RESUMO
The localization and accessibility of the group B streptococcus (GBS) surface immunogenic protein (Sip) at the surface of intact GBS cells were studied by flow cytometric assay and immunogold electron microscopy. Antibodies present in pooled sera collected from mice after immunization with purified recombinant Sip efficiently recognized native Sip at the surfaces of the different GBS strains tested, which included representatives of all nine serotypes. Examination of GBS cells by immunogold electron microscopy revealed that the Sip-specific antibodies attached preferentially to polar sites and the septal region. This result confirmed that Sip is exposed at the intact-cell surface, but it also suggests that its distribution is restricted to certain regions of the cell.
Assuntos
Antígenos de Bactérias/análise , Antígenos de Superfície/análise , Streptococcus agalactiae/imunologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Bovinos , Epitopos de Linfócito B/imunologia , Humanos , CamundongosRESUMO
The highly conserved NspA protein has been found in the outer membrane of every Neisseria meningitidis strain tested so far. Two monoclonal antibodies (MAbs) directed against this protein were used to demonstrate that biologically important epitopes of the NspA protein are exposed at the surface of serologically distinct meningococcal strains. Analysis of sera collected from mice that survived a deadly meningococcal challenge following immunization with recombinant NspA protein (rNspA) revealed the presence of cross-reactive antibodies which efficiently attached to and killed the four serogroup B strains tested. These data are additional proof that the NspA protein is exposed at the surface of intact meningococcal cells, which is an important characteristic for a vaccine candidate.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Neisseria meningitidis/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/química , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6. 84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções Estreptocócicas/prevenção & controle , Streptococcus agalactiae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/química , Vacinas Bacterianas/genética , Clonagem Molecular , Reações Cruzadas , Feminino , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência de DNA , Streptococcus agalactiae/classificação , Vacinação , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Nasal immunization was studied to determine if it could elicit an immune response capable of preventing vaginal colonization by Neisseria gonorrhoeae or of reducing its duration in the estradiol-treated mouse model. Nasal administration of gonococcal outer membrane (OM) preparations induced the development of systemic and vaginal immune responses that were directed mainly against a limited number of gonococcal OM proteins. The impact of nasal immunization on vaginal colonization by N. gonorrhoeae was evaluated by use of an experimental model, in which mice were treated with estradiol to prolong the infection. Bacterial clearance was significantly faster for mice immunized intranasally with N. gonorrhoeae OM preparations (4.0+/-2.5 days) than for control mice (8.5+/-4.3 days). The estradiol-treated mouse model may serve as a useful tool for the evaluation of potential gonococcal vaccine candidates.
Assuntos
Gonorreia/prevenção & controle , Neisseria gonorrhoeae/crescimento & desenvolvimento , Porinas/imunologia , Vagina/microbiologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/biossíntese , Eletroforese em Gel de Poliacrilamida , Feminino , Gonorreia/imunologia , Camundongos , Neisseria gonorrhoeae/imunologia , Porinas/administração & dosagem , Fatores de TempoRESUMO
The cross-bactericidal and cross-protective activities of a monoclonal antibody (MAb) named Me-7, which is directed against an antigenically highly conserved epitope on the meningococcal NspA protein, were studied. This MAb efficiently killed in vitro, in the presence of rabbit or human serum, 13 of 14 meningococcal strains tested, including 9 of 9, 2 of 3, and 2 of 2 strains of serotypes B, A, and C, respectively. MAb Me-7 also significantly reduced by more than 75% the levels of bacteremia recorded for mice challenged with 10 of 11 meningococcal strains tested. Analysis of the predicted amino acid sequence of the NspA protein from the meningococcal strain MCH88 (A:4:P1.10), which was not killed by MAb Me-7, indicated the presence of an additional glutamine residue at position 73, compared to the three other NspA sequences. The data presented in this study suggest that antibodies directed against this highly conserved outer membrane protein could protect against meningococcal infections.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Neisseria meningitidis/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Reações Cruzadas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neisseria meningitidis/genética , CoelhosRESUMO
