RESUMEN
In response to the COVID-19 pandemic, vaccines were quickly and successfully developed and deployed, saving millions of lives globally. While first generation vaccines are safe and effective in preventing disease caused by SARSCoV-2, next-generation vaccines have the potential to improve efficacy and safety. Vaccines delivered by a mucosal route may elicit greater protective immunity at respiratory surfaces thereby reducing transmission. Inclusion of viral antigens in addition to the spike protein may enhance protection against emerging variants of concern. Next-generation vaccine platforms with a new mechanism of action may necessitate efficacy trials to fulfill regulatory requirements. The Biomedical Advanced Research and Development Authority (BARDA) will be supporting Phase 2b clinical trials of candidate next-generation vaccines. The primary endpoint will be improved efficacy in terms of symptomatic disease relative to a currently approved COVID-19 vaccine. In this paper, we discuss the planned endpoints and potential challenges to this complex program.
RESUMEN
This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.
Asunto(s)
ADN Viral/administración & dosificación , ADN Viral/genética , Sistemas de Liberación de Medicamentos/métodos , Virus de la Encefalitis Equina Venezolana/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Replicón/genética , Transfección/métodos , Virus Vaccinia/genética , Virión/genética , Alphavirus/genética , Regulación Viral de la Expresión GénicaRESUMEN
A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 degrees C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells.
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Virus del Moquillo Canino/genética , Expresión Génica , Vectores Genéticos/genética , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Cricetinae , ADN Viral , ARN Polimerasas Dirigidas por ADN/genética , Virus del Moquillo Canino/aislamiento & purificación , Perros , Genes Reporteros , Humanos , Luciferasas/genética , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/genética , Recombinación Genética , Replicón , Células Tumorales Cultivadas , Células VeroRESUMEN
A modified cDNA rescue system that improves recovery of recombinant nonsegmented, negative-strand RNA viruses from cloned DNAs is described. Rescue systems based on vaccinia virus-T7 RNA polymerase vectors have been used to derive many negative-strand viruses; however, some strains can be recalcitrant to rescue possibly because of the simultaneous replication of the vaccinia virus-T7 vector. Our goal was to engineer a system where replication of the vaccinia virus-T7 vector could be blocked, yet allow for sufficient T7 RNA polymerase expression to enable genetic rescue. To that end, a recombinant modified vaccinia virus Ankara (MVA) was engineered that contained the bacteriophage T7 gene-1 under the control of a strong early promoter that would enable T7 RNA polymerase expression in the absence of MVA DNA replication. The new T7 helper, MVAGKT7, was then utilized successfully for the genetic rescue of a measles virus minigenome and full-length cDNAs, in the presence of DNA synthesis inhibitors. In addition to blocking completely MVAGKT7 replication, AraC treatment was found to enhance minigenome-encoded gene expression and the efficiency of measles virus rescue.
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Bacteriófago T7/genética , ADN Viral/biosíntesis , ARN Polimerasas Dirigidas por ADN/genética , Vectores Genéticos , Genoma Viral , Virus Vaccinia/metabolismo , Animales , Línea Celular , Replicación del ADN/efectos de los fármacos , ADN Complementario/genética , Genes Virales , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Regiones Promotoras Genéticas , Recombinación Genética , Transfección/métodos , Virus Vaccinia/genética , Proteínas Virales , Replicación ViralRESUMEN
Alphavirus replicon particles are being exploited for a variety of purposes both in vitro as gene expression vectors, and in vivo as vaccines or gene therapy vectors. There is a need for a simple and universal method of titration of replicon particles that is independent of expression of the foreign protein. We devised a method that uses modified vaccinia virus Ankara (MVA) as an indicator virus, to deliver a Venezuelan equine encephalitis virus (VEE) defective helper RNA encoding green fluorescent protein (GFP). Co-infection of cells with the MVA-based indicator and Venezuelan equine encephalitis virus replicon particles (VRP) results in expression of the GFP gene. VRP titer is readily determined by counting fluorescent cells.
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ARN Polimerasas Dirigidas por ADN/fisiología , Virus Defectuosos/genética , Virus de la Encefalitis Equina Venezolana/genética , Proteínas Luminiscentes/genética , Replicón/genética , Animales , Chlorocebus aethiops , Proteínas Fluorescentes Verdes , Virus Vaccinia/genética , Células VeroRESUMEN
Following the attacks of 11 September 2001, emergency preparedness within the U.S. Department of Health and Human Services, as well as at the Department of Defense and other federal agencies, received higher visibility, new mandates and increased funding. Emergency deployment teams increased the frequency of drills to enable better response to the health consequences of mass-casualty incidents. Interagency coordination has also continued to increase to more efficiently and effectively leverage federal resources toward emergency medical preparedness for both civilian and military populations.