Synthetic chimeric fimbrin peptides

Details Synthetic chimeric fimbrin peptides Synthetic chimeric fimbrin peptides

1998-09-04

2002-08-20

Duffy, Patricia A. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Conjugate or complex

C424S184100, C424S185100, C424S192100, C424S193100, C424S234100, C424S256100, C530S300000, C530S324000, C530S326000, C530S327000

Reexamination Certificate

active

06436405

ABSTRACT:

BACKGROUND OF THE INVENTION
Otitis media is an infection of the middle ear that occurs primarily in children. Left untreated, the disease can result in hearing loss and developmental delays. It is estimated that otitis media accounted for 31 million of the 130 million office visits for respiratory diseases in the period from 1977-87. Recent data indicate that suppurative and unspecified otitis media rank first in the list of the 30 most common diagnoses requiring a physician's office visit for patients up to age 24. Over one billion dollars per year is spent on treatment of this disease and the related loss of income for working parents is estimated to be between $300 and $600 million. Approximately 83% of all children will have had at least one episode of acute otitis media by three years of age. Non-typable strains of Haemophilus influenzae account for 25-30% of all cases of otitis media, 53% of recurrent otitis media, and are the primary pathogens isolated from 62% of cases of chronic otitis media with effusion. Although non-typable
Haemophilus influenzae
(NTHi) are primary pathogens in otitis media, neither the pathogenic mechanisms nor the host immunological response has been fully defined for this disease.
Fimbriae, which are surface appendages found on non-typable
Haemophilus influenzae
, are produced by 100% of the bacteria recovered from the middle ears and nasopharyngeal region of children with chronic otitis media. A vaccine comprised of fimbrin, a filamentous protein derived from the fimbriae of non-typable
Haemophilus influenzae
was previously developed and is useful in studying, preventing, or reducing the severity of otitis media. However, existing methodologies to isolate fimbrin protein from the bacterial outer membrane are tedious and time-consuming. Similarly, purification of fimbrin expressed by the fimbrin gene in other host vector, is also tedious due to the homology between the fimbrin protein and the outer membrane proteins of the host vector.
A strategy with other bacterial species has been to use an alternative immunogen having relatively short linear peptides. However, such alternative immunogens are of limited value due since antibodies to such alternative immunogens frequently fail to recognize the native pathogen.
It would be desirable to have an immunogenic composition to immunize animals against non-typable
Haemophilus influenzae
which does not depend on tedious purification techniques.
SUMMARY OF THE INVENTION
The present invention provides synthetic chimeric fimbrin peptides which induce an immunogenic response in animals to non-typable
Haemophilus influenzae
and that do not require tedious purification techniques. The synthetic chimeric fimbrin peptides are synthesized using commercially available peptide synthesizers. Preferably the synthetic chimeric fimbrin peptides are from about 18 to about 55, more preferably from about 35 to about 45, most preferably about 40-41 amino acids. The synthetic chimeric fimbrin peptides comprises three peptide units. The first peptide unit preferably contains 12 to 35, more preferably 15 to 30, most preferably 18 to 19 amino acids and is a subunit of the fimbrin protein. Preferably, the fimbrin subunit is comprised of the amino acids of Sequence ID No. 1 or Sequence ID No. 2. The amino acid of Sequence ID No. 1 is:
Arg-Ser-Asp-Tyr-Lys-Phe-Tyr-Glu-Asp-Ala-Asn-Gly-Thr-Arg-Asp-His-Lys-Lys SEQ ID NO: 1
The amino acid of Sequence ID No. 2 is:
Tyr-Gln-Trp-Leu-Thr-Arg-Val-Gly-Lys-Tyr-Arg-Pro-Gln-Asp-Lys-Pro-Asn-Thr SEQ ID NO: 2
The second peptide unit is a t cell epitope and preferably has from about 14 to about 22, more preferably about 15 to 21, most preferably 16 amino acids. The second peptide unit preferably has the following amino acid sequence:
Leu-Ser-Leu-Ile-Lys-Gly-Val-lle-Val-His-Arg-Leu-Glu-Gly-Val-Glu SEQ ID NO: 3
The third peptide unit is a linker peptide unit which joins the first and second peptide unit. The linking sequence preferably has from about 2 to about 15 amino acids, more preferably from about 2 to about 10 amino acids, most preferably from about 5 to about 6 amino acids. The most preferred amino acid sequence for the linker peptide unit is shown in sequence ID NO. 4. The amino acid of Sequence ID NO. 4 is:
Gly-Pro-Ser-Leu-Lys-Leu SEQ ID NO. 4
The synthetic chimeric fimbrin peptides are useful immunogens against NTHi and also useful as laboratory tools for detecting antibodies in sera. The invention also relates to an immunogenic composition containing the synthetic chimeric fimbrin peptides and a pharmacologically acceptable carrier.


