EP1330473A2 - Streptokokken-gene - Google Patents

Streptokokken-gene

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Publication number
EP1330473A2
EP1330473A2 EP01976527A EP01976527A EP1330473A2 EP 1330473 A2 EP1330473 A2 EP 1330473A2 EP 01976527 A EP01976527 A EP 01976527A EP 01976527 A EP01976527 A EP 01976527A EP 1330473 A2 EP1330473 A2 EP 1330473A2
Authority
EP
European Patent Office
Prior art keywords
gene
sit2a
microorganism
pneumoniae
iron
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01976527A
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English (en)
French (fr)
Inventor
David William Dept. Infectious Diseases HOLDEN
Jeremy Stuart Brown
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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Publication date
Priority claimed from GB0026231A external-priority patent/GB0026231D0/en
Priority claimed from GB0028345A external-priority patent/GB0028345D0/en
Priority claimed from GB0102666A external-priority patent/GB0102666D0/en
Application filed by Imperial College Innovations Ltd filed Critical Imperial College Innovations Ltd
Publication of EP1330473A2 publication Critical patent/EP1330473A2/de
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • This invention relates to genes and the proteins that they encode, to vaccines containing the proteins or functional fragments of the proteins, and to live attenuated bacterial vaccines lacking any or part of the genes. More particularly, the invention relates to their prophylactic and therapeutic uses and their use in diagnosis.
  • Streptococcus pneumoniae is second only to M. tuberculosis as a cause of mortality worldwide. S. pneumoniae frequently colonises the nasopharynx and invasive infection can develop in a variety of body compartments, including the middle ear, the lung interstitium, the blood, and cerebrospinal fluid. The organism must utilise iron sources in each of these environments, but at present it is poorly understood how S. pneumoniae acquires iron and from which substrate(s).
  • iron sources in the respiratory tract include tactoferrin, transferrin, ferritin (released from dead cells shed from the mucosal epithelium), and possibly small amounts of haemoglobin and its breakdown products (LaForce et al, 1986; Thompson et al, 1989; Schryvers and Stojiljkovic, 1999).
  • siderophores produced by other nasopharyngeal commensuals may provide an alternative iron source. S.
  • pneumoniae growth in iron-deficient medium can be complemented by haemin, haemoglobin and ferric sulphate but, in contrast to other pathogens of mucosal surfaces (Schryvers and Stojiljkovic, 999; Cornelissen and Sparling, 1994), not by transferrin and lactoferrin (Tai et al., 1993).
  • Chemical and biological assays suggest S. pneumoniae does not produce siderophores (Tai et al., 1993), but a haemin-binding polypeptide has been isolated and an undefined mutant unable to utilise haemin as an iron source was reduced in virulence (Tai et al, 1993 and 1997).
  • the molecular basis for iron uptake by S. pneumoniae has yet to be characterised, and iron transporters have not been proven to be virulence determinants in animal models for a Gram-positive pathogen.
  • Virulence determinants of Gram-negative bacteria are frequently encoded in defined areas of chromosomal DNA thought to be acquired by horizontal transmission and termed pathogenicity islands (Pis) (Carniel et al, 1996; Ralpher et al, 1997; Blanc- Potard and Groisman, 1997; Janakiraman and Slauch, 2000). Characteristically, Pis have a different GC content to host chromosomal DNA, frequently have tRNA or insertion sequences at their boundaries, contain genes encoding mobile genetic elements, and are not present in less pathogenic but related strains of bacteria (Hacker et al, 1997). Pis often contain genes encoding virulence functions specific for the host bacteria.
  • SPI-2 of S. typhimurium encodes a type III secretion apparatus which allows the bacteria to multiply within macrophages but is not present in closely related enteric pathogens (Shea et al, 1996). Consequently, acquisition of Pis is probably a major influence in the evolution of distinct Gram-negative pathogens (Hacker et al 1997; Ochman et al, 2000). In contrast, only a few Pis have been described for Gram-positive pathogens and they rarely have the classical genetic characteristics of Gram- negative Pis (Hacker et al., 1997).
