Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Patent
1994-10-05
1997-02-18
Sidberry, Hazel F.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
536 221, 536 231, 536 237, 536 241, 435 691, 435 693, A61K 39102, C07H 1900, C07H 2100, C07H 2102
Patent
active
056039387
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
This invention relates to high molecular weight proteins of non-typeable haemophilus.
BACKGROUND TO THE INVENTION
Non-typeable Haemophilus influenzae are non-encapsulated organisms that are defined by their lack of reactivity with antisera against known H. influenzae capsular antigens.
These organisms commonly inhabit the upper respiratory tract of humans and are frequently responsible for infections, such as otitis media, sinusiris, conjunctivitis, bronchitis and pneumonia. Since these organisms do not have a polysaccharide capsule, they are not controlled by the present Haemophilus influenzae type b (Hib) vaccines, which are directed towards Hib bacterial capsular polysaccharides. The non-typeable strains, however, do produce surface antigens that can elicit bactericidal antibodies. Two of the major outer membrane proteins, P2 and P6, have been identified as targets of human serum bactericidal activity. However, it has been shown that the P2 protein sequence is variable, in particular in the non-typeable Haemophilus strains. Thus, a P2-based vaccine would not protect against all strains of the organism.
There have previously been identified by Barenkamp et al (Pediatr. Infect. Dis. J., 9:333-339, 1990) a group of high-molecular-weight (HMW) proteins that appeared to be major targets of antibodies present in human convalescent sera. Examination of a series of middle ear isolates revealed the presence of one or two such proteins in most strains. However, prior to the present invention, the structures of these proteins were unknown as were pure isolates of such proteins.
SUMMARY OF INVENTION
The inventors, in an effort to further characterize the high molecular weight (HMW) Haemophilus proteins, have cloned, expressed and sequenced the genes coding for two immunodominant HMW proteins (designated HMW1 and HMW2) from a prototype non-typeable Haemophilus strain and have cloned, expressed and almost completely sequenced the genes coding for two additional immunodominant HMW proteins (designated HMW3 and HMW4) from another non-typeable Haemophilus strain.
in accordance with one aspect of the present invention, therefore, there is provided an isolated and purified gene coding for a high molecular weight protein of a non-typeable Haemophilus strain, particularly a gene coding for protein HMW1, HMW2, HMW3 or HMW4, as well as any variant or fragment of such protein which retains the immunological ability to protect against disease caused by a non-typeable Haemophilus strain. In another aspect, the invention provides a high molecular weight protein of non-typeable Haemophilus influenzae which is encoded by these genes.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1A-1G is a DNA sequence of a gene coding for protein HMW1 (SEQ ID NO: 1);
FIG. 2A & 2B is a derived amino acid sequence of protein HMW1 (SEQ ID NO: 2);
FIG. 3A-3G is a DNA sequence of a gene coding for protein HMW2 (SEQ ID NO: 3);
FIG. 4A & 4B is a derived amino acid sequence of HMW2 (SEQ ID NO: 4);
FIG. 5A shows restriction maps of representative recombinant phages which contained the HMW1 or HMW2 structural genes, the locations of the structural genes being indicated by the shaded bars;
FIG. 5B shows the restriction map of the T7 expression vector pT7-7;
FIG. 6A-6L contains the DNA sequence of a gene cluster for the hmw1 gene (SEQ ID NO: 5), comprising nucleotides 351 to 4958 (ORF a) (as in FIG. 1), as well as two additional downstream genes in the 3' flanking region, comprising ORFs b, nucleotides 5114-6748 and c nucleotides 7062-9011;
FIG. 7A-7L contains the DNA sequence of a gene cluster for the hmw2 gene (SEQ ID NO: 6), comprising nucleotides 792 to 5222 (ORF a) (as in FIG. 3), as well as two additional downstream genes in the 3' flanking region, comprising ORFs b, nucleotides 5375-7009, and c, nucleotides 7249-9198;
FIG. 8A-8F is a partial DNA sequence of a gene coding for protein HMW3 (SEQ ID NO: 7);
FIG. 9A -9F is a partial DNA sequence of a gene coding for protein HMW4 (SEQ ID NO: 8); and
FIG. 10A-10L is a comparison table for th
REFERENCES:
Pediatric Infectious Disease Journal, vol. 9, No. 5, issued May 1990, S.J. Barenkamp et al., "Development of Serum Bactericidal Activity Following Nontypable Haemophilus influenzae Acute Otitis Media", pp. 333-339, see entire document.
