  • the vaccine formulation may be applied to the mucosal tissue of the nose in any convenient manner. However, it is preferred to apply it as a liquid stream or liquid droplets to the walls of the nasal passage.
  • the intranasal composition can be formulated, for example, in liquid form as nose drops, spray, or suitable for inhalation, as powder, as cream, or as emulsion.
  • the vaccine is formulated for oral administration; the vaccine may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule.
  • Liposomes provide another delivery system for antigen delivery and presentation.
  • Liposomes are bilayered vesicles composed of phospholipids and other sterols surrounding a typically aqueous center where antigens or other products can be encapsulated.
  • the liposome structure is highly versatile with many types ranging from about 25 nm to about 500 ⁇ in size. Liposomes have been found to be effective in delivering therapeutic agents to dermal and mucosal surfaces. Liposomes can be further modified for targeted delivery by, for example, incorporating specific antibodies into the surface membrane, or altered to encapsulate bacteria, viruses or parasites.
  • Liposomes may be unilamellar or multilamellar. Liposomes may have diverse ionic charges.
  • the vaccine composition may be formulated by: encapsulating an antigen or an antigen/adjuvant complex in liposomes to form liposome-encapsulated antigen and mixing the liposome-encapsulated antigen with a carrier comprising a continuous phase of a hydrophobic substance. If an antigen/adjuvant complex is not used in the first step, a suitable adjuvant may be added to the liposome-encapsulated antigen, to the mixture of liposome-encapsulated antigen and carrier, or to the carrier before the carrier is mixed with the liposome-encapsulated antigen. The order of the process may depend on the type of adjuvant used.
  • the adjuvant and the antigen are mixed first to form an antigen/adjuvant complex followed by encapsulation of the antigen/adjuvant complex with liposomes.
  • the resulting liposome- encapsulated antigen is then mixed with the carrier.
  • liposome-encapsulated antigen may refer to encapsulation of the antigen alone or to the encapsulation of the antigen/adjuvant complex depending on the context. This promotes intimate contact between the adjuvant and the antigen and may, at least in part, account for the immune response when alum is used as the adjuvant.
  • the antigen may be first encapsulated in liposomes and the resulting liposome-encapsulated antigen is then mixed into the adjuvant in a hydrophobic substance.
  • antigen or antigen/adjuvant complex is encapsulated with liposomes and mixed with a hydrophobic substance.
  • the antigen or antigen/adjuvant complex is encapsulated with liposomes in an aqueous medium followed by the mixing of the aqueous medium with a hydrophobic substance.
  • the aqueous medium containing the liposomes may be added in aliquots with mixing to the hydrophobic substance.
  • the liposome-encapsulated antigen may be freeze- dried before being mixed with the hydrophobic substance or with the aqueous medium as the case may be.
  • an antigen/adjuvant complex may be encapsulated by liposomes followed by freeze-drying.
  • the antigen may be encapsulated by liposomes followed by the addition of adjuvant then freeze-drying to form a freeze-dried liposome-encapsulated antigen with external adjuvant.
  • the antigen may be encapsulated by liposomes followed by freeze-drying before the addition of adjuvant. Freeze-drying may promote better interaction between the adjuvant and the antigen resulting in a more efficacious vaccine, as well as maintenance of stability.
  • Formulation of the liposome-encapsulated antigen into a hydrophobic substance may also involve the use of an emulsifier to promote more even distribution of the liposomes in the hydrophobic substance.
  • Typical emulsifiers are well-known in the art and include mannide oleate (ArlacelTM A), lecithin, TweenTM 80, SpansTM 20, 80, 83 and 85.
  • the emulsifier is used in an amount effective to promote even distribution of the liposomes.
  • the volume ratio (v/v) of hydrophobic substance to emulsifier is in the range of about 5 : 1 to about 15: 1.
  • Microparticles and nanoparticles employ small biodegradable spheres which act as depots for vaccine delivery.
