DNA encoding the 15 kD outer membrane protein of Haemophilus influenzae

1. Field of the Invention

This invention relates to the 15 kD outer membrane protein of Haemophilus influenzae type b and nontypable Haemophilus influenzae.

For the sake of simplicity, Haemophilus influenzae is hereinafter referred to as H. influenzae.

2. Discussion of the Prior Art

Haemophilus influenzae type b is a major cause of meningitis and other invasive bacterial diseases in children under the age of five. Efficacious vaccines have been produced. The vaccines contain the type b capsular polysaccharide conjugated to a carrier protein. Nontypable H. influenzae cause surface mucosal infections in children and adults. Such organisms also cause invasive disease in children in the developing world and immunocompromised patients. The vaccines which have been developed to prevent disease due to type b organisms are not effective against nontypable H. influenzae.

Outer membrane proteins elicit antibodies which are protective in animal models and therefore should be considered as components in the next generation of vaccines designed to prevent both serotype b and nontypable Haemophilus disease.

The object of the invention is to clone the gene for this H. influenzae protein, and to determine the DNA sequence thereof.

Accordingly, the present invention relates to a recombinant polynucleotide comprising a nucleotide sequence for the 15 kD protein of Haemophilus influenzae type b and nontypable Haemophilus influenzae, said protein having the amino acid sequence as follows:

10       20        30         40       50        60

70         80        90      100        110      120

130      140       150        160       170      180

MetThrLysS

190       200       210       220       230      240

erSerAlaGlnIleAlaGluMetGlnThrLeuProThrIleThrAspLysThrValValT

250       260       270       280       290      300

yrSerCysAsnLysGlnThrValThrAlaValTyrGlnPheGluAsnGlnGluPrnValA

310       320        330       340       350      360

laAlaMetValSerValGlyAspGlyIleIleAlaLysAspPheThrArgAspLysSerG

370       380      390        400      410       420

lnAsnAspPheThrSerPheValSerGlyAspTyrValTrpAsnValAspSerGlyLeuT

430       440       450       460      470       480

hrLeuAspLysPheAspSerValValProValAsnLeuIleGlnLysGlyLysSerSerA

490        500       510       520       530      540

spAsnIleIleValLysAsnCysAspValAsnValLysAlaThrLysLysAlaAsnLeu*

550        560       570       580       590      600

oc

610        620       630       640       650      660

TTCTTGA (SEQ ID. NOS: 1 and 2)

Nucleotide Sequence Coding for a Common Outer Membrane Protein from H. influenzae and Monoclonal Antibodies

The 15 kD outer membrane protein described herein is conserved among type b and nontypable H. influenzae. Epitopes on the native protein are recognized by the murine monoclonal antibodies 6B11, 1A6, and 5E6. The epitopes are present on the surface of intact H. influenzae cells. The gene for the 15 kD protein has been cloned and the DNA sequence thereof has been determined. The gene, when expressed in an appropriate host/vector system, produces a recombinant protein which is reactive with the monoclonal antibodies. Since the protein is antigenically highly conserved, it should receive serious consideration for inclusion in a vaccine to prevent H. influenzae disease. Moreover monoclonal antibodies and DNA probes may be used as a diagnostic tool to detect the presence of H. influenzae.

FIG. 1 is a Western blot analysis of H. influenzae with monoclonal antibodies;

FIG. 2 is a partial restriction map of plasmid pRSM1255 which contains the gene for the 15 kD outer membrane protein from H. influenzae type b strain 1613;

FIG. 3 is a Western blot analysis of the expression of the recombinant 15 kD protein;

FIG. 4 is a dot blot immunoassay of extracts of E. coli strains LE392/pGD103 and LE392/pRSM1311; and

FIG. 5 is the amino acid sequence for the polynucleotide of the present invention. ( SEQ ID NOs 1 and 2 are set forth in this Figure. )

Monoclonal antibodies (Mabs) were obtained from two independent fusion experiments. Mab 1A6 was generated from Balb/c mice immunized with sarcosyl-insoluble proteins of nontypable H. influenzae MTL6 as described by Hamel et al [see Journal of General Microbiology (1992), 138, 161-168] and monoclonal antibodies 6B11 and 5E6, were produced from mice immunized with outer membranes extracted from nontypable H. influenzae 12085 by the lithium chloride method described by Hamel et al [see J. Med. Microbiology, (1987) 23 163-170]. Isotype analysis revealed that the 1A6, 6B11 and 5E6 hybridomas secreted immunoglobulin IG2a, IG1 and IG3 respectively.

