Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Patent
1994-10-03
2000-01-25
Stucker, Jeffrey
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
530325, 530326, 530327, 435851, 4241851, 4241901, 4242561, A61K 3812
Patent
active
060180196
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to synthetic vaccines against Haemophilus influenzae (Hi) infection. In particular, the invention relates to the use of potent T-helper cell determinants (THDs) and B-cell epitopes (BEs) of the outer membrane proteins (OMPs) P1, P2 and P6 of Hi, covalently linked to synthetic oligosaccharides containing repeating units of polyribosylribitol phosphate (sPRP) to form immunogenic synthetic PRP-peptide conjugate vaccines that can elicit high titers of anti-PRP and anti-OMP antibodies in mammals.
BACKGROUND TO THE INVENTION
Haemophilus influenzae type b (Hib) is a major cause of bacterial meningitis in children under five years of age (refs. 1, 2. The literature references are identified at the end of this disclosure). The bacterium is protected from phagocytosis by a polysaccharide capsule that is a repeating polymer of polyribosyl ribitol phosphate (PRP). Antibodies induced against the capsular polysaccharide of the organism are protective (ref. 3). Effective conjugate vaccines in which PRP is linked to different carrier proteins such as diphtheria toxoid (PRP-D), tetanus toxoid (PRP-T), CRM 197 (HbOC) and the outer membrane protein of Neisseria meninaitidis have been developed (refs. 4, 5). However, these conjugate vaccines do not protect against other invasive encapsulated H. influenzae type a and c strains and, more importantly, against non-encapsulated non-typeable H. influenzae strains that are one of the common causes of otitis media for which there is no vaccine. Therefore, the inclusion of selected non-encapsulated H. influenzae immunogens in current Hib vaccines is necessary to develop a universal Hi vaccine.
Granoff and Munson (ref. 6) have reported that antibodies directed against Hib outer membrane proteins (OMP) P1, P2 and P6 were protective in the infant rat model of bacteremia. Therefore, a promising strategy for designing a universal H. influenzae vaccine with enhanced protective ability would be to use either purified OMPs or their protective epitopes as additional immunogens and carriers for PRP. The gene coding for P1 has been cloned from several different Hib subtypes (refs. 7, 8). The comparative analysis of P1 protein sequences from these Hib isolates revealed the existence of three hypervariable regions. Indeed, the P1-specific MAbs reported by Hansen's group recognize only 50% of the Hib isolates tested (refs. 7, 9). For the P2 protein, although the nucleotide sequences of the P2 gene isolated from two different Hib subtypes (1H and 3L) were found to be identical (refs. 10, 11), some amino acid variability was found among the P2 sequences of two other Hib subtypes (2L and 6U) (ref. 11). In contrast, analysis of antigenic determinants, gene sequences and restriction fragment length polymorphisms experiments indicated that the P6 protein was highly conserved among all strains of Hi (ref. 12).
Recent studies showed that a murine P1-specific monoclonal antibody (MAb 7C8) and rabbit antisera raised against purified P1 from either typeable or non-typeable H. influenzae strains were protective in animal models (refs. 9, 13, 14). Murphy and Bartos (ref. 15) also reported that a monoclonal antibody recognizing a surface-exposed epitope of a non-typeable H. influenzae P2 protein had bactericidal activity in vitro. Anti-P1 and anti-P2 monoclonal antibodies were found to cross-react with typeable and non-typeable strains of H. influenzae (refs. 16 to 18). However, there are still serious concerns with the use of whole native Hib OMPs as an efficacious universal vaccine against both typeable and non-typeable Hi. Firstly, children who recover from otitis media caused by non-typeable Hi generally develop bactericidal antibodies against variable antigens, such as P2 and lipooligosaccharides. Secondly, the P1 and P2 cross-protective epitopes described above have not yet been identified. Thirdly, it was reported (ref. 12) that the epitope(s) recognized by anti-P6 bactericidal antibodies are expressed in small amounts on the bacterial surface, and recu
REFERENCES:
Munson et al, "Molecular Cloning, Expression, and Primary Sequence of Outer Membrane Protein P2 of Haemophilus influenzae Type b", Infection and Immunity, vol. 57, No. 1(Jan. 1989), pp. 88-94.
Hansen et al, "Structural and Antigenic Conservation of the P2 Porin Protein Among Strains of Haemophilus influenzae Type b", Infection and Immunity, vol. 57, No. 11(Nov. 1989), pp. 3270-3275.
Martin et al, "Mapping of B-Cell Epitopes on the Outer Membrane P2 Porin Protein of Haemophilus influenzae by Using Recombinant Proteins and Synthetic Peptides", Infection and Immunity, vol. 59, No. 4 (Apr. 1991), pp. 1457-1464.
Hamel et al, "Localization of Conserved B-Cell Epitopes Among Encapsulated and Non-Encapsulated Haemophilus influenzae P2 Porin Proteins Using Synthetic Peptides", Journal Of General Microbiology, vol. 138, Part 1(Jan. 1992), pp. 161-168.
Jemmerson "Effects of Conformation, Amino Acid Sequence, and Carrier Coupling on the Immunological Recognition of Peptide and Protein Antigens" in: Zegers et al., Immunological Recognition of Peptides in Medicine and Biology (New York, CRC, 1995), pp. 213-225.
Chong Pele
Kandil Ali
Klein Michel
Sia Charles
Connaught Laboratories Limited
Stucker Jeffrey
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