  • the major advantage that polymer microspheres possess over other depot-effecting adjuvants is that they are extremely safe and have been approved by the Food and Drug Administration in the US for use in human medicine as suitable sutures and for use as a biodegradable drug delivery system (Langer R. Science. 1990; 249(4976): 1527-33).
  • the rates of copolymer hydrolysis are very well characterized, which in turn allows for the manufacture of microparticles with sustained antigen release over prolonged periods of time (O'Hagen, et al., Vaccine. 1993;l l(9):965-9).
  • CCS/C ® is a synthetic polycationic sphingolipid derived from D-erythro ceramide to which spermine is covalently attached, thereby forming Ceramide Carbamoyl Spermine (CCS).
  • CCS Ceramide Carbamoyl Spermine
  • CCS/C Ceramide Carbamoyl Spermine
  • VaxiSome Based on its structure and components (ceramide, C0 2 and spermine), CCS is predicted to be biocompatible and biodegradable. In vitro and in vivo studies suggest that the CCS/C formulation up-regulates levels of CD40 and B7 co- stimulatory molecules, which are essential in antigen presentation and T-helper cell activation. As a result, VaxiSome is a potent liposomal adjuvant/delivery system for stimulating enhanced immune responses via the Thl and Th2 pathways.
  • microparticles elicits long-lasting immunity, especially if they incorporate prolonged release characteristics.
  • the rate of release can be modulated by the mixture of polymers and their relative molecular weights, which will hydrolyze over varying periods of time.
  • the formulation of different sized particles (1 ⁇ to 500 ⁇ ) may also contribute to long- lasting immunological responses since large particles must be broken down into smaller particles before being available for macrophage uptake. In this manner a single- injection vaccine could be developed by integrating various particle sizes, thereby prolonging antigen presentation.
  • an adjuvant or excipient may be included in the vaccine formulation.
  • Alum for example, is a preferred adjuvant for human use.
  • the choice of the adjuvant will be determined in part by the mode of administration of the vaccine.
  • One preferred mode of administration is intramuscular administration.
  • Another preferred mode of administration is intranasal administration.
  • intranasal adjuvants include chitosan powder, PLA and PLG microspheres, QS-21, AS02V, calcium phosphate nanoparticles (CAP); mCTA/LTB (mutant cholera toxin E112K with pentameric B subunit of heat labile enterotoxin), and detoxified E. coli derived heat- labile toxin.
  • the adjuvant used may also be, theoretically, any of the adjuvants known for peptide- or protein-based vaccines.
  • inorganic adjuvants in gel form aluminium hydroxide/aluminium phosphate, Warren et al., 1986; calcium phosphate, Relyvelt, 1986
  • bacterial adjuvants such as monophosphoryl lipid A (Ribi, 1984; Baker et al., 1988) and muramyl peptides (Ellouz et al., 1974; Allison and Byars, 1991; Waters et al., 1986)
  • particulate adjuvants such as the so-called ISCOMS ("immunostimulatory complexes", Mowat and Donachie, 1991 ; Takahashi et al., 1990; Thapar et al., 1991), liposomes (Mbawuike et al.
  • adjuvants based on oil emulsions and emulsifiers such as MontanideTM (Incomplete Freund's adjuvant, Stuart-Harris, 1969; Warren et al., 1986), SAF (Allison and Byars, 1991), saponins (such as QS-21 ; Newman et al., 1992), squalene/squalane (Allison and Byars, 1991); synthetic adjuvants such as non-ionic block copolymers (Hunter et al., 1991), muramyl peptide analogs (Azuma, 1992), synthetic lipid A (Warren et al., 1986; Azuma, 1992), synthetic polynucleotides (Harrington et al., 1978) and polycationic adjuvants (WO 97/30721).
  • MontanideTM Incomplete Freund's adjuvant, Stuart-Harris, 1969; Warren et al., 1986
  • SAF Allison and Byars, 1991
  • Adjuvants for use with immunogens of the present invention include aluminum or calcium salts (for example hydroxide or phosphate salts).
  • a particularly preferred adjuvant for use herein is an aluminum hydroxide gel such as AlhydrogelTM.