REFERENCES:
patent: 5110908 (1992-05-01), Deich et al.
patent: 94/26304 (1994-11-01), None
Burgess et al, The Journal of Cell Biology, 111: 2128-2138, 1990.*
Jobling et al, Molecular Microbiology 5(7): 1755-1767, 1991.*
Rudinger in “Peptide Hormones”, Ed Parsons, J.A Baltimore, University Park Press pp. 1-6, 1976.*
Lazar et al, Molecular & Cellular Biology 8(3): 1247-1252, 1988.*
“Protection Against Histopathological Changes of Tympanic Membrane (TM) and Middle Ear Mucosa (MM) in a Chinchilla Model of Otitis Media by Immunization with Fimbrial Protein Isolated from NontypableHaemophilus influenzae(NTHi) Strain #1128” by L. Bakaletz, et al., American Society for Microbiology, 93rd General Meeting, May 16-20, 1993, Atlanta, Georgia.
“The Effect of Immunization with Fimbrial Protein in a Chinchilla Model of NontypableHaemophilus influenzae-Induced Experimental Otitis Media” by L. Bakaletz, et al., Second Extraordinary International Symposium on Recent Advances in Otitis Media, Mar. 31-Apr. 3, 1993, Oita, Japan.
“An Investigation of the Molecular Basis of the Adherence of NontypableHaemophilus influenzaeto Mucosal Epithelium” by L. Bakaletz, et al., Molecular Biology of Hearing and Deafness Conf., LaJolla, CA, May 1-4, 1992.
“Passive Immunization of Chinchillas with Anti-NTHi Fimbrial Serum and Protection Against Experimental Otitis Media” by L. Bakaletz, et al., Fifth International Symposium, Recent Advances in Otitis Media, May 20-24, 1991, Columbus, Ohio.
“Serological Relatedness of Fimbriae Expressed by NTHi Isolates Recovered from Children with Chronic Otitis Media” by L. Bakaletz, et al., Fifth International Symposium, Recent Advances in Otitis Media, May 20-24, 1991, Columbus, Ohio.
“Isotype Specific Antibody Response Against OMPs and Fimbriae of NontypableHaemophilus influenzaeIsolated from Patients with Chronic Otitis Media” by L. Bakaletz, et al., Fifth International Symposium, Recent Advances in Otitis Media, May 20-24, 1991, Columbus, Ohio.
“Immunological Responsiveness of Chinchillas to Isolated Fimbrial Proteins of NontypableHaemophilus influenzaeDuring Experimental Otitis Media” by L. Bakaletz, et al., Eleventh Midwinter Research Meeting, Association for Research in Otolaryngology, Jan. 31-Feb. 4, 1988, Clearwater Beach, Florida.
“Inhibition of Adherence of NontypableHaemophilus influenzaeto Human Oropharyngeal Cells”, by L. Bakaletz, et al., Twelfth Midwinter Research Meeting, Association for Research in Otolaryngology, Feb. 5-9, 1989, St. Petersburg Beach, Florida.
“Role of Degree of of Fimbriation on Ability of NontypableHaemophilus influenzaeto Colonize the Nasophrynx and Middle Ears of the Chinchilla” by L. Bakaletz, et al., Thirteenth Midwinter Research Meeting, Association for Research in Otolaryngology, Feb. 4-8, 1990, St. Petersburg Beach, Florida.
“Chinchilla Immunological Responsiveness to Isolated Outer Membrane and Fimbrial Proteins of NontypableHaemophilus influenzae” by L. Bakaletz, et al., Thirteenth Midwinter Research Meeting, Association for Research in Otolaryngology, Feb. 4-8, 1990, St. Petersburg Beach, Florida.
“Colonization of the Chinchilla Middle Ear and Nasopharynx by Fimbriated Isolates of NontypableHaemophilus influenzae” by L. Bakaletz, et al., 90th Annual Meeting of the American Society for Microbiology, May 13-17, 1990, Anaheim, California.
“Presumptive Identification of the NTHi Adhesin for Human Oropharyngeal and Chinchilla Middle Ear Epithelial Cells” by L. Bakaletz, et al., Fifth International

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