A low-molecular-weight protein named NspA (neisserial surface protein A) was recently identified in the outer membrane of all Neisseria meningitidis strains tested. Antibodies directed against this protein were shown to protect mice against an experimental meningococcal infection. Hybridization experiments clearly demonstrated that the nspA gene was also present in the genomes of the 15 Neisseria gonorrhoeae strains tested. Cloning and sequencing of the nspA gene of N. gonorrhoeae B2 revealed an open reading frame of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a calculated molecular weight of 18,316 and a pI of 10.21. Comparison of the predicted amino acid sequence of the NspA polypeptides from the gonococcal strains B2 and FA1090, together with that of the meningococcal strain 608B, revealed an identity of 93%, suggesting that the NspA protein is highly conserved among pathogenic Neisseria strains. The level of identity rose to 98% when only the two gonococcal predicted NspA polypeptides were compared. To evaluate the level of antigenic conservation of the gonococcal NspA protein, monoclonal antibodies (MAbs) were generated. Four of the seven NspA-specific MAbs described in this report recognized their corresponding epitope in 100% of the 51 N. gonorrhoeae strains tested. Radioimmunobinding assays clearly indicated that the gonococcal NspA protein is exposed at the surface of intact cells.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , DNA Bacteriano , Genes Bacterianos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neisseria gonorrhoeae/imunologia , Neisseria meningitidis/imunologia , Homologia de Sequência de AminoácidosRESUMO
A new surface protein, named NspA, which is distinct from the previously described Neisseria meningitidis outer membrane proteins was identified. An NspA-specific mAb, named Me-1, reacted with 99% of the meningococcal strains tested indicating that the epitope recognized by this particular mAb is widely distributed and highly conserved. Western immunoblotting experiments indicated that mAb Me-1 is directed against a protein band with an approximate molecular mass of 22,000, but also recognized a minor protein band with an approximate molecular mass of 18,000. This mAb exhibited bactericidal activity against four meningococcal strains, two isolates of serogroup B, and one isolate from each serogroup A and C, and passively protected mice against an experimental infection. To further characterize the NspA protein and to evaluate the protective potential of recombinant NspA protein, the nspA gene was identified and cloned into a low copy expression vector. Nucleotide sequencing of the meningococcal insert revealed an ORF of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a predicted molecular weight of 18,404 and a isoelectric point of 9.93. Three injections of either 10 or 20 microg of the affinity-purified recombinant NspA protein efficiently protected 80% of the mice against a meningococcal deadly challenge comparatively to the 20% observed in the control groups. The fact that the NspA protein can elicit the production of bactericidal and protective antibodies emphasize its potential as a vaccine candidate.
Assuntos
Antígenos de Bactérias/uso terapêutico , Proteínas da Membrana Bacteriana Externa/uso terapêutico , Vacinas Bacterianas/uso terapêutico , Infecções Meningocócicas/prevenção & controle , Vacinação , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Genes Bacterianos , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Neisseria meningitidis/patogenicidade , Proteínas Recombinantes/uso terapêutico , Análise de Sequência de DNAAssuntos
Anticorpos Antivirais/genética , Diversidade de Anticorpos , Citomegalovirus/imunologia , Rearranjo Gênico do Linfócito B , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Infecções por Citomegalovirus/imunologia , Humanos , Hibridomas/imunologiaRESUMO
