RESUMO
Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1-infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.
Assuntos
Antirretrovirais/imunologia , Anticorpos Neutralizantes/imunologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Imunoterapia/métodos , Animais , Antirretrovirais/farmacologia , Anticorpos Neutralizantes/farmacologia , Primers do DNA/genética , DNA Viral/metabolismo , Dependovirus , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Carga Viral/efeitos dos fármacosRESUMO
BACKGROUND: Newly synthesized HIV-1 particles assemble at the plasma membrane of infected cells, before being released as free virions or being transferred through direct cell-to-cell contacts to neighboring cells. Localization of HIV-1 Gag precursor at the cell membrane is necessary and sufficient to trigger viral assembly, whereas the GagPol precursor is additionally required to generate a fully matured virion. HIV-1 Nef is an accessory protein that optimizes viral replication through partly defined mechanisms. Whether Nef modulates Gag and/or GagPol localization and assembly at the membrane and facilitates viral cell-to-cell transfer has not been extensively characterized so far. RESULTS: We report that Nef increases the total amount of Gag proteins present in infected cells, and promotes Gag localization at the cell membrane. Moreover, the processing of p55 into p24 is improved in the presence of Nef. We also examined the effect of Nef during HIV-1 cell-to-cell transfer. We show that without Nef, viral transfer through direct contacts between infected cells and target cells is impaired. With a nef-deleted virus, the number of HIV-1 positive target cells after a short 2h co-culture is reduced, and viral material transferred to uninfected cells is less matured. At later time points, this defect is associated with a reduction in the productive infection of new target cells. CONCLUSIONS: Our results highlight a previously unappreciated role of Nef during the viral replication cycle. Nef promotes HIV-1 Gag membrane localization and processing, and facilitates viral cell-to-cell transfer.
Assuntos
Membrana Celular/virologia , HIV-1/fisiologia , Montagem de Vírus , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Humanos , Internalização do VírusRESUMO
Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Betacoronavirus/química , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19 , Linhagem Celular , Infecções por Coronavirus/terapia , Citofagocitose , Epitopos , Humanos , Imunização Passiva , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores de Coronavírus , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Adulto Jovem , Soroterapia para COVID-19Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por HIV/terapia , HIV-1/fisiologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Células HeLa , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/virologia , Internalização do VírusRESUMO
The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir.
Assuntos
Anticorpos Neutralizantes/fisiologia , Anticorpos Antivirais/fisiologia , Linfócitos T CD4-Positivos/fisiologia , HIV-1/fisiologia , Animais , Linhagem Celular , HumanosRESUMO
The neutralizing activity of anti-HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4(+) lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células HEK293 , Células HeLa , Humanos , Concentração Inibidora 50 , Microscopia de Fluorescência , Fatores de Tempo , Vírion/fisiologiaRESUMO
Retroviruses like HIV-1 and HTLV-1 can be transmitted efficiently by direct contact between infected and target cells. For HIV-1, various modes of cell-to-cell transfer have been reported, including virological synapses, polysynapses, filopodial bridges, and nanotube-like structures. So far, only synapses and biofilms have been described for HTLV-1 transmission. Recently, Van Prooyen et al. [1] identified an additional mode of HTLV-1 transmission through cellular conduits induced by the viral accessory protein p8.
Assuntos
Infecções por HTLV-I/transmissão , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Linhagem Celular , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
In human cells, endogenous TRIM5alpha strongly inhibits N-tropic strains of murine leukemia virus (N-MLV) but does not target the closely related B-MLV. We have used a shRNA-based loss-of-function screen to isolate factors other than TRIM5alpha involved in the restriction of N-MLV. In one of the isolated clones, the shRNA expressed was found to target the murine double minute-2 mRNA. Knocking down MDM2 increased N-MLV and EIAV infection of human cells by 2- to 5-fold while having little effect on B-MLV. Similarly, knocking down MDM2 in African green monkey cells diminished the restriction of both N-MLV and HIV-1. Dual knockdown experiments showed that MDM2 was involved in the restriction mediated by TRIM5alpha. Moreover, MDM2 knockdown decreased the sensitivity of N-MLV infection to treatment with MG132 and As(2)O(3), two known TRIM5alpha pharmacological inhibitors. Altogether, our data suggest that MDM2 is a general but nonessential modulator of TRIM5alpha-mediated antiretroviral functions.