Patent Publication Number: US-7592011-B2

Title: Genes and proteins, and their use

Description:
REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. application Ser. No. 10/096,162, filed Mar. 11, 2002 and issued on May 10, 2005 as U.S. Pat. No. 6,890,539, which claims priority to U.K. Publication No. GB0105922D under 35 U.S.C. §365, filed Mar. 09, 2001 and is a continuation-in-part of U.S. application Ser. No. 09/868,352, filed Sep. 10, 2001 under 35 U.S.C. §371(c) and issued on Nov. 02, 2004 as U.S. Pat. No. 6,812,021, which is the national stage filing of International Application No. PCT/GB99/04377, filed Dec. 22, 1999, each of which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to the identification of a bacterial gene and protein, and its use. More particularly, it relates to its use in therapy, for immunisation and in screening for drugs. 
     BACKGROUND TO THE INVENTION 
     Group B  Streptococcus  (GBS), also known as  Streptococcus agalactiae , is the causative agent of various conditions. In particular, GBS causes: 
     Early Onset Neonatal Infection. 
     This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate. 
     Late Onset Neonatal Infection. 
     This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and endopthalmitis. 
     Adult Infections. 
     These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterised by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis. 
     Urinary Tract Infections. 
     GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections. 
     Veterinary Infections. 
     GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance. 
     GBS infections can be treated with antibiotics. However, immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine. 
     SUMMARY OF THE INVENTION 
     The present invention is based on the identification of a gene in GBS, and also related organisms, the product of which may be localised on the outer surface of the organism and therefore may be used as a target for immuno-therapy. 
     According to one aspect of the invention, a peptide is encoded by a gene identified herein as pho2-2, obtainable from Group B  Streptococcus , or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated. 
     The term “functional fragment” is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide. For example, a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies. 
     A gene fragment may be used to encode the active peptide. Alternatively, the gene fragment may have utility in gene therapy, targetting the wild-type gene in vivo to exert a therapeutic effect. 
     A peptide according to the present invention may comprise the amino acid sequence identified herein as SEQ ID NO. 2, or a functional fragment thereof. 
     Because of the extracellular or cell surface location, the peptide of the present invention may be a suitable candidate for the production of therapeutically-effective vaccines against GBS. The term “therapeutically-effective” is intended to include the prophylactic effect of vaccines. For example, a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection. The vaccine may be administered to females prior to or during pregnancy to protect mother and neonate against infection by GBS. 
     According to another aspect of the invention, the peptide or gene may be used for screening potential antimicrobial drugs or for the detection of virulence. 
     A further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain. 
     Although the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention. In particular, the peptide or the vaccines may be used in the treatment of chronic mastitis, especially in cows. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
       The invention is described with reference to the accompanying drawing where: 
         FIG. 1  is a graphic illustration of the average level of protection offered by anti-pho2-2 (SEQ ID NO:2) anti-sera against a challenge dose of GBS. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     The present invention is described with reference to Group B Streptococcal strain M732. However, all the GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732. Organisms likely to contain the peptide include, but are not limited to,  S. pneumoniae, S. pyogenes, S. suis, S. milleri , Group C and Group G  Streptococci  and  Enterococci . Vaccines to each of these may be developed in the same way as described for GBS. 
     Preferably, the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptide identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity. With regard to the polynucleotide sequence identified herein, related polynucleotides that may be useful in the various aspects of the invention may have greater than 40% identity with the sequence identified herein. More preferably, the polynucleotide sequences have greater than 60% sequence identity. Most preferably, the polynucleotide sequences have greater than 80% sequence identity, e.g. 95% identity. 
     The terms “similarity” and “identity” are known in the art. The use of the term “identity” refers to a sequence comparison based on identical matches between correspondingly identical positions in the sequences being compared. The term “similarity” refers to a comparison between amino acid sequences, and takes into account not only identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, in addition to sequence similarity. 
     Levels of identity between gene sequences and levels of identity or similarity between amino acid sequences can be calculated using known methods. In relation to the present invention, publicly available computer based methods for determining identity and similarity include one BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol., 1990; 215:403-410), the BLASTX program available from NCBI, and the Gap program from Genetics Computer Group, Madison Wis. The levels of similarity and identity provided herein, were obtained using the Gap program, with a Gap penalty of 12 and a Gap length penalty of 4 for determining the amino acid sequence comparisons, and a Gap penalty of 50 and a Gap length penalty of 3 for the polynucleotide sequence comparisons. 