Referring to FIG. 1, Western Immunoblotting analysis of outer membrane preparations was performed. Outer membrane preparations were fractionated on 16% SDS-PAGE, transferred to nitrocellulose, and probed with Mab 1A6 (lane 1 ), 6B11 (lane 2 ), 5E6 (lane 3 ) and porin-specific Mab P2-18 (lane 4 ). The analysis indicated that the monoclonal antibodies were directed against a protein with an apparent mass of 15 kD. Antibody accessibility radioimmunoassay [see Proulx et al, Infection and Immunity (1991)59, 963-970] indicated that monoclonal antibodies bound to surface-exposed epitopes on both type b and nontypable H. influenzae isolates.

CONSERVATION OF 15 KD-EPITOPES

A total of 193 H. influenzae isolates were tested by dot blot immunoassay for their reactivity with monoclonal antibodies 1A6, 6B11 and 5E6.

MOLECULAR CLONING OF THE GENE FOR THE 15 KD OUTER MEMBRANE PROTEIN

A lambda EMBL3 genomic library of DNA from H. influenzae strain 1613 was immunologically screened with murine monoclonal antibody 6B11 as described by Munson and Grass [see Infection and Immunity (1988) 561 2235-2242]. An immunologically reactive clone was isolated by plaque purification. A liquid lysate was prepared and DNA was purified from a Promega lambda DNA kit according to the manufacturer's directions. The Haemophilus insert was identified as a SalI fragment of approximately 16 kb. DNA from the lambda clone was partially digested with Sau3A and fragments of approximately 3-6 kb were isolated by preparative agarose gel electrophoresis. The 3-6 kb fragments were ligated into the low copy number vector pGD103 [see Deich et al, Journal of Bacteriology (1988) 489- 498] which had been digested sequentially with BamHI and alkaline phosphatase. The ligation mixture was transformed into E. coli strain LE392 and the cells were plated on LB agar containing 35 μg/ml of kanamycin. Immunologically reactive colonies were identified by screening with murine Mab 6B11. Strain LE392/pRSM1255 was saved for further study. As shown in FIG. 2 plasmid pRSM1255 has an insert of approximately 3.8 kb. Strain LE392/pRSM1255 produces the full size protein as determined by Western blot (see FIG. 3 ). Membrane preparations were fractionated by SDS-PAGE, transferred to nitrocellulose and probed with murine monoclonal antibody 6B11. Lane 1 is the total membrane preparation of H. influenzae strain 1613; lane 2 is the total membrane preparation of E. coli strain LE392/pGD103 and lane 3 is the total membrane preparation of E. coli strain LE392/pRSM1255. The full size 15 Kd protein is produced by E. coli strain LE392/pRSM1255.

In order to further subclone the gene for sequencing, pRSM1255 was partially digested with Sau3A, fragments of approximately 0.5 to 1.5 kb were isolated, ligated into BamHI-treated pGD103 and transformed into E. coli strain LE392. An immunologically positive clone, designated LE392/pRSM1311 was saved for further analysis. Western blot analysis employing Mab 6B11 indicated that the full size protein was produced by this strain (data not shown). The Haemophilus insert in pRSM1311 is approximately 0.6 kb in size. Extracts of E. coli strain LE392/pRSM1311 react with all three murine Mabs (FIG. 4 ). 5 μg of cell extracts were applied to the nitrocellulose and probed with Mabs 3B11, 1A6, and 5E6.

The insert was cloned into M13mp18 and M13mp19 as a SalI to EcoRI fragment and sequenced in both directions. A 369 codon open reading frame ( SEQ ID NO: 3 ) was identified encoding a protein composed of 123 amino acids ( SEQ ID NO: 2 ) and having a molecular weight of 13,460. The open reading frame is notable for a lysine at position 3 and a cysteine at position 26 suggesting that the protein is a lipoprotein with a 25 amino acid leader peptide. ( The sequence of the protein without the leader peptide is set forth in SEQ ID NO: 4 . )

DNA and protein sequence analysis were done using Gen Bank (a trademark of NIH) and EMBL (from European Molecular Biology Organization) data bases. The nucleotide ( SEQ ID NO: 1 ) and derived amino acid sequence ( SEQ ID NO: 2 ) of the 15 kD outer membrane protein of H. influenzae type b, strain 1613 is set out in Table 3.

