RESUMEN
Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients. However, increasing potency of an antibody by selection from libraries may lead to a decrease in the breadth of neutralization. In an attempt to solve this problem, we subjected a random mutagenesis library of the scFv X5 to sequential rounds of selection on non-homologous HIV-1 envelope glycoproteins (Envs) dubbed sequential antigen panning (SAP). By using SAP, we identified two scFv antibodies, m6 and m9, that were tested with a panel of 33 diverse primary HIV-1 infectious isolates in an assay based on a reporter cell-line expressing high levels of CD4, CCR5 and CXCR4. The IC(50) was less than 50 microg/ml for 21 (m6) and 19 (m9) out of 29 isolates from group M (subtypes A-C, F, G and CRF-01AE) and one isolate from group N; three isolates from group O were not significantly inhibited at 50 microg/ml. The average IC(50) values for the two antibodies were significantly (p<0.001, n=29) lower compared to scFv X5. Their inhibitory activity does not appear to be related to the HIV-1 subtype, coreceptor usage or the disease stage. m9 inhibited infection of peripheral blood mononuclear cells by the primary isolates JRCSF, 89.6 and BR020 with IC(90) of 4, 6 and 25 microg/ml, respectively; for a single-round infection by pseudovirus, the IC(90) for JRSCF, 89.6, YU2 and HXBc2 was 15, 5, 15 and 5 microg/ml, respectively. In these two assays the IC(90) for m9 was, on average, two- to threefold lower than for scFv X5. These results demonstrate that both the potency and the breadth of HIV-1 neutralization of one of the few known potent broadly cross-reactive human monoclonal antibodies, scFv X5, could be improved significantly. However, only experiments in animal models and clinical trials in humans will show whether these new scFvs and the approach for their identification have potential in the development of prophylactics and therapeutics for HIV-1 infections.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , VIH-1/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Mutagénesis , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos CD4/inmunología , Evaluación Preclínica de Medicamentos/métodos , Anticuerpos Anti-VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Fragmentos de Inmunoglobulinas/genética , Concentración 50 Inhibidora , Mutación , Biblioteca de PéptidosRESUMEN
Identification of broadly cross-reactive human monoclonal antibodies (mAbs) has major implications for development of vaccines, inhibitors and research tools. Here we describe a sequential antigen panning (SAP) methodology that may facilitate the selection of such antibodies. An HIV-specific antibody Fab (m18) was selected from a human Fab phage-display library by SAP against several recombinant soluble HIV envelope glycoproteins (Envs) and Env-sCD4 complexes. This Fab bound to a variety of recombinant soluble Envs (gp140s) from primary HIV isolates representing different clades, and inhibited cell fusion and virus entry mediated by Envs of primary HIV isolates. The methodology and the results may have implications for development of HIV vaccines and inhibitors, as well as for identification of antibodies to conserved epitopes on rapidly mutating viruses and cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Reacciones Cruzadas , Productos del Gen env/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Fusión de Membrana , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
A new human monoclonal antibody (hmAb), designated m16, was selected by sequential antigen panning (SAP) of a human phage display library against recombinant soluble HIV-1 envelope glycoproteins (Envs) (gp140s) and their complexes with soluble CD4. It bound with high (nM) affinity to gp120 and gp140; the binding was further enhanced by interactions of the Envs with CD4. m16 inhibited cell fusion mediated by the Envs of 9 HIV-1 isolates from clades A, B, E and G with potency on average comparable to that of the broadly neutralizing human monoclonal antibody Fab X5. The identification of a new hmAb with broad neutralizing activity that exhibits differential inhibitory profile suggests a potential for its use as a component of anti-HIV-1 treatments.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Afinidad de Anticuerpos , Antígenos CD4/inmunología , Fusión Celular , Productos del Gen env/inmunología , Genotipo , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Pruebas de Neutralización , Biblioteca de Péptidos , Unión Proteica , Proteínas Recombinantes/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.
Asunto(s)
Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/química , VIH-1/metabolismo , Animales , Sitios de Unión de Anticuerpos , Línea Celular , Femenino , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Humanos , Pruebas de Neutralización , Unión Proteica/inmunología , Conformación Proteica , Conejos , TermodinámicaRESUMEN
DNA plasmids and recombinant adenovirus serotype-5 (rAd5) vectors are being studied in human clinical trials as HIV-1 vaccine candidates. Each elicits robust T-cell responses and modest antibody levels. Since protein immunization alone elicits antibody but not CD8 T-cell responses, we studied protein boosting of DNA and rAd5 HIV-1 vaccine vectors. A single Env protein immunization provided a marked boost in antibody titer in guinea pigs primed with either DNA or rAd5 vaccines, and the resulting antibody binding and neutralization levels were similar to those attained after thee sequential protein immunizations. Since both T-cell immunity and neutralizing antibodies are thought to be required for protection against HIV-1, it may be possible to establish a balanced T-cell and antibody response with appropriate vectored vaccines and improve the neutralizing antibody titer with protein boosting.