  • Sit1, Sit2 and Sit3 ABCD genes are shown in the accompanying sequence listing.
  • the Sit2 operon is located in a pathogenicity island required for in vivo growth.
  • Other genes that encode putative virulence determinants were also identified, and are referred to herein as MS1 to 11 and ORFI to 14.
  • a peptide is encoded by any of the gene sequences identified herein as SitlA, B or C, Sit2B, C or D, Sit3A, B, C or D, ORF1 to 14, and MS1 to 11 , or a functional fragment thereof, for therapeutic or diagnostic use.
  • an attenuated microorganism comprises a mutation that disrupts expression of any of the gene sequences identified above and also SitlD and Sit2A.
  • a vaccine composition comprises any of the gene sequences identified above, with an optional pharmaceutically acceptable diluent, carrier or adjuvant.
  • a vaccine composition comprises a peptide encoded by SitlD and a peptide encoded by Sit2A, or a functional fragment thereof capable of elliciting an immune response.
  • This combination vaccine ellicits a very good immune response compared to other known peptide vaccines.
  • a peptide of the invention is used in a screening assay for the identification of an antimicrobial drug, or in a diagnostic assay for the detection of a streptococcal microorganism. Description of the Figures The invention is described with reference to the accompanying figures, wherein:
  • Figure 1 is a schematic representation of the Sit1 and Sit2 loci, where clear boxes represent ORFs flanking the Sit loci; black boxes represent putative iron-binding receptors; grey boxes represent putative ATPases; diagonal shading represent putative permeases; and arrows represent site of insertions in mutant strains;
  • Figure 2 is a schematic representation of PPM , where (A) and (B) show the GC content plot for PPI1 , with the dashed line representing mean GC content of S. pneumoniae DNA (38.9%), and (C) shows ORFs flanking and contained within PPM , where ORFs present in a species are marked by a cross and ORFs absent from a species are marked by a minus mark;
  • Figure 3 is a graphic representation of the growth of sit mutants measured by optical density, where (A) shows growth in THY broth, (B) in Chelex-THY broth, (C) in Chelex-THY broth + 10 ⁇ M FeCI 2 , (D) in Chelex-THY broth + 10 ⁇ M FeCI 3 and (E) in Chelex-THY broth + 10 ⁇ M haemoglobin, and where 0 represent the wild-type strain; ⁇ a Sit1A ⁇ strain; O a Sit2A ⁇ strain; A a Sit1A /Sit2A ⁇ strain and where (F) shows the growth of the Sit1A ' lSit2A ⁇ strain in Chelex-RPMIm with X representing no supplement; H represents 10 ⁇ M FeCI 3 ; O represents 10 ⁇ M haemoglobin; ⁇ represents 25 ⁇ M MnSO 4 and 25 ⁇ M ZnSO 4 supplements; Figure 4 illustrates the sensitivity to strepton
  • Figure 5 is a graphic representation of 55 FeCI 3 uptake, where
  • FIG. 6 is a graphic representation of the survival of groups of 10 mice inoculated with Sit mutant strains where (A) represents intranasal (IN) inoculation and (B) represents intraperitoneal (IP) inoculation;
  • Figure 7 is a graphic representation of the % survival of mice treated with various proteins and infected with S. pneumoniae.
  • Figure 8 is a graph showing the survival rates for mice which were administered serum from mice immunised with various proteins of the invention, wherein series 1 represents alum only, series 2 is SitlD, series 3 is Sit2A, series 4 is pdb, series 5 is pdb + SitlD, series 6 is pdb + Sit2A and series 7 is SitlD + Sit2A.
  • the peptides (proteins) and genes of the invention were identified as putative virulence determinants using signature tagged mutagenesis (Hensel et al, 1995).
