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-11679151-B2
Publication Date
2023-06-20
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 vaccines based on influenza hemagglutinin (HA) proteins specifically targeting the stem region of the HA protein. These vaccines comprise modified influenza HA stem-region proteins stabilized in the pre-fusion conformation that elicit broadly neutralizing antibodies against the influenza virus. The vaccines include nanoparticles that display HA trimers on their surface, formed by fusion proteins consisting of a monomeric subunit such as ferritin joined to an immunogenic portion of the HA stem region. The fusion proteins self-assemble into nanoparticles, presenting the HA stem regions to induce protective immune responses against influenza.
The problem solved is the difficulty in eliciting broad heterosubtypic protective immunity against influenza virus due to the immunodominance and variability of the HA globular head. Current vaccines mainly induce strain-specific immune responses against the variable HA head that undergoes antigenic drift and shift, limiting their efficacy. Furthermore, existing vaccine production methods are time-consuming and limited in capacity, such as production in embryonated eggs. The invention addresses these limitations by providing a vaccine that removes or replaces most of the variable HA head region with a short linker, stabilizes the conserved stem domain to improve immunogenicity, and presents the stem domain in its native trimeric form on nanoparticles to induce broadly protective antibodies.
The invention also relates to protein constructs and nucleic acid molecules encoding such fusion proteins, methods for producing the nanoparticles through self-assembly of the fusion proteins, and their use in vaccination and serological assays to detect anti-influenza antibodies. Through iterative structure-based design, the inventors optimized stabilized HA stem immunogens that efficiently form trimers and self-assembling nanoparticles, which induce protective humoral immune responses, including broadly neutralizing antibodies, providing improved universal protection against influenza.
Claims Coverage
The claims include one independent claim directed to a nucleic acid molecule encoding an influenza HA protein construct with specific modifications to the HA stem domain, deletion or replacement within the head region, and enhanced intramolecular bonding to improve stability. Key inventive features cover structural alterations, linker compositions, and fusion to monomeric subunits to form nanoparticles displaying stabilized HA stem trimers.
Nucleic acid encoding an HA protein construct with HA head region replacement and stabilization
A nucleic acid molecule encoding an HA protein with at least 95% of the HA head region removed and replaced by a short linker (less than 10 amino acids), including modifications such as deletion of the internal loop region or replacement with a different linker, and substitutions in amino acid pairs forming noncovalent bonds to strengthen intramolecular interactions compared to wild-type HA.
Substitutions to strengthen bond between specific amino acid pairs in HA stem
Substitution of both amino acid residues in a pair that normally form a salt bridge in the wild-type HA, specifically residues corresponding to K1 and E53 in SEQ ID NO:149, to amino acids that form stronger noncovalent bonds such as hydrophobic or hydrogen bonds, thereby stabilizing the protein construct.
First and second stem region sequences joined by linker
The protein construct comprises two stem region sequences flanking the missing head, covalently linked by a linker sequence. The first sequence comprises at least 20 contiguous amino acids upstream of the head region, and the second comprises at least 20 contiguous amino acids downstream. The first and second sequences are sourced from influenza HA proteins of specific strains such as A/New Caledonia/20/1999, A/California/04/2009, and others.
Fusion to monomeric subunit protein to enable nanoparticle formation
The HA protein domain is joined to a monomeric subunit protein (e.g., ferritin or lumazine synthase) that directs self-assembly of the protein construct into a nanoparticle, displaying HA stem trimers on the nanoparticle surface.
Additional amino acid substitutions for stabilization
Incorporation of specific mutations in the HA stem domain at chosen residues (positions 36, 45, 47, 49, 339, 340, 341, 342, 361, 372, 394, 402, 437, 438, 445, 446, 448, 449, 450, and 452 relative to SEQ ID NO:8) to further improve protein stability and immunogenicity.
The claims encompass nucleic acid molecules encoding HA stem-only constructs with specific deletions, substitutions, and linker sequences fused to monomeric subunits to form nanoparticles, providing stabilized HA stem trimers designed to elicit broad protective immune responses against influenza.
Stated Advantages
The invention offers an easily manufactured influenza vaccine that is potent and elicits broadly neutralizing antibodies against diverse influenza virus strains.
It provides broad heterosubtypic protection due to targeting of the conserved HA stem region, overcoming limitations of current strain-specific vaccines.
The nanoparticle display of HA stem trimers increases immunogenicity by presenting the pre-fusion conformation of HA in a native, trimeric form.
The vaccines avoid unwanted immune responses to the variable immunodominant HA head region, focusing the immune response on conserved broadly neutralizing epitopes.
The self-assembling nanoparticle platform allows scalable production and improved stability compared to traditional vaccines produced in eggs.
Documented Applications
Vaccination of individuals against influenza virus by administering nanoparticles displaying stabilized HA stem trimers to induce broadly protective immune responses.
Diagnostics and assays for detecting anti-influenza antibodies in biological samples using nanoparticles presenting HA stem proteins to identify immune status or exposure to influenza virus.
Methods of producing stabilized HA stem protein constructs and self-assembling nanoparticles for use in vaccines and antibody detection assays.
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