Journal of Clinical Microbiology, vol. 29, No. 11, issued Nov. 1991, A. C. Caputa et al., "110 Kilodalton Recombinant Protein which is Immunoreactive with Sera from Humans, Dogs, and Horses with Lyme Borreliosis", pp. 2416-2423, see entire document.
Joint Meeting of the American Pediatric Society and the Society for Pediatric Research, 7-10 May 1990, S. J. Barenkamp, "Cloning and Expression of Genes for Nontypable Haemophilus influenzae (NTH) High Molecular Weight (HMW) Outer Membrane Proteins which are Targets of Bactericidal Antibody", Abstract 983, Pediatric Research, vol. 27, (4 part 2).
The Journal of Infectious Diseases, vol. 165 (Suppl.), issued Aug 1992, S. J. Barenkamp, "Outer Membrane Protein and Lipopolysaccharides of Nontypeable Haemophilus influenzae", pp. S181-S184, see entire document.
Infection and Immunity, vol. 60(4), issued Apr. 1992, S. J. Barenkamp et al., "Cloning Expression and DNA Sequence Analysis of Genes Encoding Nontypable Haemophilus influenzae High-Molecular-Weight Surface-Exposed Proteins Related to Filamentous Hemegglutinin of Bordetella pertussis" pp. 1302-1313, see entire document.
Infection and Immunity, voll 56(1), Issued Jan. 1988, E. J. Hansen, "Immune Enhancement of Pulmonary Clearance on Nontypable Haemophilus Influenzae," pp. 182-190, see entire document, especialty FIGS. 3 and 4.
Infection and Immunity, vol. 52(2), issued May 1986, S. J. Barenkamp, "Protection by Serum Antibodies in Experimental Nontypable Haemophilus influenzae Otitis Media", pp. 572-578, see FIGS. 1 and 2.
Proceedings of the National Academy of Sciences USA, vol. 80, issued Mar. 1983, R. A. Young et al., "Efficient Isolation of Genes by Using Antibody Probes", pp. 1194-1198, see entire document.
Infection and Immunity, vol. 45(3), issued Sep. 1984, R. Schneerson et al., "Serum Antibody Responses of Juvenile and Infant Rhesus Monkeys Injected with Haemophilus influenzae Type b and Pneumococcus Type 6A Capsular Polysaccharide-Protein Conjugates", pp. 582-591, see entire document.
Journal of Molecular Biology, vol. 157, issued 1982, J. Kyfe et al., "A Simple Method for Displaying the Hydropathic Character of a Protein", pp. 105-132, see entire document.
Proceedings of the National Academy of Sciences, vol. 78(6), issued Jun. 1981, T. P. Hopp et al. "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", pp. 3824-3828, see entire document.
Pediatr. Infect. Dis. J., 9:333-339, 1990, Stephen J. Barenkamp and Frank F. Bodor, "Development of Serum Bacterial Activity Following Nontypable Haemophilus influenzae Acute Otitis Media".
Barenkamp et al., Infection & Immunity, 60:1302-1313, 1992.
Barenkamp et al. Pediatric Infect Dis Journ. 9: 333-339, 1990.
Barenkamp, Abstract 983, Pediatric Research vol. 27.
Young et al., PNAS 80:1194-1198, 1983.
Houghten et al. Vaccine 86 pp. 21-25.
Green et al, Infection and Immunity 61:1950-1957 1993.
Erwin et al. Can Journ of Microbiology 34: 723-729, 1988.
Thomas et al. Infection & Immunity 58:1909-1913, 1990.
Barenkamp, Pediatric Research vol. 20, N67A, Abstract 985, 1991.
Sidberry Hazel F.
St. Louis University
Washington University
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