RESUMO
OBJECTIVE: To determine whether passive transfer of a monoclonal antibody specific for the principal neutralizing determinant in the V3 region of HIV-1IIIB gp120 can protect mice with severe combined immunodeficiency (SCID) transplanted with normal human peripheral blood leukocytes (hu-PBL), designated hu-PBL-SCID mice, from subsequent challenge with the homologous viral strain. DESIGN AND METHODS: hu-PBL-SCID mice were given intraperitoneal injections of an anti-HIV-1 neutralizing murine monoclonal antibody (BAT123), its mouse-human chimeric form (CGP 47 439), or a control murine antibody (PNTU), at a dose of 40 mg/kg. The mice were then challenged intraperitoneally with 10 mouse infectious doses of HIV-1IIIB. Three weeks later the mice were killed, and spleen cells and peritoneal lavage collected for determination of infection by coculture for viral isolation and by detection of HIV-1 DNA using polymerase chain reaction (PCR). RESULTS: All three antibodies had similar serum half-lives of 9-12 days. No toxicity was observed in the animals. HIV-1 was recovered by coculture from five out of the six mice given PNTU, and by PCR from two out of the six mice given PNTU, but was not recovered by either technique from any of the 12 mice given BAT123 or CGP 47 439. CONCLUSION: BAT123 and CGP 47 439, which are specific for the principal neutralizing determinant of HIV-1IIIB, protect hu-PBL-SCID mice from infection by this viral strain. Our findings support the use of the hu-PBL-SCID mouse as an in vivo model for studying protection against HIV-1 infection by passive immunization with anti-HIV-1 neutralizing antibodies.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Epitopos , Anticorpos Anti-HIV/administração & dosagem , Infecções por HIV/imunologia , Imunização Passiva , Camundongos , Camundongos SCID , Testes de NeutralizaçãoRESUMO
We previously reported that passive transfer of a murine V3-specific monoclonal antibody (BAT123) to hu-PBL-SCID mice challenged with HIV-1LAI confers postexposure protection from infection. The role of the Fc fragment of this antibody as well as the involvement of the complement system in protection were evaluated in vivo. When we compared the postexposure protection offered by BAT123 and CGP 47439, a chimeric form of BAT123 in which the murine Fc domain has been replaced by a human IgG1 Fc domain, CGP 47439 failed to provide postexposure protection against HIV-1LAI despite having similar pharmacokinetics and in vitro neutralizing activity. Furthermore, when hu-PBL-SCID mice were treated with cobra venom factor, which inactivates serum complement activity, the postexposure protective ability of BAT123 was abrogated. These findings suggest that the complement system is involved in the passive protection against HIV-1 infection conferred by the murine monoclonal antibody BAT123 in hu-PBL-SCID mice.
Assuntos
Proteínas do Sistema Complemento/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Imunização Passiva , Fragmentos de Peptídeos/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Ensaio de Atividade Hemolítica de Complemento , Proteínas Inativadoras do Complemento/farmacologia , Modelos Animais de Doenças , Venenos Elapídicos/farmacologia , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Camundongos SCID , Testes de Neutralização , Proteínas Recombinantes de Fusão/imunologia , Baço/virologiaRESUMO
We have studied antibody reactivity with monomeric and oligomeric forms of the gp120 envelope glycoprotein from the macrophage-tropic primary virus, HIV-1 JR-FL. We find that the correlation between oligomer reactivity and virus neutralization is not absolute for MAbs to epitopes overlapping the CD4-binding site on gp120. An MAb (205-46-9) with very limited neutralizing ability for JR-FL binds about as avidly to oligomeric JR-FL envelope glycoproteins as the strongly neutralizing IgG1b12 MAb does. In addition, neutralizing and nonneutralizing sera from HIV-1-infected people are similar in their reactivities to oligomeric JR-FL envelope glycoproteins; the correlation between oligomer reactivity and virus neutralization is weak. Although oligomer reactivity of an anti-gp120 antibody is necessary for virus neutralization, it is not always sufficient to cause it.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Oligopeptídeos/imunologia , Anticorpos Monoclonais/imunologia , Humanos , Testes de NeutralizaçãoRESUMO
Human hybridoma cell lines secreting IgG specific for the major allergen in the pollen of short ragweed, Amb a I, were established from patients who had been receiving antigen injections for immunotherapy. Recombinant Ig genes were then constructed by cloning the heavy and light chain variable region genes of the human hybridoma cell line and joining them to the human alpha or kappa constant region genes in mammalian expression vectors. Amb a I-specific IgA was expressed in two mouse myeloma cell lines, NS0 and Sp2/0. In both systems, transfected alpha and kappa chains were assembled into IgA monomers or into dimers covalently linked by the endogenous murine J chains. We propose that recombinant IgA monoclonal antibodies specific for airborne allergens may be applied to the mucosal surface of the nasal linings or of the lower airway of sensitized individuals to inhibit the entry of allergenic molecules across the mucosal epithelium and, therefore, to prevent the development of allergic responses.
