DNA encoding the 15 kD outer membrane protein of Haemophilus...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

Reissue Patent

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C536S022100, C536S023100, C536S023700, C530S300000, C424S184100, C424S185100, C424S256100, C424S200100, C435S006120, C435S320100, C435S252200

Reissue Patent

active

RE037768

ABSTRACT:

BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
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
GATCCCACCTTGTTTATTCCAATAATGGAACTTTATTTTATTAAAGGTATCTAAGTAGCA

        70         80        90      100        110      120
CCCTATATAGGGATTAATTAACGAGGTTTAATAATGAACTTTAACTAAAATTTTACCAGC

        130      140       150        160       170      180
ATTTGCTGCTGTAGTCTGTATTATCTGCTTGTGCAAAGGATGCACCTGAAATGACAAAAT
                                               MetThrLysS

        190       200       210       220       230      240
CATCTGCGCAAATAGCTGAAATGCAAACACTTCCAACAATCACTGATAAAACAGTTGTAT
erSerAlaGlnIleAlaGluMetGlnThrLeuProThrIleThrAspLysThrValValT

        250       260       270       280       290      300
ATTCCTGCAATAAACAAACGGTGACTGCTGTGTATCAATTTGAAAACCAAGAACCAGTTG
yrSerCysAsnLysGlnThrValThrAlaValTyrGlnPheGluAsnGlnGluPrnValA

       310       320        330       340       350      360
CTGCAATGGTAAGTGTGGGCGATGGCATTATTGCCAAAGATTTTACTCGTGATAAATCAC
laAlaMetValSerValGlyAspGlyIleIleAlaLysAspPheThrArgAspLysSerG

        370       380      390        400      410       420
AAAATGACTTTACAAGTTTCGTTTCTGGGGATTATGTTTGGAATGTAGATAGTGGCTTAA
lnAsnAspPheThrSerPheValSerGlyAspTyrValTrpAsnValAspSerGlyLeuT

        430       440       450       460      470       480
CGTTAGATAAATTTGATTCTGTTGTGCCTGTCAATTTAATTCAAAAAGGTAAATCTAGCG
hrLeuAspLysPheAspSerValValProValAsnLeuIleGlnLysGlyLysSerSerA

       490        500       510       520       530      540
ATAATATCATCGTCAAAAATTGTGATGTAAACGTAAAAGCAACTAAAAAAGCAAATTTAT
spAsnIleIleValLysAsnCysAspValAsnValLysAlaThrLysLysAlaAsnLeu*

       550        560       570       580       590      600
AATTAATCCCAAATGAGCAGCATAATTGCTGGTTATTTATCTTCCTCGAGGGGAGATTTT
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.


REFERENCES:
Young et al. PNAS. 80: 1194-1198. 1993.*
Dugourd et al. Abstracts of Gen Meet ASM p. 88, Abstract B-373.*
Green et al. Infection and Immunity 55: 2878-2883, 1987.*
Grass et al., Abstracts of Gen Meeting of ASM P. 105, Abst. D-57.*
Humel et al. J. Med. Microbiol. 23: 163-70, 1987, Abstract only.

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