WO2013040648A1 - Immunogène glpo pneumococcique - Google Patents

Immunogène glpo pneumococcique Download PDF

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WO2013040648A1
WO2013040648A1 PCT/AU2012/001139 AU2012001139W WO2013040648A1 WO 2013040648 A1 WO2013040648 A1 WO 2013040648A1 AU 2012001139 W AU2012001139 W AU 2012001139W WO 2013040648 A1 WO2013040648 A1 WO 2013040648A1
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glpo
protein
immunogenic
fragment
pneumococcal
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PCT/AU2012/001139
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English (en)
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James Paton
Layla MAHDI
Abiodun OGUNNIYI
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Adelaide Research & Innovation Pty Ltd
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Priority claimed from AU2011903930A external-priority patent/AU2011903930A0/en
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Publication of WO2013040648A1 publication Critical patent/WO2013040648A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03021—Glycerol-3-phosphate-oxidase (1.1.3.21)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to use of GlpO (alpha-glycerophosphate oxidase) in prevention, treatment or diagnosis of Streptococcal disease.
  • GlpO alpha-glycerophosphate oxidase
  • Streptococcus pneumoniae (pneumococcus) is a major cause of invasive diseases such as meningitis, septicaemia and pneumonia. Approximately, one million children under 5 years of age die of pneumococcal disease annually.
  • Nasopharyngeal colonization by S. pneumoniae is common. Probably all humans are colonized with this organism at least once early in life. Especially in circumstances of crowding, as in day-care centres, nursing homes, hospitals and jails, the risk of colonization with pneumococci is high.
  • the first pneumococcal vaccines contained purified capsular polysaccharide antigen from 14 different types of pneumococcal bacteria.
  • a 23-valent polysaccharide vaccine replaced the 14-valent vaccine.
  • This contains polysaccharide antigen from 23 types of pneumococcal bacteria which cause 88% of bacteremic pneumococcal disease.
  • cross- reactivity occurs for several capsular types which account for an additional 8% of bacteremic disease.
  • pneumococcal polysaccharide based vaccine to be successful, the capsular types responsible for most pneumococcal infections would have to be made adequately immunogenic. This approach may be difficult, because the twenty- three polysaccharides are not all adequately immunogenic, even in adults.
  • the 23-valent polysaccharide vaccine has a reported efficacy of
  • this 7-valent vaccine covers infections caused by only some pneumococcal serotypes and the replacement over time of these serotypes by resistant ones is likely, particularly in areas of high disease burden.
  • the cost of manufacture of a conjugate vaccine is also relatively high.
  • pneumococcal proteins include pneumolysin toxoid (Pd), Pneumococcal surface protein A (PspA), Pneumococcal surface adhesin A (PsaA),
  • Pneumococcal surface protein C Pneumococcal surface protein C (PspC; also called Choline binding protein A (CbpA)), LytB glucosaminidase, LytC muramidase, PrtA serine protease, PhtD (Poly-histidine triad protein D), Pneumococcal vaccine antigen A (PvaA), neuraminidase A (NanA) and autolysin.
  • the inventors have devised a method of identifying immunogenic proteins that are candidates for reducing bacterial infections of the CNS, particularly meningitis, by screening up-regulated bacterial gene products in the brain of meningitis affected individuals.
  • Such targets should be particularly valuable because the susceptibility of relevant infectious bacteria such as Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis, whereas current vaccines target more specifically the primary infection to thereby prevent septicaemia. It will be understood that a combination vaccine eliciting one or more antibodies that primarily inhibit the primary infection and one or more antibodies that inhibit the secondary infection, will likely be more effective than a combination of antibodies targeting the primary infection.
  • the method has resulted in the identification of GlpO of Streptococcus pneumoniae as a candidate and demonstration that a vaccine to GlpO is protective for the meningitis phase of pneumococcal infection.
  • the invention encompasses an immunogenic composition comprising GlpO protein or an immunogenic fragment thereof, this may be used either on its own or preferably in combination with other immunogens that are protective against pneumococcal infections.
  • the invention also encompasses a method of making such immunogenic composition or a vaccine comprising the immunogenic composition.
  • the GlpO is enzymatically inactive or at least has a reduced cytotoxicity as a result of enzymatic activity that is reduced relative to unaltered GlpO.
  • the invention additionally encompasses a vaccine, which vaccine comprises a GlpO protein or fragment thereof, or a polynucleotide encoding the same.
  • a further aspect of the invention encompasses a method of immunizing a mammal against a streptococcal infection by administering an immunogenic composition or vaccine set out herein.
  • a yet further aspect of the invention encompasses the use of a anti GlpO antibody or active fragment thereof in the manufacture of a medicament for the treatment or prevention of a streptococcal infection.
  • A regulated gene expression in WCH16
  • B regulated gene expression in WCH43
  • C combined gene expression WCH16 and WCH43 analyses. Yellow to blue scales represents fold difference in differential regulation; yellow,
  • Horizontal line represents median. Data were analyzed using unpaired Mest (one-tailed). *, P ⁇ 0.05.
  • D Competition experiments between wild-type WCH43 and its isogenic mutants in the brain of mice at 48 h post-infection. 5-12 CD1 mice were infected i.n. with equal numbers (approx. 1 ⁇ 10 7 CFU each) of wild-type and mutant. Each datum point represents the ratio of recovered mutant bacteria to wild-type. The horizontal solid line represents a 1 1 ratio of recovered mutant bacteria. The horizontal broken line denotes the median value of the ratio of recovered mutant bacteria for each comparison (*** PO.001 ; one sample r-test; two-tailed).
  • E Adherence to human brain microvascular endothelial cells (HBMEC).
  • A Flow cytometry using polyclonal anti-GlpO serum on wild-type WCH43 and its isogenic AglpO mutant showing surface labeling of the wild-type, but not the AglpO mutant.
  • B Active immunization with alum, GlpO, PdT and GlpO+PdT and challenge with 3 x 10 4 CFU of WCH43. Data were analyzed using Mann-Whitney IZ-test (two-tailed). *, P ⁇ 0.05; **, P ⁇ 0.01.
  • C Passive immunization with anti-GlpO and challenge with 5 x 10* CFU of WCH43.
  • mice were immunized with high titre (32,000) antiserum 1 h prior to challenge, using serum from alum-immunized mice as a control.
  • Numbers of bacteria from blood (1 ml) and the brain were then determined from each mouse at 30 h post challenge. Horizontal line indicates median CFU of bacteria.
