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1.
PLoS One ; 12(1): e0170530, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28107435

RESUMEN

The only evidence of vaccine-induced protection from HIV acquisition in humans was obtained in the RV144 HIV vaccine clinical trial. One immune correlate of risk in RV144 was observed to be higher titers of vaccine-induced antibodies (Abs) reacting with a 23-mer non-glycosylated peptide with the same amino acid sequence as a segment in the second variable (V2) loop of the MN strain of HIV. We used NMR to analyze the dynamic 3D structure of this peptide. Distance restraints between spatially proximate inter-residue protons were calculated from NOE cross peak intensities and used to constrain a thorough search of all possible conformations of the peptide. α-helical folding was strongly preferred by part of the peptide. A high-throughput structure prediction of this segment in all circulating HIV strains demonstrated that α-helical conformations are preferred by this segment almost universally across all subtypes. Notably, α-helical conformations of this segment of the V2 loop cluster cross-subtype-conserved amino acids on one face of the helix and the variable amino acid positions on the other in a semblance of an amphipathic α-helix. Accordingly, some Abs that protected against HIV in RV144 may have targeted a specific, conserved α-helical peptide epitope in the V2 loop of HIV's surface envelope glycoprotein.


Asunto(s)
Vacunas contra el SIDA/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Conformación Proteica en Hélice alfa , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Secuencia Conservada/genética , Anticuerpos Anti-VIH/inmunología , VIH-1/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Pliegue de Proteína
2.
Mol Cell Biol ; 35(11): 2024-34, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25825525

RESUMEN

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by liver X receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRα serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRα at S198 is nonphosphorylated. In bone marrow-derived macrophages (BMDMs), we also observed induction of CCR7 by ligands that promote nonphosphorylated LXRα S198, and this was lost in LXR-deficient BMDMs. LXRα occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing nonphosphorylated LXRα (RAW-LXRα S198A) compared to RAW264.7 cells expressing wild-type (WT) phosphorylated LXRα (RAW-LXRα WT). Expression profiling of ligand-treated RAW-LXRα S198A cells compared to RAW-LXRα WT cells revealed induction of cell migratory and anti-inflammatory genes and repression of proinflammatory genes. Modeling of LXRα S198 in the nonphosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with the presence of sites for protein interaction. Therefore, gene transcription is regulated by LXRα S198 phosphorylation, including that of antiatherogenic genes such as CCR7.


Asunto(s)
Expresión Génica/genética , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/genética , Fosforilación/genética , Serina/genética , Animales , Aterosclerosis/genética , Línea Celular , Humanos , Ligandos , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Receptores CCR7
3.
Nanomedicine (Lond) ; 9(11): 1613-24, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24195674

RESUMEN

AIMS: Antibodies are the principal mediator of immunity against reinfection with viruses. Antibodies typically neutralize viruses by binding to virion particles in solution prior to attachment to susceptible cells. Once viruses enter cells, conventional antibodies cannot inhibit virus infection or replication. It is desirable to develop an efficient and nontoxic method for the introduction of virus-inhibiting antibodies into cells. MATERIALS & METHODS: In this article, we report a new method for the delivery of small recombinant antibody fragments into virus-infected cells using a dendrimer-based molecular transporter. RESULTS & CONCLUSION: The construct penetrated virus-infected cells efficiently and inhibited virus replication. This method provides a novel approach for the immediate delivery of inhibitory antibodies directed to virus proteins that are exposed only in the intracellular environment. This approach circumvents the current and rather complicated expression of inhibitory antibodies in cells following gene transfer.


Asunto(s)
Anticuerpos/química , Nanomedicina/métodos , Virión/química , Animales , Anticuerpos Monoclonales/química , Transporte Biológico , Proteínas de la Cápside/química , Citoplasma/metabolismo , Dendrímeros/química , Ensayo de Inmunoadsorción Enzimática , Técnicas de Transferencia de Gen , VIH-1/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Riñón , Macaca mulatta , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Pruebas de Neutralización , Péptidos/química , ARN Interferente Pequeño/metabolismo , Rotavirus/metabolismo , Virus/química
4.
J Virol ; 88(1): 469-76, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24155406

RESUMEN

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.


Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Epítopos/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión de Anticuerpos , Biopolímeros/inmunología , Microscopía por Crioelectrón , Cartilla de ADN , Epítopos/química , Espectrometría de Masas , Datos de Secuencia Molecular
5.
PLoS One ; 8(5): e61101, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23671563

RESUMEN

Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.


Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Infecciones por Rotavirus/inmunología , Rotavirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Antígenos Virales/genética , Antígenos Virales/metabolismo , Linfocitos B/inmunología , Linfocitos B/virología , Células CACO-2 , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Rotavirus/metabolismo , Rotavirus/fisiología , Infecciones por Rotavirus/virología , Homología de Secuencia de Aminoácido , Transcripción Genética , Virión/genética , Virión/inmunología , Virión/metabolismo , Replicación Viral/genética , Replicación Viral/inmunología
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