RESUMO
COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.
Assuntos
COVID-19/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Expressão Gênica , Células HEK293 , Humanos , Insetos/citologia , Ligação Proteica , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunização/métodos , Modelos Animais , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/genética , COVID-19/virologia , Feminino , Furões , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. METHODS: Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. RESULTS: Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. CONCLUSIONS: This work provides a framework able to track down SARS-CoV-2 genomic variability.
Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sítios de Ligação/genética , COVID-19/epidemiologia , Vacinas contra COVID-19/genética , Biologia Computacional , Mineração de Dados , Variação Genética , Humanos , Fenômenos Imunogenéticos , Modelos Moleculares , Mutação , Pandemias/estatística & dados numéricos , Domínios Proteicos , Receptores Virais , SARS-CoV-2/imunologia , Software , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Translacional BiomédicaRESUMO
COVID-19 has rapidly spread all over the world, progressing into a pandemic. This situation has urgently impelled many companies and public research institutes to concentrate their efforts on research for effective therapeutics. Here, we outline the strategies and targets currently adopted in developing a vaccine against SARS-CoV-2. Based on previous evidence and experience with SARS and MERS, the primary focus has been the Spike protein, considered as the ideal target for COVID-19 immunotherapies.