  • Data were analyzed usjng unpaired f-test (one-tailed). *, P ⁇ 0.05.
  • D Ratios of bacteria in the brain vs blood compared between alum-immunized and anti-GlpO immunized mice, as in (B) above. Data were analyzed using unpaired f-test (one-tailed). **, P ⁇ 0.01.
  • CD1 mice were infected i.n. with equal numbers (approx. 1 ⁇ 10 7 CFU each) of wild-type and mutant. Each datum point represents the ratio of recovered mutant bacteria to wild-type.
  • the horizontal broken line represents a 1 :1 ratio of recovered mutant bacteria.
  • the horizontal solid line denotes the median value of the ratio of recovered mutant bacteria for each comparison (.*** P ⁇ 0.001 ; ** P ⁇ 0.01 ; * P ⁇ 0.05; one sample f-test; two-tailed).
  • CD1 mice were infected i.n. with equal numbers (approx. 1 x 10 7 CFU each) of wild-type and mutant. Each datum point represents the ratio of recovered mutant bacteria to wild-type.
  • the horizontal solid line denotes the median value of the ratio of recovered mutant bacteria for each comparison (*** P ⁇ 0.001 ; one sample f-test; two-tailed)
  • ELISA titres for each derivative is indicated in parenthesis, and was determined as the reciprocal of the dilution of the antiserum giving 50% absorbance at A405nm- Wild type gave a titre of 30,000, C1 30,000, C2 20,000, C3 20,000; C4 7,000, C5 3,500, N1 20,000, N2 3,500, N3 7,000 and N4 3,500.
  • Streptococcus pneumoniae [SEQ ID No. 1], Streptococcus faecium [SEQ ID No. 2], Streptococcus Mitis [SEQ ID No. 3], Streptococcus oralis [SEQ ID No. 4], Streptococcus gordonii [SEQ ID No. 5], Streptococcus sanguinis [SEQ ID No. 6], Streptococcus agalactiae [SEQ ID No. 7], Streptococcus pyogenes [SEQ ID No. 8], Streptococcus suis [SEQ ID No. 9] and Streptococcus uberis [SEQ ID No. 10].
  • GlpO is known to act as the principal immunogen in Mycoplasma infections and thus immunogenic compositions with Mycoplasma GlpO are taught in WO2007/006712 (Frey et al.).
  • Mycoplasma's are very rudimentary organisms that contain no cell wall and are unable to synthesize certain fundamental molecules such as purines. They are therefore obligate parasites. Their mode of infection is quite different to bacteria such as Streptococci. Many
  • Mycoplasma species do not invade their hosts as a complete organism, rather their lack of cell wall is postulated to allow them to fuse with host cells. Given the difference in mode of infection it is unexpected that GlpO of a
  • Streptococcus would have a protective effect, and more so for meningitis.
  • the present invention thus related to the use and composition comprising a GlpO that elicits an immune response protective against a streptococcus, in particular for Streptococcus pnuemoniae.
  • GlpO that elicits an immune response protective against a streptococcus, in particular for Streptococcus pnuemoniae.
  • Closely related GlpO's are found in Streptococcal species and other closely related bacteria.
  • Figure 10 The sequence of Figure 10 being the amino acid sequence of S. pneumoniae GlpO [NCBI-GI: 15901992; GenBank Acc: AE005672; UniProt: P35596].
  • the GlpO may be any one of those produced by a Streptococcus species, including those from Streptococcus pneumoniae, Streptococcus gordonii, Streptococcus sanguinis, Streptococcus thermophilus, Streptococcus suis, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, Enterococcus faecium, Rhodococcus sp.
  • the sequence may be any one selected from the sequences or organisms shown in Figure 11 ([SEQ ID Nos. 1 - 0].
  • the GlpO need not be identical to the amino acid sequence of the GlpO set out in Figure 11 for S. pneumoniae [or the Streptococcal species, preferably S. pneumoniae]. Various modifications can be made to the protein sequence without altering its immunogenicity, and protective capacity of antibodies raised thereto. Conservative substitutions may be made in other parts of the GlpO protein sequence.
  • Such conservative substitution might be made within the following eight groups of amino acids 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).
  • the GlpO protein is enzymically inactive.
  • it may have its flavine adenine dinucleotide (FAD) binding sequence inactivated.
  • the consensus sequence for the FAD binding is GGGITG [SEQ ID No. 1 ], and thus recombinant GlpOAFAD protein, was constructed in the examples hereto and to be immunogenic by active immunisation/challenge ( Figure 4B).
  • the GlpOAFAD protein has 38 amino acids deleted from the N terminus of the wild type GlpO. A corresponding deletion of the 38 N-terminal amino acids of GlpO molecules from S. mitis, S. oralis, S gordonii, S. sanguinis, S. pyogenes, S.
  • deletion of 44 N-terminal amino acids of S. faecium will similarly be enzymically inactive and immunogenic.
  • Other deletions, in particular to the FAD binding consensus sequence may be made, and if only to delete the GGGITG [SEQ ID No. 1 ] consensus sequence. It is anticipated that other deletions will also still be inactive and it has been found that substantial immunogenicity is still present in a 120-amino acid N-terminal deletion. Similarly deletion of 240 amino acids from the C terminus also provides for substantial immunogenicity.
  • the FAD can be inactivated in other ways, for example by smaller deletion of at least two amino acids of the GGGITG sequence, or by conservative substitution of the GGGITG amino acids, or at least one, and preferably two or more these amino acids might be substituted by alanine.
  • the FAD binding site above has at least one substitutions selected from the group consisting of, one or more of the glycine residues is replaced by an alanine, the isoleucine is replaced by either a leucine, methionine, or a valine, and the threonine is replaced by a serine. Inactivation of the FAD binding sequence is desirable when GlpO is used in immunisation because its presence is found to have a cytotoxic effect.
  • the inactivation of FAD binding of a GlpO is readily tested, for example and preferably by its loss of cytotoxicity.
  • testing of mutants can be by way of the method set out herein to determine its cytotoxicity to HBMEC (Human Brain Microvascular Endothelial Cells).
  • identity is measured using well known computer programs.
  • a program such as CLUSTAL to compare amino acid sequences.
  • This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It can calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment.
  • a program like BLASTP will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score.
  • the GlpO has at least 70% identity, preferably 80 or 85% and more preferably at least 90% identity and still more preferably at least 95, 98 or 99% identity to the GlpO, in one form the GlpO being a sequence set out in Figure 10 [SEQ ID Nos. 1 - 10].