Four human hybridoma antibodies directed against the human cytomegalovirus (CMV) were characterized with respect to their immunoglobulin gene usage and expression of rheumatoid factor (RF) associated idiotypes and variable region epitopes. The aims of these experiments were: (1) to characterize the immunoglobulin gene usage of four antibodies directed against a single protein of a human pathogen; and (2) to examine how this humoral response may be linked to the production of RFs, autoantibodies found in the majority of patients with rheumatoid arthritis (RA). All four anti-CMV antibodies were of the gamma heavy chain isotype and were specific for the immunodominant 65 kDa viral matrix phosphoprotein (pp65). The four anti-pp65 antibodies expressed different light (L) and heavy (H) chain variable region gene combinations. These were: VkIII/VH3, V lambda 1/VH3, V lambda 1/VH4 and V lambda 3/VH3, respectively for the HCV-2, HCV-3, HCV-63 and HCV-65 hybridoma cell lines. Although none had RF activity, each of these antibodies expressed a unique set of RF-associated determinants, implying different three-dimensional configurations of the variable regions of these antibodies. The HCV-2 antibody, however, had the most extensive similarities to human RFs since it not only expressed the greatest number of RF-associated determinants but also had a protein sequence that was very homologous to RFs of the "Po" idiotypic family. Furthermore, predicted germline gene usage by anti-CMV antibodies and RFs suggest that some are encoded by identical or similar genes and that the different specificities are achieved by somatic mutations in the L and H chain complementarity determining regions (CDRs) and genetic diversity in the H chain CDR3.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Citomegalovirus/imunologia , Fosfoproteínas/imunologia , Fator Reumatoide/química , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Sequência de Bases , Epitopos/imunologia , Humanos , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Idiótipos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Dados de Sequência Molecular , Fator Reumatoide/imunologia , Homologia de Sequência de AminoácidosRESUMO
In this study, it has been determined that immunoglobulin G1 (IgG1) and IgG3 monoclonal antibodies directed to the lipooligosaccharide A of Bordetella pertussis were able to protect mice from fatal aerosol infection. No correlation was found between the bactericidal activity in vitro in the presence of complement and the protection in mice, since a bactericidal IgG3 did not elicit protection. In addition, no significant difference in protective capacity was observed with bactericidal and nonbactericidal IgG1 antibodies, indicating that bactericidal activity is not a requirement for protection mediated by certain anti-lipooligosaccharide A antibodies. A reduction in protection in C5-deficient mice was observed, suggesting a significant role for complement in certain host defense mechanisms against B. pertussis infection.
Assuntos
Anticorpos Monoclonais/farmacologia , Bordetella pertussis/imunologia , Lipopolissacarídeos/imunologia , Coqueluche/prevenção & controle , Animais , Anticorpos Antibacterianos/farmacologia , Especificidade de Anticorpos , Antígenos de Bactérias , Bordetella pertussis/isolamento & purificação , Imunoglobulina G/farmacologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Traqueia/microbiologia , Coqueluche/imunologia , Coqueluche/microbiologiaRESUMO
Structural analysis of the human immunoglobulin repertoire holds promise for determining the basis of variable region gene usage in response to a variety of auto and exogenous antigens. Here we report the nucleotide sequences of the heavy and light chain variable regions expressed by three human monoclonal antibodies specific for two clinically relevant bacterial pathogens, Bordetella pertussis and Haemophilus influenzae type b. The cell lines were derived by in vitro stimulation of lymphocytes from spleen or tonsillar tissue, respectively, and bind to different antigens from the two organisms. The single B. pertussis antibody is of the IgM lambda isotype and utilizes the single VH6 gene segment in combination with a V lambda 2 gene and demonstrates limited somatic mutation, yet is highly indicative of an antigen-driven immune response. One H. influenzae antibody is of the IgG2 lambda isotype and expresses a VH3 gene segment with a V lambda 1 gene, while the second cell line produces an IgG3 lambda antibody expressing a combination of VH2/V lambda 3. Both molecules show evidence of somatic mutation. The D gene segments of the heavy chains vary in length and display limited sequence homology with known germline D segments. As demonstrated previously, JH4 predominates (two JH4 and one JH3) and all three utilize the J lambda 3 gene segment. In addition, we have isolated and sequenced a number of germline VH2 gene segments in an attempt to better understand the nature of the VH2 germline repertoire. In addition to contributing to the understanding of the human antibody repertoire, such clinically relevant molecules may prove to be a source of passive immunotherapy for those at risk to developing disease.
