Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1998-03-20
2001-04-24
Graser, Jennifer (Department: 1641)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S184100, C424S185100, C424S190100, C530S350000, C435S069100, C435S069300, C435S071100, C435S320100, C435S173300, C536S023100, C536S023700, C536S024300, C536S024320
Reexamination Certificate
active
06221365
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a 63,000 Dalton protein produced by
Haemophilus influenzae
designated NucA, and to the nucA gene which encodes that protein.
BACKGROUND OF THE INVENTION
Nontypable
Haemophilus influenzae
(NTHi) is a strictly human commensal organism found in the upper respiratory tracts of up to 80% of healthy adults (Bibliography entry 1). It is a leading cause of otitis media and respiratory infections in children, including pneumonia and sinusitis (2). Since NTHi strains are unencapsulated, the existing vaccines for
H. influenzae
, which are based on the capsular structure of Type b (Hib), are ineffective. Hence a vaccine specific for NTHi organisms is needed and potential vaccine components have focused on surface exposed antigens like outer-membrane proteins.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to isolate and purify an additional protein from
H. influenzae
and to test whether the protein is a viable vaccine candidate in appropriate model systems.
It is a further object of this invention to isolate and clone the gene encoding such a protein and to express the protein recombinantly.
These and other objects of the invention as discussed below are achieved by the isolation and purification of a protein from
H. influenzae
which is designated NucA, as well as peptides of NucA protein comprising an epitope or epitopes thereof. The isolated and purified NucA protein of
H. influenzae
has the amino acid sequence of amino acids 26-603 of SEQ ID NO:2 or a biologically equivalent amino acid sequence thereof. Amino acids 1-25 of SEQ ID NO:2 are the signal peptide, which is cleaved during processing of the mature protein. The NucA protein has a molecular weight of approximately 63,000 Daltons as measured on a 12% SDS-PAGE gel and possesses 5′-nucleotidase activity.
In one embodiment of the invention, the NucA protein is obtained by isolation and purification from the
H. influenzae
organism. In a preferred embodiment of the invention, the NucA protein is recombinantly expressed by an isolated and purified nucA DNA sequence which encodes that protein.
The invention includes an isolated and purified DNA sequence comprising a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence encoding the NucA protein of
H. influenzae
having the amino acid sequence of amino acids 26-603 of SEQ ID NO:2 or a biologically equivalent amino acid sequence thereof.
Examples of such biologically equivalent NucA sequences are those wherein the amino acid sequence of amino acids 26-603 of SEQ ID NO:2 is modified by one or more of the following amino acid residue changes selected from the group consisting of lysine
79
to glutamic acid, asparagine
186
to lysine, serine
262
to glycine, valine
294
to alanine, glutamic acid
305
to glutamine, lysine
327
to arginine, threonine
337
to alanine, asparic acid
360
to tyrosine, arginine
376
to histidine or valine
436
to isoleucine.
The invention further includes such a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence having the nucleotide sequence of nucleotides 304-2037 of SEQ ID NO:1.
In order to obtain expression of the NucA protein, the DNA sequence is first inserted into a suitable plasmid vector. A suitable host cell is then transformed or transfected with the plasmid. In an embodiment of this invention, the host cell is
Escherichia coli
strain Inv&agr;F′. The host cell is then cultured under conditions which permit the expression of said NucA protein by the host cell.
In another embodiment of this invention, the isolated and purified NucA protein or a peptide of NucA protein comprising an epitope or epitopes thereof, is used to prepare a vaccine composition which elicits a protective immune response in a mammalian host. The vaccine composition may further comprise an adjuvant, diluent or carrier. Examples of such adjuvants include aluminum hydroxide, aluminum phosphate, MPL™, Stimulon™ QS-21, and IL-12. The vaccine composition is administered to a mammalian host in an immunogenic amount sufficient to protect the host against disease caused by
H. influenzae.
REFERENCES:
Yamanaka et al. 1993. The Journal of Pediatrics. 122(2): 212-218, Feb. 1993.*
Hopp et al. 1981. Proc. Natl. Acad. Sci. USA. 78(6): 3824-3828, Jun. 1981.*
Geysen et al. 1988. Journal of Molecular Recognition. 1(1): 32-42, 1988.*
Murphy et al. 1989. Pediatr. J. Infect. Dis. 8(1): S66-S68, 1989.*
Suzuki et a. 1993. J. Biochem. 133(5): 607-613, 1993.*
Fleischmann et al. Jul. 28, 1995. Science. 269: 496-512, Jul. 28, 1995.*
Fleischmann et al. Nov. 1, 1995. EMBL Database entry 5NTDHAEIN, Accession No. P44569, Nov. 1, 1995.*
Fleischmann et al. Nov. 1, 1995. Swiss-Prot37 Database entry 5NTDHAEIN, Accession No. P44569, Nov. 1, 1995.*
Wood, W. Methods in Enzymology. vol.152. Guide to Mol.Cloning. Chapter 48, pp. 443-448, 1987.*
Cruse et al. Illustrated Dictionary of Immunology. 1995., pp. 102-103, 1995.*
Rodden et al., Arch. Biochem. Biophys., 153, 837-844 (1972).
American Cyanamid Company
Gordon Alan M.
Graser Jennifer
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