Assuntos
Alérgenos/imunologia , Antígenos/uso terapêutico , Imunoglobulina A/imunologia , Imunoterapia , Pólen/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Sequência de Bases , Linhagem Celular , Estudos de Viabilidade , Humanos , Hipersensibilidade/terapia , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/imunologiaAssuntos
Anticorpos Monoclonais/farmacologia , Fator D do Complemento/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Animais , Ativação do Complemento/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Masculino , Miocárdio/imunologia , CoelhosRESUMO
Monoclonal antibody BAT123 was passively transferred into SCID mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID) to study passive antibody protection against human immunodeficiency virus type 1 (HIV-1) infection. BAT123 is specific for the third variable loop of the gp120 of HIV-1LAI. Animals were protected against subsequent infection with LAI strain, but not other virus strains, when BAT123 (1 mg/kg; 25 micrograms/mouse) was given 1 h before virus inoculation. This resulted in a peak serum concentration of 16 micrograms/mL of the antibody, which should be easily attainable in humans. In addition, postexposure protection was observed when the antibody was given within 4 h of virus inoculation. No therapeutic effect was observed, however, when BAT123 was administered after infection had been established. These results indicate that passive antibody prophylaxis against HIV-1 infection may be possible in certain clinical situations.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Imunização Passiva , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Anti-HIV/administração & dosagem , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , Humanos , Camundongos , Camundongos SCID , Fragmentos de Peptídeos/imunologia , Especificidade da Espécie , Baço/virologiaRESUMO
Two murine monoclonal antibodies, G3.519, recognizing the CD4-binding region, and BAT123, a variable region of gp120 of human immunodeficiency virus, were chemically coupled to pokeweed antiviral protein isolated from seeds (PAP-S). The immunoconjugates were purified by Sephacryl S-200 gel filtration and Mono S ion exchange chromatography. Immunoconjugate G3.519-PAP-S specifically killed human T cells, H9, infected with three diverse HIV-1 strains, HTLV-IIIB, -IIIMN, and -IIIRF. Inhibition of thymidine incorporation by the immunoconjugate was concentration-dependent, with the ID50 ranging from 1.4 x 10(-10) M to 1.7 x 10(-9) M. Immunoconjugate BAT123-PAP-S was effective in killing H9 cells infected with HTLV-IIIB (ID50 = 4.3 x 10(-11) M) and -IIIMN (ID50 = 4.7 x 10(-10) M), but not -IIIRF. Both immunoconjugates did not inhibit thymidine incorporation in uninfected H9 cells up to a concentration of 5.3 x 10(-8) M, and their cytotoxic activities could be competitively blocked by the respective unconjugated antibodies. The immunoconjugates retained the ability to neutralize HIV virions to infect T cells and to prevent the syncytium formation. These in vitro studies suggest that the use of immunoconjugates capable of killing HIV-infected T cells and neutralizing virus may provide an alternative treatment for HIV-infected persons.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunotoxinas/imunologia , N-Glicosil Hidrolases , Linfócitos T/microbiologia , Anticorpos Monoclonais/uso terapêutico , Sobrevivência Celular , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Imunotoxinas/isolamento & purificação , Técnicas In Vitro , Testes de Neutralização , Proteínas de Plantas/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1 , Especificidade da Espécie , Linfócitos T/citologiaRESUMO
A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Vacinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Hibridomas , Testes de Neutralização , CoelhosRESUMO