  • Calcium phosphate nanoparticles (CAP) is another potential adjuvant.
  • the immunogen of interest can be either coated to the outside of particles, or encapsulated on the inside (He et al., 2000, Clin. Diagn. Lab. Immunol., 7,899-903).
  • a contemplated emulsion can be an oil-in-water emulsion or a water-in-oil emulsion.
  • emulsions comprise an oil phase of squalene, squalane, peanut oil or the like, as are well known, and a dispersing agent.
  • Non-ionic dispersing agents are preferred, and such materials include mono- and di-C 12 -C 24 -fatty acid esters of sorbitan and mannide such as sorbitan mono-stearate, sorbitan mono-oleate and mannide mono-oleate.
  • Such emulsions are for example water-in-oil emulsions that comprise squalene, glycerol and a surfactant such as mannide mono-oleate (ArlacelTM A), emulsified with the chimer protein particles in an aqueous phase.
  • a surfactant such as mannide mono-oleate (ArlacelTM A)
  • Alternative components of the oil-phase include alpha-tocopherol, mixed-chain di- and tri-glycerides, and sorbitan esters.
  • Well- known examples of such emulsions include MontanideTM ISA-720, and MontanideTM ISA 703 (Seppic, Castres, France.
  • Other oil-in-water emulsion adjuvants include those disclosed in WO 95/17210 and EP 0 399 843.
  • small molecule adjuvants are also contemplated herein.
  • One type of small molecule adjuvant useful herein is a 7-substituted-8-oxo- or 8-sulfo-guanosine derivative described in U.S. Pat. No. 4,539,205, U.S. Pat. No. 4,643,992, U.S. Pat. No. 5,01 1,828 and U.S. Pat. No. 5,093,318. 7-allyl-8-oxoguanosine(loxoribine) has been shown to be particularly effective in inducing an antigen-(immunogen-) specific response.
  • a useful adjuvant includes monophosphoryl lipid A (MPL ® ), 3-deacyl monophosphoryl lipid A (3D-MPL ® ), a well-known adjuvant manufactured by Corixa Corp. of Seattle, formerly Ribi Immunochem, Hamilton, Mont.
  • the adjuvant contains three components extracted from bacteria: monophosphoryl lipid (MPL) A, trehalose dimycolate (TDM), and cell wall skeleton (CWS) (MPL+TDM+CWS) in a 2% squalene/TweenTM 80 emulsion.
  • This adjuvant can be prepared by the methods taught in GB 2122204B.
  • MPL ® adjuvant called aminoalkyl glucosamide phosphates (AGPs) such as those available from Corixa Corp under the designation RC-529TM adjuvant ⁇ 2-[(R)-3-tetra-decanoyloxytetradecanoylamino]-ethyl- 2-deoxy-4-0-phosphono-3 -O- [(R)-3 -tetradecanoyloxytetra-decanoyl] -2- [(R)-3 -tetra- , decanoyloxytet-radecanoyl-amino]-p-D-glucopyranoside triethylammonium salt ⁇ .
  • AGPs aminoalkyl glucosamide phosphates
  • RC-529 adjuvant is available in a squalene emulsion sold as RC-529SE and in an aqueous formulation as RC-529AF available from Corixa Corp. (see, U.S. Pat. No. 6,355,257 and U.S. Pat. No. 6,303,347; U.S. Pat. No. 6,113,918; and U.S. Publication No. 03-0092643).
  • adjuvants include synthetic oligonucleotide adjuvants containing the CpG nucleotide motif one or more times (plus flanking sequences).
  • the adjuvant designated QS21 available from Aquila Biopharmaceuticals Inc., is an immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina (e.g., QuilTM A), and the method of its production is disclosed in U.S. Pat. No. 5,057,540.
  • Derivatives of QuilTM A for example QS21 (an HPLC purified fraction derivative of QuilTM) and other fractions such as QA17 are also disclosed.