  • the proteins and genes of the present invention may be suitable candidates for the production of therapeutical ly-effective vaccines against Gram- positive bacterial pathogens and S. pneumoniae.
  • the term "therapeutically- effective" is intended to include the prophylactic effect of the vaccines.
  • a recombinant protein may be used, as an antigen for direct administration to an individual.
  • the protein may be isolated directly from a Gram-positive bacterial pathogen or from S. pneumoniae or expressed in any suitable expression system, e.g. Escherishia coli. It is preferably administered with an adjuvant, e.g. alum.
  • the vaccine composition comprises a combination of peptides of the invention, e.g. a peptide encoded by SitlD and a peptide encoded by Sit2A, or a functional fragment thereof capable of elliciting an immune response.
  • This double protein vaccine is shown to offer improved protection compared to the known protective antigen non-toxic pneumolysin (Pdb).
  • the protein may be a mutant protein in comparison to wild-type protein, a fragment of the protein or a chimeric protein comprising different fragments or proteins, provided an effective immune response is generated.
  • protein fragments are at least 20 amino acids in size, more preferably, at least 30 amino acids and most preferably, at least 50 amino acids in size.
  • An alternative approach is to use a live attenuated Gram-positive bacterium or a live attenuated S. pneumoniae vaccine. This may be produced by deleting or disrupting the expression of a gene of the invention.
  • the S. pneumoniae strain comprises additional virulence gene mutations.
  • the mutated microorganisms of the invention may be prepared by known techniques, e.g. by deletion mutagenesis or insertional inactivation of a gene of the invention.
  • the gene does not necessarily have to be mutated, provided that the expression of its product is in some way disrupted.
  • a mutation may be made upstream of the gene, or to the gene regulatory systems.
  • the preparation of mutant microorganisms having a deletion mutation are shown in WO-A-96/17951.
  • a suicide plasmid comprising a mutated gene and a selective marker is introduced into a microorganism by conjugation.
  • the wild-type gene is replaced with the mutated gene via homologous recombination, and the mutated microorganism is identified using the selective marker.
  • the attenuated microorganisms may be used as carriers of heterologous antigens or therapeutic proteins/ polynucleotides.
  • a vector expressing an antigen may be inserted into the attenuated strain for delivery to a patient.
  • Conventional techniques may be used to carry out this embodiment.
  • Suitable heterologous antigens will be apparent to the skilled person, and include any bacterial, viral or fungal antigens and allergens, e.g. tumour- associated antigens.
  • suitable viral antigens include: hepatitis A, B and C antigens, herpes simplex virus HSV, human papilloma virus HPV, respiratory syncytial virus RSV, (human and bovine), rotavirus, norwalk, HIV, and varicella zooster virus (shingles and chickenpox).
  • Suitable bacterial antigens include those from: ETEC, Shigella, Campylobacter, Helicobacter, Vibrio cholera, EPEC, EAEC, Staphylococcus aureus toxin, Chlamydia, Mycobacterium tuberculosis, Plasmodium falciparum, Malaria and Pseudomonas spp.
  • the heterologous antigen may be expressed in the host cell utilising a eukaryotic DNA expression cassette, delivered by the mutant.
  • the heterologous antigen may be expressed by the mutant bacterium utilising a prokaryotic expression cassette.
  • the microorganism may alternatively be used to deliver a therapeutic heterologous peptide or polynucleotide to a host cell.
  • cytokines are suitable therapeutic peptides (proteins), which may be delivered by the microorganisms for the treatment of patients infected with hepatitis.
  • the delivery of a polynucleotide is desirable for gene therapy, for example, anti-sense nucleotides, such as anti-sense RNA, or catalytic RNA, such as ribozymes.
  • the gene that encodes the heterologous product may be provided on a recombinant polynucleotide that contains the regulatory apparatus necessary for the expression of the gene, e.g. promoter, enhancers etc.