  • GlpO also contemplated by the invention for example where the GlpO also includes moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide, for example tagging with six terminal histidine residue to assist with purification, or other similar tagging, some of which are set out below.
  • the GlpO can be modified in other ways for example by terminal NH 2 acylation for example by acetylation, or thipglycolic acid amidation, terminal carbdxy amidation, for example with ammonia or methylamine to provide stability, increased hydrophobicity for linking or binding to a support or other molecule.
  • the GlpO does not contain a methionine (Met) starting residue, although alternatively the Met might be present.
  • GlpO will not incorporate a leader or secretory sequence (signal sequence).
  • the signal portion of GlpO may be determined according to established molecular biological techniques, such as Signal P (version 3) or SIG Pred, as used in the examples.
  • the invention contemplates an immunogen that elicits an anti GlpO immune response need not be the complete GlpO, but only needs to be an
  • immunogenic fragment thereof.
  • the fragment may be used on its own in the immunogenic composition.
  • the GlpO fragment may be coupled to a carrier, such as for example KLH.
  • An "immunogenic fragment” is a fragment of the full length GlpO that is still an immunogen.
  • the GlpO fragment may be used on its own, or perhaps stabilised by conjugation with a stabilising moiety, however in certain specific embodiments the GlpO fragment may be part of a fusion protein that comprises the GlpO fragment and an unrelated sequence.
  • a fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein.
  • Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
  • Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments.
  • Still further fusion partners include affinity tags, which facilitate purification of the PI
  • Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
  • a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system.
  • DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector.
  • the 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both
  • a peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • the linker sequence may generally be from 1 to about 50 amino acids in length.
  • Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • Embodiments of this invention relate to a fragment of GlpO comprised in a precursor polypeptide designed for expression in a host arid having heterologous pre and pro-polypeptide regions fused to the amino terminus of the fragment and an additional region fused to the carboxyl terminus of the fragment.
  • the fusion protein may, in certain forms include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N- or C-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • regions may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize or purify polypeptides.
  • EP-A-0464 533 discloses fusion proteins comprising various portions of constant region of
  • the polypeptide of the invention or a fragment thereof may be fused with co- protein, which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein, which will have immunogenic and protective properties.
  • This fused recombinant protein preferably further comprises an antigenic co-protein, such as Glutathione-S- transferase (GST), beta-galactosidase, bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH) relatively large co-proteins which solubilise the protein and facilitate production and purification thereof.
  • GST Glutathione-S- transferase
  • beta-galactosidase beta-galactosidase
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein.
  • polypeptides which have fused thereto other compounds which alter the polypeptides biological or pharmacological properties i.e. polyethylene glycol (PEG) to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro-sequences; and
  • PEG polyethylene glycol
  • hetero and homo polypeptide multimers of the polypeptide fragments, analogues and derivatives include, for example, one or more polypeptides that have been cross-linked with cross-linkers such as avidin/biotin, gluteraldehyde or
  • polymeric forms also include polypeptides containing two or more tandem or inverted contiguous sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
  • the GlpO or immunogenic fragment thereof preferably comprises a sequence including SWAGLRPLIA [SEQ ID No. 12].
  • the antigen might more preferably be selected from the group comprising two consensus sequences for glycerol- 3-phosphate (G3P)-binding (SWAGLRPLIA [residues 342-351, SEQ ID No. 12], and GGKI [residues 430-433, SEQ ID No. 13] at the C-terminal half of S. pneumoniae GlpO ( Figure 9).
  • G3P glycerol- 3-phosphate
  • GGKI glycerol- 3-phosphates 430-433, SEQ ID No. 13
  • GlpO or immunogenic fragment thereof in one specific form SWAGLRPLIA [SEQ ID No. 12].
  • the GlpO or immunogenic fragment thereof comprises GGKI [SEQ ID No. 13].
  • an immunogenic fragment might comprise at least 10 consecutive GlpO amino acids, more preferably at least 15 or at least 20 amino acids, of any of the sequences set out in figure 9, perhaps specifically the sequence for S. pneumoniae.
  • the present inventors have shown that GlpO is thought to provide strong protection against S. pneumoniae infection, however, it might be preferred to enhance the protective effect by inclusion in the immunogenic composition one or more further proteins or immunogenic fragments thereof. This may be particularly effective if the further protein or fragment thereof has a site of exposure at a location different to that of GlpO.
  • the immunogenic composition may comprise a multivalent vaccine.
  • the further immunogenic protein might be pneumolysin toxoid (PdT), a fragment of PspA comprising the N-terminal and proline-rich region, and a fragment of CbpA comprising the N-terminal and proline-rich region.
  • PdT pneumolysin toxoid
  • Figure 4B GlpO+PdT
  • a number of surface proteins or virulence factors have been shown to be immunogenic and these may be selected from the group comprising choline binding protein A (CbpA), pneumolysin (Ply), pneumococcal surface protein A (PspA), manganese-dependent superoxide dismutase (SodA), pyruvate oxidase (SpxB), Pneumococcal surface adhesin A (PsaA), LytB
  • the further immunogenic protein is non toxic pneumolysin or immunogenic variant or fragment thereof.
  • the further immunogenic protein might be one or more selected from the group consisting of PtsGal, VgcL, AdhC.
  • a number of other surface proteins have been shown to be immunogenic or are or predicted to be surface exposed proteins and will possibly be immunogenic. Accordingly there may be an immunogenic composition or use of an immunogenic composition comprising the combination of any one or more of the proteins below may have advantages over an immunogenic composition where GlpO or fragment thereof is the only immunogenic moiety.
  • the further immunogen may be selected from the group consisting of the following protein or immunogenic fragments thereof; choline binding protein A (CbpA), pneumolysin (Ply), serine protease (PrtA), pneumococcal surface protein A (PspA), manganese-dependent superoxide dismutase (SodA), or pyruvate oxidase (SpxB), Pneumococcal surface adhesin A (PsaA), LytB glucosaminidase, LytC muramidase, PrtA serine protease, PhtA and D (histidine triad A and D), Pneumococcal vaccine antigen A (PvaA), neuraminidase (NanA), autolysin (LytA), Maltose/maltodextrin-binding protein ( alX), Oligopeptide-binding protein ( ⁇ ), and Iron-compound-binding protein (PiuA)
  • the Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB, PhtD, and PhtE. The family is present in all strains of pneumococci tested.
  • Homologous proteins have also been found in other Streptococci.
