Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms

Abstract

Infections caused by Haemophilus influenzae are a major worldwide health problem. In particular, nontypeable strains of H. influenzae are a common cause of otitis media in infants and children. A vaccine to prevent these infections would result in the prevention of substantial morbidity and cost savings. A problem in identifying an appropriate vaccine antigen has been the enormous antigenic heterogeneity among nontypeable strains of H. influenzae. The present study was undertaken to characterize the conservation of the P6 outer membrane protein (approximately 16,000 daltons) among strains of H. influenzae. A total of 20 type b strains and 20 nontypeable strains of diverse geographic and clinical origins was studied. Three approaches were taken. (i) Antigenic determinants recognized by monoclonal and polyclonal antibodies were present on P6 in all 40 strains tested. The molecular weight of P6 was identical in all strains. (ii) Comparison of the DNA sequences of the P6 genes from three epidemiologically and serologically unrelated strains demonstrated 100% homology at the amino acid level and 97 to 99% homology at the nucleotide level. (iii) Restriction fragment length polymorphism analysis demonstrated that the P6 gene and flanking sequences were highly conserved among all strains. These three independent series of experiments indicated that the P6 protein is highly conserved among strains of H. influenzae. P6 should receive serious consideration for inclusion in a vaccine to prevent infections caused by nontypeable H. influenzae.

Expression of a peroxiredoxin–glutaredoxin byHaemophilus influenzaein biofilms and during human respiratory tract infection

“…Cell envelopes were subjected to SDS-PAGE and Coomassie blue staining using previously described methods [31]. Immunoblot assays were performed as described previously [32]. After incubating with a murine antiserum against PGdx, blots were developed with peroxidase conjugated secondary antibody and color developer [32].…”

“…Immunoblot assays were performed as described previously [32]. After incubating with a murine antiserum against PGdx, blots were developed with peroxidase conjugated secondary antibody and color developer [32].…”

The major heat‐modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis

“…The blots were probed with oligonucleotides that were end-labelled with p^P]-ATP (New England Nuclear Corp.) by using T4 polynucleotide kinase (Promega Corp.) Hybridizations were carried out at 37°C and washes were performed at 48°C. The hybridization and wash buffers were described previously (Nelson et al, 1991). Autoradiography was pertormed at -70°C.…”

Development of immunochromatographic colloidal gold test strip for rapid detection ofHaemophilus influenzaein clinical specimens

“…The outer membrane protein (OMP) P6 of H. influenzae is a peptideglycan-associated lipoprotein with 16 600 Da (Murphy et al 1986a,b). The OMP P6 exhibited the high conservation of residues among strains, and stability of antigenicity (Nelson et al 1991). Consequently, OMP P6 is considered as an ideal diagnostic target for H. influenzae.…”