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1.
FEMS Immunol Med Microbiol ; 47(3): 343-50, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16872370

RESUMO

The CopB outer membrane protein has been considered a vaccine candidate for the prevention of infections due to Moraxella catarrhalis. Monoclonal antibody 10F3 recognizes whole cells of about 70% of clinical isolates, suggesting that this epitope is reasonably conserved. To determine whether CopB has other surface epitopes, we analyzed M. catarrhalis isolates using polyclonal sera against recombinant CopB proteins from a 10F3 positive isolate and a 10F3 negative isolate, and polyclonal sera against synthetic peptides that contained the sequence corresponding to the 10F3 epitope region of three different isolates. Extensive cross-reactivity was observed with the anti-CopB sera towards purified recombinant CopB proteins in Western blot and antigen ELISA, implying that antigenic regions common to both proteins were present. However, anti-CopB sera resembled anti-CopB peptide sera in exhibiting similar binding specificity to whole cells, segregating M. catarrhalis isolates into four CopB groups. We subsequently cloned and sequenced the copB genes from representative isolates. The deduced CopB amino acid sequences and the degree of sequence identity also demonstrated the existence of the same four CopB groups. Each of the four groups had a unique sequence in the 10F3 epitope region and a fifth group had the epitope deleted. The polymorphism of the major surface epitope prompts further consideration regarding the utility of CopB as a vaccine component as well as the design of an efficacious CopB-based vaccine to achieve broad protection against Moraxella infection.


Assuntos
Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Moraxella catarrhalis/imunologia , Polimorfismo Genético , Animais , Proteínas da Membrana Bacteriana Externa/genética , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Soros Imunes/imunologia , Camundongos , Moraxella catarrhalis/isolamento & purificação , Proteínas Recombinantes/imunologia
2.
J Biol Chem ; 277(14): 11664-9, 2002 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-11799130

RESUMO

Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K(d) = 1.8 microm). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg(336) by activated protein C or factor IXa is inactivating. A truncated A1 (A1(336)) showed similar affinity for A2 subunit (K(d) = 0.9 microm) and stimulated its cofactor activity to approximately 50% that observed for native A1. However, A1(336) was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1(336). Fluorescence energy transfer demonstrated that, although Fl-A1(336)/A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.


Assuntos
2-Naftilamina/análogos & derivados , Arginina/química , Fator VIIIa/química , 2-Naftilamina/química , Animais , Anisotropia , Sítios de Ligação , Western Blotting , Catálise , Bovinos , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fator X/química , Fator Xa/química , Humanos , Cinética , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Espectrometria de Fluorescência
3.
Infect Immun ; 72(12): 6961-8, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15557618

RESUMO

Nontypeable Haemophilus influenzae is a major causative agent of bacterial otitis media in children. H. influenzae Hap autotransporter protein is an adhesin composed of an outer membrane Hapbeta region and a moiety of an extracellular internal 110-kDa passenger domain called Hap(S). The Hap(S) moiety promotes adherence to human epithelial cells and extracellular matrix proteins, and it also mediates bacterial aggregation and microcolony formation. A recent work (D. L. Fink, A. Z. Buscher, B. A. Green, P. Fernsten, and J. W. St. Geme, Cell. Microbiol. 5:175-186, 2003) demonstrated that Hap(S) adhesive activity resides within the C-terminal 311 amino acids (the cell binding domain) of the protein. In this study, we immunized mice subcutaneously with recombinant proteins corresponding to the C-terminal region of Hap(S) from H. influenzae strains N187, P860295, and TN106 and examined the resulting immune response. Antisera against the recombinant proteins from all three strains not only recognized native Hap(S) purified from strain P860295 but also inhibited H. influenzae Hap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization. These observations demonstrate that the C-terminal region of Hap(S) is capable of eliciting cross-reacting antibodies that reduce nasopharyngeal colonization, suggesting utility as a vaccine antigen for the prevention of nontypeable H. influenzae diseases.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae/imunologia , Fragmentos de Peptídeos/imunologia , Serina Endopeptidases/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Aderência Bacteriana , Sítios de Ligação , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Nasofaringe/microbiologia
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