To define the domains in the envelope glycoprotein important for antibody neutralization of the human immunodeficiency virus type 1 (HIV-1), monoclonal antibodies (mAbs) were generated by immunizing mice with purified glycoprotein gp120 of the IIIB isolate. One mAb, G3-4, reacted with the gp120 of homologous (IIIB) and heterologous (RF) isolates. In addition, mAb G3-4 efficiently neutralized both IIIB and RF viruses in vitro, as well as four of nine primary HIV-1 isolates. In competition immunoassays, mAb G3-4 and soluble CD4 were found to inhibit one another in binding to gp120. However, no competition was seen between mAb G3-4 and mAbs directed to the third variable region or the fourth conserved region of gp120. In particular, mAb G3-4 did not compete with our human mAb 15e, which identifies a discontinuous epitope on gp120 involved in group-specific neutralization of HIV-1 and in gp120-CD4 binding. Epitope-mapping studies on mAb G3-4 with synthetic or unglycosylated recombinant peptides were negative, suggesting that its epitope may be discontinuous. Indeed, this hypothesis was confirmed by showing the loss of mAb G3-4 serologic reactivity when gp120 was first denatured. We conclude that the site recognized by mAb G3-4 represents another discontinuous epitope on gp120 important for neutralization of HIV-1.
Assuntos
Anticorpos Monoclonais , Antígenos CD4/imunologia , Epitopos/análise , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Complexo Antígeno-Anticorpo , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/análise , Imunoglobulina G , Cadeias kappa de Imunoglobulina , Masculino , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Testes de NeutralizaçãoRESUMO
In preparing for testing a pharmaceutical grade preparation of chimeric (mouse/human) antibody CGP 47,439 in HIV-1 infected individuals, it was administered to Macaca fascicularis (cynomolgus) monkeys to study tolerability, immunogenicity and pharmacokinetics. Four groups of monkeys, three males and three females per group, received respectively four infusions of 0, 1.43, 4.3, and 14.3 mg of CGP 47,439/kg body weight at one-week intervals. The chimeric antibody induced no fever, was tolerated well throughout the 50-day observation period, elicited no tissue damage and no anti-antibody response. The pharmacokinetic profile was similar at all dose levels with a mean T1/2 alpha of 14.2 h (range 11.8-19.3 h) and a mean T1/2 beta of 172.6 h (range 137.2-220.5h). Following four successive antibody infusions serum concentrations of CGP 47,439 increased without reaching a steady state, and its measured concentrations were comparable to the simulated values. Collectively the study has provided safety and pharmacokinetic data that would allow human studies with this antibody in AIDS patients.
Assuntos
Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/imunologia , Proteínas Recombinantes de Fusão , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Anticorpos Anti-HIV/toxicidade , Antígenos HIV/imunologia , Humanos , Macaca fascicularis , Masculino , Camundongos , Testes de Neutralização , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/toxicidadeRESUMO
We have analyzed a panel of eight murine monoclonal antibodies (MAbs) that depend on the V2 domain for binding to human immunodeficiency virus type 1 (HIV-1) gp120. Each MAb is sensitive to amino acid changes within V2, and some are affected by substitutions elsewhere. With one exception, the MAbs were not reactive with peptides from the V2 region, or only poorly so. Hence their ability to bind recombinant strain IIIB gp120 depended on the preservation of native structure. Three MAbs cross-reacted with strain RF gp120, but only one cross-reacted with MN gp120, and none bound SF-2 gp120. Four MAbs neutralized HIV-1 IIIB with various potencies, and the one able to bind MN gp120 neutralized that virus. Peptide serology indicated that antibodies cross-reactive with the HxB2 V1 and V2 regions are rarely present in HIV-1-positive sera, but the relatively conserved segment between the V1 and V2 loops was recognized by antibodies in a significant fraction of sera. Antibodies able to block the binding of V2 MAbs to IIIB or MN gp120 rarely exist in sera from HIV-1-infected humans; more common in these sera are antibodies that enhance the binding of V2 MAbs to gp120. This enhancement effect of HIV-1-positive sera can be mimicked by several human MAbs to different discontinuous gp120 epitopes. Soluble CD4 enhanced binding of one V2 MAb to oligomeric gp120 but not to monomeric gp120, perhaps by inducing conformational changes in the oligomer.
