WO2002077020A2

WO2002077020A2 - Virulence genes in h. influenzae

Info

Publication number: WO2002077020A2
Application number: PCT/GB2002/001305
Authority: WO
Inventors: Mark Anthony Herbert; Mary Eileen Deadman; Derek William Hood; Edward Richard Moxon
Original assignee: Isis Innovation Limited
Priority date: 2001-03-22
Filing date: 2002-03-18
Publication date: 2002-10-03
Also published as: AU2002246243A1; WO2002077020A3

Abstract

Virulence genes in H influenzae are identified using signature tagged mutagenesis (STM). The genes and their encoded products may be useful in the production of vaccines or antibiotics to prevent or treat H. influenzae infection.

Description

VIRULENCE GENES IN H. INFLUENZAE

Field of the Invention

This invention relates to virulence genes and proteins, and their use. More particularly, it relates to genes and proteins/peptides obtained from Haemophilus influenzae, and their use in therapy and in screening for drugs . Background of the Invention

Haemophilus influenzae are Gram-negative bacteria that were first identified during the influenza ("flu") pandemic of 1890. At the time they were erroneously thought to be the cause of the disease (hence their name) , but as is now known, influenza is of viral, and not bacterial, origin.

There are six antigenically distinct capsular types of H. influenzae, referred to as a to f . Ninety-five percent of systemic infections in childhood are caused by the serotype b; these include meningitis, sepsis and epiglottitis . Bacterial meningitis and epiglottitis due to H. influenzae are life-threatening diseases with a lethality of 5 percent or more. There are also non-capsular type H. influenzae and these cause pneumonia, sinusitis and otitis media .

Some H. influenzae infections can be treated with antibiotics. However, due to the problems associated with resistance to antibiotics, and antibiotic-allergic patients, there is a need for further therapeutics which may be useful in treating or preventing infection. The complete genome sequence for H. influenzae strain K 20 was published in Fleischmann et al . , Science, 1995; 269:496-512. Summary of the Invention The present invention is based on the discovery of virulence genes in H. influenzae .

According to a first aspect of the invention, a peptide of the invention is encoded by a gene comprising any of the nucleotide sequences identified herein as SEQ ID NOS. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 of H. influenzae or a homologue thereof in a Gram negative bacterium, or a functional fragment thereof, for therapeutic or diagnostic use .

The peptides may have many therapeutic uses for treating H. influenzae infections, including use in vaccines for prophylactic application.

According to a second aspect, a polynucleotide encoding a peptide defined above, may also be useful for therapy or diagnosis.

According to a third aspect, the genes that encode the peptides may be utilised to prepare attenuated microorganisms. The attenuated microorganisms will usually have a mutation that disrupts the expression of one or more of the genes identified herein, to provide a strain that lacks virulence. These microorganisms will also have use in therapy and diagnosis.

According to a fourth aspect, the peptides, genes and attenuated microorganisms according to the invention may be used in the treatment or prevention of a condition associated with infection by H. influenzae or Gram-negative bacteria.

The invention provides the possibility of vaccines against all serotypes and non-typable H. influenzae . Description of the Invention