  • Preferred members of the family comprise PhtA, PhtB and PhtD. More preferably, it comprises PhtA or PhtD.
  • PhtA is disclosed in WO 98/ 8930 (therein referred to as Sp36).
  • PhtD is disclosed in WO 00/37105 (therein referred to as Sp036D).
  • PhtB is disclosed in WO 00/37105 (therein referred to as Sp036B).
  • Another member of the PhtB family is the C3-Degrading Polypeptide, as disclosed in WO 00/17370.
  • An immunologically functional equivalent is the protein Sp42 disclosed in WO 98/18930.
  • PhtE is disclosed in WO00/30299 and is referred to as BVH-3.
  • SpsA is a Choline binding protein (Cbp) disclosed in WO 98/39450, and is synonymous with CbpA and PspC.
  • the Lyt family of proteins are membrane associated and connected with cell lysis. The family comprises LytA, B and C. LytA is disclosed in Ronda et at., Eur J Biochem, 164:621 (1987). LytB is disclosed in WO 98/18930 (therein referred to as Sp46). LytC is disclosed in WO 98/18930 (therein referred to as Sp91). A preferred member of that family is LytC. '
  • Sp125 is disclosed in WO 98/18930 (therein referred to as ZmpB).
  • Sp101 is disclosed in WO 98/06734 (therein referred to as #y85993).
  • Sp133 is disclosed in WO 98/06734 (therein referred to as #y85992).
  • Sp128 and Sp130 are disclosed in WO 00/76540.
  • Pneumolysin also referred to as Ply; preferably detoxified by chemical treatment or mutation
  • Ply preferably detoxified by chemical treatment or mutation
  • PsaA and transmembrane deletion variants thereof Berry & Paton, Infect Immun 1996 December; 64(12):5255
  • PspA and transmembrane deletion variants thereof U.S. Pat. No. 5,804,193, WO 92/14488, WO 99/53940
  • PspC and transmembrane deletion variants thereof WO 97/09994, WO 99/53940
  • Cbp Choline binding protein
  • Choline Binding Protein family is selected from the group consisting of Choline Binding Proteins as identified in WO 97/41151 , PbcA, SpsA, PspC, CbpA, CbpD, and CbpG.
  • CbpA is disclosed in WO 97/41151.
  • CbpD and CbpG are disclosed in WO 00/29434.
  • PspC is disclosed in WO 97/09994.
  • PbcA is disclosed in WO 98/21337.
  • the Choline Binding Proteins may be selected from the group consisting of CbpA, PbcA, SpsA and PspC.
  • the present invention also contemplates combination vaccines which provide protection against a range of different pathogens.
  • Many Paediatric vaccines are now given as a combination vaccine so as to reduce the number of injections a child has to receive.
  • other antigens from other pathogens may be formulated with the vaccines of the invention.
  • the vaccines of the invention can be formulated with (or administered separately but at the same time) the well known trivalent combination vaccine comprising Diphtheria toxoid (DT), tetanus toxoid (TT), and pertussis components;
  • the combined vaccine may also comprise other antigens, such as Hepatitis B surface antigen (HBsAg), Polio virus antigens (for instance inactivated trivalent polio virus ⁇ IPV),
  • Viral antigens may also be included in the immunogenic composition, for example, from influenza (attenuated, split, or subunit [e.g., surface
  • glycoproteins neuraminidase NA
  • haemagglutinin HA
  • Varicella e.g., attenuated, glycoproteins l-V, etc.
  • MMR measles, mumps, rubella
  • the present immunogenic composition comprises both GlpO or fragment thereof and pneumolysin.
  • Pneumolysin belongs to the group of Cholesterol-binding Cytolysins (CBCs) that bind to the cholesterol of host cell membranes prior to formation of large 30-50mer ring structures that create lytic pores. The mechanism of pore- formation is not fully understood and there is much debate over the sequence of events. However, the ability to form pores means that native pneumolysin is highly toxic, which is a problem in terms of the development of immunogenic compositions. It is desirable to use a non toxic form of pneumolysin.
  • CBCs Cholesterol-binding Cytolysins
  • pneumolysoids The toxicity of pneumolysin can be significantly reduced by site-directed mutagenesis to create pneumolysin toxoids, known as pneumolysoids.
  • Most mutations have previously been created in or near the highly conserved 11 amino acid region near the C terminus. This site has been shown to be involved in binding to the host cell.
  • a number of such mutated forms of PLY are described in International Patent Application WO 90/06951 ; each of the mutations described in this publication is towards the C terminus of the protein.
  • Other mutants of pneumolysin are described in US patent specification 7820789.
  • pneumolysin used in the present invention is a non-toxic form.
  • fusion protein might additionally comprise the further
  • immunogenic protein or immunogenic fragments thereof.
  • the immunogenic composition might additionally comprise capsular antigens of one or more serotypes known for S. pneumoniae. Capsular antigens of one or more serotypes known for S. pneumoniae.
  • the immunogenic composition might comprise a conjugate vaccine wherein one or more polysaccharides representing one or more serotypes, in particular one or more of the well known capsular antigens of S. pneumoniae is conjugated with GlpO or fragments thereof or the one or more further immunogenic proteins.
  • the immunogenic composition might comprise a conjugate of GlpO and one or more further immunogenic proteins or fragments thereof.
  • Such saccharides, oligosaccharides, polysaccharides, peptides, polypeptides or proteins are each conjugated to the immunogenic protein or fragment thereof in any suitable manner, including, but not limited to: (1) direct coupling via protein functional groups (e.g., thiol-thiol linkage, amine-carboxyl linkage, amine-aldehyde linkage; enzyme direct coupling); (2) homobifunctional coupling of amines (e.g., using bis-aldehydes); (3) homobifunctional coupling of thiols (e.g., using bis-maleimides); (4) homobifunctional coupling via photoactivated reagents (5) heterobifunctional coupling of amines to thiols (e.g., using maleimides); (6) heterobifunctional coupling via photoactivated reagents (
  • beta. -carbonyldiazo family (7) introducing amine-reactive groups into a poly- or oligosaccharide via cyanogen bromide activation or carboxymethylation; (8) introducing thiol-reactive groups into a poly- or oligosaccharide via a heterobifunctional compound such as maleimido- hydrazide; (9) protein-lipid conjugation via introducing a hydrophobic group into the protein and (10) protein-lipid conjugation via incorporating a reactive group into the lipid. Also, contemplated are heterobifunctional "non-covalent coupling" techniques such the Biotin-Avidin interaction.
