Recombinant neuraminidase and uses thereof
Inventors
Palese, Peter • Garcia-Sastre, Adolfo • Krammer, Florian
Assignees
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Publication Number
US-12655408-B2
Publication Date
2026-06-16
Expiration Date
Abstract
In one aspect, provided herein are recombinant neuraminidases comprising an ectodomain of influenza virus neuraminidase with amino acid substitutions or insertions of cysteines in the stalk domain to generate a more stable, tetrameric influenza virus neuraminidase. In specific embodiments, the influenza virus neuraminidase further comprises influenza virus neuraminidase transmembrane and cytoplasmic domains. In another aspect, provided herein are recombinant neuraminidase comprising a globular head domain of influenza virus neuraminidase and a tetramerization domain, wherein the recombinant neuraminidase lacks influenza virus neuraminidase stalk, transmembrane and cytoplasmic domains. In another aspect, provided herein are methods of immunizing against influenza virus using such recombinant neuraminidases or compositions thereof.
Core Innovation
The invention relates to recombinant neuraminidase constructs defined by specific amino acid sequences, including SEQ ID NO: 56, 58, 27, 60, and 62. The constructs include influenza virus neuraminidase globular head domain forms and related recombinant neuraminidase polypeptide sequence variants associated with N1 subtype and N2 subtype sequences, including PR8 N1 cysteine mutants and HK14 N2 cysteine mutants with specified cysteine substitutions and cysteine insertions.
The recombinant neuraminidase also comprises a globular head domain together with a tetramerization domain while lacking an influenza virus neuraminidase stalk domain, transmembrane domain, and cytoplasmic domain. The tetramerization domain comprises a paramyxovirus phosphoprotein tetramerization domain, specifically a Sendai virus phosphoprotein tetramerization domain comprising the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 25. Additional polypeptide domains such as purification/cleavage tags, including His tags, a thrombin site, and a TEV-cleavable site, as well as optional signal peptides, are also described.
The constructs are characterized as forming tetrameric or oligomeric NA forms, with additional cysteines in the NA stalk region described as affecting NA quaternary structure and NA enzymatic activity. Structural similarity, epitope-related behavior, neutralizing antibody binding, and non-contiguous epitopes are discussed, and example background includes recombinant MPP-based NA constructs, cysteine mutants, and tetrameric recombinant NA vaccine candidates with reported in vivo protection outcomes and NA activity/antibody recognition results.
Claims Coverage
The independent claims cover two inventive features: sequence-defined recombinant neuraminidase constructs and a tetrameric recombinant neuraminidase architecture with a paramyxovirus phosphoprotein tetramerization domain. The claims focus on defined amino acid sequences and on retaining the influenza neuraminidase globular head while omitting the stalk, transmembrane, and cytoplasmic domains.
Sequence-defined recombinant neuraminidase amino acid sequences
A recombinant neuraminidase comprising the amino acid sequence of SEQ ID NO: 56, 58, 27, 60, or 62.
Tetrameric influenza neuraminidase globular head with Sendai phosphoprotein tetramerization domain
A recombinant neuraminidase comprising an influenza virus neuraminidase globular head domain and a tetramerization domain, wherein the recombinant neuraminidase lacks an influenza virus neuraminidase stalk domain, transmembrane domain and cytoplasmic domain, wherein the tetramerization domain comprises a paramyxovirus phosphoprotein tetramerization domain, and wherein the paramyxovirus phosphoprotein tetramerization domain is a Sendai virus phosphoprotein tetramerization domain that comprises the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 25.
Overall, the claim scope is anchored on recombinant neuraminidases defined by specified amino acid sequences and on tetrameric designs that pair an influenza neuraminidase globular head domain with a paramyxovirus phosphoprotein tetramerization domain while lacking stalk, transmembrane, and cytoplasmic domains.
Stated Advantages
Additional cysteines in the NA stalk region increase tetrameric or oligomeric NA forms and affect NA enzymatic activity.
Structural similarity and epitope-related behavior are assessed in relation to neutralizing antibody binding and non-contiguous epitopes.
Reported in vivo protection outcomes and NA activity/antibody recognition results are discussed for tetrameric recombinant NA vaccine candidates.
Documented Applications
Murine vaccination/challenge is discussed using recombinant MPP-based NA constructs, with discussion supporting protective immune responses.
Immunogenic compositions comprising the recombinant neuraminidase are provided, including immunogenic compositions that further include a trivalent inactivated influenza vaccine (TIV), a quadrivalent inactivated influenza virus vaccine (QIV), or a recombinant influenza virus vaccine.
Recombinant influenza virus constructs that include the recombinant neuraminidase are covered.
Vaccine and immunogenic composition concepts, including live/inactivated/split vaccines, are described.
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