  • the prokaryotic or eukaryotic expression cassette may be incorporated in a vector, e.g. a multi-copy plasmid.
  • the heterologous gene may be targeted to a gene endogenous to the microorganism, including the gene to be mutated, so that the heterologous gene becomes incorporated into the genome of the microorganism, and uses the endogenous or cloned regulatory apparatus for its expression.
  • the protein (or fragments thereof) of the present invention may also be used in the production of monoclonal and polyclonal antibodies for use in passive immunisation.
  • the protein or corresponding polynucleotide may be used as a target for screening potentially useful drugs, especially antimicrobials.
  • Suitable drugs may be selected for their ability to bind to the protein or DNA to exert their effects.
  • Suitable drugs may be selected for their ability to affect the expression of Gram-positive pathogenicity island genes required for in vivo growth, for example the Sit genes, thereby reducing or altering the ability of the bacterium to survive in vivo or in a particular environment.
  • Assays for screening for suitable drugs and which make use of the protein or polynucleotides of the invention will be apparent to those skilled in the art.
  • proteins, polynucleotides, attenuated mutants and antibodies raised against the proteins and attenuated mutants are described for use in the diagnosis or treatment of individuals, veterinary uses are also considered to be within the scope of the present invention.
  • promoter sequences associated with the genes identified herein may be used to regulate expression of heterologous genes.
  • the promoters may be achieved either by incorporating the promoters in a vector system, e.g. a conventional gene expression vector.
  • the heterologous gene may be inserted into the bacterial chromosome such that the promoter regulates expression.
  • the present invention relates to pathogenicity islands, containing genes required for the growth of Gram-positive pathogens in vivo.
  • the example is described with reference to S. pneumoniae strain 0100993, however all pathogenic S. pneumoniae strains should have the same genes.
  • Southern analysis and PCR have been utilised to demonstrate that Sit1 and Sit2 homologues are present in all S. pneumoniae capsular types tested.
  • Sit1 probes hybridise to genomic fragments of other Streptococci e.g. S. mitis, S. oralis, S. sanguis, S. milleri. Vaccines to each of these may be developed in the same way as described for S. pneumoniae.
  • the mutant microorganisms may be present in a composition together with any suitable excipient.
  • the compositions may comprise any suitable adjuvant.
  • the microorganisms may be produced to express an adjuvant endogenously.
  • Suitable formulations will be apparent to the skilled person.
  • the formulations may be developed for any suitable means of administration. Preferred administration is via the oral, mucosal (e.g. nasal) or systemic routes.
  • the peptides that may be useful for the production of vaccines have greater than 40% similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably the peptides have greater than 80% sequence similarity, e.g.
  • nucleotide sequences that may be useful for the production of vaccines have greater than 40%) identity with the nucleotide sequences identified herein. More preferably, the nucleotide sequences have greater than 60% sequence identity. Most preferably the nucleotide sequences have greater than 80% sequence identity, e.g. 95% identity.
  • similarity and “identity” are known in the art. In the art, identity refers to the relatedness between polynucleotide or polypeptide sequences as determined by comparing the sequences, and particularly identical matches between nucleotides or amino acids in correspondingly identical positions in the sequences being compared.
  • Similarity refers to the relatedness of polypeptide sequences, and takes account not only of identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, even if there is little apparent identity.
  • Gap length penalty 3 available as the "Gap” program from Genetics Computer Group, Madison Wl.
  • Table 1 lists the genes of the present invention and the appropriate SEQ
  • S. pneumoniae strain 0100993, isolated from a patient with pneumonia was used for all experiments.
  • S. pneumoniae strains were cultured on Columbia agar supplemented with 5% horse blood, in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY), or using a modified version of RPMI medium at 37 °C and 5% C0 2 .
  • RPMIm was made by adding to the tissue culture medium RPMI (type 1640, Gibco) 0.4% bovine serum albumin factor V (BSA, Sigma), 1 % vitamin solution and 2 mM glutamine.
