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US-12616745-B2 - Influenza virus backbone

US12616745B2US 12616745 B2US12616745 B2US 12616745B2US-12616745-B2

Abstract

The invention provides an influenza virus that demonstrates enhanced growth in Vero cells. The influenza virus includes PB1, PB2, PA, NP, and NS gene segments encoding proteins having amino acid sequences with selected amino acids. Optionally, at least one of the PB1, PB2, and PA gene segments includes a cytosine to uracil promoter mutation at nucleotide position 4. The invention also provides a pharmaceutical formulation containing the influenza virus, as well as a method of eliciting an immune response in a mammal by administering the influenza virus to the mammal, and a method for generating the influenza virus.

Inventors

  • Yasuko Hatta
  • Michael J. Moser
  • Pamuk Bilsel

Assignees

  • FLUGEN, INC.

Dates

Publication Date
20260505
Application Date
20200605

Claims (20)

  1. 1 . An influenza virus comprising PB1, PB2, PA, NP, and NS gene segments, wherein (a) the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 7, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 15, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO: 17, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 6, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO: 19, or (b) the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 9, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 10, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO: 17, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 8, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO: 19.
  2. 2 . The influenza virus of claim 1 , wherein the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 7, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 15, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO: 17, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 6, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO: 19.
  3. 3 . The influenza virus of claim 2 , wherein the PB1 gene segment has the nucleotide sequence represented by SEQ ID NO: 2.
  4. 4 . The influenza virus of claim 2 , wherein the NP gene segment has the nucleotide sequence represented by SEQ ID NO: 1.
  5. 5 . The influenza virus of claim 2 , wherein the PB2 gene segment has the nucleotide sequence represented by SEQ ID NO: 14.
  6. 6 . The influenza virus of claim 2 , wherein (a) a leucine at position 40 , a tryptophan at position 180 , and an asparagine at position 464 in SEQ ID NO: 7 or (b) a leucine at position 116 and a lysine at position 294 in SEQ ID NO: 6 are conserved in after at least ten serial passages in a Vero cell line.
  7. 7 . The influenza virus of claim 2 , wherein (a) a leucine at position 40 , a tryptophan at position 180 , and an asparagine at position 464 in SEQ ID NO: 7 or (b) a leucine at position 116 and a lysine at position 294 in SEQ ID NO: 6 are conserved after at least ten serial passages in a Vero cell line that stably expresses the M2 ion channel protein of influenza A virus.
  8. 8 . The influenza virus of claim 2 , wherein the influenza virus is an influenza A virus, and (a) a leucine at position 40 , a tryptophan at position 180 , and an asparagine at position 464 in SEQ ID NO: 7 or (b) a leucine at position 116 and a lysine at position 294 in SEQ ID NO: 6 are conserved after at least ten serial passages in a Vero cell line that stably expresses the BM2 ion channel protein of influenza B virus.
  9. 9 . The influenza virus of claim 1 , wherein (b) the PB1 gene segment encodes a PB1 protein having the amino acid sequence of SEQ ID NO: 9, the PB2 gene segment encodes a PB2 protein having the amino acid sequence of SEQ ID NO: 10, the PA gene segment encodes a PA protein having the amino acid sequence of SEQ ID NO: 17, the NP gene segment encodes an NP protein having the amino acid sequence of SEQ ID NO: 8, and the NS gene segment encodes an NS1 protein having the amino acid sequence of SEQ ID NO: 19.
  10. 10 . The influenza virus of claim 9 , wherein the PB1 gene segment has the nucleotide sequence represented by SEQ ID NO: 4.
  11. 11 . The influenza virus of claim 9 , wherein the PB2 gene segment has the nucleotide sequence represented by SEQ ID NO: 5.
  12. 12 . The influenza virus of claim 9 , wherein the NP gene segment has the nucleotide sequence represented by SEQ ID NO: 3.
  13. 13 . The influenza virus of claim 9 , wherein (a) a leucine at position 40 , a tryptophan at position 180 , and a serine at position 607 in SEQ ID NO: 9, (b) a valine at position 504 , an isoleucine at position 467 , and a valine at position 529 in SEQ ID NO: 10, or (c) a leucine at position 116 and an arginine at position 311 in SEQ ID NO: 8 are conserved after at least ten serial passages of the virus in a Vero cell line.
  14. 14 . The influenza virus of claim 9 , wherein (a) a leucine at position 40 , a tryptophan at position 180 , and a serine at position 607 in SEQ ID NO: 9 , (b) a valine at position 504 , an isoleucine at position 467 , and a valine at position 529 in SEQ ID NO: 10, or (c) a leucine at position 116 and an arginine at position 311 in SEQ ID NO: 8 are conserved after at least ten serial passages in a Vero cell line that stably expresses the M2 ion channel protein of influenza A virus.
  15. 15 . The influenza virus of claim 1 , wherein at least one of the PB1, PB2, and PA genes segments comprises a cytosine to uracil promoter mutation at nucleotide position 4.