The present invention is based on the discovery of polynucleotide sequences encoding peptides which are implicated in virulence. The peptides and polynucleotide sequences of the invention are therefore useful for the preparation of therapeutic agents to treat infection. It should be understood that references to therapy or treatment also include preventative treatments, e.g. vaccination. Furthermore, while the products of the invention are intended primarily for treatment of infections in human patients, veterinary applications are also considered to be within the scope of the invention. The present invention is described with reference to Haemophilus influenzae. However, other Gram-negative bacterial strains are likely to include related peptides or proteins having amino acid sequence identity or similarity to those identified herein. Organisms likely to contain the peptides include, but are not limited to the species actinobacillus, pleuropneumoniae and other haemophilus species . Preferably, the peptides that may be useful in the various aspects of the invention have greater than a 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. at least 90 or 95% similarity. With regard to the polynucleotide sequences identified herein, related polynucleotides that may be useful in the various aspects of the invention may have greater than 40% identity with the sequences 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 polynucleotide 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 the 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 WI . 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 (or polynucleotide sequence) according to the invention, it is possible to use the gene sequence to search for related genes or peptides in other microorganisms. This may be carried out 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 related molecules 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. Active fragments of the peptides are those that retain the biological function of the peptide. For example, when used to elicit an immune response, the fragment will be of sufficient size, such that antibodies generated from the fragment will discriminate between that peptide and other peptides on the bacterial microorganism. Typically, the fragment will be at least 30 nucleotides (10 amino acids) in size, preferably 60 nucleotides (20 amino acids) and most preferably greater than 90 nucleotides (30 amino acids) in size. It should also be understood, that in addition to related molecules from other microorganisms, the invention encompasses modifications made to the peptides and polynucleotides identified herein which do not significantly alter the biological function. It will be apparent to the skilled person that the degeneracy of the genetic code can result in polynucleotides with minor base changes from those specified herein, but which nevertheless encode the same peptides. Complementary polynucleotides are also within the invention, as are derived RNA, e.g. mRNA sequences. Conservative replacements at the amino acid level are also envisaged, i.e. different acidic or basic amino acids may be substituted without substantial loss of function. 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, to provide effective immunisation against infection. The preparation of vaccine formulations will be apparent to the skilled person. The attenuated microorganisms may be prepared with a mutation that disrupts the expression of any of the genes identified herein. The skilled person will be aware of methods for disrupting expression of particular genes . Techniques that may be used include insertional inactivation or gene deletion techniques. In addition to mutations of the gene sequence, the skilled person will appreciate that gene expression may be disrupted by mutations to the gene regulatory apparatus, e.g. promoter sequences upstream of the gene. Attenuated microorganisms according to the invention may also comprise additional mutations in other genes, for example in a second gene identified herein or in a separate gene required for growth of the microorganism, e.g. an aro mutation. Attenuated microorganisms may also be used as carrier systems for the delivery of heterologous antigens, therapeutic proteins or nucleic acids (DNA or RNA) . In this embodiment, the attenuated microorganisms are used to deliver a heterologous antigen, protein or nucleic acid to a particular site in vivo . Introduction of a heterologous antigen, peptide or nucleic acid into an attenuated microorganism can be carried out by conventional techniques, including the use of recombinant constructs, e.g. vectors, plasmids etc, which comprise polynucleotides that express the heterologous antigen or therapeutic protein, and also include suitable promoter sequences. Alternatively, the gene that encodes the heterologous antigen or protein may be incorporated into the genome of the organism and the endogenous promoters used to control expression.

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 would be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type and/or health of the subject etc. In a separate embodiment, the products of the invention may be used in screening assays, e.g. for the identification of potential antimicrobial drugs or for the detection for virulence. Routine screening assays are known to those skilled in the art, and can be adapted using the products of the invention in the appropriate way. For example, the products of the invention may be used as the target for a potential drug, with the ability of the drug to inactivate or bind to the target indicating its potential antimicrobial activity. The various products of the invention may also be used in veterinary applications.

The following is a description of the experimental procedure used to identify the virulence genes. Bacterial strains and culture conditions The strain Rd-b+:02 (Zwahlen et al . , Microb. Pathog., 1986; 1(5) : 465-473) (referred to herein as Rd-b+) was used in the experiments. Rd-b+ is a type b encapsulated and virulent form of Rd (Wilcox and Smith, J. Bacteriol., 1975; 122: 443-453), derived by transformation of the strain Rd with whole chromosomal DNA from the strain Eagan (Anderson et al . , J. Clin. Invest., 1972; 51(1): 39-44). It has LPS and capsule characteristics similar to Eagan, but is presumed to have a similar number of genes and the same genomic plan as Rd. STM methodology