Assuntos
Anticorpos Monoclonais , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Divisão Celular , Linhagem Celular , Dissulfetos/análise , Epitopos/análise , Citometria de Fluxo , Proteína gp120 do Envelope de HIV/biossíntese , HIV-1/metabolismo , Humanos , Camundongos , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Neutralização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Timidina/metabolismo , TransfecçãoRESUMO
The pharmacokinetics of mouse V/human C (gamma 1, kappa) chimeric monoclonal antibody CGP 47 439 specific for the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) was studied in patients with stage IV HIV-1 disease in an open-labeled phase I/IIA trial. Twelve male patients were enrolled and nine completed the study. Patients were divided into three groups according to the extent of CGP 47 439 to bind to gp120 from their viral isolates: undetectable for group 1, modestly reactive for group 2, and strongly reactive for group 3. A first dose of 1, 10, or 25 mg was administered by intravenous infusion to group 1, group 2 and group 3 patients, respectively. The patients then received seven doses of 50, 100, or 200 mg, respectively, every three weeks. CGP 47 439 serum concentrations were determined by an ELISA using monoclonal antibody AB19-4 specific for the idiotope of CGP 47 439. Half an hour after infusion only 25.5-36.1% of the administered antibody was found in the serum, reflecting its rapid distribution in the extravascular space and possibly binding to gp120 antigen in some of the patients. The terminal elimination half-life (T1/2) was 16.2 days in group 1 patients, 9.7 days in group 2 and in group 3 patients 7.5 days and 9.1 days. An antibody response to CGP 47 439 was not a factor in determining elimination rates, because only very low and transient responses were found in three patients. These results suggest that the reactivity of CGP 47 439 with HIV-1 gp120 contributed to its elimination in HIV-1 infected patients.
Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1 , Proteínas Recombinantes de Fusão/farmacocinética , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/sangue , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Murine monoclonal antibody (MAb) G3-519 has been shown to recognize a conserved neutralizing epitope in the fourth constant (C4) region of the external glycoprotein gp120 of HIV-1. Inasmuch as this antibody effectively neutralized the infectivity of diverse HIV-1 isolates, it has been selected to be developed for passive immunization against HIV-1 infection in humans. In order to minimize the problem of immunogenicity of murine antibodies and to confer additional accessory immune functions, we have constructed mouse/human chimeric and humanized forms of the antibody. The chimeric antibody was constructed by cloning the murine variable regions and replacing the mouse constant regions with those from human Ig gamma 1,kappa. The humanized antibody was constructed using the human KAS variable region framework sequences as template. Engineering was guided by a three dimensional model of the murine variable region. The murine, chimeric and humanized forms of the antibody exhibited similar reactivity with the peptidic antigen in ELISA, and comparably neutralized the infectivity of HIV-1 in vitro. Taken together, our results show that the chimeric and humanized forms of G3-519 essentially retain the binding activity of the mouse parental antibody. Clinical development is planned to assess the prophylactic and therapeutic usefulness of these reshaped antibodies in humans.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Clonagem Molecular , DNA/genética , Genes de Imunoglobulinas , Vetores Genéticos , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , Humanos , Hibridomas/imunologia , Imunização Passiva , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Murine mAb BAT123, which was made against the envelope glycoprotein gp120 of HTLV-IIIB strain of HIV type 1 (HIV-1), is capable of neutralizing HTLV-IIIB in vitro. It also inhibits the fusion between uninfected CD4+ cells and HIV-1-infected cells to form syncytia. As a step to explore the potential utility of the anti-HIV antibody in vivo, we have constructed a mouse-human chimeric antibody by rDNA techniques. The chimeric antibody, which bears the variable domains of mouse antibody BAT123 and constant domains Cr1 and C kappa of human Ig retains the Ag specificity of BAT123 as determined by its reactivity with HIV-1-infected H9 cells, gp120 in Western blot analysis, and the oligopeptide recognized by BAT123. The antiviral activities of the chimeric antibody in neutralizing HIV-1 infection as well as inhibiting the syncytia formation are also found identical to those of the parent murine antibody. Moreover, in the presence of human blood mononuclear cells, the chimeric antibody but not BAT123 (mouse IgG1) induces antibody-dependent cellular cytotoxicity. The findings point to the potential usefulness of the chimeric antibody in treating patients infected with HIV-1.