Assuntos
Anticorpos Monoclonais/genética , Antígenos de Bactérias/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/análise , Diversidade de Anticorpos/genética , Sequência de Bases , Bordetella pertussis/imunologia , Linhagem Celular , Haemophilus influenzae/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/análise , Região Variável de Imunoglobulina/análise , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência do Ácido NucleicoRESUMO
Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Antígenos Virais/imunologia , Citomegalovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Especificidade de Anticorpos , Epitopos , Idiótipos de Imunoglobulinas/imunologia , Camundongos , Testes de Neutralização , CoelhosRESUMO
We have developed a novel enzyme immunoassay (EIA) for the specific detection of Chlamydia trachomatis utilizing a monoclonal anti-idiotypic antibody to an antibody directed against a chlamydia specific epitope on 60 kDa heat-shock protein (HSP60). The basis of the assay is the inhibition of the binding of idiotype to anti-idiotype by antigen present in test samples. Two configurations of the assay were developed: a blocking EIA and a competition EIA. Greater sensitivity was observed using the competition EIA, with the assay detecting purified recombinant HSP60 and purified chlamydia in a concentration-dependent manner from 0.01 to 10 micrograms protein and from 0.5 to 12 micrograms total protein, respectively. The assay is highly specific and offers several potential advantages over currently available EIAs for the detection of this pathogen.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/imunologia , Técnicas Imunoenzimáticas , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Ligação Competitiva/imunologia , Chaperonina 60 , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/genética , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de FusãoAssuntos
Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Lipopolissacarídeos/imunologia , Oligossacarídeos/imunologia , Fatores de Virulência de Bordetella , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos de Bactérias/química , Bacteriólise , Sequência de Carboidratos , Humanos , Imunização Passiva , Idiótipos de Imunoglobulinas/imunologia , Lipopolissacarídeos/química , Camundongos , Dados de Sequência Molecular , Coqueluche/imunologia , Coqueluche/prevenção & controleRESUMO
A novel enzyme-linked immunosorbent assay (ELISA) was developed for human cytomegalovirus (HCMV) utilizing a monoclonal anti-idiotype specific for CMVB1, an antibody to HCMV. Samples of HCMV were measured by their inhibition of the binding of CMVB1 to anti-idiotype. The ELISA detected HCMV in a concentration-dependent manner from 20 to 0.6 x 10(3) PFU/ml, with 50% inhibition at approx. 3 x 10(3) PFU/ml. These data demonstrate the potential of anti-idiotype antibodies as the basis of simple and rapid diagnostic tests for infectious agents.
Assuntos
Anticorpos Anti-Idiotípicos , Anticorpos Antivirais , Antígenos Virais/análise , Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Antígenos Virais/imunologia , Citomegalovirus/isolamento & purificação , Humanos , Sensibilidade e EspecificidadeRESUMO
This study describes, for the first time, the production and use of an "internal-image" anti-idiotypic monoclonal antibody (MAb) to elicit a rotavirus-specific antibody response. An immunoglobulin G2a MAb, designated RQ31 (MAb1), specific for the outer capsid protein VP4 of bovine Q17 rotavirus and capable of neutralizing viral infection in vitro was used to generate an anti-idiotypic MAb (MAb2). This MAb2, designated RQA2, was selected by enzyme-linked immunosorbent assay (ELISA) using F(ab')2 fragments of RQ31. RQA2 (MAb2) inhibited the binding of RQ31 (MAb1) to the virus but had no effect on the binding of other rotavirus-specific MAbs. The MAb2 also inhibited virus neutralization mediated by MAb1 in a dose-dependent fashion. Naive guinea pigs immunized with the MAb2 produced anti-anti-idiotypic antibodies (Ab3) that reacted with bovine Q17 rotavirus in an ELISA and neutralized rotavirus infection in vitro. The Ab3 response was characterized as MAb1-like because the Ab3 recognizes only the Q17 and neonatal calf diarrhea virus rotavirus strains in ELISA, as did RQ31 (MAb1). The Ab3 response also possessed two other characteristics of RQ31: the abilities to bind the 1.36 (double-capsid) but not the 1.38 (single-capsid) purified rotavirus fraction in ELISA and to immunoprecipitate the VP4 rotavirus protein.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/biossíntese , Proteínas do Capsídeo , Rotavirus/imunologia , Animais , Capsídeo/imunologia , Bovinos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Cobaias , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de NeutralizaçãoRESUMO
Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Enterovirus/imunologia , Animais , Autorradiografia , Western Blotting , Células Cultivadas , Epitopos/imunologia , Imunofluorescência , Macaca mulatta , Testes de Neutralização , Ensaio de Radioimunoprecipitação , Ensaio de Placa ViralRESUMO