  • the immunological composition comprises the immunogenic protein this will be prepared using a recombinant source of the immunogenic protein or fragment thereof.
  • Genes of interest can be cloned from a streptococcal source, for example a strain of S. pneumoniae using a primer design based on the sequence published in a sequence database. Primers can be selected by computer analysis to provide for binding uniquely to the gene of interest. Primers can be designed by assessment of published polynucleotide sequences for the proteins concerned.
  • the primers to clone GlpO were derived from the S. pneumoniae TIGR4 (serotype 4) genome as deposited in the Kyoto
  • the immunogenic protein, or fragment thereof is thus preferably cloned into a vector. Substitutions may be made in the sequence to take into account codon preferences of the type of cell in which production is to take place.
  • Polynucleotide encoding the immunogenic protein is ligated into position in the vector so as to be linked with a promoter suitable for expression of the immunogenic proteins.
  • One or more control elements may impact on the promoter, which control elements can be used to manipulate the extent of transcription and/or translation the polynucleotide sequence encoding the immunogenic protein.
  • the polynucleotide with the appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et a/., (supra).
  • the vector may be a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector, a single or double-stranded RNA or DNA viral vector.
  • Vectors will generally comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • Expression may be inducible or expression only in certain types of cells or both inducible and cell-specific.
  • Preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, for example, temperature and nutrient additives.
  • a variety of vectors suitable to this aspect of the invention are well known and employed routinely by those of skill in the art.
  • a great variety of expression vectors can be used to express a polypeptide of the invention.
  • Such vectors include, among other, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention.
  • polynucleotides to express a polypeptide in a host may be used for .
  • the DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription.
  • appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription.
  • promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs.
  • prokaryotic promoters suitable for expression of
  • polynucleotides and polypeptides in acpordance with the present invention are the E. coli lad and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.
  • eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus "(RSV), and metallothionein promoters, such as the mouse metallothionein-l promoter.
  • CMV immediate early promoter the HSV thymidine kinase promoter
  • the early and late SV40 promoters the promoters of retroviral LTRs, such as those of the Rous sarcoma virus "(RSV)
  • metallothionein promoters such as the mouse metallothionein-l promoter.
  • Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
  • Polynucleotides of the invention encoding the heterologous structural sequence of the immunogenic protein or fragment thereof generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression.
  • the polynucleotide will be positioned so that the transcription start site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.
  • a transcription termination signal appropriately disposed at the 3' end of the transcribed region may also be included in the polynucleotide construct.
  • secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into fhe extracellular environment appropriate secretion signals may be incorporated into the expressed polypeptide.
  • These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N- or C- terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • regions may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art.
  • expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initiating codon at the beginning and a termination codon at the end.
  • constructs may contain control regions that regulate as well as enable expression. Generally such regions will operate by controlling transcription, such as transcription factors, repressor binding sites and termination, among other.
  • Vectors for propagation and expression generally will include selectable markers and amplification regions, such as, for example, those set forth in Sambrook er a/., (supra)
  • Polynucleotides can be introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, transvection,
  • microinjection cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et a/., (supra).
  • Representative examples of appropriate cells which host said vectors include bacterial cells, such as staphylococci, £.
  • coli coli, streptomyces and Bacillus subtilis ceils
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells, and plant cells.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well known to those skilled in the art.
  • Mammalian expression vectors may comprise expression sequences, such as an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non- transcribed sequences that are useful or necessary for expression.
  • expression sequences such as an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non- transcribed sequences that are useful or necessary for expression.
  • polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity
  • chromatography hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification.
  • Well-known techniques for refolding protein may be employed to regenerate the active conformation when the polypeptide is denatured during isolation and or purification.
  • the purified protein or fragment thereof may then be modified if desired, for example coupled with a carrier protein, or conjugated with one or more antigenic polysaccharide components.
  • a immunogenic formulation is then prepared dependent on the route by which the formulation is to be introduced.
  • the formulation will typically include a carrier or excipient and may also include an adjuvant to enhance the extent or nature of the reaction, preferably the reaction is biased to a humoral immune reaction.
  • an adjuvant is commonly used as 0.001 to 50 wt % solution in phosphate buffered saline, and the antigen is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % (see, e.g., Examples below or in applications cited herein).
  • the antigen is present in an amount in the order of micrograms to milligrams, or, about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.
  • compositions of the invention include liquid preparations for introduction into a body orifice, for example, oral, nasal, anal, vaginal, peroral, intragastric; application to a mucosal surface, for example, perlingual, alveolar, gingival, olfactory or respiratory; these composition may take the form of suspensions, syrups or elixirs.
  • the compositions may be preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration, for example, by injectable administration and may take the form of sterile suspensions or emulsions.
  • the immunogenic protein or fragment thereof may be in admixture with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose or the like.
  • a suitable carrier such as sterile water, physiological saline, glucose or the like.
  • the compositions can contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavouring agents, colours, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, may be consulted to prepare suitable preparations.
  • the vaccination in one form is parenteral, to elicit strong reaction in the systemic system of the target animal. This is preferred because the GlpO is thought to impact most importantly in S. pneumoniae crossing the blood brain barrier. It has however been shown that GlpO also acts in the nasal mucosa, and in another preferred form the immunogenic composition is applied to a mucosal surface, perhaps respiratory mucosal surface perhaps bringing to greater prominence a form of localised mucosal immunity. Other forms of vaccination, such as per oral may also be used, particularly because of the convenience of not requiring the attendance of medical staff.
  • compositions of the invention in one form are provided as liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH.
  • compositions of the invention can be in the "solid” form of pills, tablets, capsules, caplets and the like, including "solid" preparations that are time-released or that have a liquid filling, for example, gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut.
  • compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser.
  • Aerosols are usually under pressure by means of a hydrocarbon.
  • Pump dispensers can preferably dispense a metered dose or, a dose having a particular particle size.
  • Compositions of the invention can contain pharmaceutically acceptable flavours and/or colours for rendering them more appealing, especially if they are administered orally.
  • the viscous compositions may be in the form of gels, lotions, ointments, creams and the like and will typically contain a sufficient amount of a thickening agent.
  • the compositions may also be in solid or gelatinous form which are then easily administered as a swallowed pill for oral ingestion.
  • Solutions, suspensions and gels normally contain a major amount of water (preferably purified water) in addition to the antigen and optional adjuvant. Minor amounts of other ingredients, such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents (e.g., methylceliulose), colors and/or flavors may also be present.
