Influenza virus backbone
An influenza virus, amino acid technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of low efficiency and poor yield of vaccines
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[0110] The invention provides the following embodiments:
[0111] (1) Influenza virus comprising PB1, PB2, PA, NP and NS gene segments, wherein: (a) the PB1 gene segment encodes a PB1 protein having an amino acid sequence comprising selected amino acids, wherein the selected The amino acid comprises leucine at position 40 and tryptophan at position 180, and at least one of asparagine at position 464 or serine at position 607, and wherein the PB1 gene segment optionally comprising a cytosine to uracil promoter mutation at nucleotide position 4; (b) the PB2 gene segment encodes a PB2 protein having an amino acid sequence comprising a selected amino acid, wherein the selected amino acid comprising valine at position 504 and optionally isoleucine at position 467 and valine at position 529, and wherein the PB2 gene segment optionally comprises at nucleotide position 4 (c) said PA gene segment encodes a PA protein having an amino acid sequence comprising a selected amino acid compr...
Embodiment 1
[0154] This example compares the growth of viruses with different backbones in Vero cells.
[0155] The high-yield PR8 ("PR8-HY") backbone described in Ping et al., Nature Communications, 6:8148 (2015) (which comprises ) of the mutated backbone gene segment) was used to extract from two different influenza viruses representing two influenza A virus subtypes in seasonal vaccines, namely A / Massachusetts / 15 / 2013 (MA15; H1N1) and A / Brisbane / 10 / 2007 (Bris10, H3N2) Generation of M2SR virus encoding HA and NA.
[0156] Specifically, cDNAs encoding the HA and NA gene segments from these viruses were transfected together with cDNAs encoding the PR8-HY backbone gene segment and the M2SR M gene segment (SEQ ID NO: 11). As described in Example 8, HA derived from MA15 was Vero adapted (MA15V). The two viruses produced were HY-M2SR-MA15V and Bris10 M2SR-HY. Viruses were generated by standard virus rescue techniques and expanded in MDCK cells stably expressing M2 (ie, M2CK cells), as des...
Embodiment 2
[0166] This example shows the production of viruses capable of enhanced growth in Vero cells. To generate these viruses, two M2SR viruses containing NA and Vero-adapted HA derived from A / Massachusetts / 15 / 2013 (i.e. MA15VM2SR virus) or derived from A / California / 07 / 2009 were serially passaged in M2VeroA cells (ie, CA07 M2SR virus) and contain the PR8-HY backbone (ie, HY-M2SR-MA15V and HY-M2SR-CA07).
[0167] Viruses were serially diluted 10-fold and adsorbed onto M2VeroA cells in TC-6 plates using standard influenza virus techniques. However, prior to virus infection, the cell culture medium was removed and the cells were washed with PBS. After adsorption for 60 min at 35°C, virus growth medium containing trypsin / TPCK was added. Cultures were incubated at 35°C for 4-7 days. Culture supernatants were harvested from the highest dilution well showing cytopathic effect and HA activity. Cytopathic effect (CPE) was determined by visual inspection of the monolayer under low power o...
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