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-11147867-B2
Publication Date
2021-10-19
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 antibodies against the stem region of the influenza HA protein. These vaccines comprise modified influenza HA stem-region proteins in the pre-fusion conformation, and portions thereof, designed to induce the production of neutralizing antibodies. Additionally, the invention provides nanoparticle-based vaccines that express influenza HA proteins on their surface, wherein the nanoparticles comprise fusion proteins composed of a monomeric subunit (such as ferritin) joined to an antigenic or immunogenic portion of the HA stem region. These fusion proteins self-assemble to form HA-displaying nanoparticles suitable for vaccinating individuals against influenza virus.
The problem being solved arises from the limitations of existing influenza vaccines, which primarily induce immune responses against the variable and immunodominant head region of the HA protein. This leads to strain-specific protection that is undermined by antigenic drift and shift, necessitating yearly vaccine updates and leaving gaps in protection against antigenically diverse and evolving influenza viruses. Moreover, current vaccine production in embryonated eggs is slow and capacity limited, and alternative platforms such as virus-like particles (VLPs) have similar limitations in potency and breadth of protective immunity. The conserved HA stem region is poorly immunogenic and difficult to target robustly by vaccination, which motivates the need for vaccine designs that focus on the conserved HA stem to elicit broadly protective antibodies.
Claims Coverage
The independent claims cover protein constructs comprising modified influenza HA protein domains with head region deletions/replacements, linked to monomeric subunits forming nanoparticles, and include specific mutations improving stability and antigenicity.
Protein construct comprising influenza hemagglutinin lacking most of the head region replaced by a short linker
A protein construct where the 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 (often less than 5 contiguous amino acids from the head).
Alterations enhancing stability of the HA stem region
Alterations include: a deletion of the internal loop amino acid region (corresponding to residues N403-W435 in SEQ ID NO:8) with ends joined directly or by a second linker sequence; and substitution of at least one amino acid pair forming a noncovalent bond in the folded HA to increase bond strength compared to wild-type, particularly substitutions at positions corresponding to K1 and E53 of SEQ ID NO:149.
Protein construct comprising two stem region sequences covalently linked by a short linker
The HA protein domain comprises a first amino acid sequence from upstream of the head region and a second amino acid sequence from downstream of the head region covalently linked by a short linker sequence, each sequence comprising at least 20 contiguous amino acids (often from specified influenza strains).
Protein construct joined to a monomeric subunit protein enabling nanoparticle formation
The modified HA protein domain is fused to a monomeric subunit protein such as ferritin or lumazine synthase, allowing the protein construct to self-assemble into nanoparticles that display trimeric HA on their surface.
Additional mutations at defined positions in the HA stem and monomeric subunit for functional optimization
The protein construct or nanoparticle may include further mutations at specified HA positions (e.g., 36, 45, 47, 49, 339, 340, 341, 342, 361, 372, 394, 402, 437, 438, 445, 446, 448, 449, 450, 452) or ferritin positions (e.g., 18, 20, 68) to enhance stability, antigenicity, or assembly.
The claims cover protein constructs of the influenza HA stem domain with extensive modifications including substantial removal/replacement of the head region, specific stabilizing mutations, covalent linkage to a monomeric subunit protein for nanoparticle formation, and further functionality-enhancing mutations, thus enabling vaccines that display stabilized stem trimers to elicit broadly protective influenza antibodies.
Stated Advantages
The invention provides a vaccine platform that is easily manufactured and potent.
The vaccines elicit broadly neutralizing antibodies against the conserved stem region of influenza HA, offering broad heterosubtypic protection.
The nanoparticles stabilize the HA stem in its pre-fusion trimeric conformation, reducing unfavorable antibody responses to exposed non-native epitopes.
The vaccine platform overcomes limitations of current vaccines such as strain-specific efficacy, manufacturing constraints, and limited induction of broadly neutralizing antibodies.
Ferritin nanoparticles allow multivalent antigen presentation that induces stronger B cell responses, including T-cell independent antibody responses.
Documented Applications
Vaccines to elicit robust and broadly protective immune responses against a wide range of influenza virus strains, including seasonal and pandemic influenza viruses.
Nanoparticle-based assays to detect anti-influenza antibodies in samples such as blood, plasma, serum, saliva, and other body fluids.
Methods of vaccinating individuals using the nanoparticles comprising the stabilized HA stem constructs to induce broadly neutralizing stem-directed antibodies.
Use in prime/boost vaccination protocols for improving influenza immunity.
Passive immunization methods by transfer of antibodies elicited by the nanoparticle vaccines to protect against lethal influenza virus challenge.
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