  • pH adjusters e.g., a base such as NaOH
  • emulsifiers or dispersing agents e.g., a base such as NaOH
  • buffering agents e.g., preservatives
  • wetting agents e.g., methylceliulose
  • jelling agents e.g., methylceliulose
  • colors and/or flavors may also be present.
  • the compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal fluid.
  • compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • sodium chloride is preferred particularly for buffers containing sodium ions.
  • Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent.
  • Methylceliulose is preferred because it is readily and economically available and is easy to work with.
  • suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like.
  • a pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives, including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride, may also be employed
  • compositions must be selected to be chemically inert with respect to the immunogenic protein and fragment thereof and optional adjuvant. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.
  • the immunologically effective compositions of this invention are prepared by mixing the ingredients following generally accepted procedures.
  • the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
  • the pH may be from about 3 to 7.5.
  • the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the subject being vaccinated.
  • Adjuvants that may be usefully employed in the preparation of vaccines include the following: oil adjuvants, for example, Freund's complete and incomplete adjuvants, mineral salts, alum, silica, kaolin, and carbon, polynucleotides and certain natural substances of microbial origin.
  • the vaccination is by way of a single dose, however it may be beneficial to have multiple spaced apart doses. Suitable regimes for initial administration and booster doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan.
  • the compositions can be administered alone, or can be co-administered or sequentially administered with other compositions of the invention or with other prophylactic or therapeutic compositions.
  • the compositions might additionally comprise other commonly applied vaccine antigens that are protective against diptheria tetanus, hepatitis, polio and pertussis.
  • Vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection as described in P. R. Murray (Ed, in chief), E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken. Manual of Clinical Microbiology, ASM Press, Washington, D.C. sixth edition, 1995, 1482p.
  • vaccine compositions of the present invention are used for the treatment or prophylaxis of meningitis, otitis media, bacteremia or pneumonia.
  • vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection, in particular S. pneumoniae, but may also be effective against group A streptococcus (S. pyogenes), group B streptococcus (GBS or S. agalactiae), Streptococcus gordonii, Streptococcus sanguinis, Streptococcus themiophilus,
  • Streptococcus suis Streptococcus agalactiae
  • Streptococcus pyogenes Streptococcus mutans
  • Streptococcus dysgalactiae Streptococcus uberis
  • Enterococcus faecalis Enterococcus faecium and, Rhodococcus sp.
  • vaccines are administered to those individuals at risk of streptococcus infection such as infants, elderly and
  • mammals include mammals.
  • the mammal is human.
  • the present invention also encompasses within its scope the preparation and use of DNA vaccines.
  • Vaccination methods and compositions of this type are well known in the art (see, for example, chapter 11 of "Molecular
  • the immunogenic protein or fragment thereof may be modified to comprise a signal sequence or other secretory sequence to facilitate expression on surfaces of cells in which it is introduced.
  • a promoter that is suitable for expression in a mammalian cell, and/or mammalian control sequences are also introduced so as to positively impact on expression in the patient's cell.
  • immune enhancers including adjuvants or cloning in frame other immune enhancing cytokines, together with the DNA vaccines is also within the scope of the present invention.
  • DNA vaccine vectors are specifically designed to stimulate humoral immune responses by intramuscular injection.
  • the antigenic peptide produced on the surface of muscle cells is taken up by antigen presenting cells (APCs), processed and presented to the immune system T helper cells through the major histocompatibility complex (MHC) class II molecules.
  • APCs antigen presenting cells
  • MHC major histocompatibility complex
  • the use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscle, delivery of DNA complexed with specific protein carriers, coprecipitation of DNA with calcium phosphate, encapsulation of DNA in various forms of liposomes, particle bombardment, or in vivo infection using cloned retroviral vectors.
  • the immunogenic protein or fragment thereof on introduction of a
  • polynucleotide encoding the same is expressed by at least some of the cells of the individual being treated and an immune reaction is elicited.
  • an anti-GlpO antibody preparation may be introduced into the individual.
  • one or more further antibodies specific for the one or more further immunogenic protein, set out hereinbefore may also be coadministered.
  • the further antibody is specific for pneumolysin.
  • Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against streptococcus infection in a test model.
  • an animal model is the mouse model described in the examples herein.
  • the antibody may be a whole antibody or an antigen- binding fragment thereof and may belong to any immunoglobulin class.
  • the antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment.
  • the term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques.
  • the antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the Streptococcus pneumoniae immunogenic protein but is preferably specific for one.
  • the antibody may include, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. It is within the scope of the present invention that the antibody may be chimeric, i.e. that different parts thereof stem from different species or at least the respective sequences are taken from different species.
  • Antibodies generated against the immunogenic protein corresponding to a sequence of the present invention can be obtained by direct injection of the immunogenic protein into an animal or by administering the immunogenic protein to an animal, preferably a non-human. The antibody so obtained will then bind the immunogenic protein itself.
  • any technique known in the art which provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein. C., Nature 256: 495-497 (1975). Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies directed at the immunogenic protein. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
  • phage display technology could be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Fab or from naive libraries.
  • the affinity of these antibodies can also be improved by chain shuffling.
  • each domain may be directed against a different epitope, termed 'bispecific antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.
  • antibodies against the polypeptide of the present invention may be employed to inhibit and/or treat infections, particularly bacterial infections and especially infections arising from S. pneumoniae.
  • the antibody or derivative thereof is modified to make it less immunogenic in the individual.
  • the antibody may most preferably be "humanized", wherein the complementarity determining region(s) of the hybridpma-derived antibody has been
  • Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, bacteremia and meningitis in children and adults (1). It is the commonest cause of bacterial meningitis in the United States and many countries worldwide (2, 3). Despite effective antimicrobial therapy, pneumococcal meningitis remains highly lethal and has substantial long-term sequelae (4, 5). The availability and use of pneumococcal conjugate vaccines has decreased the incidence of invasive diseases caused by vaccine types. However, this has been offset by significant increases in both carriage and disease caused by non-vaccine serotypes, which have occupied the niche vacated by the vaccine types (6, 7). Therefore, pneumococcal disease continues to be an important health issue worldwide.
  • CNS central nervous system
  • mice with either CbpA, Ply, or both did not afford adequate protection against pneumococcal meningitis (our unpublished observations), suggesting that virulence factors other than CbpA and Ply make an important contribution.
  • Microarray technology is a very powerful tool that provides a global view on integrated cellular processes that are active during infection. This is followed by the combined application of gene cloning, recombinant protein technology and in vitro functional assays to validate target selection for vaccine development.