  • Plasmid DNA was isolated from E. coli using Qiagen Plasmid Kits (Qiagen) using the manufacturer's protocols. Standard protocols were used for cloning, transformation, restriction digests, and ligations of plasmid DNA (Sambrookef al, 1989). Nylon membranes for Southern hybridisations were prepared and probed using 32 P-dCTP labelled probes made using the RediPrime random primer labelling kit (Amersham International Ltd, Bucks, UK) as previously described (Holden et al, 1989). S. pneumoniae chromosomal DNA was isolated using Wizard genomic DNA isolation kits (Promega). Computer analysis of nucleic acid sequences
  • Preliminary S. pneumoniae sequence data was obtained from The Institute for Genomic Research website (http://www.tigr.org) and analysed and manipulated using MacVector (International Biotechnologies, Inc.).
  • BLAST2 searches of available nucleotide and protein databases and of incomplete microbial genomes were performed using the NCBI website (http://www.ncbi.nlm.nih.gov/blast/).
  • Dendrograms were constructed using Multalin (http://www. toulouse.inra.fr/multalin. html) and TreeViewPPC. Sequence GC content was analysed using Artemis (Genome Research Ltd) and graphs of GC content made with the Window application of the Wisconsin Sequence Analysis Package (Genetics Computer Group). Construction of mutant strains
  • Plasmids, primers and S. pneumoniae strains constructed and used for this work are shown in Table 2.
  • S. pneumoniae mutant strains were constructed by insertional duplication mutagenesis. Internal portions of the target genes were amplified by PCR using primers designed from the available genomic sequence, and ligated into plD701 (cm resistance, derived from pEVP3, or pACH74 erythromycin resistant.
  • plD701 cm resistance, derived from pEVP3, or pACH74 erythromycin resistant.
  • pPC5 an internal portion of SitlA from bp 320 to 711 was amplified using primers Smt6.1 and Smt6.2, digested with Xbal and ligated into the Xbal Site of plD701.
  • pPC12 an internal portion of Sit2A from bp 84 to 428 was amplified using primers IRP1.1 and IRP1.2, digested with Xbal and ligated into the Xbal Site of plD701.
  • pPC25 for construction of the double mutant an internal portion of SitlA from bp 320 to 711 was amplified using primers Smt6.3 and Smt6.4, digested with Kpnl and ligated into the Kpnl Site of pACH74.
  • Vectors designed to insert plasmid DNA 265 bp downstream of the stop codon of SitlD (plasmid pPC4) and 234 bp downstream of Sit2D (plasmid pPC29) were constructed by ligating PCR products generated by primer pairs Smt3.3/Smt3.4 and IRP1.7/IRP1.8 respectively into the Xbal Site of plD701.
  • the inserts of the disruption vectors were sequenced to confirm that they contained the predicted genomic sequences for their respective PCR primers.
  • S. pneumoniae strains were transformed using a modified protocol requiring induction of transformation competence with Competence Stimulating Peptide 1 (CSP1 ) (Havarstein et al., 1995).
  • CSP1 Competence Stimulating Peptide 1
  • 8 ml cultures of S. pneumoniae grown to an OD 580 between 0.012 and 0.020 in THY broth pH 6.8 were collected by centrifugation at 20000 g at 4°C and resuspended in 1 ml THY broth pH 8.0 supplemented with 1 mM CaCI 2 and 0.2% BSA. Competence was induced by addition of 400 ng of CSP1 followed by the addition of 10 ⁇ g of circular transforming plasmid.
  • the transformation reactions were incubated at 37°C for 3 hours then plated on selective medium and incubated for 24 to 48 hours at 37°C in 5% CO 2 .
  • Individual mutants were made by transformation of the wild- type S. pneumoniae strain with the appropriate plasmid, and the double Sit1A7Sit2A ⁇ strain was constructed by transformation of the Sit2A ' strain with pPC25. The identity of the mutations carried by mutant strains was confirmed by PCR and Southern hybridisation.