Two structurally and immunologically different components of Bordetella pertussis endotoxin can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining: a major A band and a faster-migrating minor B band. Certain mutant strains of B. pertussis express only the B band, while the wild-type strains produce both lipooligosaccharides (LOS). Two monoclonal antibodies (MAbs) directed against the minor LOS B band were generated, allowing the study of this surface molecule on different strains of Bordetella. These two MAbs, designated BL-8 and BL-9, reacted strongly with phenol-water-purified LOS obtained from a B. pertussis LOS B mutant strain. Sodium periodate treatment of the purified LOS prevented binding of the MAbs, indicating the carbohydrate nature of the epitope(s). Western immunoblotting experiments revealed that the epitope(s) recognized by these MAbs is conserved on all B. pertussis and Bordetella bronchiseptica Vir- (avirulent) variant strains tested but is not present on Bordetella parapertussis and B. bronchiseptica Vir+ (virulent) wild-type strains. Further studies showed that although present in the lipopolysaccharide B band expressed by Vir- strains, the epitope(s) recognized by the MAbs is not accessible on the surface of intact B. bronchiseptica cells. For B. pertussis, the density and accessibility of this epitope(s) are dependent on the virulence-associated or LOS phenotype expressed by the strain. Our data demonstrate that the expression and accessibility of the epitope(s) are significantly greater on the LOS B variant strains and LOS AB Vir- strains compared with fresh B. pertussis clinical isolates. For these latter strains, which are Vir+, this epitope(s) was barely detectable on the surface of intact bacteria, despite Western blot analyses that revealed specific reactions between the MAbs and the LOS B band. The two LOS B-specific MAbs had no bacteriolytic activity against a LOS AB wild-type strain, while the control MAb BL-2, which is specific for the B. pertussis LOS A band, significantly reduced the number of living bacteria in the same assay. Moderate lytic activity against a mutant strain expressing only the LOS B band was observed for MAb BL-8 but not for MAb BL-9 or BL-2. These data demonstrate that the type, amount, and surface exposure of the LOS are related to the phenotype expressed by a specific B. pertussis strain. In addition, the LOS B MAbs also reveal the antigenic conservation of carbohydrate epitopes among B. pertussis and B. bronchiseptica strains.
Assuntos
Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Lipopolissacarídeos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Bacteriólise/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas , Camundongos , Camundongos Endogâmicos BALB C , RadioimunoensaioRESUMO
Synthetic peptides, anti-idiotypic antibodies (anti-Id) and human and murine monoclonal antibodies (mAbs) were used to further define a major antigenic domain on the outer membrane P1 protein (OMP) of Haemophilus influenzae type b (Hib). Synthetic peptides were elaborated from the known primary sequences of the P1 protein of prototype Hib strains MinnA (OMP subtype 1H) and 8358 (OMP subtype 6U). By peptide mapping, antibodies are categorized into three groups: A, B and C. A first epitope on the P1 from strain MinnA was identified by the reactivity of one set of murine anti-P1 mAbs with the two overlapping peptides 11H and 13H, corresponding to amino acid residues 384-412 and 400-437, respectively. On the basis of their reactivity with both peptides, these mAbs were designated as group A. Anti-Id obtained from mice immunized with two group A mAbs reacted specifically with all group A mAbs. A second epitope on the same P1 protein was identified by the reactivity of the peptide 13H with another distinct set of murine anti-P1 mAbs assigned to group B. This group of mAbs did not recognize the peptide 11H. Murine anti-Id which were prepared against one group B mAb inhibited the attachment of this mAb to outer membrane preparations, whereas the binding of the other group B mAbs was not affected, suggesting that these mAbs represent a heterologous group of mAbs. The epitope(s) recognized by two human anti-P1 mAbs was (were) distinct from the ones recognized by murine mAbs since no reactivity with the peptides was observed. Similarly, the binding of the two human mAbs to the P1 antigen was not inhibited by anti-Id raised against group A or B mAbs. Interestingly, an epitope on a different P1 protein recovered from strain 8358 was identified by the reactivity of group C murine mAbs with the peptide 13U, which occupies the same position on the P1 protein as 13H but differs from the latter by 10 amino acid residues. Our studies demonstrated the presence of several distinct surface-exposed B-cell epitopes within the antigenic domain which was defined previously on the P1 protein of Hib MinnA. Furthermore, we showed the immunodominance of this region on two different P1 proteins. None of the mAbs, however, had a bacteriolytic or protective activity against Hib strains. We suggest that the surface-exposed immunodominant region on the OMP P1 of Hib do not induce protective antibodies against Hib infection.