  • Protective vaccine components are often derived from proteins that are expressed under the specific disease conditions. However, because many virulence factors and antigens are expressed only in vivo, approaches that rely on in v/fro-grown bacteria are likely to miss important protective antigens. Hence, the main focus of the present study was to identify novel factors involved in the pathogenesis of pneumococcal meningitis, to have a better understanding of the underlying molecular mechanisms of the disease.
  • the pneumococcal strains used in this study were serotype 2 (D39), serotype 4 (WCH43) and serotype 6A (WCH16). Serotype-specific capsule production was confirmed by Banlung reaction, as described previously (28). Opaque-phase variants of the three strains, selected on Todd-Hewitt broth supplemented with 1% yeast extract (THY)- catalase plates (47), were used in all animal experiments. Before infection, the bacteria were grown statically at 37°C in serum broth to Aeoo of 0.16 (equivalent to approx. 5 x 10 7 CFU/ml). Mice.
  • mice Intranasal challenge of mice and harvesting of bacteria for gene expression analyses.
  • WCH16 and WCH43 challenges groups of 40 mice were used. The mice were anesthetized by i.p. injection of pentobarbital sodium
  • mice were sacrificed by CO2
  • RNA for microarray experiments was isolated and purified from either blood borne bacteria or a mixture of bacteria that have either crossed over to the brain or those still in transition (attached to the microvascular
  • RNA samples were checked for purity and integrity as described previously (26, 27). Bacterial-RNA samples from a minimum of five mice that satisfied these criteria from a specific niche were pooled. The RNA was then purified further using a Qiagen RNeasy minikit. RNA was further enriched for prokaryotic RNA using the MicrobEw/cA? kit (Ambion); bacterial mRNA from brain samples was further enriched using the MicrobExpress kit (Ambion). The amount of RNA recovered following purification/enrichment was determined by ⁇ 260/280 measurements.
  • Microarray analysis of bacterial RNA was performed on whole genome S. pneumoniae PCR arrays obtained from the Bacterial Microarray Group at St George's Hospital Medical School, London
  • the array was designed using TIGR4 base strain annotation (49) and extra target genes from strain R6 (50).
  • the array design is available in BpG@Sbase (Accession No. A-BUGS-14;
  • RNA samples were reverse-transcribed using Superscript III (Invitrogen) and then labeled with either Alexa Fluor 546 or Alexa Fluor 647 dye.
  • the foreground intensities were log2 transformed and a single ratio (Alexa Fluor 647/Alexa Fluor 546) value was obtained for each probe. Ratio values were normalized using the print-tip Loess normalization routine (54). The replicate arrays were normalized to each other to give similar ranges of mRNA expression values. For each probe across the arrays a linear model was fitted to determine a final expression value for each mRNA probed and associated statistics (55). These statistics were used to rank the mRNAs from those most likely to be differentially expressed to the least likely using false-discovery rate values of p ⁇ 0.05.
  • RNA from infected blood and brains was performed on a total of 9 independent hybridizations from three separate assays, including one dye reversal per comparison for each strain. Relative Quantitation real-time RT-PCR. For a subset of selected
  • pneumococcal genes that were significantly upregulated in the brain by microarray analysis, gene expression were validated by one-step RT-PCR kit (Invitrogen) in a LightCycler®480 II (Roche) as described previously (27). The relative gene expression was analyzed using the 2 " ⁇ CT method (56). The reference gene was 16S rRNA. The primer pairs used for gene expression analysis are listed in Table S2. All data were obtained from three biological replicates. Construction of mutants and assessment of bacterial growth in vitro. Defined, non-polar mutants of genes of interest were constructed in WCH43 (serotype 4) and in some cases, also in WCH16 and D39.
  • Mutants were constructed by overlap extension PGR as described previously (57) and validated by PCR and sequencing to be in-frame deletion mutation replacements. All PCR procedures were performed with the Phusion High Fidelity Kit (FINNZYMES). The primer pairs used for construction and validation of the mutants are listed in Table S2. In order to evaluate the growth rate of the mutants in comparison to the wild type, bacterial strains were grown in serum broth (SB) and A $ oo monitored overnight on a Spectramax M2 spectrophotometer (Millenium Science).
  • mice from each separate infection experiment were sacrificed, and samples from the blood and brain were processed as described previously.
  • mutant and wild type bacteria were mixed at an input ratio of 1 :1 , and 20 mice were challenged i.n. with 50 ⁇ bacterial suspension containing approx. 1 x 10 7 CFU.
  • mice from each mixed infection experiment were sacrificed, and samples from the nasopharynx, lungs, blood, and brain were processed as described previously.
  • HBMEC Human brain microvascular endothelial cell line
  • FCS heat-inactivated fetal calf serum
  • HPEPES heat-inactivated fetal calf serum
  • glpOAFAD open reading frames were PCR amplified from WCH43 genomic DNA with forward and reverse primers (Table S2), which incorporate Sph ⁇ and Sa/I restriction sites.
  • the PCR fragment was digested with the same enzymes and cloned into the corresponding restriction sites in pQE-31 (QIAGEN Inc.) to generate recombinant plasmid.
  • High-level expression in a lipid A (IpxM) mutant of E. coli BL21 (DE3) (59) transformed with pREP4 (QIAGEN) was induced with 2 mM isopropyl-(3-D-thiogalactopyranoside (IPTG) for 3 h.
  • the cells were harvested by centrifugation at 6,000 * g for 10 min and then purified on a nickel-nitrilotriacetic acid column essentially as described previously (60).
  • GlpO and glpOAFAD were purified under denaturing conditions, and dialyzed extensively by buffer exchange according to QIAGEN recommendations. Each protein was judged to be >98% pure by SDS-PAGE and staining with Coomassie brilliant blue R-250.
  • Recombinant glpOAFAD was used for immunization, as it has been shown that the FAD fragment is not essential for immunogenicity (59). However the full recombinant GlpO protein containing the FAD fragment was used for cytotoxicity assays.
  • mice Two weeks after the third immunization, mice were challenged with approx. 3 ⁇ 10 4 CFU of WCH43. Mice were closely monitored for survival over 14 days. Differences in survival of mice between groups were analyzed by the Mann-Whitney U-test (two- tailed). Overall survival rates of mice between groups were compared by the Fisher's Exact test (two-tailed).
  • ELISA titer for anti-GlpO serum was determined from pooled sera from GlpO-immunized mice as described previously (60).
