The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.
Vaccination is believed to be the most effective method for the prevention of infectious diseases. Thus it is imperative to develop cost effective and scalable process for the production of vaccines so as to make them affordable for mass use. In this study, performance of a novel disposable iCELLis fixed bed bioreactor system was investigated for the production of some viral vaccines like Rabies, Hepatitis-A and Chikungunya vaccines in comparison to conventional systems like the commercially available packed bed system and roller bottle system. Vero and MRC-5 cell substrates were evaluated for growth parameters in all the three systems maintaining similar seeding density, multiplicity of infection (MOI) and media components. It was observed that Vero cells showed similar growth in all the three bioreactors whereas MRC-5 cells showed better growth in iCELLis Nano system and roller bottle system. Subsequently, the virus infection and antigen production studies also revealed that for Hepatitis-A and Chikungunya iCELLis Nano bioreactor system was better to the commercial packed bed bioreactor and roller bottle systems. Although for rabies antigen production commercially available packed bed bioreactor system was found to be better. This study shows that different bioreactor platforms may be employed for viral vaccine production and iCELLis Nano is one of such new convenient and a stable platform for production of human viral vaccines.
The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.
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