  • nitrocellulose filters were prefiltered with 40 ⁇ M FeCI 3 and 55 FeCI 3 medium filtered through 0.2 ⁇ M membranes (Sartorius) before use. Reaction filters were placed in 10 ml of Optisafe scintillation fluid (Wallac) and counted using the H settings of a Beckman LS 1801 scintillation counter (Beckman). In vivo studies using mice models of S. pneumoniae infection
  • mice Outbred male white mice (strain CD1 , Charles Rivers Breeders) weighing from 18 to 22 g were used for all animal experiments except for the SitlD and Sit2A immunisation experiment.
  • Inocula consisted of appropriately diluted defrosted stocks of S. pneumoniae strains cultured in THY broth and stored at - 70°C. Strains being compared by competitive infection were mixed in proportions calculated to result in each strain contributing 50%) of the cells in the inoculum.
  • mice were anaesthetised by inhalation of halothane (Zeneca) and inoculated intranasally (IN) with 40 ul of 0.9% saline containing between 5 x 10 5 to 5 x 10 6 bacteria.
  • mice were inoculated by intraperitoneal injection with 100 ul of 0.9% 0 saline containing 5 x 10 1 (for survival curves) or 1 x 10 3 (for competitive infections) bacteria. Three to five mice were inoculated per competitive infection experiment and sacrificed after 24 hours (IP inoculations) or 48 hours (IN inoculations). Target organs were recovered, homogenised in 0.5 ml 0.9% saline and dilutions plated and incubated overnight at 37°C in 5% CO 2 on non-selective and selective medium.
  • CI competitive indices
  • Mice inoculated with a pure inocula of each strain for survival curves were sacrificed when the mice exhibited the following clinical signs of disease; severely ruffled fur, hunched posture, poor mobility, weight loss, and (for IN inoculation only) coughing and tachypnoea.
  • Cls were compared to 1.0 (the predicted CI if there is no difference in virulence between the two strains tested) using Student's t test, and survival curves were compared using the log rank method.
  • a signature-tagged mutagenesis screen of S. pneumoniae strain in a mouse model of pneumonia identified a strain attenuated in virulence which contains a mutation in a gene (smtA) whose predicted amino acid product has 31 %) identity and 53%o similarity to CeuC, a component of a Campylobacter coli iron uptake ABC transporter (Richardson and Park, 1995).
  • smtA is the second gene of a four gene locus encoding a likely ABC transporter with high degrees of identity to iron uptake ABC transporters ( Figure 1 ). This four gene locus was renamed SH1ABC and D (streptococcal iron transporter 1). Searches of the following genome sequence.
  • Sit2A is the first gene of a second four gene locus, Sit2ABC and D, with high degrees of identity to iron uptake ABC transporters ( Figure 1 ). Both the Sit1 and Sit2 loci conform to the reported organisation of loci encoding ABC transporters and contain one gene encoding putative ATPases, one gene encoding putative lipoprotein iron receptors, and two genes encoding putative transmembrane permease proteins (Fig.
  • the Sit1 locus is flanked by an ORF whose derived amino acid sequence has 43%o identity to UDP galactose epimerase of Lactococcus lacti (terminates 392 bp upstream of SitlA, transcribed in the same direction) and a small ORF whose derived amino acid sequence has high degrees of similarity to ORFs of unknown function (starts 230 bp downstream of SitlD, transcribed in the opposite direction) (Fig. 1 ).
  • the Sit2 locus is flanked by an ORF whose derived amino acid sequence has 42% identity to a Bacillus subtilis RNA methyltransferase homolog (terminates 1353 bp upstream of Sit2A, transcribed in the same direction), and an ORF whose derived amino acid sequence has 25% identity to a Staphylococcus aureus recombinase (starts 1997 bp downstream of Sit2D, transcribed in the same orientation) (Fig. 1 ).