  • Muramyl dipeptide adjuvants are also contemplated and include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine [CGP 11637, referred to as nor-MDP], and N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(r-2'-dipalmityol-s-n-glycero-3-hydroxyphosphoryloxy) ethylamine [(CGP) 1983 A, referred to as MTP-PE].
  • MTP-PE N-acetyl- muramyl dipeptide analogues are described in U.S. Pat. No. 4,767,842.
  • adjuvant mixtures include combinations of 3D-MPL and QS21 (EP 0 671
  • oil-in-water emulsions comprising 3D-MPL and QS21 (WO 95/17210, PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0 689 454 Bl), QS21 formulated in cholesterol-containing liposomes (WO 96/33739), or immunostimulatory oligonucleotides (WO 96/02555).
  • Adjuvant SBAS2 now AS02
  • Alternative adjuvants include those described in WO 99/52549 and non-particulate suspensions of polyoxyethylene ether (UK Patent Application No. 9807805.8).
  • an adjuvant that contains one or more agonists for toll-like receptor-4 (TLR-4) such as an MPL ® adjuvant or a structurally related compound such as an RC- 529 ® adjuvant or a Lipid A mimetic, alone or along with an agonist for TLR-9 such as a non-methylated oligodeoxynucleotide-containing the CpG motif is also optional.
  • TLR-4 toll-like receptor-4
  • a heat-shock protein, fragment or peptide is also an optional adjuvant, as a carrier protein or peptide, in a mixture, or as part of a fusion polypeptide expressed or synthesized together with at least one polypeptide according to the invention.
  • U.S. Patent Nos. 5,736,146 and 5,869,058 provide peptides derived from humans and E. coli heat-shock protein 60 (hsp60) as carriers for vaccination against viral and bacterial pathogens.
  • hsp60 E. coli heat-shock protein 60
  • Defined peptides present uniquely effective characteristics in conjugate vaccines due to the following reasons: i. HSP60 epitopes provide natural T-cell help; Humans are born with a high frequency of T cells responsive to HSP60, so no induction is needed and youngsters respond.
  • HSP60-peptide conjugates function as built-in adjuvants activating innate TLR-4 receptors on APC; the HSP60-conjugate vaccine administered in
  • HSP60-peptide conjugates do not induce the production of competing antibodies and therefore do not suppress vaccination responses, even with multiple administrations.
  • Boosting to the HSP60-epitope occurs naturally, since HSP60 is up-regulated at the site of any immune response (infection or tumor); the vaccination effect does not decline for prolonged periods. Immune memory is robust and effective.
  • Detoxified pneumolysin known as a carrier protein and as an adjuvant (for example Michon et al., Vaccine, 18, 1732-1741, 1998), or fragment or analog thereof, can be also used in conjunction or conjugation of the polypeptides of the present invention.
  • Another type of adjuvant mixture comprises a stable water-in-oil emulsion further containing aminoalkyl glucosamine phosphates such as described in U.S. Pat. No. 6,113,918.
  • aminoalkyl glucosamine phosphates such as described in U.S. Pat. No. 6,113,918.
  • the aminoalkyl glucosamine phosphates the molecule known as RC-529 ⁇ (2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl-2-deoxy-4-0-phosphono-3-0- [(R)-3-tetradecanoyloxy-tetradecanoyl]-2-[(R)-3— tetradecanoyloxytetra- decanoylamino]-p-D-glucopyranoside triethylammonium salt.) ⁇ is most preferred.
  • a preferred water-in-oil emulsion is described in
  • Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, host animal and immunogen. Typical amounts can vary from about 1 ⁇ g to about 10 mg per immunization. Those skilled in the art know that appropriate concentrations or amounts can be readily determined.
  • Vaccine compositions comprising an adjuvant based on oil in water emulsion is also included within the scope of the present invention.
  • the water-in-oil emulsion may comprise a metabolisable oil and a saponin, such as for example as described in US 7,323,182.
  • the vaccine compositions of the present invention may contain one or more adjuvants, characterized in that it is present as a solution or emulsion which is substantially free from inorganic salt ions, wherein said solution or emulsion contains one or more water soluble or water-emulsifiable substances which are capable of making the vaccine isotonic or hypotonic.