TABLE3

MW for gene product = i3460.    Number of amino acids = 123

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130       140       150       160       170      180

Met

ThrLys

S

190       200       210       220       230      240

erSerAlaGlnIleAlaGluMetGlnThrLeuProThrIleThrAspLysThrValValT

250       260       270       280       290      300

yrSerCysAsnLysGlnThrValThrAlaValTyrGlnPheGluAsnGlnGluProValA

310       320       330       340       350      360

laAlaMetValSerValGlyAspGlyIleI1eAlaLysAspPheThrArgAspLysSerG

370       380       390       400       410      420

lnAsnAspPheThrSerPheValSerGlyAspTyrValTrpAsnValAspSerGlyLeuT

430       440       450       460       470      480

hrLeuAspLysPheAspSerValValProValAsnLeuIleGlnLysGlyLysSerSerA

490       500       510       520       530      540

spAsnIleIleValLysAsnCysAspValAsnValLysAlaThrLysLysAlaAsnLeu*

550       560       570       580       590      600

oc

610       620       630       640       650      660

The sequence was found to have no significant homology to any known proteins including two previously described Haemophilus outer membrane lipoproteins of similar size.

The research described herein was supported in part by United States Public Health Service grant R01AI17572 from the National Institutes of Health.

The sequence for the polynucleotide claimed in this application is as follows:

The amino acid sequence for the outer membrane protein of haemophilus influenzae claimed in this application is as follows:

GATCCCACCT TGTTTATTCC AATAATGGAA CTTTATTTTA TTAAAGGTAT CTAAGTAGCA 60

CCCTATATAG GGATTAATTA ACGAGGTTTA ATAATGAACT TTAACTAAAA TTTTACCAGC 120

ATTTGCTGCT GTAGTCTGTA TTATCTGCTT GTGCAAAGGA TGCACCTGAA ATGACAAAAT 180

CATCTGCGCA AATAGCTGAA ATGCAAACAC TTCCAACAAT CACTGATAAA ACAGTTGTAT 240

ATTCCTGCAA TAAACAAACG GTGACTGCTG TGTATCAATT TGAAAACCAA GAACCAGTTG 300

CTGCAATGGT AAGTGTGGGC GATGGCATTA TTGCCAAAGA TTTTACTCGT GATAAATCAC 360

AAAATGACTT TACAAGTTTC GTTTCTGGGG ATTATGTTTG GAATGTAGAT AGTGGCTTAA 420

CGTTAGATAA ATTTGATTCT GTTGTGCCTG TCAATTTAAT TCAAAAAGGT AAATCTAGCG 480

ATAATATCAT CGTCAAAAAT TGTGATGTAA ACGTAAAAGC AACTAAAAAA GCAAATTTAT 540

AATTAATCCC AAATGACCAG CATAATTGCT GGTTATTTAT CTTCCTCGAG GGGAGATTTT 600

TTCTTGA 607

Met Thr Lys Ser Ser Ala Gln Ile Ala Glu Met Gln Thr Leu Pro Thr

1 5 10 15

Ile Thr Asp Lys Thr Val Val Tyr Ser Cys Asn Lys Gln Thr Val Thr

20 25 30

Ala Val Tyr Gln Phe Glu Asn Gln Glu Pro Val Ala Ala Met Val Ser

35 40 45

Val Gly Asp Gly Ile Ile Ala Lys Asp Phe Thr Arg Asp Lys Ser Gln

50 55 60

Asn Asp Phe Thr Ser Phe Val Ser Gly Asp Tyr Val Trp Asn Val Asp

65 70 75 80

Ser Gly Leu Thr Leu Asp Lys Phe Asp Ser Val Val Pro Val Asn Leu

85 90 95

Ile Gln Lys Gly Lys Ser Ser Asp Asn Ile Ile Val Lys Asn Cys Asp

100 105 110

Val Asn Val Lys Ala Thr Lys Lys Ala Asn Leu

115 120

(1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES: 4

(2) INFORMATION FOR SEQ ID NO:1:

(A) LENGTH: 607 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(B) LOCATION: 171..539

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GATCCCACCT TGTTTATTCC AATAATGGAA CTTTATTTTA TTAAAGGTAT C TAAGTAGCA 60