  • the degrees of identity and similarity between the derived amino acid sequence of the putative metal-binding receptors of Sit1, SitlD, and Sit2, Sit2A is low at 22% and 53%).
  • SitlD and Sit2A have the highest degree of identities to separate groups of iron compound binding lipoproteins.
  • SitlD and Sit2A both contain motifs matching the consensus sequence for the lipoprotein signal peptide cleavage site (Sutcliffe and Russell, 1995).
  • the derived amino acid sequences of the putative ATPases, SitlC and Sit2D contain motifs commonly found in ATP-binding proteins (Linton and Higgins, 1998).
  • strains carrying mutations of SitlA and Sit2A were constructed by insertional duplication mutagenesis. Insertions were placed in the first genes of each loci to ensure complete inactivation of operon function. A third mutant containing insertions in both SitlA and Slt2A was also constructed. To ensure the mutant phenotypes were not due to polar effects of the insertions on genes downstream of the Sit loci, two further mutants, PPC4 and PPC29, were constructed containing insertions 73 and 100 bp downstream of the stop codons of SitlD and Sit2D respectively (Fig. 1 ).
  • Sit mutants on iron-deficient medium The growth of the Sit mutant strains were compared with the wild-type strain in an undefined complete medium, THY, and in THY which had been treated with Chelex-100 to remove cations (Fig. 3). There were no discernible differences in growth between all the mutant strains and the wild-type strain in THY. Compared to growth in THY all strains had impaired growth in Chelex-THY, 0 and in this medium the double mutant Sit1A/Sit2A ⁇ strain had decreased growth compared to wild type and the single Sit strains (Fig. 3).
  • the wild-type and single SitlA ' and Sit2A ⁇ strains' growth defects in chelex-THY were reversed by supplementation with 40 ⁇ M ferric or ferrous chloride, partially reversed by supplementation with 10 ⁇ M haemoglobin but not by 5 ⁇ g ml "1 of lactoferrin or 5 5 ⁇ g ml "1 of ferritin.
  • Growth of the double mutant Sit1A ⁇ ISit2A strain in chelex-THY was restored to levels similar to that of the wild-type strain by the addition of ferric or ferrous chloride, suggesting that this strain's growth defect compared to the wild-type strain in chelex-THY is due to iron depletion.
  • the Sit loci seem to have different roles during infection, with Sit2 being of greater importance for both pulmonary and systemic infection.
  • the mutant strain containing an insertion immediately downstream of the Sit1 locus, pPC4 was not reduced in virulence, confirming the reduced virulence of the SitlA " strain was not due to a polar effect. Due to its relative instability, the mutant strain containing an insertion immediately downstream of the Sit2 locus, pPC29, was not tested in vivo.
  • the closest homolog of the ORF immediately downstream of the Sit2 locus is a putative recombinase carried by the S. aureus mec mobile genetic element, mec is an 'antibiotic resistance island' which confers methicillin 5 resistance, and the recombinase may catalyse recombination of mec into S. aureus chromosomal DNA (Ito et al, 1999).
  • Sit2 locus has a lower GC content than the upstream DNA sequence, with a striking drop in GC content occurring within the C terminus of the ORF immediately upstream of the Sit2 locus (ORF B).
  • the GC content of 250,000 bp of S. pneumoniae chromosomal DNA on the contig containing Sit1 was 38.9%, similar to the estimate calculated by chemical methods (38.5%, Hardie, 1986).
  • Analysis of the GC content of consecutive 800 5 bp segments of 41.6 kb of DNA including the Sit2 locus identified an area of approximately 27,000 bp with a mean GC content of 32.6%, nearly 7%> lower than the surrounding sequence (p ⁇ 0.001 , Fig. 2). This area was termed PPI1 (Pneumococcal Pathogenicity Island 1).
  • the boundaries of PPI1 are marked by sharp decreases in GC content (Fig.