  • the water soluble or water-emulsifiable substances may be, for example, selected from the group consisting of: maltose; fructose; galactose; saccharose; sugar alcohol; lipid; and combinations thereof.
  • the polypeptides, multimers, and fusion proteins of the present invention may comprise according to several specific embodiments a proteosome adjuvant.
  • the proteosome adjuvant comprises a purified preparation of outer membrane proteins of meningococci and similar preparations from other bacteria. These proteins are highly hydrophobic, reflecting their role as transmembrane proteins and porins. Due to their hydrophobic protein-protein interactions, when appropriately isolated, the proteins form multi-molecular structures consisting of about 60-100 nm diameter whole or fragmented membrane vesicles. This liposome-like physical state allows the proteosome adjuvant to act as a protein carrier and also to act as an adjuvant.
  • proteosome adjuvant has been described in the prior art and is reviewed by Lowell GH in "New Generation Vaccines", Second Edition, Marcel Dekker Inc, New York, Basel, Hong Kong (1997) pages 193-206.
  • Proteosome adjuvant vesicles are described as comparable in size to certain viruses which are hydrophobic and safe for human use.
  • the review describes formulation of compositions comprising non-covalent complexes between various antigens and proteosome adjuvant vesicles which are formed when solubilizing detergent is selectably removed using exhaustive dialysis technology.
  • Vaccine compositions comprising different immunogenic polypeptides can be produced by mixing or linking a number of different polypeptides according to the invention with or without an adjuvant.
  • an immunogenic polypeptide according to the present invention may be included in a vaccine composition comprising any other S. pneumoniae protein or protein fragment, including mutated proteins such as detoxified pneumolysin, or they can be linked to or produced in conjunction with any such S. pneumoniae protein or protein fragment.
  • Vaccine compositions according to the present invention may include, for example, influenza polypeptides or peptide epitopes, conjugated with or coupled to at least one immunogenic S. pneumoniae polypeptide according to the invention.
  • the antigen content is best defined by the biological effect it provokes. Naturally, sufficient antigen should be present to elicit the production of measurable amounts of protective antibody.
  • a convenient test for the biological activity of an antigen involves the ability of the antigenic material undergoing testing to deplete a known positive antiserum of its protective antibody. The result is reported in the negative log of the LD 50 (lethal dose, 50%) for mice treated with virulent organisms which are pretreated with a known antiserum which itself was pretreated with various dilutions of the antigenic material being evaluated.
  • a high value is therefore reflective of a high content of antigenic material which has blocked the antibodies in the known antiserum, thus reducing or eliminating the neutralizing effect of the antiserum on the virulent organism.
  • the antigenic material present in the final formulation is at a level sufficient to increase the negative log of LD 50 by at least 1 preferably 1.4 compared to the result from the virulent organism treated with untreated antiserum.
  • the absolute values obtained for the antiserum control and suitable vaccine material are, of course, dependent on the virulent organism and antiserum standards selected.
  • the following method may be also used to achieve the ideal vaccine formulation: starting from a defined antigen, which is intended to provoke the desired immune response, in a first step an adjuvant matched to the antigen is found, as described in the specialist literature, particularly in WO 97/30721.
  • the vaccine is optimized by adding various isotonic-making substances as defined in the present inventions, preferably sugars and/or sugar alcohols, in an isotonic or slightly hypotonic concentration, to the mixture of antigen and adjuvant, with the composition otherwise being identical, and adjusting the solution to a physiological pH in the range from pH 4.0 to 10.0, particularly 7.0-7.5.
  • the substances or the concentration thereof which will improve the solubility of the antigen/adjuvant composition compared with a conventional, saline-buffered solution are determined.
  • the improvement in the solubility characteristics by a candidate substance is a first indication that this substance is capable of bringing about an increase in the immunogenic activity of the vaccine.
  • APCs antigen presenting cells
  • the immunomodulatory activity of the formulation is measured in animal tests.