The STM technique has been well described for other pathogens (Hensel etal., Science, 1995; 269(5222): 400-403; Mei et l., Mol. Microbiol., 1997; 26(2): 399-407). For the screening of H. influenzae mutants in vivo as per the STM protocol, the five-day old infant rat model of H. influenzae bacteraemia (Moxon, Haemophilus Influenzae, eds. Sell and Wright, Elsevier 1982 pp 59-71) was adapted. In brief, banks of 24 transposon mutants in microtitre plate array format were recovered from -70°C storage and subcultured to fresh BHI broth containing kanamycin. Mutants were pooled, inoculated into 20 ml BHI broth containing nalidixic acid (5 μg/ml) and kanamycin (10 μg/ml) and cultured to OD_A490 0.3. Bacteria were pelleted, resuspended in phosphate buffered saline containing 0.1% gelatin, diluted to 2xl0⁵ CFU/100 μl, and this dose administered via intraperitoneal injection in duplicate into infant rats. The same dose was plated on to BHI agar containing kanamycin and nalidixic acid to provide the "inoculum" colonies. At 30 hours, the rats were bled and a 10 μl ventral tail vein blood sample was then plated on to BHI agar containing kanamycin and nalidixic acid, to provide the "recovered" colonies. Resulting "inoculum" and "recovered" colonies were pooled after 16 hours incubation at 37°C, the DNA extracted, and tags amplified, labeled and probed against the colony blots. Mutants that were present in the inoculum (tag hybridization to the inoculum blot) but not present in the recovered bacteria (tag hybridization consistently absent on two recovery blots) were re-cultured from -70°C storage and the genes which contained an insertion mutation were identified and sequenced.

The genes identified by this procedure are shown in Table 1. Table 1

The HI numbers refer to the annotation given in the genome sequence available at : www.tigr.org/tigr-scripts/CMR2/ The sequences are shown in the accompanying sequence listing.

Seven of the gene hits were originally annotated as hypothetical proteins. However, low threshold homology matches to proteins in the microbial databases, through Interpro (www.ebi.ac.uk/interpro/), enabled putative functions to be assigned to some of these (Table 1) .

One of the H. influenzae bacteraemia (HB) genes had a transposon insertion in HI1018 that encodes for the insertion element IS1016. In the sequenced non-pathogenic strain Rd, IS1016 has no obvious function (Fleischmann et al . , Science, 1995; 269 (5223): 496-512), however, in the pathogenic type b encapsulated strain Eagan, the IS1016 element flanks the type b capsulation locus (Kroll et al . , Cell, 1991; 53(3): 347-356) and is involved in a dose- dependent duplication of that locus during invasive disease. Other genes that may be directly involved in pathogenesis include HI0364, encoding the cell wall biosynthetic enzyme PBP-7; HI1537, encoding lic-lA, an enzyme involved in the addition of phosphorylcholine to LPS (Weiser et al . , Infect. Immun, 1990; 58: 3455-3457); and HI1732, encoding hia, an adhesin and putative auto-transporter found in some NTHi (St Geme et al.,, J. Bacteriol., 2000; 182: 6005-6018).

The products of five genes are involved in transcription or DNA processing, including HI0814 (tRNA synthetase) , HI0877 (GTP binding protein) , HI1056 (encoding a putative DNA restriction or methylation enzyme) , HI1459 (sigma 70) and HI1528 (encoding topoisomerase IV) ; and two gene products have roles in protein modification, HI1152 (encoding a protein modification and repair enzyme) and HI1368 (a zinc protease) . One gene, HI0898, encodes a multidrug resistance protein (EmrA) , which was reported through STM as important for E. coli colonisation of the gastrointestinal tract (Martindale et al . , Mol. Microbiol., 2000; 37(6): 1293-1305), possibly acting by extrusion of toxic host molecules .