     Having characterised a gene according to the invention, it is possible to use the gene sequence to establish homologies in other microorganisms. In this way it is possible to determine whether other microorganisms have similar outer surface products. Sequence homologies may be established by searching in existing databases, e.g. EMBL or Genbank. 
     A fragment of the gene sequence disclosed herein may be used to prepare a vaccine product, or as a probe in a diagnostic method, or to identify homologues in other microorganisms. Preferably the fragment will be at least 20 nucleic acids, more preferably at least 30 nucleic acids, and most preferably at least 70 nucleic acids. 
     When used to identify homologues, the gene sequence, or fragment thereof, will preferably hybridise to the putative homologue under stringent hybridisation conditions. Stringent hybridisation conditions are those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104. According to this, hybridisation under stringent conditions means that a positive hybridisation signal is still observed after washing for 1 hour with 1×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and most preferably at 68° C. 
     Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptide or its active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Peptide fragments may be used to identify those parts of the protein that have the most favourable antigenic epitopes. The fragments can be generated by methods known to those skilled in the art. For example, partial digests of the complete protein can be made and tested. Alternatively, synthetic peptide fragments can be made and tested. The fragments may be tested to establish which fragments elicit the strongest immune response. 
     A therapeutic antibody of the invention will have affinity for the protein (or peptide) of the invention and preferably should not cross-react with unrelated proteins or proteins in the patient. 
     Suitable antibodies which may be active against the peptide of the invention, include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art. 
     The preparation of vaccines is known to those skilled in the art. Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B  Streptococci  or other microorganisms that contain related proteins. The preparation of vaccine formulations will be apparent to the skilled person. 
     More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc. 
     The products of the invention may also be used in assays to screen potential antimicrobial drugs. For example, the protein of the invention may be used as a target for antimicrobial therapy, to localise a drug to the infecting microbe. 
     The products of the present invention were identified as follows: 
     A partial gene library of GBS (strain M732) chromosomal DNA was prepared using the plasmid vectors pFW-phoA1, pFW-phoA2 and pFW-phoA3 (Podbielski, A. et al. 1996. Gene 177:137-147). These plasmids possess a constitutive spectinomycin adenyltransferase antibiotic resistance marker, which confers a high level of spectinomycin resistance and is therefore easily selected. Furthermore, these vectors contain a truncated (leaderless)  Escherichia coli  phoA gene for alkaline phosphatase. The three vectors differ only with respect to the reading frame in which the leaderless phoA gene exists, as compared to an upstream in-frame BamHI restriction enzyme site. Because this truncated  E. coli  phoA gene lacks the appropriate leader sequence for export of this enzyme across the bacterial membrane, extracellular alkaline phosphatase activity is absent when these plasmids are propagated in an  E. coli  phoA mutant (e.g. strain DH5α). The chromogenic alkaline phosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate), does not enter intact bacterial cells and therefore only exported or surface associated alkaline phosphatase activity can be detected. When exported or surface associated alkaline phosphatase activity is present, the chromogenic XP substrate is cleaved to yield a blue pigment and the corresponding bacterial colonies can be identified by their blue colour. 
     Plasmid DNA was digested to completion with BamHI and dephosphorylated using shrimp alkaline phosphatase. GBS genomic DNA was partially digested with Sau3AI, size fractionated on a sucrose gradient and fragments &lt;1 kb in size were ligated into the prepared pFW-phoA vectors.  E. coli  strain DH5α was chosen as the cloning host since it lacks a functional phoA gene. Recombinant plasmids were selected on Luria agar containing 100 μg/ml of spectinomycin and 40 μg/ml of the chromogenic XP substrate.  E. coli  transformants harbouring plasmids containing GBS insert DNA that complements the export signal sequence of the leaderless phoA gene were identified by the blue colour of the colonies. Approximately 30000 different recombinant plasmids containing GBS insert DNA were screened in this manner and 83 recombinant plasmids, which complemented the leaderless phoA, were chosen for further study. 
     From these experiments, several clones were selected each containing a plasmid containing a gene (or part thereof), which complemented the leaderless phoA. 
     A clone was selected containing a plasmid designated pho2-2. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 1 and 2, respectively. 
     A comparison of the amino acid sequence of pho2-2 was performed. 
     Homologues to the GBS pho2-2 gene product can be identified in  Enterococcus faecalis, Escherichia coli  (malK and afuC),  Bacillus subtilis  (glnO),  Haemophilus influenzae  (yebM and potA),  Streptococcus pyogenes, Streptococcus pneumoniae  and  Salmonella typhimurium  (malK). The  E. faecalis, S. pyogenes  and  S. pneumoniae  homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In all other cases, homologues represented ATP-binding transport proteins that are part of ABC type transporters. Many of the components of ABC type transporters are membrane or cell surface associated, as these systems are involved in the transport of macromolecules from the extracellular environment to the intracellular compartment. 
