Haemophilus adhesin protein

Details Haemophilus adhesin protein Haemophilus adhesin protein

1999-12-08

2001-04-17

Graser, Jennifer (Department: 1641)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S071100, C435S243000, C435S252300, C536S023100, C536S023700, C424S256100

Reexamination Certificate

active

06218147

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the isolation of lipid-binding adhesins and, more specifically, to the isolation and identification of a lipid-binding adhesin from
Haemophilus influenzae.
BACKGROUND OF THE INVENTION
Various journal articles referred to herein are identified by number in parentheses and are listed, with full citations, at the end of the specification.
Haemophilus influenzae
is a respiratory pathogen which colonises human, mucosal surfaces and is associated with otitis media, sinusitis, conjunctivitis, bronchitis, pneumonia, meningitis, epiglottitis and cellulitis [1, 2]. The natural habitat and reservoir for this organism is the upper respiratory mucosal surfaces, primarily the nasopharynx [3]. Mucosal surfaces are ports of entry and major sites of many infectious agents. Many pathogens, including viruses [4, 5], bacteria [6, 7] and bacterial toxins [8] bind to specific carbohydrate moieties on the mucosal surfaces, enabling colonization and infection and potentially mediating a toxic effect on the host cells. These carbohydrate moieties may be present in either glycolipids or glycoproteins. For example, many respiratory pathogens recognise gangliotriaosylceramide (GalNAc&bgr;1-4gal&bgr;1-4glc cer [Gg
3
]), gangliotetraosylceramide (gal&bgr;1-3galNAc&bgr;1-4gal&bgr;1-4glc cer [Gg
4
]) [7, 9-15] and sulfatoxygalactosylceramide [SGC] [16, 17]. Respiratory pathogens have also been identified which recognize phosphatidylethanolamine (PE). PE, Gg
3
and Gg
4
are also recognised by such pathogens as
Pseudomonas aeruginosa
, Burholderia cepacia Chlamydia trachomatis, C. pneumoniae, Neisseria meningitis, enteropathogenic
Escherichia coli,
and
H. pylori
(9-14, 26, 29, 32).
There is a need for new means of combatting infections due to these pathogens, as antibiotic resistance becomes increasingly common. A vaccine to stimulate the production of antibodies which would interfere with the attachment of these pathogens to their target cells would be of great assistance in combatting such infections.
SUMMARY OF THE INVENTION
The present invention provides a purified and isolated adhesin protein of about 46 kDa from strains of
Haemophilus influenzae.
The adhesin binds specifically to phosphatidylethanolamine (PE), gangliotriaosylceramide (Gg
3
) and gangliotetrasylceramide (Gg
4
). This adhesin protein is referred to herein as “adhesin”.
The invention also provides purified and isolated nucleic acid molecules encoding an adhesin protein of a strain of
H. influenzae
or a fragment or analogue of the adhesin.
The adhesin protein, fragments or analogues thereof, nucleic acid molecules encoding the adhesin or encoding fragments or analogues thereof and vectors containing such nucleic acid molecules are useful in immunogenic compositions for immunising against diseases caused by pathogens which bind to their target cells by means of an adhesin which binds to phosphatidylethanolamine (PE), gangliotriaosylceramide (Gg
3
) and gangliotetraosylceramide (Gg
4
).
The nucleic acid molecules provided herein are also useful for amplifying a specific DNA target sequence using PCR, and as probes to locate specific, unique DNA sequences.
The nucleic acid molecules provided herein are also useful for production of the encoded adhesin protein or fragments or analogs thereof, free of other Haemophilus proteins, by expression of the nucleic acids in a recombinant DNA expression system.
The invention also provides antibodies raised against the purified adhesin of the invention. These antibodies are useful for the treatment of patients infected by
H. influenzae
and other pathogens which also bind to phosphatidylethanolamine (PE), gangliotriaosylceramide (Gg
3
) and gangliotetrasylceramide (Gg
4
).
In accordance with one embodiment of the present invention, there is provided a substantially pure adhesin protein which binds specifically to phosphatidylethanolamine (PE), gangliotriaosylceramide (Gg