CCCTATATAG GGATTAATTA ACGAGGTTTA ATAATGAACT TTAACTAAAA T TTTACCAGC 120

ATTTGCTGCT GTAGTCTGTA TTATCTGCTT GTGCAAAGGA TGCACCTGAA A TG ACA 176

Met Thr

1

AAA TCA TCT GCG CAA ATA GCT GAA ATG CAA A CA CTT CCA ACA ATC ACT 224

Lys Ser Ser Ala Gln Ile Ala Glu Met Gln T hr Leu Pro Thr Ile Thr

5 10 15

GAT AAA ACA GTT GTA TAT TCC TGC AAT AAA C AA ACG GTG ACT GCT GTG 272

Asp Lys Thr Val Val Tyr Ser Cys Asn Lys G ln Thr Val Thr Ala Val

20 25 30

TAT CAA TTT GAA AAC CAA GAA CCA GTT GCT G CA ATG GTA AGT GTG GGC 320

Tyr Gln Phe Glu Asn Gln Glu Pro Val Ala A la Met Val Ser Val Gly

35 40 45 50

GAT GGC ATT ATT GCC AAA GAT TTT ACT CGT G AT AAA TCA CAA AAT GAC 368

Asp Gly Ile Ile Ala Lys Asp Phe Thr Arg A sp Lys Ser Gln Asn Asp

55 60 65

TTT ACA AGT TTC GTT TCT GGG GAT TAT GTT T GG AAT GTA GAT AGT GGC 416

Phe Thr Ser Phe Val Ser Gly Asp Tyr Val T rp Asn Val Asp Ser Gly

70 75 80

TTA ACG TTA GAT AAA TTT GAT TCT GTT GTG C CT GTC AAT TTA ATT CAA 464

Leu Thr Leu Asp Lys Phe Asp Ser Val Val P ro Val Asn Leu Ile Gln

85 90 95

AAA GGT AAA TCT AGC GAT AAT ATC ATC GTC A AA AAT TGT GAT GTA AAC 512

Lys Gly Lys Ser Ser Asp Asn Ile Ile Val L ys Asn Cys Asp Val Asn

100 105 110

GTA AAA GCA ACT AAA AAA GCA AAT TTA TAATTAAT CC CAAATGACCA 559

Val Lys Ala Thr Lys Lys Ala Asn Leu

115 120

GCATAATTGC TGGTTATTTA TCTTCCTCGA GGGGAGATTT TTTCTTGA 607

(2) INFORMATION FOR SEQ ID NO:2:

(A) LENGTH: 123 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Thr Lys Ser Ser Ala Gln Ile Ala Glu M et Gln Thr Leu Pro Thr

1 5 10 15

Ile Thr Asp Lys Thr Val Val Tyr Ser Cys A sn Lys Gln Thr Val Thr

20 25 30

Ala Val Tyr Gln Phe Glu Asn Gln Glu Pro V al Ala Ala Met Val Ser

35 40 45

Val Gly Asp Gly Ile Ile Ala Lys Asp Phe T hr Arg Asp Lys Ser Gln

50 55 60

Asn Asp Phe Thr Ser Phe Val Ser Gly Asp T yr Val Trp Asn Val Asp

65 70 75 80

Ser Gly Leu Thr Leu Asp Lys Phe Asp Ser V al Val Pro Val Asn Leu

85 90 95

Ile Gln Lys Gly Lys Ser Ser Asp Asn Ile I le Val Lys Asn Cys Asp

100 105 110

Val Asn Val Lys Ala Thr Lys Lys Ala Asn L eu

115 120

(2) INFORMATION FOR SEQ ID NO:3:

(A) LENGTH: 369 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATGACAAAAT CATCTGCGCA AATAGCTGAA ATGCAAACAC TTCCAACAAT C ACTGATAAA 60

ACAGTTGTAT ATTCCTGCAA TAAACAAACG GTGACTGCTG TGTATCAATT T GAAAACCAA 120

GAACCAGTTG CTGCAATGGT AAGTGTGGGC GATGGCATTA TTGCCAAAGA T TTTACTCGT 180

GATAAATCAC AAAATGACTT TACAAGTTTC GTTTCTGGGG ATTATGTTTG G AATGTAGAT 240

AGTGGCTTAA CGTTAGATAA ATTTGATTCT GTTGTGCCTG TCAATTTAAT T CAAAAAGGT 300

AAATCTAGCG ATAATATCAT CGTCAAAAAT TGTGATGTAA ACGTAAAAGC A ACTAAAAAA 360

GCAAATTTA 369

(2) INFORMATION FOR SEQ ID NO:4:

(A) LENGTH: 98 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Cys Asn Lys Gln Thr Val Thr Ala Val Tyr G ln Phe Glu Asn Gln Glu

1 5 10 15

Pro Val Ala Ala Met Val Ser Val Gly Asp G ly Ile Ile Ala Lys Asp

20 25 30

Phe Thr Arg Asp Lys Ser Gln Asn Asp Phe T hr Ser Phe Val Ser Gly

35 40 45

Asp Tyr Val Trp Asn Val Asp Ser Gly Leu T hr Leu Asp Lys Phe Asp

50 55 60

Ser Val Val Pro Val Asn Leu Ile Gln Lys G ly Lys Ser Ser Asp Asn

65 70 75 80

Ile Ile Val Lys Asn Cys Asp Val Asn Val L ys Ala Thr Lys Lys Ala

85 90 95

Asn Leu