  • SEQ ID NOS 26-75 of Table 1 have no homology to human sequences and retain 100% homology to all S. pneumoniae strains (NCBI, March 2009). Table 1.
  • NP 358460 LLGSIMIKATTLEPRFGNPTPRVAETPAGMLNAIG 64 dihydroorotate LQNPGLEWLAEKLPWLEREYPNLPIIANVAGFS dehydrogenase IB KQEYAAVSHGISK
  • Polypeptide arrays and polypeptide libraries are used to synthesize the peptides of table 1 and derivatives and analogs of these peptides.
  • the peptides are synthesized using different linkers, matrixes and absorption methods, using methods known in the art (for example US 2002/0006672; Gaseitsiwe et al., Plos One 3, e3840, 1- 8, 2008; Biissow et al., Am J Pharmacogenomics 2001 ; 1, 1-7; Andresen et al., Proteomics 6, 1376-1384, 2006, Jan Marik and Kit S. Lam, Methods in Molecular Biology, vol. 310: Chemical Genomics: Reviews and Protocols, Ed. E. D.
  • Polypeptides are obtained for screening either in a solution or absorbed or linked to a matrix.
  • the peptide arrays are screened using sera obtained from infants at various ages as described for example in Ling et al., Clin Exp Immunol 2004, 138, 290-8.
  • sera are collected longitudinally from healthy children attending day-care centers at different ages (for example 18, 30 and 42 months). Starting at 12 months of age, nasopharyngeal swabs are taken from the children on a bimonthly schedule over the 2.5 years of the study.
  • Pneumococcal isolates are characterized by inhibition with optochin and a positive slide agglutination test (Phadebact, Pharmacia Diagnostics). In addition, sera are collected from healthy adults.
  • Artificial genes encoding polypeptides comprising sequences selected to be immunogenic and age dependent with or without carrier polypeptides, are constructed to encode chimeric proteins of up to 1000 amino acids.
  • the structure of the chimeric proteins is constructed to minimize homology to human sequences based on potential neoantigens at the fusion junction of peptides in the construct.
  • One set of constructs comprises 2-5 different polypeptides, each in 1-5 repeats, with a spacer of 0-20 Glycine and/or Alanine residues between each peptide, and an optional a detoxified pneumolysin as a carrier protein.
  • DNA sequences include (in italics) the restriction sites 5' Nde I (CAT ) and Bpul 102 I 3' ( TAAGC TTGCT GAGC):
  • PS19 comprising SEQ ID NOs: 41-42 derived from elongation factor Tu family protein, linked by an Ala- Ala-Ala (aaa) spacer:
  • GAGGA ACGCC TGCAG GCTGA ACTGC AGACT GATGT AAGCC TGCGT GTAGA
  • PS20 comprising SEQ ID Nos. 58 and 56 derived from the protein Aminopeptidase C, linked by an Ala- Ala-Ala (aaa) spacer:
  • PS25 comprising SEQ ID NOs. 32, 33, 34, 29, 30 and 31 derived from Elongation factor G, linked by an Ala- Ala- Ala (aaa) spacer: 551 amino acids sequence (SEQ ID NO:80):
  • TCTAC ACCAA CGACC TGGGT ACGGA TATCC TGGAG GAAGA TATCC CGGCT
  • GAGTA CCTGG ACCAG GCACA GGAAT ATCGC GAAAA ACTGG CTGCA GCCGA
  • AAGAC ACGAC CACTG GTGAC TCTCT GACGG ATGAG AAAGC GGCTG CGAAA
  • AACTG GCTGA AGAGG ACCCG ACCTT CCGTG TCGAA ACTAA CGTTG AAACC
  • PS26 comprising SEQ ID NOs. 62, 63 and 61 derived from Carbamoyl phosphate synthase large subunit, linked by an Ala-Ala-Ala (aaa) spacer:
  • GGTCA CTCAG TGCCT GATCG AACGT TCTAT TGCGG GTTTC GCGGC TGCCC
  • PS30 comprising SEQ ID NOs. 50, 51, 49 derived from Elongation factor Ts, and SEQ ID NO. 54 derived from Phosphoglycerate kinase and 57 derived from Aminopeptidase C, linked by an Ala-Ala-Ala (aaa) spacer:
  • GGTAA ACCGG AGAAA ATCTG GGACA AAATT ATCCC AGGCA AAATG GATCG
  • TGTCA GCCTG TTTGA GAAAT ACGGT GTCGT GCCGA AAAGC GTCTA TCCGG
  • PS31 comprising SEQ ID Nos. 55 derived from 30S ribosomal protein S2,SEQ ID Nos. 38, 36 and 37 derived from glyceraldehyde-3 -phosphate dehydrogenase, and SEQ ID NO. 35 derived from L-lactate dehydrogenase, linked by an Ala- Ala- Ala (aaa) spacer:
  • ACGCA CGCAG CGTTC ACGCC TACAT CATGG GTGAG CACGG TGACT CTGAA
  • PS32 comprising SEQ ID Nos. 26, 27 and 28 derived from phosphoglucomutase/phosphomannomutase family protein, SEQ ID No. 39 derived from UDP-glucose 4-epimerase, SEQ ID Nos. 69 and 70 derived from ABC transporter, ATP- binding protein, and SEQ ID No. 75 derived from Hypothetical protein SP_0565, linked by an Ala-Ala-Ala (aaa) spacer:
  • TTCAA CCTGA TTGCA GGTAT CCTGG AGGTC CAGTC TGGTC GTATT GTTCT
  • PS33 comprising SEQ ID NOs. 43, 44, 46 and 47 derived from Bifiinctional GMP synthase/glutamine amidotransferase protein, and SEQ ID NO: 48 derived from glutamate dehydrogenase, linked by an Ala-Ala-Ala (aaa) spacer:
  • GAGCA GACGG TACTG ATGTC TCATG GCGAC GCTGT AACGG AAATC CCAGC
  • GTAAA GGCGA AGCTG ACCAG GTAAT GGATA TGCTG GGCGG TAAAT TCGGT
  • GCTCA GGGTA CCCTG TATAC CGACG TGATC GAATC CGGTA CGGAT ACTGC
  • GGCGG TGGCA AAGGT GGCAG CGATT TCTAA TAAAA GCTTG CTGAG C PS34 comprising SEQ ID No. 68 and 67 derived from Elongation factor Tu, SEQ ID Nos. 52 and 53 derived from Phosphoglycerate kinase, and SEQ ID NO: 65 derived from dihydroorotate dehydrogenase IB, linked by an Ala- Ala- Ala (aaa) spacer:
  • GAGCA GACGG TACTG ATGTC TCATG GCGAC GCTGT AACGG AAATC CCAGC
  • GTAAA GGCGA AGCTG ACCAG GTAAT GGATA TGCTG GGCGG TAAAT TCGGT
  • GCTCA GGGTA CCCTG TATAC CGACG TGATC GAATC CGGTA CGGAT ACTGC
  • PS35 comprising SEQ ID NOs. 71, 72 and 74 derived from endopeptidase O, and SEQ ID NO: 64 derived from dihydroorotate dehydrogenase IB, linked by an Ala- Ala- Ala (aaa) spacer:
  • Immunogenic polypeptides are produced and used individually, as multimers, or in different combinations as parts of fusion polypeptides with or without a carrier or adjuvant sequence, and are tested with or without an external adjuvant for their vaccine potential in several in-vitro, ex-vivo and in-vivo models. Cross protection against capsularly and genetically unrelated bacterial strains is also tested. In certain cases, antibodies produced against selected peptides and polypeptides are used. The following models are used to test the efficacy:
  • mice in vaccinated mice, the extent of nasopharyngeal, lung, blood and spleen colonization of S. pneumoniae tagged with luciferase is monitored using the bioluminescence live-imaging system(IVIS live-imaging system).
  • IVIS live-imaging system bioluminescence live-imaging system
  • mice immunized with a polypeptide formulated with adjuvant and with adjuvant alone as control are inoculated intraperitoneally (i.p.) or intravenous (i.v.) with a lethal dose of S. pneumoniae serotype 3 strain WU2.
