RESUMEN
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins.
Asunto(s)
Células Dendríticas/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Lectinas Tipo C/metabolismo , Manosa/metabolismo , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Interleucina-10/biosíntesis , Activación de Linfocitos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Linfocitos T/inmunologíaRESUMEN
The generation of an antibody response capable of neutralizing a broad range of clinical isolates remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Envelope glycoprotein (Env)-based vaccine candidates will also need to take into account the extensive genetic diversity of circulating HIV-1 strains. We describe here the generation of soluble, stabilized, proteolytically cleaved, trimeric forms of Env (SOSIP gp140 proteins) based on contemporary Env subtype A viruses from East Africa. We discuss issues associated with the construction, purification, and characterization of such complex proteins; not all env sequences allow the expression of trimeric proteins. However, stabilized trimers from one such protein, KNH1144 SOSIP gp140, were successfully made. These proteins are now being prepared for preclinical immunogenicity studies.
Asunto(s)
Productos del Gen env , Anticuerpos Anti-VIH/sangre , Vacunas contra el SIDA , África Oriental , Animales , Línea Celular , Dimerización , Diseño de Fármacos , Productos del Gen env/química , Productos del Gen env/inmunología , Productos del Gen env/aislamiento & purificación , Productos del Gen env/metabolismo , VIH-1/clasificación , Humanos , Ratones , Pruebas de Neutralización , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
Endoproteolytic processing of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoproteins is an obligate part of the biosynthetic pathway that generates functional, fusion-competent Env complexes, which are then incorporated into infectious virions. We have examined the influence of cleavage on Env-specific antibody reactivity, Env incorporation into pseudovirions, and the infectivity and neutralization sensitivity of Env-pseudotyped viruses. To do so, we have used both incompletely processed wild-type (Wt) Env and engineered, cleavage-defective Env mutants. We find that there is no simple association between antibody reactivity to cell surface-expressed Env, and the ability of the same antibody to neutralize virus pseudotyped with the same Env proteins. One explanation for the absence of such an association is the diverse array of Env species present on the surface of transiently transfected cells. We also confirm that cleavage-defective mutants are antigenically different from Wt Env. These findings have implications for the use of Env binding assays as predictors of neutralizing activity, and for the development of cleavage-defective Env trimers for use as subunit immunogens.