Stabilized influenza hemagglutinin stem region trimers and uses thereof
Inventors
Mascola, John R. • BOYINGTON, Jeffrey C. • YASSINE, Hadi M. • Kwong, Peter D. • Graham, Barney S. • Kanekiyo, Masaru
Assignees
US Department of Health and Human Services
Publication Number
US-12268737-B2
Publication Date
2025-04-08
Expiration Date
2035-05-27
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Abstract
Vaccines that elicit broadly protective anti-influenza antibodies. Some vaccines comprise nanoparticles that display HA trimers from influenza virus on their surface. The nanoparticles are fusion proteins comprising a monomeric subunit (e.g., ferritin) joined to the stem region of an influenza HA protein. The fusion proteins self-assemble to form the HA-displaying nanoparticles. The vaccines comprise only the stem region of an influenza HA protein joined to a trimerization domain. Also provided are fusion proteins, and nucleic acid molecules encoding such proteins, and assays using nanoparticles of the invention to detect anti-influenza antibodies.
Core Innovation
The invention provides novel influenza vaccines based on hemagglutinin (HA) protein stem regions that are easily manufactured, potent, and elicit broadly neutralizing antibodies against conserved HA stem epitopes. The vaccines comprise modified influenza HA stem-region proteins stabilized in the pre-fusion conformation and portions thereof, useful for inducing neutralizing antibodies. These proteins can be presented on nanoparticles formed by fusion proteins comprising a monomeric subunit such as ferritin joined to the HA stem, which self-assemble into nanoparticles displaying HA trimers on their surface.
The invention addresses the problem that traditional influenza vaccines mainly induce strain-specific antibodies targeting the highly variable HA globular head, resulting in limited protection against evolving viral strains. The HA stem is highly conserved and a target for broadly neutralizing antibodies, but it is poorly immunogenic due to immunodominance of the head and the instability of stem-only proteins. Existing vaccine platforms have manufacturing limitations and do not robustly elicit broadly neutralizing stem-directed antibodies, highlighting the need for a universal vaccine platform.
To overcome these challenges, the invention provides protein constructs that replace the HA head region with short linker sequences, stabilize the HA stem domain through specific mutations, and fuse the stem constructs to self-assembling monomeric subunits such as ferritin to form nanoparticles displaying native-like HA stem trimers. These nanoparticles retain correct antigenic conformations, induce effective B-cell responses by engaging germline precursors of broadly neutralizing antibodies, and elicit protective immunity against diverse influenza strains including heterosubtypic viruses.
Claims Coverage
The patent includes one independent claim directed to a method of vaccinating an individual against influenza using a nucleic acid molecule encoding a modified HA stem protein construct fused to a monomeric subunit for nanoparticle formation. The claim recites key inventive features related to HA head removal, linker sequences, specific mutations for stability, and nanoparticle display.
HA head region replacement with short linker sequence
The influenza HA protein domain lacks at least 95% of the head region amino acid sequence, replaced by a first linker sequence less than 10 amino acids in length.
Deletion or replacement of the internal loop region
Alterations include deletion of the amino acid region corresponding to N403-W435 of the internal loop in SEQ ID NO:8, with direct joining of remaining ends, or replacement of this region with a second linker sequence.
Substitution of amino acids to strengthen noncovalent bonding
Substitution of at least one amino acid residue in a pair that form a noncovalent bond in the folded HA protein to increase bond strength relative to wild-type, specifically substitutions corresponding to K1 and E53 of SEQ ID NO:149.
Incorporation of HA stem sequences covalently linked by a linker
The HA protein domain comprises first and second amino acid sequences from the stem region upstream and downstream of the head region, covalently linked by the first linker sequence, each comprising at least 20 contiguous amino acids.
Fusion to monomeric subunit for nanoparticle formation
The HA protein domain is joined to a monomeric subunit protein that enables the protein construct to self-assemble into a nanoparticle displaying HA trimers.
Additional mutations at specified HA positions
Inclusion of other mutations at amino acid positions corresponding to selected residues in SEQ ID NO:8 to improve stability or antigenicity.
The claims cover a nucleic acid-based vaccination method employing protein constructs of influenza HA in which the immunodominant head region is largely removed and replaced by a short linker, combined with stabilizing mutations and fused to monomeric subunits, enabling nanoparticle formation that displays stabilized HA stem trimers capable of inducing broadly protective immune responses against diverse influenza strains.
Stated Advantages
The vaccine platform is easily manufactured and produces stable, potent immunogens that preserve native-like HA stem structures.
It elicits broadly neutralizing antibodies targeting the conserved HA stem, providing heterosubtypic protection against diverse influenza strains.
Fusion to self-assembling nanoparticles enhances immunogenicity by multivalent antigen display.
The immunogen engages precursor B cell receptors of broadly neutralizing stem antibodies, facilitating effective immune responses.
Protection is mediated by antibody functions beyond neutralization, such as antibody-dependent cell-mediated cytotoxicity (ADCC).
Documented Applications
Use as a universal influenza vaccine to protect individuals against a broad range of influenza viruses, including heterologous and antigenically divergent strains.
Use of nanoparticles displaying HA stem trimers as immunogens in vaccines administered by various routes to humans and animals.
Use in detection assays to measure anti-influenza antibodies in samples by forming antibody/nanoparticle complexes using nanoparticles displaying HA stem regions.
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