DE69935080T2 - GROUP B STREPTOCOCCUS PROTEINS AND THEIR USE - Google Patents

Description

Territory of invention

These The invention relates to the identification of bacterial genes and proteins and their use. In particular, it relates to their use for therapy, for the immunization and the screening of drugs.

background the invention

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is the causative agent various diseases. In particular, GBS causes:

Early neonatal infection.

These Infection usually begins in utero and causes severe sepsis and pneumonia in infants which, when left untreated, is deadly and even when treated with a mortality rate of 10 to 20% is.

Late neonatal infection.

These Infection occurs in the stage shortly after birth until age from about 3 months up. It causes sepsis, which in 90% of the cases is made difficult by meningitis. There are also other focal infections, including Osteomyelitis, septic arthritis, abscesses and endopthalmitis, on.

Infection in adults.

These seem stronger to spread and occur most often in women who have recently given birth, who are older and immune deficiencies exhibit. These infections are due to sepsis and focal infections, including Osteomyelitis, septic arthritis, abscesses and endopthalmitis, characterized.

Urinary tract infections.

GBS is a cause of urinary tract infections and is in pregnancies for about 10% of all infections are responsible.

Infections in animals.

GBS causes chronic mastitis in cows. These in turn leads to a reduced milk production and is therefore of considerable economic Importance.

GBS infections can be treated with antibiotics. However, an immunization is preferable. It is therefore desirable to develop an immunogen that is therapeutically effective Vaccine could be used.

Summary the invention

The The present invention is based on the identification of a gene in GBS and also related organisms, whose product is on the outer surface of the Organism can be localized and therefore as the target of immunotherapy can serve.

Under In one aspect of the invention, a peptide comprises the present invention with SEQ ID NO: 23 marked amino acid sequence or a homologue or functional fragment thereof for therapeutic use, e.g. if it is isolated.

Of the Term "functional Fragments "means present part of the gene or peptide, the activity or the Maintains the efficacy of the entire gene or peptide. For example may be a functional fragment of the peptide as an antigenic determinant used in a vaccine or for the production of antibodies is usable.

One Gene fragment can be used to encode the active peptide. In another embodiment The fragment can be used in gene therapy in the case of the wild-type gene is targeted in vivo to have a therapeutic effect cause.

One Peptide of the present invention may be present as SEQ ID NO: 23 labeled amino acid sequence include.

by virtue of its extracellular or cell surface localization could the peptide of the invention suitable candidate for the production of a therapeutically effective Vaccine against GBS. The term "therapeutically effective" is intended to be prophylactic Effect of vaccines. For example, a vaccine may be a peptide of the invention or funds for its expression to treat an infection. The vaccine May be given to women before or during pregnancy be through to the mother and the newborn against infection To protect GBS.

Under In another aspect of the invention, the peptide or the gene for screening potential antimicrobial agents or used for the detection of virulence.

One Another aspect of the invention is the use of one of presently defined products for treatment or prevention one with infection by a group B streptococcal strain related Illness.

Even though the protein is described for use in the treatment of patients The present invention also encompasses veterinary uses the products of the invention. In particular, you can the peptides or vaccines in the treatment of chronic mastitis, especially in cows, be used.

description the invention

The The present invention is related to the group B Streptococcus strain M732 described. All GBS strains and many other bacterial strains however, probably contain related peptides or proteins, the one amino acid homology with the peptide of M732. Organisms that are likely containing the peptides close S. pneumoniae, S. pyogenes, S. suis, S. milleri, group C and group C streptococci and Enterococci, but are not limited thereto. Versus each of these tribes can in the same way as described above for GBS, vaccine be developed.

Preferably have the peptides that are suitable for the production of vaccines, a sequence similarity from bigger than 40% with the peptide identified herein. More preferred the peptides have a sequence similarity from bigger than 60% up. Most preferably, the peptides have sequence similarity of 80%, e.g. 95% similarity on.

By the characterization of a gene according to the invention makes it possible the gene sequence for detecting homologies in other microorganisms to use. In this meadow it is possible to determine if others Organisms similar Products on the outer surface. Sequence homologies can by searching in existing databases, e.g. EMBL or Genbank, be determined.

Inventive peptides or proteins can purified and isolated by means known in the art become. In particular, it is due to the identification of the gene sequence possible, use recombinant techniques to make the gene in a suitable To express host. Active or effective fragments and homologs can be identified, and these may be suitable for therapies. For example can Peptides or their active fragments as antigenic determinants be used in a vaccine to elicit an immune response. You can also for the production of antibodies, for passive immunization or for diagnostic applications are used. Suitable antibodies include monoclonal antibody or fragments thereof, including Single-chain fv fragments, a. Methods for producing antibodies are a person skilled in the art.

