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-11969466-B2
Publication Date
2024-04-30
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 hemagglutinin (HA) protein-based vaccines that are easily manufactured, potent, and elicit broadly neutralizing influenza antibodies against the stem region of the HA protein. Specifically, the invention includes modified influenza HA stem-region proteins stabilized in the pre-fusion conformation, which can be used to induce neutralizing antibodies. These vaccines also comprise nanoparticle constructs displaying the influenza HA protein on their surface; the nanoparticles are formed from fusion proteins comprising a monomeric subunit, such as ferritin, joined to an antigenic portion of the HA stem region, and these fusion proteins self-assemble into nanoparticles presenting HA trimers.
The problem addressed stems from limitations of current influenza vaccines, which primarily target the highly variable and immunodominant HA head domain, leading to strain-specific immunity and necessitating frequent vaccine updates due to antigenic drift and shift. The HA stem region is more conserved but less immunogenic and has been difficult to effectively target for broadly protective antibody responses. Furthermore, existing vaccine platforms such as inactivated or live attenuated viruses face manufacturing and efficacy challenges, and alternatives like virus-like particles (VLPs) or recombinant HA proteins have limitations in producing broad and potent immunity.
The invention solves these issues by genetically removing most or all of the globular head from HA and stabilizing the remaining stem domain, which is then fused to nanoparticle scaffolds like ferritin to create self-assembling particles that display trimers of the stabilized stem on their surface. This design increases immunogenicity toward the conserved stem region and enables elicitation of broadly neutralizing antibodies across influenza virus subtypes, potentially providing universal protection. The stabilized stem domain contains certain mutations to enhance trimeric stability and antigenic fidelity. The nanoparticles elicit protective immune responses in animal models against heterologous influenza strains, including pandemic H5N1 virus.
Claims Coverage
The independent claims cover methods of vaccinating individuals using nanoparticles composed of protein constructs with specific HA protein domain modifications.
Method of vaccination using a nanoparticle with a stabilized HA stem lacking most of the head region
The protein construct comprises an HA protein domain missing at least 95% of the HA head domain, replaced by a short linker less than 10 amino acids. The HA protein domain includes at least one alteration from: deletion of the internal loop region (N403-W435) joined directly, replacement of this internal loop with a second linker, or substitution of amino acid pairs forming noncovalent bonds to strengthen interactions over the wild-type HA.
Specific substitutions enhancing HA stem domain stability by altering amino acid pairs in the HA sequence
The substitutions are made in paired amino acid residues that form a noncovalent bond in the folded HA protein, such as substituting both amino acids in the interaction pair corresponding to K1 and E53 of SEQ ID NO:149 to increase bond strength, thereby stabilizing the HA stem.
Composition of the HA protein domain from stem region sequences upstream and downstream of the head region
The HA protein domain consists of a first amino acid sequence from at least 20 residues upstream of the head domain and a second amino acid sequence from at least 20 residues downstream of the head domain, covalently linked by the linker sequence. These sequences are selected from known influenza strains including H1, H2, H3, H5 subtypes and influenza B strains.
Joining of the HA protein domain to a monomeric subunit protein forming self-assembling nanoparticles
The HA protein domain is joined to a monomeric subunit protein such as ferritin or lumazine synthase which facilitates self-assembly of the protein construct into nanoparticles that display HA trimers on the surface capable of eliciting immune responses.
Additional mutations in the HA protein domain enhancing stability and immunogenic presentation
The protein construct may include additional mutations at multiple specific amino acid positions in the HA stem region corresponding to positions in SEQ ID NO:8, such as positions 36, 45, 47, 49, 339, 340, 341, 342, and others, to optimize immunogenicity and structural fidelity.
The independent claims define a method of vaccinating using nanoparticles comprising protein constructs with genetically stabilized influenza HA stem domains lacking the head region, comprising linker sequences and mutations enhancing protein stability, joined to monomeric subunits that enable nanoparticle formation and display of trimeric HA stem for broad immunogenicity.
Stated Advantages
The invention provides an influenza vaccine that is easily manufactured and potent.
It elicits broadly heterosubtypic protective antibodies that target the conserved HA stem region.
The fusion with self-assembling nanoparticles enhances immunogenicity, leading to robust immune responses.
The stabilized stem protein in the pre-fusion conformation preserves native epitopes, improving antibody response quality.
The vaccine offers protection against multiple influenza virus strains, including pandemic and evolving seasonal viruses.
Documented Applications
Use of nanoparticles displaying stabilized influenza HA stem region trimers as vaccines to elicit broadly neutralizing antibodies against influenza viruses.
Methods of vaccinating individuals against influenza by administering said nanoparticles.
Use of the nanoparticles and protein constructs in assays to detect anti-influenza antibodies, including diagnostic applications.
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