Assuntos
Anticorpos Monoclonais/genética , Especificidade de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos , Quimera , Produtos do Gene env/imunologia , HIV-1/imunologia , Cadeias kappa de Imunoglobulina/genética , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Epitopos/análise , Produtos do Gene env/genética , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Antígenos HIV/imunologia , Proteína gp160 do Envelope de HIV , HIV-1/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Cadeias kappa de Imunoglobulina/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Precursores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologiaRESUMO
Two monoclonal antibodies designated BAT085 and G3-136 were raised by immunizing BALB/c mice with gp120 purified from human immunodeficiency virus type 1 (HIV-1) IIIB-infected H9 cell extracts. Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection of CEM-SS cells, whereas G3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primary HIV-1 isolates in the infection of activated human peripheral blood mononuclear cells. In indirect immunofluorescence assays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB or RF, but not MN. Using sequence-overlapping synthetic peptides of HIV-1 IIIB gp120, the binding site of BAT085 and G3-136 was mapped to a peptidic segment in the V2 region (amino acid residues 169 to 183). The binding of these antibodies to immobilized gp120 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gp120. In a competition enzyme-linked immunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding to gp120. Deglycosylation of gp120 by endo-beta-N-acetylglucosaminidase H or reduction of gp120 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gp120 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -independent manner.
Assuntos
Anticorpos Monoclonais , Sítios de Ligação de Anticorpos , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Sequência de Aminoácidos , Ligação Competitiva , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Epitopos/imunologia , Variação Genética , Proteína gp120 do Envelope de HIV/análise , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/síntese química , Peptídeos/imunologia , Conformação ProteicaRESUMO
A phase I/IIA clinical trial with the chimeric mouse-human monoclonal antibody CGP 47,439 to the principal neutralization determinant in the V3 region of human immunodeficiency virus type 1 (HIV-1) strain IIIB envelope protein gp120 is reported. The trial was an uncontrolled single-center, open-label, multidose tolerability, immunogenicity, and pharmacokinetic study in homosexual men with advanced HIV disease. Patient groups were formed on the basis of the reactivity of the antibody with the gp120 of their HIV-1 isolates. Intravenous infusions of 1, 10, and 25 mg of antibody were followed by seven escalated doses of 50, 100, and 200 mg, every 3 weeks. The antibody was well tolerated; no toxicity was observed. Some patients showed a transient but insignificant antibody response to the antibody with no apparent adverse reactions or accelerated elimination of it. Substantial serum levels of the antibody were maintained with a mean t1/2 beta of 8-16 days. A virus burden reduction was observed in some patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/terapia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Anti-HIV/uso terapêutico , Proteína gp120 do Envelope de HIV/imunologia , HIV-1 , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Adulto , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Contagem de Linfócito CD4 , Estudos de Coortes , Anticorpos Anti-HIV/efeitos adversos , Proteína do Núcleo p24 do HIV/sangue , HIV-1/isolamento & purificação , HIV-1/fisiologia , Homossexualidade Masculina , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Testes de Neutralização , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
We have probed the structure of the C4 and V3 domains of human immunodeficiency virus type 1 gp120 by immunochemical techniques. Monoclonal antibodies (MAbs) recognizing an exposed gp120 sequence, (E/K)VGKAMYAPP, in C4 were differentially sensitive to denaturation of gp120, implying a conformational component to some of the epitopes. The MAbs recognizing conformation-sensitive C4 structures failed to bind to a gp120 mutant with an alteration in the sequence of the V3 loop, and their binding to gp120 was inhibited by both V3 and C4 MAbs. This implies an interaction between the V3 and C4 regions of gp120, which is supported by the observation that the binding of some MAbs to the V3 loop was often enhanced by amino acid changes in an around the C4 region.