3
) and gangliotetraosylceramide (Gg
4
).
In accordance with a further aspect of the invention an adhesin protein is provided comprising the amino acid sequence
MKKLLKISAISAALLSAPMMANADVLASVKPLGFIVSSIADGVTGTQVLVPA GASPHDYNLKLSDIQKVKSADLVVWIGEDIDSFLDKPISQIERKKVITIADL ADVKPLLSKAHHEHFHEDGDHDHDHKHEHKHDHKHDHDHDHDHKHEHKHDHE HHDHDHHEGLTTNWHVWYSPAISKIVAQKVADKLTAQFPDKKALIAQNLSDF NRTLAEQSEKITAQLANVKDKGFYVFHDAYGYFNDAYGLKQTGYFTINPLVA PGAKTLAHIKEEIDEHKVNCLFAEPQFTPKVIESLAKNTKVNVGQLDPIGDK VTLGKNSYATFLQSTADSYMECLAK.
In accordance with another aspect of the invention, a recombinant adhesin protein or fragment or analogue thereof is provided produced by expression of the nucleotide sequence in an appropriate vector.
In accordance with another aspect of the invention is an immunogenic composition comprising at least one active component selected from the group consisting of:
(a) a purified and isolated nucleic acid molecule encoding an adhesin protein which binds specifically to PE, Gg
3
and Gg
4
or a fragment or analogue of said protein;
(b) a purified and isolated nucleic acid molecule having the nucleotide sequence of Table 1 or a nucleotide sequence complementary thereto;
(c) an adhesin protein which binds specifically to PE, Gg
3
and Gg
4
or an effective analogue or fragment thereof; and
(d) a recombinant adhesin protein and a pharmaceutically acceptable carrier, said at least one active component producing an immune response when administered to a mammal.
In accordance with another aspect of the invention is an immunogenic composition formulated as a vaccine for administration to a mammal to protect the mammal against diseases caused by bacterial pathogens having the adhesin protein as a surface protein.
In accordance with another aspect of the present invention, a method is provided of protecting a susceptible mammal against a disease caused by a bacterial pathogen having as a surface protein, an adhesin protein which binds specifically to phosphatidylethanolamine (PE), gangliotriaosylceramide (Gg
3
) and gangliotetraosylceramide (Gg
4
), the method comprising administering to the mammal an effective amount of an immunogenic composition. The immunogenic composition comprises at least one active component selected from the group consisting of:
(a) a purified and isolated nucleic acid molecule encoding an adhesin protein which binds specifically to PE, Gg
3
and Gg
4
or a fragment or analogue of said protein;
(b) a purified and isolated nucleic acid molecule having the nucleotide sequence of Table 1 or a nucleotide sequence complementary thereto;
(c) an adhesin protein which binds specifically to PE, Gg
3
and Gg
4
or an effective analogue or fragment thereof; and
(d) a recombinant adhesin protein and a pharmaceutically acceptable carrier, said at least one active component producing an immune response when administered to a mammal.
According to another aspect of the invention is an antibody or antiserum specific for an isolated and purified adhesin protein, a recombinant adhesin protein or an immunogenic composition containing adhesin protein.
According to another aspect of the invention is a method for producing a substantially pure adhesin protein which bind specifically to PE, Gg
3
, Gg
4
comprising the steps of.
(a) providing a bacterial strain which produces said adhesin protein;
(b) growing said bacterial strain to provide a cell mass;
(c) harvesting the cell mass by centrifugation;
(d) extracting the cell mass to provide an adhesin-containing extract;
(e) applying the adhesin-containing extract to a PE affinity matrix;
(f) eluting unbound proteins from the matrix; and
(g) releasing the adhesin protein from the matrix to provide a substantially pure adhesin protein.
In accordance with another aspect of the invention, an isolated nucleic acid molecule is provided encoding an adhesin protein which binds specifically to phosphatidylethanolamine (PE), gangliotriaosylceramide (Gg
3
) and gangliotetraosylceramide (Gg
4
).


REFERENCES:
patent: 5646259 (1997-07-01), St. Geme, II

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