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2018
All influenza vaccines currently sold in Canada require one fertilized chicken egg to produce roughly one dose of vaccine. During pandemic influenza outbreaks, the limited availability of eggs stresses the ability of this method to deliver vaccine in a timely manner (1). Unlike eggs, cell lines grow exponentially, resulting in virtually limitless substrate for cultivating influenza vaccines. This ability to rapidly scale production during periods of increased demand is ideal for effectively responding to pandemic influenza outbreaks. While promising, cell-based influenza vaccine production suffers from low volumetric yield (~10-fold lower) compared to egg-based methods (2).
Scientific Reports
A pooled genome-wide screening strategy to identify and rank influenza host restriction factors in cell-based vaccine production platformsCell-derived influenza vaccines provide better protection and a host of other advantages compared to the egg-derived vaccines that currently dominate the market, but their widespread use is hampered by a lack of high yield, low cost production platforms. Identification and knockout of innate immune and metabolic restriction factors within relevant host cell lines used to grow the virus could offer a means to substantially increase vaccine yield. In this paper, we describe and validate a novel genome-wide pooled CRISPR/Cas9 screening strategy that incorporates a reporter virus and a FACS selection step to identify and rank restriction factors in a given vaccine production cell line. Using the HEK-293SF cell line and A/PuertoRico/8/1934 H1N1 influenza as a model, we identify 64 putative influenza restriction factors to direct the creation of high yield knockout cell lines. In addition, gene ontology and protein complex enrichment analysis of this list of putative restriction factors o...
The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and ...
Biotechnology and Bioengineering
From functional genomics of vero cells to CRISPR‐based genomic deletion for improved viral production rates2022 •
Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high‐throughput vaccine manufacturing platforms is the lack of understanding of virus‐host interactions during infection and cell‐based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology‐based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70‐fold and an 87‐fold increase of total viral particles productions in ISG15−/− Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV‐GFP.
2020 •
The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells...
Journal of virology
A genome-wide small interfering RNA screen identifies host factors required for vesicular stomatitis virus infection2014 •
Viruses are dependent on their host cells for replication and thus have evolved in intimate association with them. The identification of host factors required for viral infection has led to advances in both viral and cellular biology. Vesicular stomatitis virus (VSV), a negative-sense RNA virus, replicates in all eukaryotic cells in culture, suggesting that the host requirements for its replication are ubiquitous. In this study, we performed a genome-wide small interfering RNA screen of human cells in culture and identified multiple cellular genes that influence the entry and replication of VSV. From a list of >300 genes, we selected the most promising candidates to perform further analysis to assign their functions to either the entry or intracellular replication step of infection. We implicate 3 new factors in VSV entry and 20 new factors in viral gene expression. These proteins have diverse cellular roles, including S-adenosylmethionine synthesis, respiration, and host transla...
At each stage of the HIV life cycle, host cellular proteins are hijacked by the virus to establish and enhance infection. We adapted the virus packageable HIV-CRISPR screening technology at a genome-wide scale to comprehensively identify host factors that affect HIV replication in a human T cell line. Using a smaller, targeted HIV Dependency Factor (HIVDEP) sub-library, we then performed screens across multiple HIV strains representing different clades and with different biological properties to define which T cell host factors are strain-specific versus which ones are important across all HIV strains and different clades. Nearly 90% genes selected across multiple host pathways validated in subsequent assays as bona fide host dependency factors including numerous proteins not previously reported to play role in HIV biology such as UBE2M, MBNL1, FBXW7, PELP1, SLC39A7, and others. Our ranked list of screen hits across multiple viral strains form a resource of HIV dependency factors fo...
The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus–host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid–host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bin...
BMC Systems Biology
BiologicalNetworks - tools enabling the integration of multi-scale data for the host-pathogen studies2011 •
Journal of Biology
Decoding the multifaceted HIV1 virus-host interactome2009 •
ABSTRACT: Recently in BMC Medical Genomics, Tozeren and colleagues have uncovered virus-host interactions by searching for conserved peptide motifs in HIV and human proteins. Their computational model provides a novel perspective in the interpretation of high-throughput data on the HIV-host interactome.
BMC Bioinformatics
GPS-Prot: A web-based visualization platform for integrating host-pathogen interaction data2011 •
BackgroundThe increasing availability of HIV-host interaction datasets, including both physical and genetic interactions, has created a need for software tools to integrate and visualize the data. Because these host-pathogen interactions are extensive and interactions between human proteins are found within many different databases, it is difficult to generate integrated HIV-human interaction networks.ResultsWe have developed a web-based platform, termed GPS-Prothttp://www.gpsprot.org, that allows for facile integration of different HIV interaction data types as well as inclusion of interactions between human proteins derived from publicly-available databases, including MINT, BioGRID and HPRD. The software has the ability to group proteins into functional modules or protein complexes, generating more intuitive network representations and also allows for the uploading of user-generated data.ConclusionsGPS-Prot is a software tool that allows users to easily create comprehensive and in...
2014 •
BioMed Research International
Current and Emerging Cell Culture Manufacturing Technologies for Influenza Vaccines2015 •
Journal of virology
Genome-wide small interfering RNA screens reveal VAMP3 as a novel host factor required for Uukuniemi virus late penetration2014 •
Retrovirology
Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function2011 •
Microbiology and molecular biology reviews : MMBR
Multiple Inhibitory Factors Act in the Late Phase of HIV-1 Replication: a Systematic Review of the Literature2018 •
2014 •
2013 •
International journal of molecular sciences
Systematic approaches towards the development of host-directed antiviral therapeutics2011 •
The Journal of biological chemistry
Characterization of the influence of mediator complex in HIV-1 transcription2014 •
2015 •
Journal of Experimental Medicine
IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation2013 •
2019 •
Journal of Virology
Cellular Transcription Factor ZASC1 Regulates Murine Leukemia Virus Transcription2010 •
Journal of Biomolecular Screening
Automated Genome-Wide Visual Profiling of Cellular Proteins Involved in HIV Infection2011 •
Antimicrobial Agents and Chemotherapy
Combinatorial CRISPR-Cas9 and RNAi attack on HIV-1 DNA and RNA can lead to cross-resistanceBiomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Thymoquinone: A novel strategy to combat cancer: A review2018 •
2014 •
2012 •
Virology Journal
Identification of novel host-oriented targets for Human Immunodeficiency Virus type 1 using Random Homozygous Gene Perturbation2009 •
2015 •
2009 •
Molecular & cellular proteomics : MCP
In Vivo and in Vitro Proteome Analysis of Human Immunodeficiency Virus (HIV)-1-infected, Human CD4(+) T Cells2017 •
Journal of Virology
Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Matrix Proteins Specify Different Capabilities To Modulate B Cell Growth2014 •
2011 •
Nucleic Acids Research
HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression2015 •
2009 •
2009 •
Clinical Chemistry and Laboratory Medicine
Exploring synergies between academia and vaccine manufacturers: a pilot study on how to rapidly produce vaccines to combat emerging pathogens2000 •