Asunto(s)
Vacunas contra el SIDA/inmunología , Adenoviridae/inmunología , VIH-1/inmunología , Inmunización Secundaria/métodos , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Adenoviridae/genética , Animales , Formación de Anticuerpos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Productos del Gen env/genética , Productos del Gen env/inmunología , Cobayas , VIH-1/genética , Plásmidos/genética , Plásmidos/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
The ability to readily elicit broadly neutralizing antibodies to HIV-1 remains elusive. We and others have hypothesized that interaction of the viral envelope glycoprotein (Env, gp120-gp41) with its receptor molecules could enhance the exposure of conserved epitopes that may facilitate the elicitation of broadly neutralizing antibodies. The Env-CD4-coreceptor complexes mediate HIV-1 entry into cells and serve as a major target for inhibitors of this process. To begin to evaluate their potential also as vaccine immunogens we produced relatively large amounts of complexes of purified recombinant soluble truncated Env, gp140(89.6) or gp120(89.6), with CD4 and CCR5 or CXCR4. We found that gp140(gp120)-CD4-CCR5 complexes are stable and immunogenic in mice transgenic for human CD4 and CCR5. They elicited anti-gp120 and anti-gp140 antibodies that inhibited an heterologous primary HIV-1 isolate (JR-FL) with two- to threefold higher neutralizing activity than those elicited by gp120 and gp140. The antibodies elicited by the complexes competed better with the antibodies X5 and CG10 but not with b12 for binding to gp120 and gp120-CD4 complexes compared to those elicited with gp140(120) alone. These findings suggest that stable purified Env-CD4-CCR5(CXCR4) complexes can be produced in relatively large amount sufficient for their further characterization that may help in the development of novel vaccines candidates.
Asunto(s)
Antígenos CD4/biosíntesis , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/química , VIH-1/inmunología , Receptores CCR5/biosíntesis , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/biosíntesis , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Plásmidos/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution. There are two crystallographically independent Fab fragments in the asymmetric unit. The crystallographic R value for the final model is 0.22. The antibody-combining site features a long (22 amino acid residues) CDR H3 with a protruding hook-shaped motif. The X5 structure and site-directed mutagenesis data suggest that X5 amino acid residues W100 and Y100F in the CDR H3 motif may be critical for the binding of Fab X5 to gp120. X5 bound to a complex of a CD4 mimetic and gp120 with approximately the same kinetics and affinity as to a CD4-gp120 complex, suggesting that specific interactions between CD4 and X5 are unlikely to contribute to the binding of X5 to gp120-CD4 complexes. Binding of X5 to alanine scanning mutants of gp120JR-CSF complexed with CD4 suggested a critical role of the highly conserved amino acid residues at positions 423 and 432. The X5 structure and fine mapping of its epitope may assist in the elucidation of the mechanisms of viral entry and neutralization, and the development of HIV-1 inhibitors and vaccines.
Asunto(s)
Anticuerpos Monoclonales/química , Epítopos/química , Epítopos/inmunología , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Mapeo Peptídico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Antivirales/química , Antivirales/genética , Antivirales/metabolismo , Sitios de Unión/genética , Antígenos CD4/química , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Reacciones Cruzadas/genética , Cristalización , Cristalografía por Rayos X , Epítopos/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/patogenicidad , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Mapeo Peptídico/métodos , Estructura Terciaria de Proteína/genética , Resonancia por Plasmón de SuperficieRESUMEN
HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor, typically CCR5. Here we provide evidence that purified gp120(JR-FL)-CD4-CCR5 complexes exhibit an epitope recognized by a Fab (X5) obtained by selection of a phage display library from a seropositive donor with a relatively high broadly neutralizing serum antibody titer against an immobilized form of the trimolecular complex. X5 bound with high (nM) affinity to a variety of Envs, including primary isolates from different clades and Envs with deleted variable loops (V1, -2, -3). Its binding was significantly increased by CD4 and slightly enhanced by CCR5. X5 inhibited infection of peripheral blood mononuclear cells by a selection of representative HIV-1 primary isolates from clades A, B, C, D, E, F, and G with an efficiency comparable to that of the broadly neutralizing antibody IgG1 b12. Furthermore, X5 inhibited cell fusion mediated by Envs from R5, X4, and R5X4 viruses. Of the five broadly cross-reactive HIV-1-neutralizing human monoclonal antibodies known to date, X5 is the only one that exhibits increased binding to gp120 complexed with receptors. These findings suggest that X5 could possibly be used as entry inhibitor alone or in combination with other antiretroviral drugs for the treatment of HIV-1-infected individuals, provide evidence for the existence of conserved receptor-inducible gp120 epitopes that can serve as targets for potent broadly cross-reactive neutralizing antibodies in HIV-1-infected patients, and have important conceptual and practical implications for the development of vaccines and inhibitors.