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 ...
The proteome of the malaria parasite Plasmodium falciparum is notable for the pervasive occurrence of homopeptides or low-complexity regions (i.e., regions that are made from a small subset of amino-acid residue types). The most prevalent of these are made from residues encoded by adenine/thymidine (AT)-rich codons, in particular asparagine. We examined homopeptide occurrences within protein domains in P. falciparum. Homopeptide enrichments occur for hydrophobic (e.g., valine), or small residues (alanine or glycine) in short spans (<5 residues), but these enrichments disappear for longer lengths. We observe that short asparagine homopeptides (<10 residues long) have a dramatic relative depletion inside protein domains, indicating some selective constraint to keep them from forming. We surmise that this is possibly linked to co-translational protein folding, although there are specific protein domains that are enriched in longer asparagine homopeptides (≥10 residues) indicating a functional linkage for specific poly-asparagine tracts. Top gene ontology functional category enrichments for homopeptides associated with diverse protein domains include “vesicle-mediated transport”, and “DNA-directed 5′-3′ RNA polymerase activity”, with various categories linked to “binding” evidencing significant homopeptide depletions. Also, in general homopeptides are substantially enriched in the parts of protein domains that are near/in IDRs. The implications of these findings are discussed.
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