Recombinant high growth rate influenza virus comprising mutations in M1, NS2, and PB2
Inventors
Muster, Thomas • Aspelund, Amy • Wolschek, Markus
Assignees
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Publication Number
US-12350325-B2
Publication Date
2025-07-08
Expiration Date
Abstract
The present invention provides high growth influenza reassortant virus and high growth influenza reassortant virus vectors comprising amino acid modifications in the PB2, PB1, M1 and/or NS2 proteins which exhibit highly increased growth rates compared to unmodified influenza virus. Further provided are pharmaceutical compositions comprising reassortant virus and viral vectors comprising said modifications and their use for vaccination purposes.
Core Innovation
The invention provides a recombinant influenza B virus with increased growth rate in Vero cells as compared to wild type influenza virus. The recombinant virus is delNS1 influenza, meaning it lacks a functional NS1 protein, and is defined to include SEQ ID NOs: 2, 6 and 10. It comprises an M gene segment producing an M1 protein having an amino acid sequence SEQ ID NO:8, and an NS gene segment producing an NS2 protein having one of the amino acid sequences SEQ ID NO:12 or 34.
The described embodiments connect specific internal protein sequence features to increased growth in Vero cells. Growth enhancement is supported for mutation combinations involving PB2, M1, and NS2, including a substitution at position 427 such as G427S, a substitution at position 89 such as T89S/serine, and substitutions at positions 75 and/or 76 such as K75R and/or R76G. The document also outlines related influenza A variants having analogous deINS1 internal mutations, and includes optional PB1 substitution embodiments such as D67N.
The invention further addresses the construction and use of influenza virus vectors for vaccine and pharmaceutical compositions. It includes reverse genetics system and reassortant formats, including reassortants described as 6:2, 6:1:1, and 7:1, with HA and NA gene segments sourced from seasonal/pandemic strain origins. Supporting growth-curve findings in the document indicate that particular mutation combinations yield higher titers versus controls, in the context of the delNS1 and internal protein sequence constraints.
Claims Coverage
The independent claim covers a delNS1 recombinant influenza B virus designed to show increased growth rate in Vero cells versus wild type, with the core construct defined by specified M, NS, and optional PB2 sequence features. Dependent claims further add specific options for PB1 substitutions, HA/NA constraints, and phenotype attributes such as attenuation and replication deficiency, as well as vaccination use.
DelNS1 recombinant influenza B virus with increased Vero growth
A recombinant influenza B virus with increased growth rate in Vero cells as compared to wild type influenza virus comprising SEQ ID NOs: 2, 6 and 10, and lacking a functional NS1 protein (delNS1 influenza).
M gene segment defining M1 protein sequence
An M gene segment resulting in an M1 protein comprising amino acid sequence SEQ ID NO:8.
NS gene segment defining NS2 protein sequence options
An NS gene segment comprising one or more nucleotide modifications resulting in an NS2 protein comprising one of amino acid sequences SEQ ID NO:12 or 34.
Optional PB2 gene segment defining PB2 protein sequence
Optionally, a PB2 gene segment resulting in a PB2 protein comprising amino acid sequence SEQ ID NO:4.
PB1 substitution at defined positions
A PB1 gene segment with nucleotide modifications resulting in a PB1 protein comprising an asparagine amino-acid substitution at either position 67 or position 678.
Attenuated or replication deficient reassortant phenotype with HA/NA modification context
A recombinant, reassortant influenza virus that is attenuated or replication deficient and/or contains one or more HA and/or NA gene modifications relative to a wild type influenza virus defined by SEQ ID NOs: 2, 6 and 10.
NS2 protein fixed as SEQ ID NO:12
An NS2 protein defined as SEQ ID NO:12.
Group 1 or Group 2 HA gene constraint
The recombinant influenza virus includes Group 1 HA or Group 2 HA genes.
Vaccination to treat influenza virus infection
Treating an influenza virus infection by administering an immunogenically effective amount of a vaccine that comprises the virus of claim 1.
Overall, the claim set centers on a delNS1 recombinant influenza B virus whose internal gene-segment sequence definitions constrain M1 and NS2 (and optionally PB2) to achieve increased growth rate in Vero cells, with dependent claims further narrowing sequence options including PB1 substitution sites, HA gene-group constraints, and vaccination use.
Stated Advantages
Increased growth rate in Vero cells as compared to wild type influenza virus.
Documented Applications
Vaccination to treat an influenza virus infection using a vaccine comprising the virus of claim 1.
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