  • PPM of approximately 4000 and 3200 bp length have a GC content approaching that of the S. pneumoniae chromosome (Fig. 2).
  • the putative transposase immediately downstream of the 3' end of PPM belongs to the insertion sequence family IS605, which includes the IS200 transposons of Gram-negative pathogens 0 (Mahillon and Chandler, 1998).
  • IS605 transposons have been reported in S. pneumoniae (Oggioni and Claverys, 1999) and are characterised by a low frequency of transposition and hairpin loops 5' to the transposase, but do not contain terminal inverse repeats (IR) (Beuzon and Casadesus, 1997; Mahillon and Chandler, 1998).
  • PPM contains two ORFs associated with mobile genetic elements, the recombinase described above and a relaxase (ORF11 ).
  • ORF11 relaxase
  • chromosomal DNA around the Sit1 locus has a mean GC content of 40.1 % with no regions whose GC content significantly varies from the mean for S. pneumoniae.
  • ORF1 is a recombinase
  • ORF10 is a relaxase
  • ORF13 is a likely ATPase
  • ORF11 is a possible transcription factor
  • Sit2A has high degrees of similarity only to ORFs from non-streptococcal species whereas SitlD has high degrees of identity to an ORF from Streptococcus mutans (35 % identity and 52% similarity over 332 residues).
  • BALBc were mice given 10 ug of protein (expressed in pQE30 expression vectors in E. coli and purified using the His-tag) via IP, on three occasions separated by 7 - 10 days, and then challenged 2 weeks after the last immunisation with i0,000 S. pneumoniae cells inoculated IP.
  • Alum was used as a negative control, and the non-toxic pneumolysin variant, termed Pdb, was used as a positive control (known to be protective).
  • the other proteins were SitlD, Sit2A, SitlD combined with Sit2A, SitlD combined with Pdb, and Sit2A combined with Pbd. Essentially, both SitlD and Sit2A are as protective as Pdb, and the combination of SitlD and Sit2A is very protective (80% 0 long term survivors compared to 0% in the alum group). Combinations of Pdb and either SitlD or Sit2A had no additional protective benefit over the individual proteins (Fig. 7).
  • the serum from immunised mice was given IP to another group of na ⁇ ve mice, and then these mice were challenged with 3000 bacteria.
  • the results showed a clear benefit for the group receiving the combined Sit1DlSit2A antisera.
  • the clear positive result with the Sit1DISit2A antisera confirms that the protective effect of immunisation with SitlD and Sit2A is a serum, i.e. antibody, dependent phenomena (Fig. 8).
  • Salmonella typhimurium maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J 19: 3235-3249.
  • the Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival. EMBO J 16: 5376-85.
  • the pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2. Mol Microbiol 13: 697-708.

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GB0714963D0 (en) 2007-08-01 2007-09-12 Novartis Ag Compositions comprising antigens
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AU2010258677B2 (en) 2009-06-10 2016-10-06 Glaxosmithkline Biologicals S.A. Benzonaphthyridine-containing vaccines
CN102844047B (zh) 2009-09-02 2017-04-05 诺华股份有限公司 含tlr活性调节剂的免疫原性组合物
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WO2011057148A1 (en) 2009-11-05 2011-05-12 Irm Llc Compounds and compositions as tlr-7 activity modulators
AU2010339921B2 (en) 2009-12-15 2016-08-11 Glaxosmithkline Biologicals S.A. Homogeneous suspension of immunopotentiating compounds and uses thereof
EA031379B1 (ru) 2010-03-23 2018-12-28 Новартис Аг Соединения (липопептиды на основе цистеина) и композиции в качестве агонистов tlr2, применяемые для лечения инфекционных, воспалительных, респираторных и других заболеваний
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US11098349B2 (en) * 2015-11-04 2021-08-24 The Translational Genomics Research Institute Systems and methods of diagnosing and characterizing infections
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