RESUMO
Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the "HCDR4", located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , Receptores de IgG/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/genética , Linhagem Celular , Separação Celular , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Receptores de IgG/genéticaRESUMO
INTRODUCTION: Respiratory syncytial virus (RSV) is a major etiological agent of lower respiratory tract infection in infants. Genotypes of this virus and the role of the infants' serum antibodies have yet to be fully clarified. This knowledge is important for the development of effective therapeutic and prophylactic measures. OBJECTIVES: To evaluate the types and genotypes of RSV causing respiratory tract infection in infants, to analyze the association of subtype-specific serum antibodies with the occurrence of infection and to evaluate the presence of subtype-specific antibodies in the infants' mothers and their association with the profile of the childrens' serum antibodies. METHODS: This was a prospective study on infants hospitalized with respiratory infection. Nasopharyngeal secretions were collected for viral investigation using indirect immunofluorescence and viral culture and blood was collected to test for antibodies using the Luminex Multiplex system. RESULTS: 192 infants were evaluated, with 60.9% having RSV (73.5%- A and 20.5% B). Six genotypes of the virus were identified: A5, A2, B3, B5, A7 and B4. The seroprevalence of the subtype-specific serum antibodies was high. The presence and levels of subtype-specific antibodies were similar, irrespective of the presence of infection or the viral type or genotype. The mothers' antibody profiles were similar to their infants'. CONCLUSIONS: Although the prevalence of subtype-specific antibodies was elevated, these antibodies did not provide protection independently of virus type/genotype. The similarity in the profiles of subtype-specific antibodies presented by the mothers and their children was consistent with transplacental passage.
Assuntos
Anticorpos Antivirais/sangue , Especificidade de Anticorpos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/virologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/genética , Brasil/epidemiologia , Métodos Epidemiológicos , Feminino , Humanos , Lactente , Masculino , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sinciciais Respiratórios/genética , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/prevenção & controleRESUMO
INTRODUCTION: Respiratory syncytial virus (RSV) is a major etiological agent of lower respiratory tract infection in infants. Genotypes of this virus and the role of the infants' serum antibodies have yet to be fully clarified. This knowledge is important for the development of effective therapeutic and prophylactic measures. OBJECTIVES: To evaluate the types and genotypes of RSV causing respiratory tract infection in infants, to analyze the association of subtype-specific serum antibodies with the occurrence of infection and to evaluate the presence of subtype-specific antibodies in the infants' mothers and their association with the profile of the childrens' serum antibodies. METHODS: This was a prospective study on infants hospitalized with respiratory infection. Nasopharyngeal secretions were collected for viral investigation using indirect immunofluorescence and viral culture and blood was collected to test for antibodies using the Luminex Multiplex system. RESULTS: 192 infants were evaluated, with 60.9 percent having RSV (73.5 percent- A and 20.5 percent B). Six genotypes of the virus were identified: A5, A2, B3, B5, A7 and B4. The seroprevalence of the subtype-specific serum antibodies was high. The presence and levels of subtype-specific antibodies were similar, irrespective of the presence of infection or the viral type or genotype. The mothers' antibody profiles were similar to their infants'. CONCLUSIONS: Although the prevalence of subtype-specific antibodies was elevated, these antibodies did not provide protection independently of virus type/genotype. The similarity in the profiles of subtype-specific antibodies presented by the mothers and their children was consistent with transplacental passage.
