RESUMEN
Protease-activated receptor 2 (PAR-2) is expressed in various tissues, including lung, and when activated, promotes inflammation, differentiation, and migration of dendritic cells. We found that combining influenza virosomes containing hemagglutinin and neuraminidase with a PAR-2 agonist peptide (PAR-2AP) in an intranasal prime boost approach increased survival of mice challenged weeks later with lethal influenza virus over that by virosome or PAR-2AP prime boost alone. No weight loss occurred from influenza challenge after virosome-plus-PAR-2AP prime boost compared with either virosomes or PAR-2AP alone. Thus, virosomes plus PAR-2AP prevented morbidity as well as mortality. Through adoptive transfer, CD8+ lung T cells but not CD4+ T cells from virosomes plus PAR-2AP-primed mice protected from lethal influenza virus challenge and enhanced survival with less weight loss and faster recovery. Virosome-plus-PAR-2AP prime boost resulted in greater percentages of T effector memory phenotype cells (Tem) in lung, and higher frequencies of CD8 Tem and T central memory cells displayed effector functions in response to virus challenge in vivo. Virosome-plus-PAR-2AP prime boost also resulted in greater percentages of Ag-specific CD8+ T cells, both Tem and T central memory cells, in lungs of animals subsequently challenged with live influenza virus. Our findings indicate that PAR-2AP, a short peptide, may be a new and useful mucosal adjuvant.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Receptor PAR-2/agonistas , Virosomas/inmunología , Traslado Adoptivo/métodos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Perros , Femenino , Memoria Inmunológica/efectos de los fármacos , Pulmón/inmunología , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Orthomyxoviridae/inmunología , Virosomas/efectos de los fármacosRESUMEN
Recombinant foot-and-mouth disease virus-like particles (VLPs) can be expressed in a number of expression systems including plants. However, yields in plants have formerly been shown to be low, possibly due to their acid and/or heat lability, previously shown to affect VLP yields produced in other systems. This work describes the introduction of mutations into the FMDV structural protein-encoding gene (P1-2A) which have been previously shown to increase acid and thermostability. VLPs expressed in plants using the mutant constructs had negative rather than positive effects on yield and temperature and acid stability compared to the control.
Asunto(s)
Virus de la Fiebre Aftosa/genética , Expresión Génica , Nicotiana/genética , Proteínas Estructurales Virales/genética , Virosomas/genética , Ácidos , Animales , Virus de la Fiebre Aftosa/efectos de los fármacos , Virus de la Fiebre Aftosa/efectos de la radiación , Calor , Proteínas Mutantes/genética , Virosomas/efectos de los fármacos , Virosomas/efectos de la radiaciónRESUMEN
Dengue virus (DV) is a compact, icosahedrally symmetric, enveloped particle, covered by 90 dimers of envelope protein (E), which mediates viral attachment and membrane fusion. Fusion requires a dimer-to-trimer transition and membrane engagement of hydrophobic 'fusion loops'. We previously characterized the steps in membrane fusion for the related West Nile virus (WNV), using recombinant, WNV virus-like particles (VLPs) for single-particle experiments (Chao et al., 2014). Trimerization and membrane engagement are rate-limiting; fusion requires at least two adjacent trimers; availability of competent monomers within the contact zone between virus and target membrane creates a trimerization bottleneck. We now report an extension of that work to dengue VLPs, from all four serotypes, finding an essentially similar mechanism. Small-molecule inhibitors of dengue virus infection that target E block its fusion-inducing conformational change. We show that ~12-14 bound molecules per particle (~20-25% occupancy) completely prevent fusion, consistent with the proposed mechanism.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Internalización del Virus/efectos de los fármacos , Antivirales/síntesis química , Membrana Celular/metabolismo , Modelos Biológicos , Multimerización de Proteína , Rodaminas/síntesis química , Rodaminas/farmacología , Ácidos Sulfónicos/síntesis química , Ácidos Sulfónicos/farmacología , Proteínas del Envoltorio Viral/metabolismo , Virosomas/efectos de los fármacosRESUMEN
UNLABELLED: Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level 4 (BSL-4) agent, however, EBOV is restricted to costly BSL-4 laboratories for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we report an EBOV-like particle (EBOVLP)-based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside BSL-4 facilities. Cotransfection of HEK293T cells with four plasmids encoding the proteins VP40, NP, and GP of EBOV and firefly luciferase (Fluc) resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner and was recognized by a conformation-dependent anti-EBOV monoclonal antibody (MAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that could be blocked by the administration of EBOV neutralizing MAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines. IMPORTANCE: Ebola virus (EBOV) researches have been limited to costly biosafety level 4 (BSL-4) facilities due to the lack of animal models independent of BSL-4 laboratories. In this study, we reveal that a firefly luciferase-bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology is capable of delivering the reporter protein into murine target cells both in vitro and in vivo Moreover, we demonstrate that the reporter delivery can be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody, 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs, and vaccines. The system may also be useful for mechanistic study of the viral entry process.
Asunto(s)
Antivirales/aislamiento & purificación , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/efectos de los fármacos , Endocitosis , Genes Reporteros , Luciferasas/análisis , Virosomas/metabolismo , Animales , Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Ebolavirus/genética , Luciferasas/genética , Ratones , Virosomas/efectos de los fármacos , Virosomas/genética , Virosomas/inmunologíaRESUMEN
Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50-100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using viral structural proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the structural proteins. In both approaches, the key to produce high titer VLP (3x10(6)foci-forming unit/ml) is to use low temperature (30 degrees C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against viral envelope protein and by an inhibitor of viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
Asunto(s)
Antivirales/aislamiento & purificación , Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Virosomas/efectos de los fármacos , Animales , Bioensayo/métodos , Línea Celular , Chlorocebus aethiops , Cricetinae , Virus del Dengue/genética , Electroporación , Genes Reporteros , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Macaca mulatta , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virosomas/genéticaRESUMEN
RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments-hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10(6) to 10(2) copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.
Asunto(s)
Sustancias Macromoleculares/metabolismo , Reacción en Cadena de la Polimerasa/normas , ARN Viral/metabolismo , Ribonucleasas/metabolismo , Virosomas/biosíntesis , Virosomas/química , Escherichia coli/genética , Hepacivirus/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Levivirus/genética , ARN Viral/genética , Estándares de Referencia , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/genética , Virosomas/efectos de los fármacos , Virosomas/genéticaRESUMEN
The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20-22 degrees C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10-40 degrees C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.