A pooled genome-wide screening strategy to identify and rank influenza host restriction factors in cell-based vaccine production platforms

Abstract

Cell-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 genomewide 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 offers broader insights into the primary host cell determinants of viral yield in cell-based vaccine production systems. Overall, this work will advance efforts to address the public health burden posed by influenza. Cell-based influenza vaccine production. Mitigating the disease burden of influenza virus is an ongoing public health challenge, with 290,000-650,000 deaths annually and tenfold as many hospitalizations worldwide 1,2. The efficacy of influenza vaccines, which must be reformulated annually to adjust for antigenic drift and changes in dominantly circulating strains, has fluctuated between 10 and 60% since 2005 3. The vast majority (85-90%) of these vaccines are currently produced using embryonated chicken eggs, but a growing body of evidence suggests significant improvements in vaccine efficacy could be made by switching to cell-based manufacturing platforms 4. Passaging influenza through eggs induces antigenic drift as the virus adapts to an avian host 4-6. As a result, egg-based vaccines exhibit a 15-20% decrease in protection rate compared to similarly formulated cell-based vaccines 6-8. Cell-based platforms can also drastically reduce production lead time by accelerating seed stock reassortment via reverse genetics 9. This, in turn, reduces the chance of major changes in circulating strains occurring between initial strain selection and vaccine release, an issue that rendered the 2014-2015 seasonal vaccine largely ineffective 10,11. Additionally, the manufacturing capacity of cell-based vaccines is not constrained by the availability of billions of pathogen-free, synchronously fertilized chicken eggs, which would facilitate rapid response to influenza pandemics if they arise. Other advantages of cell-based vaccines include a lack of allergen contamination and better growth of certain strains 8. For all the benefits of cell-based influenza vaccines, current production platforms generally exhibit 4 to 10 fold lower volumetric yield than egg-based counterparts and are 40-100% more expensive 8,12. Given that influenza vaccine ...

Only a small fraction of cells produce assembled capsids during transfection‐based manufacturing of adeno‐associated virus vectors

“…HEK‐293SF cells were maintained in serum‐free suspension as previously described (D. M. Sharon et al, 2020). All transfections were performed at a cell density of 10 6 cell/ml using linear polyethylenimine with a mean molecular weight of 25,000 Da (Polysciences) complexed with plasmid DNA at a 1:2 ratio.…”

The use of base editing technology to characterize single nucleotide variants

“…Moreover, specific phenotypes caused by cell cycle effects or cell subpopulations may be masked because of the low-resolution readout [76] . Recent screens have expanded beyond cell survival or growth-based assays, including techniques that rely on fluorescence-activated cell sorting (FACS) to physically separate different populations of cells based on differences in cell morphology, gene expression levels, and virus infectivity [8] , [12] , [73] , [79] , [88] , [92] . While these advancements have enabled researchers to use CRISPR screens to study more complex phenotypes, only a single phenotype can be studied at once.…”

Adding a Chemical Biology Twist to CRISPR Screening

“…Many FACS-based pooled screens typically involve the tagging of a protein of interest with a fluorescent protein [53,54] or immunostained with a fluorescent antibody. [24] The CRISPR-perturbed cells are then sorted into different populations based on their fluorescence levels (Figure 2B).…”