The Production of attenuated microorganism vaccines is one Specialist known. Vaccine compositions may be treated with suitable carriers or Adjuvants, e.g. Alum, formulated according to requirement or desire and therapeutically used to provide effective immunization against group B streptococci or other related organisms. The preparation of vaccine formulations is known to a person skilled in the art.

As a person skilled in the art can in general, a suitable amount of an active ingredient of the Invention for therapeutic use, as well as suitable carriers or Excipients, and routes of administration are selected. These factors be known according to Criteria such as the type / severity of the disease to be treated, the type / Health status of the subject, etc. selected or determined.

The products according to the invention were identified as follows:
A partial gene library of chromosomal GBS DNA (strain M732) was prepared using the plasmid vectors pFW-phoA1, pFW-phoA2 and pFW-phoA3 (Podbielski, A. et al., 1996, Gene 177: 137-147). These plasmids contain a constitutive spectinomycin adenyltransferase antibiotic resistance marker which confers high spectinomycin resistance and is therefore easy to select. Furthermore, they contain a truncated (without leader sequence) phoA gene from Escherichia coli for alkaline phosphatase. The three vectors differ only in the reading frame of the leaderless phoA gene compared to an upstream in-frame BamHI restriction enzyme site. Since this truncated E. coli phoA lacks the appropriate leader sequence for the export of this enzyme across the bacterial membrane, no extracellular alkaline phosphatase activity is present when these plasmids are propagated in an E. coli phoA mutant (eg strain DH5α). The chromogenic alkaline phosphatase substrate XP (5-bromo-4-clor-3-indolylphosphate) does not enter intact bacterial cells, therefore only exported or surface-associated alkaline phosphatase activities can be detected. If there is an exported or surface-associated alkaline phosphatase activity, the chromogenic XP substrate is cleaved to give a blue pigment and the corresponding bacterial colonies identified by their blue color.

The Plasmid DNA was complete digested with BamHI and using crab alkaline phosphatase dephosphorylated. Genomic GBS DNA was partially digested with Sau3AI, size fractionated on a sucrose-graded, and fragments <1kB in size were inserted into the prepared pFW-phoA vectors ligated. The E. coli strain DH5α was selected as cloning host, because he lacks a functional phoA gene. Recombinant plasmids was on Luria agar containing 100 μg / ml Spectinomycin and 40 μg / ml of the chromogenic XP substrate. E. coli transformants with plasmids containing the GBS insert DNA containing the export signal sequence phoA gene without Leader sequence were complimented by the blue colouration of the Identified colonies. About 30,000 different recombinant plasmids which Containing GBS insert DNA were screened in this way, and 83 recombinant plasmids carrying the phoA without leader sequence were selected for further study.

Out In these experiments, several clones were selected, each of which containing a plasmid containing a gene (or a part thereof), which the phoA is complemented without leader sequence.

To Identification of the gene in each clone then becomes possible full To obtain gene sequence as follows.

Under Use of the identified sequenced gene fragments were Oligonucleotide primer for sequencing genomic DNA selected. These Primers were chosen such that was sequenced in the "out" direction, starting from the sequence obtained. After reading, the preserving sequence was checked to determine if the 5 'and 3' ends of the gene reached were. The presence of these features was by comparison was detected with homologous sequences, and with respect to the 5 'end, the Presence of an AUG start codon (or equivalent) checked with a precedent Shine-Dalgarno consensus sequence, and regarding the 3'-end the presence of a translation termination (stop) codon was checked.

To the identification of the complete Genes were selected for amplification of the complete product. The The primers used contained restriction enzyme recognition sequences (NcoI at the 5 'end and Eco0109I at the 3'-end) subsequent cloning of the product into the Lactococcus expression system used to enable.

The PCR was performed using the primer and the pCR-2.1 cloning vector (InVitrogen) cloned products performed. After confirmation of the Existence of the cloned fragment, the DNA was using the restriction enzymes NcoI and Eco0109I excised.

The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, OP et al. (1998) J. Biotech 64: 15-21). This vector contains a replication origin of Lactococcus, a cloramphenicol resistance marker, an inducible nisin promoter, and a multiple cloning interface was replaced by replacing the multiple cloning site with two 10x His tag sequences flanked at the 5 'end by a NocI site, divided in the middle by a multiple cloning site (including an Eco0109I site), and a stop (termination ) Codon at the 3 'end of the His tag sequences, altered.

