RESUMO
Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Epitopos/imunologia , Glicoproteínas/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Linhagem Celular , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Imunização , Imunoglobulina G/imunologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes. Identification of the protective determinants of the human antibody response to DENV is a vital requirement for the design and evaluation of future preventative therapies and treatments. Here, we describe the isolation of a neutralizing antibody from a DENV1-infected patient. The human antibody 14c10 (HM14c10) binds specifically to DENV1. HM14c10 neutralizes the virus principally by blocking virus attachment; at higher concentrations, a post-attachment step can also be inhibited. In vivo studies show that the HM14c10 antibody has antiviral activity at picomolar concentrations. A 7 Å resolution cryoelectron microscopy map of Fab fragments of HM14c10 in a complex with DENV1 shows targeting of a discontinuous epitope that spans the adjacent surface of envelope protein dimers. As found previously, a human antibody specific for the related West Nile virus binds to a similar quaternary structure, suggesting that this could be an immunodominant epitope. These findings provide a structural and molecular context for durable, serotype-specific immunity to DENV infection.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Antivirais/uso terapêutico , Vírus da Dengue/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Microscopia Crioeletrônica , Dengue/tratamento farmacológico , Vírus da Dengue/imunologia , Vírus da Dengue/ultraestrutura , Epitopos/imunologia , Humanos , CamundongosRESUMO
Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required.