     Having identified the gene in each clone it is then possible to obtain the full-length gene sequence, as follows. 
     Using the identified and sequenced gene fragment, oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an ‘outward’ direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5′ and 3′ termini of the gene had been reached. The presence of these features was identified by checking against homologous sequences, and for the 5′ end the presence of an AUG start codon (or accepted equivalent) preceded by a Shine-Dalgarno consensus sequence, and for the 3′ end, the presence of a translation termination (Stop) codon. 
     Upon identification of the full-length gene, primers were designed for amplification of full-length product. Primers used included restriction enzyme recognition sites (NcoI at the 5′ end and EcoO109I at the 3′ end) to allow subsequent cloning of the product into the Lactococcal expression system used. 
     PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (Invitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoO109I. 
     The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). This vector, harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10× His tags, flanked on the 5-most end with an NcoI site, split in the middle with a multicloning site (including an EcoO109I site), and a stop (termination) codon at the 3′ end of the His tags. 
     The gene of interest was inserted so that a 10× His tag was in the 3′ position relative to the coding region. Following transformation of the recombinant plasmid into  L. lactis  (strain NZ9000-Kuipers, O. P. et al. (1998) supra), a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole. 
     To identify fractions containing the His-tagged recombinant protein, an aliquot from each fraction was analysed by SDS-PAGE, Western blotted and probed with anti-His antibodies. 
     The recombinant protein obtained was then used to immunise New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models. 
     Using the sera obtained from the animal studies, immunosorption studies were carried out. 
     Group B  Streptococcus  was grown in 20 ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in 5 ml PBS. 50 μl aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4° C. overnight to allow for adsorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3% BSA in PBS for 1hr at 37° C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50 μl of these dilutions were added to the wells of the plate, in duplicate. The plate was covered and incubated for 1 hr at 37° C. The plate was washed, then 50 μl anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37° C. for an hour, the plate was washed again. 50 μl substrate (PNPP) was added to each well, and the reaction allowed to proceed for 30 min before the adsorbance was read at 405 nm. 
     The results showed that the antibody bound to the whole cells indicating that pho2-2 resides on the outer surface of the cell. 
     Animal protection studies were also carried out to test the effectiveness of protection on the immunised rabbits. 
     Anti-sera against the pho2-2 outer surface protein and a further protein (pho3-9) identified in the same screen were raised in rabbits and the IgG from each anti-sera was purified using Protein A column chromatography. Newborn pups from time-mated Sprague Dawley rats were ‘immunised’ with 50 μl of the purified IgG intraperitoneally and returned to different mothers for at least 5 hours before they were challenged with the GBS. 
     The A909 strain of GBS was used as the challenge. The strain was streaked on to a blood agar plate, and allowed to grow over the weekend at room temperature. A single colony of GBS was used to produce the inoculum in THB. Following overnight growth, the GBS bacteria were centrifuged, and an appropriate volume of PBS added to produce a final dilution of 1×10 6  CFU/ml GBS (5×10 4  GBS/50 μl). The innoculum was kept on ice until use in the challenge assay in the rat pups. 
     The GBS was administered subcutaneously to each rat and the time of challenge recorded. All pups were monitored for approximately 63 hours after the GBS challenge. 
     Table A shows the percentage of pups that survived 63 hours after challenge with GBS. This data has been represented in a graph in  FIG. 1 . 
     
       
         
           
               
               
               
               
             
               
                 TABLE A 
               
               
                   
               
               
                 Study 
                   
                   
                   
               
               
                 date 
                 PBS 
                 Pho 2-2 
                 Pho 3-9 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Feb. 27, 2001 
                 14 
                 52 
                 41 
                 21 
               
               
                 Mar. 27, 2001 
                 20 
                 — 
                 67 
                 53 
               
               
                 Apr. 3, 2001 
                 17 
                 — 
                 46 
                 — 
               
               
                 MEAN 
                 19.50 
                   
                 51.50 
                 37.00 
               
               
                 SD 
                 5.57 
                   
                 11.27 
                 22.63 
               
               
                 SE 
                 2.78 
                   
                 5.63 
                 16.00 
               
               
                   
               
            
           
         
       
     
     The pho2-2 protein offered significant protection against GBS infection compared to the PBS control and to the other outer surface protein pho3-9.