Assuntos
Anticorpos Monoclonais/metabolismo , Proteína gp120 do Envelope de HIV/química , HIV-1/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva , Células Cultivadas , Epitopos/análise , Epitopos/química , Variação Genética , Proteína gp120 do Envelope de HIV/análise , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Humanos , Cinética , Camundongos/imunologia , Modelos Estruturais , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
A panel of seven monoclonal antibodies against the relatively conserved CD4-binding domain on human immunodeficiency virus type 1 (HIV-1) gp120 was generated by immunizing mice with purified gp120. These monoclonal antibodies reacted specifically with gp120 in an enzyme-linked immunosorbent assay and Western blots (immunoblots). By using synthetic peptides as antigens in the immunosorbent assay, the epitopes of these seven monoclonal antibodies were mapped to amino acid residues 423 to 437 of gp120. Further studies with radioimmunoprecipitation assays showed that they cross-reacted with both gp120 and gp160 of diverse HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, and HTLV-IIIWMJ). They also bound specifically to H9 cells infected with HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, HTLV-IIIZ84, and HTLV-IIIZ34 in indirect immunofluorescence studies. In addition, they blocked effectively the binding of HIV-1 to CD4+ C8166 cells. Despite the similarity of these properties, the monoclonal antibodies differed in neutralizing activity against HTLV-IIIB, HTLV-IIIRF, and HTLV-IIIAL, as demonstrated in both syncytium-forming assays and infectivity assays. Our findings suggest that these group-specific monoclonal antibodies to the putative CD4-binding domain on gp120 are potential candidates for development of therapeutic agents against acquired immunodeficiency disease syndrome.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Receptores Virais/imunologia , Proteínas dos Retroviridae/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Epitopos/análise , HIV/imunologia , Proteína gp120 do Envelope de HIV , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Receptores de HIV , Receptores Virais/análiseRESUMO
A sulfated polysaccharide, curdlan sulfate (CRDS) with 1,3-beta-D-glucan as a main chain, inhibits HIV-1 infection of human peripheral blood lymphocytes (PBLs) by binding to the V3 region of gp 120. We previously showed that T cell (T)-tropic HIV-1 isolates are over 10-fold more sensitive to neutralization by CRDS than macrophage (MT)-tropic viruses, which possesses a relatively less charged amino acid composition in the V3 sequence. To analyze the interaction of CRDS with V3 and its association with neutralization sensitivity of HIV-1 isolates, we examined the effect of CRDS on the binding of neutralizing antibodies to monomeric and oligomeric gp 120 mutants of T- and MT-tropic HIV-1 clones in which the V3 loop was either deleted or substituted by V3 of another isolate. Our results showed that the presence and the amino acid composition of the V3 loop appears to determine the extent of interaction of CRDS with the V2 and CD4-binding regions on native gp 120 monomers; however, the positive charge of V3 has less effect on this interaction on oligomeric gp 120. Furthermore, our results established that only the CRDS-induced masking of V3 on oligomeric gp120 appears to be associated with the anti-HIV-1 activity of CRDS in vitro. Our findings underline the usefulness of CRDS for understanding the structural constraints on gp 120 that drive the transition from MT- to T-tropic isolates in vivo and enable the virus to use multiple fusion cofactors.