  16. 16 . The influenza virus of claim 1 , wherein the influenza virus is a recombinant influenza virus.
  17. 17 . The influenza virus of claim 1 , wherein the virus further comprises an NA gene segment and an HA gene segment.
  18. 18 . The influenza virus of claim 17 , wherein the HA gene segment encodes an HA protein having the amino acid sequence comprising at least one amino acid mutation in HA1.
  19. 19 . The influenza virus of claim 17 , wherein the HA gene segment encodes an HA protein having the amino acid sequence comprising at least one amino acid mutation in HA2.
  20. 20 . The influenza virus of claim 19 , wherein the at least one amino acid mutation in HA2 is an asparagine at position 107.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application 62/858,737, filed on Jun. 7, 2019, which is incorporated by reference in its entirety herein. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 73,647 bytes ASCII (Text) file named “758243SequenceListing.txt.” created Dec. 2, 2021. BACKGROUND OF THE INVENTION Influenza, i.e., the “flu,” is a highly contagious viral infection that claims the lives of hundreds of thousands of humans globally each year. There are four types of influenza viruses (i.e., A, B, C, and D) categorized based on their core proteins, although seasonal epidemics are most often caused by circulating Influenza A and B viruses. While vaccines are the best way to prevent influenza, influenza vaccines must be re-formulated often, as the influenza virus is subject to antigenic drift and antigenic shift. Moreover, because influenza viruses, particularly Influenza A, generally exhibit a high rate of mutation and evolution, influenza vaccine strains may mismatch circulating strains, resulting in minimal vaccine effectiveness. However, when circulating flu viruses are well-matched to the flu vaccine, vaccination can reduce the risk of flu-related illness by 40%-60% among the overall population. Accordingly, researchers have been studying vaccines that can induce cross-protective immunity between different influenza subtypes. One such example is a vaccine comprising a live, attenuated influenza virus that does not express a functional M2 protein (e.g., the M2SR vaccine). Influenza vaccines are preferably propagated in Madin-Darby canine kidney (MDCK) cells and African Green Monkey (Vero) cells, as mammalian-cell cultures provide advantages over egg-based production. These advantages include lower costs, faster production time, and a reduced risk of antigenic mutation in the virus. For example, the M2SR vaccine is propagated in Vero cells that stably express M2 protein. However, vaccine production in cell cultures has often resulted in undesirable yields. Moreover, MDCK cells are generally more permissive than Vero cells, such that vaccine production in Vero cells is comparatively inefficient. Modifications to the virus backbone, i.e., the six internal gene segments consisting of PB1, PB2, PA, NP, M, and NS, have been shown to boost vaccine production. For example, the high-yield vaccine backbone “PR8-HY” developed and described in Ping et al., Nature Communications, 6: 8148 (2015), comprises specific amino acid mutations in the PB1, PB2, PA, NP, and NS1 proteins, and was expected to improve the titers of pandemic and seasonal influenza vaccines in both cell and egg culture systems. However, these modifications described in the art have been ineffective in increasing viral growth in Vero cells, particularly under preferred manufacturing conditions. Therefore a need exists to enhance viral growth in Vero cells, such that vaccines, like the M2SR vaccine, can be produced more efficiently and effectively. BRIEF SUMMARY OF THE INVENTION The invention provides an influenza virus having enhanced growth in Vero cells. The influenza virus comprises gene segments encoding proteins, e.g., the PB1, PB2, PA, NP, and NS1 proteins, having amino acid sequences comprising selected amino acids. For example, the PB1 protein comprises a leucine at position 40 and a tryptophan at position 180, and at least one of an asparagine at position 464 or a serine at position 607. The PB2 protein comprises a valine at position 504, and optionally an isoleucine at position 467 and a valine at position 529. The PA protein comprises a lysine at position 401. The NP protein comprises a leucine at position 116, and at least one of a lysine at position 294 or an arginine at position 311. The NS1 protein comprises a proline at position 30 and a lysine at position 118. Furthermore, at least one of the PB1, PB2, and PA gene segments optionally comprises a cytosine to uracil promoter mutation at nucleotide position 4. The invention also provides a pharmaceutical formulation comprising the influenza virus, a method of eliciting an immune response in a mammal comprising administering the influenza virus to the mammal, and a method of generating the influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a graph of virus titer (log TCID50/ml) versus time (days post-infection) depicting the growth curve for A/Massachusetts/15/2013 M2SR viruses comprising a Vero-adapted HA protein (i.e., M2SR-MA15V viruses) comprising UW-PR8 (“HG”) and PR8-HY (“HY”) backbones in Vero cells. FIG. 1B is a graph of virus titer (TCID50/ml) versus time (days post-infection) depicting the growth curve for A/Brisbane/10/2007 M2SR viruses (i.e., Bris10 M2SR) comprising HG and HY backbones in Vero cells. FIG. 1C