INTRODUÇÃO: O vírus sincicial respiratório é um dos principais agentes etiológicos das infecções do aparelho respiratório inferior em lactentes. Os genótipos deste vírus e o papel dos anticorpos séricos ainda não estão esclarecidos. Este conhecimento é importante para o desenvolvimento de medidas terapêuticas e profiláticas. OBJETIVOS: Avaliar: os tipos e genótipos do vírus sincicial que causam infecção respiratória em lactentes e a associação dos anticorpos séricos subtipo-específicos com a ocorrência de infecção; a presença de anticorpos subtipo-específicos nas mães e sua associação com o perfil de anticorpos da criança. MÉTODOS: Estudo prospectivo incluindo lactentes hospitalizados com infecção respiratória. Foi coletada secreção de nasofaringe para investigação viral usando imunofluorescência indireta e cultivo viral. Foi coletado sangue para pesquisa de anticorpos usando o sistema Luminex Multiplex. RESULTADOS: Avaliados 192 lactentes: 60,9 por cento com vírus sincicial (73,5 por cento - A e 20,5 por cento - B). Seis genótipos de vírus sincicial respiratório foram identificados: A5,A2,B3,B5,A7 e B4. A soroprevalência dos anticorpos subtipos-específicos foi alta. A presença e o nível de anticorpos subtipos-específicos foram semelhantes, independentemente da presença de infecção, tipo e genótipo do vírus. As mães e as crianças apresentaram perfis semelhantes de anticorpos. CONCLUSÕES: A prevalência dos anticorpos subtipos-específicos foi elevada mas estes anticorpos não conferiram proteção, independentemente do tipo/genótipo do vírus. A semelhança dos perfis de anticorpos das mães e das crianças foi compatível com transmissão transplacentária.
Assuntos
Feminino , Humanos , Lactente , Masculino , Anticorpos Antivirais/sangue , Especificidade de Anticorpos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/virologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/genética , Brasil/epidemiologia , Métodos Epidemiológicos , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sinciciais Respiratórios/genética , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/prevenção & controleRESUMO
Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.
Assuntos
Especificidade de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Vacinas contra a AIDS , Especificidade de Anticorpos/genética , Reações Cruzadas/genética , Reações Cruzadas/imunologia , Feminino , Genótipo , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Masculino , Testes de Neutralização/métodos , Fragmentos de Peptídeos/genéticaRESUMO
Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.
Assuntos
Feminino , Humanos , Masculino , Especificidade de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , /imunologia , HIV-1 , Fragmentos de Peptídeos/imunologia , Vacinas contra a AIDS , Especificidade de Anticorpos/genética , Reações Cruzadas/genética , Reações Cruzadas/imunologia , Genótipo , /genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Testes de Neutralização/métodos , Fragmentos de Peptídeos/genéticaRESUMO
Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5(+) B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.
Assuntos
Especificidade de Anticorpos , Autoantígenos/metabolismo , Sítios de Ligação de Anticorpos , Imunoglobulinas/biossíntese , Depleção Linfocítica , Tecido Linfoide/efeitos da radiação , Envelhecimento/genética , Envelhecimento/imunologia , Animais , Especificidade de Anticorpos/genética , Autoantígenos/imunologia , Sítios de Ligação de Anticorpos/genética , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Transplante de Tecido Fetal/imunologia , Imunidade Inata/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Imunoglobulinas/sangue , Imunoglobulinas/metabolismo , Fígado/imunologia , Fígado/metabolismo , Depleção Linfocítica/métodos , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Quimera por Radiação/imunologiaRESUMO
In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Especificidade de Anticorpos/genética , Antígenos Glicosídicos Associados a Tumores/imunologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Fusão Celular , Vetores Genéticos , Humanos , Hibridomas/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/isolamento & purificação , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
Uma biblioteca de expressao de cDNA de verme adulto de S. mansoni foi selecionada utilizando-se soros de macacos babuinos em fase inicial da infeccao. Os clones que reagiram positivamente com os soros de infeccao recente foram examinados quanto a reatividade contra soros normais e soros de infeccao heterologa. Com a finalidade de se conseguir melhor discriminacao entre reatividade positiva com o anticorpo especifico e aquela devido aos anticorpos anti-E. coli residuais, um clone de cDNA nao relacionado ao S. mansoni foi plaqueado em mistura com o clone positivo. O plano de fundo negativo proporcionado pelo clone nao relacionado forneceu o contraste necessario para discriminar a reacao positiva especifica...
Assuntos
Animais , Células Clonais , Schistosoma mansoni/genética , Esquistossomose mansoni/microbiologia , Especificidade de Anticorpos/genética , Autoimunidade , Análise de Sequência de DNA , Sorotipagem/classificaçãoRESUMO
We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.