Eleven HIB genes are involved in nutrient/cofactor acquisition and metabolic processes, including HI0086 (cystathionine gamma synthase required for selenoamino acid metabolism) ; HI0129 (a putative iron transporter) ; HI0406

(acetyl co-A carboxylase) ; HI0429 (glucosamine-fructose-6- phosphate aminotransferase) ; HI0432 (a putative NADH pyrophosphatase) ; HI0561 (encoding a putative oligopeptide transporter) ; HI0406, HI0527 and HI0936 (electron transport chain enzymes) ; HI1170 encoding a putative para- aminobenzoate synthase important for folate synthesis; HI1218 (L-lactate permease) , and HI1245 (L-malate oxidoreductase) . Confirmation of reduced in vivo survival Six HIB genes were independently disrupted by insertion-deletion mutagenesis: HI0129, HI0406, HI0432, HI0700, HI1218 and HI1245. Correct mutagenesis was confirmed by PCR analysis for all mutants and by Southern analysis for HI1218. Virulence of the resulting mutants was assessed in the infant rat by competitive index (CI) experiments (Sun et al . , Nat. Med., 2000; 6(11) : 1269-1273) . Mutants were diluted to 2xl0⁵ cfu/100 μl , mixed with Rd-b+ in a 1:1 ratio and intraperitoneal inoculated, each into two infant rats. At 30 hours, the animals were bled and 5 μl of a 1:200 dilution of blood was plated on to BHI agar, both plain and containing nalidixic acid and kanamycin. To confirm differences in survival of mutants and Rd-b+, 200 colonies from the plain agar plate (containing mixed Rd-b+ and mutant) were sub-cultured on to either BHI agar containing kanamycin or BHI agar containing no antibiotics. The CI was determined as the inoculated ratio divided by the recovered ratio of Rd-b+ to mutant (Rd-b+ .-mutant) ^I/ (Rd- b+:mutant)^R. Three mutants, HI0129, HI0432 andHI0700, were profoundly reduced in virulence with CI < 0.0001. The other three mutants all had CI <0.4.

Claims

I. A peptide encoded by a gene comprising any of the nucleotide sequences identified herein as SEQ ID NOS. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 of H. influenzae or a related molecule having at least 40% sequence similarity or identity at the peptide or nucleotide level in a Gram- negative bacterium, or a functional fragment thereof, for therapeutic or diagnostic use.

2. A peptide according to claim 1, wherein the sequence similarity or identity is at least 60%.

3. A peptide according to claim 1 or claim 2, wherein the sequence similarity or identity is at least 90%.

4. A peptide according to claim 1, comprising the amino acid sequence identified herein as SEQ ID NOS. 2, 4, 6, 8,

10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52.

5. A polynucleotide encoding a peptide according to any preceding claim, for therapeutic or diagnostic use.

6. A host transformed to express a peptide according to any of claims 1 to 4.

7. An attenuated microorganism comprising a mutation that disrupts the expression of any of the nucleotide sequences defined in claim 1.

8. A microorganism according to claim 7, wherein the mutation is insertional inactivation or a gene deletion.

9. A microorganism according to claim 7 or claim 8, wherein the microorganism is a H. influenzae .

10. A microorganism according to any of claims 7 to 9, for therapeutic or diagnostic use.

II. A microorganism according to any of claims 7 to 10, comprising a mutation in a further nucleotide sequence.

12. A microorganism according to any of claims 7 to 11, comprising a heterologous antigen, therapeutic peptide or nucleic acid.

13. A vaccine comprising a peptide according to any of claims 1 to 4, or the means for its expression.

14. A vaccine comprising a microorganism according to any of claims 7 to 12.

15. Use of a product according to any of claims 1 to 12, for the manufacture of a medicament for use in the treatment or prevention of a condition associated with infection by H. influenzae.

16. Use according to claim 15, wherein the condition is pneumonia .

17. Use according to claim 15 or claim 16, for veterinary treatment .

18. An antibody raised against a peptide according to any of claims 1 to 4.

19. Use of a product according to any of claims 1 to 12, in a screening assay for the identification of an antimicrobial drug.