  • the inoculum size is determined to be the lowest that cause 100% mortality in the control mice within 96-120 hours. Survival is monitored daily.
  • mice immunized with polypeptide in adjuvant, and with adjuvant alone as control are anaesthetized with isoflurane and inoculated intranasally (IN) with a lethal dose of S. pneumoniae serotype 3 strain WU2 (in 25 ⁇ PBS).
  • the inoculum's size is determined to be the lowest that causes 100% mortality in the control mice within 96-120 hours. Survival is monitored daily.
  • mice are inoculated intranasally with S. pneumoniae serotype 3 prior and after treatment ex vivo with antibodies to the polypeptide.
  • the polypeptide is mixed with S. pneumoniae strain WU2 bacteria, and the mixture is inoculated IN with 5xl0 5 to 5x10 7 CFU S. pneumoniae.
  • mice are sacrificed, and the nasopharynx and lungs excised homogenized and plated onto blood agar plates for colony number enumeration.
  • Otitis media models - Otitis media models in chinchilla and the rat (developed according to Chiavolini et al., 2008, Clinical Microbiology Reviews, 21 :666-685; Giebink, G. S. 1999, Microb. Drug Resist., 5:57-72; Hermansson et al., 1988, Am. J. Otolaryngol. 9:97-101 ; and Ryan et al., 2006, Brain Res. 1091 :3-8) are utilized to test the effectiveness of multimers according to the invention. The ability of multimers to protect these animals from developing otitis media following IN challenge is studied.
  • Example 5 Immunogenicity of PS20 protein formulated with CCS/C or CFA in CBA/N xid mice
  • Control mice were immunized with l( ⁇ g non-lectin proteins (NL) of S. pneumonia serotype 3 strain WU2 cell wall fraction as a positive control or adjuvant alone as a negative control, or with rPS20 alone to control the adjuvant effect.
  • NL non-lectin proteins
  • mice were injected with CFA as an adjuvant in the first immunization and IFA in the two following immunizations.
  • Mice were subsequently challenged intranasally (IN) with a sub-lethal dose of S. pneumoniae strain WU2 (7.5* 10 5 CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx and right lobe-lung of each mouse were homogenized and plated onto blood agar for CFU enumeration.
  • Colonization studies using CCS/C as adjuvant demonstrate that immunized mice showed reduced colonization in the nasopharynx using 3 ⁇ g PS20 (P value ⁇ 0.05, Figure 2A). In the lungs, reduced colonization was observed using 3 ⁇ g protein and to a lesser extent using 10 ⁇ g protein (P value ⁇ 0.05, Figure 2B). No significant protection was observed when the multimers were administered alone without an adjuvant.
  • mice Forty eight hours later, mice were sacrificed and nasopharynx and right lobe-lung of each mouse were homogenized and plated onto blood agar for CFU enumeration.

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Abstract

La présente invention concerne des fragments immunogènes, y compris des variantes et des analogues dérivés de protéines de Streptococcus pneumoniae (S. pneumoniae), des multimères de polypeptides et des protéines de fusion comprenant de tels polypeptides, et des vaccins comprenant de telles entités immunogènes. L'invention concerne notamment l'utilisation de tels vaccins pour provoquer une immunité protectrice contre S. pneumoniae.
PCT/IL2010/001009 2009-12-02 2010-12-01 Fragments immunogènes et multimères pour protéines de streptococcus pneumoniae WO2011067758A2 (fr)

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WO2015189422A1 (fr) * 2014-06-12 2015-12-17 Universidade Do Porto - Reitoria Vaccin pour hôtes immunodéprimés
US9393294B2 (en) 2011-01-20 2016-07-19 Genocea Biosciences, Inc. Vaccines and compositions against Streptococcus pneumoniae
US10105412B2 (en) 2009-06-29 2018-10-23 Genocea Biosciences, Inc. Vaccines and compositions against Streptococcus pneumoniae
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection

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