RESUMEN
RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.
Asunto(s)
Nanopartículas , ARN , Humanos , Ratones , Animales , Distribución Tisular , ARN/genética , Antígenos , Inmunidad Humoral , InflamaciónRESUMEN
Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.
Asunto(s)
Técnicas Citológicas/métodos , Animales , Anticuerpos Biespecíficos , Linfocitos B/inmunología , Células COS , Línea Celular , Supervivencia Celular , Chlorocebus aethiops , Técnicas Citológicas/instrumentación , Pruebas Inmunológicas de Citotoxicidad/instrumentación , Pruebas Inmunológicas de Citotoxicidad/métodos , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Inmunoterapia , Células K562 , Activación de Linfocitos , Neoplasias/inmunología , Neoplasias/terapia , Unión Proteica , Linfocitos T Citotóxicos/inmunología , Células U937RESUMEN
The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68-neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.
Asunto(s)
Anticuerpos Neutralizantes , Enterovirus Humano D , Infecciones por Enterovirus , Animales , Infecciones por Enterovirus/prevención & control , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/virología , Enterovirus Humano D/inmunología , Enterovirus Humano D/genética , Anticuerpos Neutralizantes/inmunología , Ratones , Vacunas Virales/inmunología , Modelos Animales de Enfermedad , Humanos , Vacunas de ARNm , Anticuerpos Antivirales/inmunología , FemeninoRESUMEN
Mycobacterium tuberculosis (M.tb), a bacterial pathogen that causes tuberculosis disease (TB), exerts an extensive burden on global health. The complex nature of M.tb, coupled with different TB disease stages, has made identifying immune correlates of protection challenging and subsequently slowing vaccine candidate progress. In this work, we leveraged two delivery platforms as prophylactic vaccines to assess immunity and subsequent efficacy against low-dose and ultra-low-dose aerosol challenges with M.tb H37Rv in C57BL/6 mice. Our second-generation TB vaccine candidate ID91 was produced as a fusion protein formulated with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion) or as a novel replicating-RNA (repRNA) formulated in a nanostructured lipid carrier. Protein subunit- and RNA-based vaccines preferentially elicit cellular immune responses to different ID91 epitopes. In a single prophylactic immunization screen, both platforms reduced pulmonary bacterial burden compared to the controls. Excitingly, in prime-boost strategies, the groups that received heterologous RNA-prime, protein-boost or combination immunizations demonstrated the greatest reduction in bacterial burden and a unique humoral and cellular immune response profile. These data are the first to report that repRNA platforms are a viable system for TB vaccines and should be pursued with high-priority M.tb antigens containing CD4+ and CD8+ T-cell epitopes.
RESUMEN
Epithelial to mesenchymal transition (EMT) is a key process in embryonic development and has been associated with cancer metastasis and drug resistance. For example, in EGFR mutated non-small cell lung cancers (NSCLC), EMT has been associated with acquired resistance to the EGFR inhibitor erlotinib. Moreover, "EGFR-addicted" cancer cell lines induced to undergo EMT become erlotinib-resistant in vitro. To identify potential therapeutic vulnerabilities specifically within these mesenchymal, erlotinib-resistant cells, we performed a small molecule screen of ~200 established anti-cancer agents using the EGFR mutant NSCLC HCC827 cell line and a corresponding mesenchymal derivative line. The mesenchymal cells were more resistant to most tested agents; however, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the Abl/Src inhibitor, dasatinib. Analysis of the tyrosine phospho-proteome revealed several Src/FAK pathway kinases that were differentially phosphorylated in the mesenchymal cells, and RNAi depletion of the core Src/FAK pathway components in these mesenchymal cells caused apoptosis. These findings reveal a novel role for Src/FAK pathway kinases in drug resistance and identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Resistencia a Antineoplásicos/fisiología , Transición Epitelial-Mesenquimal/fisiología , Quinasa 1 de Adhesión Focal/metabolismo , Neoplasias Pulmonares/metabolismo , Familia-src Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dasatinib , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/genética , Clorhidrato de Erlotinib , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genes erbB-1 , Humanos , Immunoblotting , Ratones , Ratones Desnudos , Mutación , Pirimidinas/farmacología , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tiazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.
Asunto(s)
Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Neovascularización Patológica/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/enzimología , Permeabilidad Capilar/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Ligandos , Ratones , Modelos Biológicos , Receptores de Netrina , Unión Proteica/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacos , Sus scrofa , Factor A de Crecimiento Endotelial Vascular/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic alpha-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2'-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for beta-catenin. Sequencing of the tumor DNA encoding beta-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.