The gene of interest was inserted such that a 10 x His tag sequence on the 3'-side with respect to the coding region. After the transformation of the recombinant plasmid in L. lactis (strain NZ9000 - Kuipers, O. P. et al. (1998) supra), a 400 ml liquid culture was prepared and the Translation of the protein induced by addition of nisin to the culture. After an incubation for The cells were harvested for 2 hours and lysed by bead heating. The resulting lysate was clarified by centrifugation and then via a Metal affinity column (Talon, Clontech). The pillar was washed several times before the bound proteins with imidazole were eluted.

to Identify fractions that have the His-tagged recombinant Contained an aliquot of each fraction by SDS-PAGE, Western blot and hybridization with anti-His antibodies analyzed. The recombinant protein obtained was then used to immunize New Zealand rabbits are used, with preimmune sera prior to immunization were won. After the boost immunization, the rabbits became killed and the serums won. These sera were used in Western blots, ELISA and animal welfare models.

Under Use of the sera obtained from the animal experiments were immunosorbent studies carried out.

Group B Streptococcus was cultured in 20ml Todd Hewitt's medium (THB) for eight hours, harvested and resuspended in 5 ml PBS. Aliquots of 50 μl of it were made for coating wells of a 96-well plate (Nunc Immuno-Sorb). This was allowed to stand at 4 ° C to the Absorbance of the bacteria to the Platter cause. The plates were washed twice with PBS, then with 3% BSA in PBS for 1 h 37 ° C blocked. The plates were washed again. There were 10-fold serial dilutions of these sera prepared in PBS, and 50 μl of these dilutions were placed in the wells of the plate, 2-fold each. The plate was covered and for 1 h at 37 ° C incubated. The plate was washed, then 50 μl rabbit alkaline phosphatase conjugated Secondary antibodies at a concentration of 1: 5000 in each well. To incubation at 37 ° C for 1 h was washed the plate again. There were 50 μl of substrate (PNPP) in each well and the reaction was run for 30 minutes, before the absorbance was measured at 405 nm.

It animal welfare examinations have also been carried out to Protection in immunized rabbits.

GBS M732 was cultured in THB until mid log phase, approximately 5 hours. The cells were counted in a counting chamber and the bacteria were diluted to a concentration of 2 x 10 ⁷ bacteria per ml in preimmune or test sera. 50 μl of it was injected intraperitoneally into 0-1 day old mice. The mice were observed for survival for 48 hours.

The The following example illustrates the invention.

example 1

It a clone was selected which was a pho3-1 designated plasmid contained. This plasmid contained a gene (or part of it), that complemented the phoA without a leader sequence. The nucleotide and deduced amino acid sequence of the gene are shown as SEQ ID NO: 22 and 23.

One Comparison of the amino acid sequence pho3-1 was performed.

Homologs of the pho3-1 gene product of GBS can be identified in Streptococcus pyogenes, Streptococcus pneumoniae, Bacillus subtilis (YutD) and Enterococcus faecalis. The homologues in S. pyogenes, S. pneumoniea, and E. faecalis were identified by genome sequence data, and no comments were made regarding the identity of the genes or gene products. The function of the yutD gene product in B. subtilis is unknown. However, it can be stated that the yutD gene is located on the B. subtilis chromosome in a region containing genes involved in cell wall synthesis. The fact that this DNA sequence complements the phoA gene without a leader sequence indicates that this gene product is located extracellularly.

SEQUENCE LISTING

Claims (9)

A peptide comprising the present invention with SEQ ID NO. 23 designated amino acid sequence, or a homologue or fragment thereof, wherein the homologue or Fragment for the production of antibodies with an affinity to the peptide is for therapeutic use. A peptide according to claim 1, which is the present SEQ ID NO. 23 designated amino acid sequence includes. A polynucleotide comprising a peptide according to claim 1 or Claim 2 encoded for therapeutic use. A vaccine which is a peptide according to claim 1 or claim 2 includes. Use of a product according to any one of claims 1 to 3 for the screening of potential drugs or for the detection of a Virulence. Use of a product according to any one of claims 1 to 3 for the manufacture of a medicament for Use in the treatment or prevention of infection by a group B streptococcal microorganism. Use according to claim 6, wherein the infection is a Focal infection is. Use according to claim 6, wherein the infection is a Urinary tract infection is. Against a peptide according to claim 1 or claim 2 directed antibody.