ZA200104818B - Genes and proteins, and their use. - Google Patents

Description

GENES AND PROTEINS, AND THEIR USE

Field of the Inventjon

This invention relates to the identification of bacterial genes and proteins, and their use. More particularly, it relates to their use in therapy, for immunisation and in screening for drugs.

Background to the Inventjon

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 series of genes in GBS, and also related organisms, the products 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 an operon including any of the genes identified herein as phol-13, pho3-21, pho2-15, pho3-18, pho3-22, pho3-3, pho3-17, pho2-2, phol-5, pho3-1, pho3-23, pho3-50, . phol-14, pho2-10, pho3-14, pho3-24 and pho3-29, 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 fragments" 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 any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35, or a functional fragment thereof.

Because of the extracellular or cell surface location, the peptides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against GBS. The term "therapeutically-effective"

R WYO 00/37646 PCT/GB99/04377 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 peptides or genes 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 peptides or the vaccines may be used in the treatment of chronic mastitis, especially in cows.

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 peptides 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 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. 95% similarity.

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.

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 peptides or their 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. Suitable ] antibodies 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 based on attenuated microorganisms 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 related microorganisms. 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 present invention were identified as follows:

A partial gene library of GBS (strain M732) chromosomal DNA was prepared using the plasmid vectors pFW- § phoAl, 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, i0 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 DH5a). 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 <1lkb in size were ligated into the prepared pFW-phoA vectors. E. coli strain DHSa was chosen as the cloning host since it lacks a functional phoA gene. Recombinant plasmids were selected on Luria agar containing 100 ug/ml of spectinomycin and 40 ug/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.

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 §' ) 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 S5'end and Eco0109I 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 (In

Vitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and Eco0109I.

The vector into which this fragment was inserted was a modified version of pN28048 (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 10X His tags, flanked on the 5- 5S most end with an Ncol site, split in the middle with a multicloning site (including an Eco0109I site), and a Stop (termination) codon at the 3'end of the His tags.

The gene of interest was inserted so that a 10X His tag was in the 3' position relative to the coding region.

Following transformation of the recombinant plasmid into

L.lactis (strain NZ9000 - Kuipers, 0. 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 i 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 20ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in 5ml

PBS. 50ul aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4°cC 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 50ul 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

S50ul 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. 50ul substrate (PNPP) was added to each i0 well, and the reaction allowed to proceed for 30min before the adsorbance was read at 405 nm.

Animal protection studies were also carried out to test the effectiveness of protection on the immunised rabbits. .

GBS M732 was grown up in THB until mid-log phase was reached - approximately 5 hours. Cells were counted in a counting chamber, and bacteria were diluted to give a concentration of 2x10’ bacteria per ml in pre-immune or test sera. 5041 of this was injected via the intraperitoneal route into 0-1 day old mice. The mice were observed for survival over 48 hours.

The following Examples illustrate the invention.

Example 1

A first clone contained a gene sequence identified herein as SEQ ID NO. 1, with an amino acid sequence identified as SEQ ID NO. 2, and classified as phol-13.

A comparison of the amino acid sequence of phol-13 was performed.

Homologues to the GBS phol-13 gene product can be identified in Streptococcus pyogenes, S. pneumoniae, S. salivarius, Escherichia coli, Yersinia enterocolitica,

Aquifex aeolicus, Helicobacter pylori and Haemophilus influenzae. The 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, the above homologues can be identified as ATP-dependent Clp protease proteolytic subunits. The catalytic activity of Clp proteases results in the hydrolysis of proteins to small peptides in the presence of ATP and magnesium (Giffard, P.M. et al. 1993.

J. Gen. Micrcbiol. 139:913-920). Furthermore, the CIlpP component of Clp proteases has been shown to be induced as part of the heat shock response (Kroh, H.E. and L.D. Simon. 1990. J. Bacteriol. 172:6026-6034) and it is probable that this subunit or the complete proteolytic domain would associated with the bacterial surface.

Immunisation studies, carried out as described above, yielded the following results. : Treatment No animals No animals surviving at time (hrs) 24 48

PBS 10 7 0)

Pre-immunised 37 13 0

Immunised 38 17 9

Example 2

A second clone was selected containing a plasmid designated phol-14. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences are shown as

SEQ ID NOS. 3 and 4, respectively.

A comparison of the amino acid sequence of phol-14 was performed.

Homologues to the GBS phol-14 gene product can be identified in Streptococcus pyogenes, Enterococcus faecalis and Streptococcus pneumoniae. These homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. Additionally, two possible homologues were also identified from Shigella flexneri (SpaR) and Yersinia pseudotuberculosis (¥scT). These latter two homologues are related proteins, believed to be anchored in the bacterial membrane (Bergman, T. et al. 1994. J. Bacteriol. 176:2619- 2626). In S. flexneri, the product of the spaR gene has been shown to be important for invasion of epithelial cells (Sasakawa, C. et al. 1993. J. Bacteriol. 175:2334-2346).

Furthermore, the product of the spaR gene is also required for surface presentation of invasion plasmid antigens. The analogous protein in Y. pseudotuberculosis is a component of the Yop secretion system and is also important for virulence in this organism.

Example 3

A third clone was selected containing a plasmid designated phol-5. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences are shown as ’

SEQ ID NOS. 5 and 6.

A comparison of the amino acid sequence of phol-5 was performed.

Homologues to the GBS phol-5 gene product can only be identified in Streptococcus pyogenes and Staphylococcus carnosus (sceA). The S. pyogenes homologue was identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.

Furthermore, little information is available on the function of the sceA gene product from S. carnosus. The sceA dene product shows some sequence similarity to the aggregation promoting protein from Lactobacillus gasseri.

Based on analysis of the sceA gene product, this molecule contains a well-conserved signal sequence and is apparently secreted or associated with the bacterial cell surface.

Example 4

A further clone was selected containing a plasmid designated pho3-3. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The

Claims (12)

1. A peptide encoded by an operon including any of the genes identified herein as phol-13, pho3-21, pho2-15, pho3- 18, pho3-22, pho3-3, pho3-17, pho2-2, phol-5, pho3-1, pho3- 23, pho3-50, phol-14, pho2-10, pho3-14, pho3-24 and pho3- 29, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof.

2. A peptide according to claim 1, comprising any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, io 6, 8, 10, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35.

3. A peptide according to claim 1 or claim 2, for therapeutic use.

4. A polynucleotide encoding a peptide according to claim - 1 or claim 2, for therapeutic use.

5. A host transformed to express a peptide according to claim 1 or claim 2.

6. A vaccine comprising a peptide according to claim 1 or claim 2, or the means for its expression.

7. Use of a product according to any of claims 1 to S, for screening potential drugs or for the detection of virulence.

8. Use of a product according to any of claims 1 to 5, for the manufacture of a medicament for use in the treatment or prevention of a condition associated with bacterial infection.

9. Use according to claim 8, wherein the infection is a Group B streptococcal infection.

10. Use according to claim 8 or claim 9, wherein the infection is a focal infection.

11. Use according to claim 8 or claim 9, wherein the infection is a urinary tract infection.

12. An antibody raised against a peptide according to claim 1 or claim 2.