  • 10 mice were immunized i.p. with high titer (32,000) anti-GlpO serum 1 h prior to i.p. challenge with 5 ⁇ 10 4 CFU of WCH43.
  • Serum from alum-immunized mice was injected i.p. into another group of 10 mice 1 h before challenge, as a control.
  • Bacteria were then enumerated from blood and brain of mice from each group at 30 h post challenge, and ratios of bacteria in the brain vs blood determined.
  • Cytotoxicity to HBMEC and H2O2 production were performed essentially as described for M. mycoides subsp. mycoides Small Colony (31). The various pneumococcal preparations for cytotoxicity assays (with or without the indicated treatments) were used to infect confluent
  • Purified recombinant GlpO was used at 30 g well.
  • Viability of HBMEC monolayers after cytotoxicity assay was analyzed using Countess® Cell Counter system (Invitrogen) according to the manufacturer's recommendations.
  • strains were grown in complete C+Y medium (61) to Cells were washed once in the same medium and then resuspended in twice the starting culture volume of fresh medium consisting of either 1 % glucose or 1 % glycerol.
  • Control tubes for each treatment contained either catalase or anti- GlpO serum.
  • Differentially expressed genes between blood and brain are identified by microarray analysis.
  • HBMEC human brain microvascular endothelial cells
  • GlpO is cytotoxic towards HBMEC, and induces H2O 2 release from glycerol metabolism in vitro.
  • an ortholog of pneumococcal GlpO from Mycoplasma mycoides subsp. mycoides Small Colony has been reported to be cytotoxic for embryonic calf nasal epithelial cells (31 , 32).
  • pneumococcal meningitis is still the commonest bacterial meningitis in children in the USA, mostly due to non-vaccine serotypes (3). Therefore, the development of effective, multivalent vaccines providing universal protection represents the best prospect for managing pneumococcal diseases, including meningitis, septicemia and pneumonia, in the 21 st Century.
  • the critical determinants that enable certain strains of pneumococci to cross the blood-brain barrier to cause meningitis are largely unknown, but presumably involve expression of a distinct array of bacterial genes.
  • progression from the blood to the brain will require niche-specific alterations in virulence gene expression, and that surface-exposed virulence factors that are up-regulated during this transition could be protective immunogens against meningitis.
  • glpO alpha- glycerophosphate oxidase
  • H2O2 alpha- glycerophosphate oxidase
  • GlpO alpha- glycerophosphate oxidase
  • glycerol 3-phosphate oxidation involves the formation of H 2 O2 which is important for damaging host cells (31), and glycerol metabolism via GlpO is a major source of the cytotoxic H2O2.
  • GlpO is involved in production and translocation of toxic H2O2 into the host cell, causing inflammation and cell death (32).
  • Interaction of bacteria with the endothelium of the BBB affects invasion of bacteria, recruitment of leukocytes, and local disruption of barrier functions.
  • pneumococci are able to induce rapid apoptosis of neurons by caspase independent mechanisms, mostly initiated by H 2 O 2 (35).
  • the pneumococcus lacks catalase, therefore produces excessive amounts of H 2 0 2 (36).
  • Pneumococcal-derived H 2 0 2 appears to increase intracellular reactive oxygen species (ROS) and Ca 2+ and triggers the release of apoptosis-inducing factors resulting in rapid and massive damage to mitochondria (37).
  • ROS reactive oxygen species
  • H202and ROS may be important in the pathogenesis of pneumococcal meningitis, as they might have a role in peroxidation of brain lipids.
  • Analysis of the Ag/pO mutant revealed that the corresponding enzyme and the formation of H2O2 are important for the toxic effects on the host cells.
  • the AglpO mutant is less cytotoxic than the isogenic wild-type strain, there was still residual host cell damage, suggesting that other factors also play an important role.
  • a strong candidate is pneumolysin, which has been shown to be cytotoxic to eukaryotic cells (38, 39).
  • SpxB which has also been implicated in pneumococcal meningitis (40), and the NADH oxidase (Nox), shown to be important in respiratory tract and otitis media infection models (41).
  • Nox the NADH oxidase
  • pneumoniae proteins essential to the development of meningitis through comparative analysis of transcript levels between discrete in vivo niches during pathogenesis. Indeed, the genes that were picked to be significantly up-regulated in the brain relative to blood had hitherto been uncharacterized, highlighting the robustness of the technique in identifying novel, potentially important factors crucial to the pathogenic process, which other methodologies have missed.
  • novel in vivo transcriptomic strategy described in this paper can be adapted to the discovery of virulence genes that contribute to the development of meningitis caused by other pathogens such as Haemophilus influenzae type B and Neisseria meningitidis.

Abstract

L'invention concerne une composition immunogène qui peut être un vaccin comprenant GlpO ou un fragment de celui-ci, approprié pour l'utilisation dans le traitement ou la prévention d'une infection streptococcique, en particulier une méningite. Le GlpO ou le fragment de celui-ci est de préférence inactif du point de vue enzymatique. Dans une forme préférée, la composition immunogène comprend en outre un immunogène supplémentaire connu comme étant protecteur contre une infection streptococcique, ledit immunogène supplémentaire étant de préférence une protéine. Une telle protéine supplémentaire particulièrement illustrée est une pneumolysine non toxique.
PCT/AU2012/001139 2011-09-23 2012-09-21 Immunogène glpo pneumococcique WO2013040648A1 (fr)

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AU2011903930A AU2011903930A0 (en) 2011-09-23 PNEUMOCCOCAL GlpO IMMUNOGEN

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CN108164586A (zh) * 2018-01-22 2018-06-15 西南医科大学 合成多肽及其应用
EP3439693A4 (fr) * 2016-04-05 2020-05-27 The Research Foundation For The State University Of New York University at Buffalo Nouvelles formulations de vaccin antipneumococcique

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WO2007006712A2 (fr) * 2005-07-07 2007-01-18 University Of Bern Vaccin de sous-unite de mycoplasma
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3439693A4 (fr) * 2016-04-05 2020-05-27 The Research Foundation For The State University Of New York University at Buffalo Nouvelles formulations de vaccin antipneumococcique
US11103568B2 (en) 2016-04-05 2021-08-31 The Research Foundation For The State University Of New York Pneumococcal vaccine formulations
CN108164586A (zh) * 2018-01-22 2018-06-15 西南医科大学 合成多肽及其应用
CN108164586B (zh) * 2018-01-22 2021-03-26 西南医科大学 合成多肽及其应用

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