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-10363301-B2
Publication Date
2019-07-30
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 potent, easily manufactured, and elicit broadly neutralizing antibodies targeting the conserved stem region of the influenza HA protein. The invention details modified influenza HA stem-region proteins stabilized in the pre-fusion conformation, useful for inducing neutralizing antibody production. Additionally, it provides novel nanoparticle-based vaccines composed of fusion proteins where a monomeric subunit, such as ferritin, is joined to an antigenic or immunogenic portion of the influenza HA stem region, enabling self-assembly into nanoparticles displaying HA trimers on their surface.
The problem addressed is the limitation of existing influenza vaccines, which primarily elicit strain-specific antibodies targeting the highly variable HA globular head, resulting in limited protection against evolving strains. The HA stem region, although highly conserved, is poorly immunogenic. Current vaccines produced in embryonated eggs have production and capacity limitations, with efficacy restricted by antigenic drift and shift. There is a need for influenza vaccines that can provide broad heterologous protection, including against seasonal and pandemic strains, and that avoid immunodominant responses to the HA head.
The invention solves these problems by removing the immunodominant HA head region and stabilizing the HA stem domain genetically and structurally, presenting the stem in a native-like trimeric pre-fusion conformation fused to self-assembling nanoparticles like ferritin. This approach increases immunogenicity against the conserved stem, eliciting broadly heterosubtypic antibody responses that confer protection against diverse influenza virus subtypes. The use of fusion proteins that self-assemble into nanoparticles displaying multiple trimeric HA stem antigens provides a novel platform for universal influenza vaccine development.
Claims Coverage
The patent contains multiple inventive features focused on a nanoparticle vaccine comprising a modified influenza hemagglutinin protein lacking the head region, fused to a monomeric subunit protein forming self-assembling nanoparticles. These features address structural modifications for enhanced stability, composition of the HA domain, and applications in vaccination and detection of anti-influenza antibodies.
Nanoparticle comprising an HA protein lacking at least 95% of the head region
The nanoparticle comprises a protein construct with an influenza HA protein domain lacking at least 95% of the head region amino acid sequence replaced by a short linker sequence less than 10 amino acids in length.
Stabilizing alterations in the HA stem domain
The HA protein comprises alterations including deletion of a specific region corresponding to amino acids N403-W435 of SEQ ID NO:8, and substitution of amino acid residues corresponding to K1 and E53 of SEQ ID NO:149 to increase bond strength in the folded protein compared to the wild type.
Linker sequence composition
The linker sequence replacing the missing head region comprises less than 5 contiguous amino acids from the HA head region, allowing proper protein conformation and assembly.
Fusion to monomeric subunit protein enabling nanoparticle formation
The HA protein domain is joined to a monomeric subunit protein such as ferritin, allowing the protein construct to self-assemble into a nanoparticle displaying HA trimers on the surface.
Composition of first and second HA stem amino acid sequences
The HA protein domain comprises a first amino acid sequence upstream of the head region and a second amino acid sequence downstream, each with at least 20 contiguous amino acid residues from the stem region, covalently linked by the linker sequence.
Source and sequence identity of HA stem regions
The first and second amino acid sequences originate from influenza HA proteins of specified strains including A/New Caledonia/20/1999, A/California/04/2009, A/Singapore/1/1957, among others, with specified minimum sequence identity percentages.
Additional HA mutations at defined amino acid positions
The HA protein comprises mutations at amino acid positions corresponding to specific residues (e.g., 36, 45, 47, 49, 339, 340, 341, 342, 361, 372, 394, 402, 437, 438, 445, 446, 448, 449, 450, and 452) of SEQ ID NO:8 to improve properties.
Multivalent nanoparticle formulation
The nanoparticle can be a multivalent nanoparticle comprising more than one type of HA protein to broaden immune response.
Kit comprising the nanoparticle
A kit including the described nanoparticle for use in vaccination or assay applications.
Method of vaccination with the nanoparticle
Methods for vaccinating an individual against influenza virus by administering the nanoparticle to elicit an immune response.
Method of detecting anti-influenza antibodies
Methods for detecting anti-influenza antibodies in a sample by contacting the sample with the nanoparticle and detecting nanoparticle/antibody complexes.
Method of identifying individuals with anti-influenza antibodies
Identifying individuals having anti-influenza antibodies by detecting nanoparticle/antibody complexes formed upon contacting nanoparticle with the individual's sample.
Method of determining vaccine response
Measuring the response of an individual to a vaccine by detecting anti-influenza antibody/nanoparticle complexes in post-vaccination samples and comparing to pre-vaccination levels.
Specific amino acid substitutions for stability enhancement
Substitution of amino acids corresponding to K1 with leucine, isoleucine, methionine or glutamine and E53 with leucine, isoleucine, methionine or glutamine to strengthen interactions in the folded protein.
The claims cover a nanoparticle influenza vaccine comprising a hemagglutinin protein domain with the head region removed and replaced by a short linker, stabilized by specific deletions and amino acid substitutions, fused to a monomeric subunit protein such as ferritin, capable of self-assembly into nanoparticles displaying HA trimers, with defined applicability in inducing broad immune responses and in diagnostic assays.
Stated Advantages
Elicits broadly neutralizing antibodies against the highly conserved HA stem region, overcoming the immunodominance and variability of the HA head.
The nanoparticle vaccine is potent and easily manufactured as fusion proteins self-assemble into stable nanoparticles.
Provides broadly heterosubtypic protective immunity, including protection against diverse seasonal and pandemic influenza strains.
Induces functional antibody-mediated protection even in the absence of strong neutralization titers, potentially via ADCC or complement-mediated mechanisms.
Documented Applications
Use as a vaccine for immunizing individuals against influenza virus infection.
Use in eliciting broadly protective antibodies against multiple influenza virus subtypes.
Use in methods of detecting anti-influenza antibodies in biological samples.
Use in identifying individuals having been exposed to influenza virus based on antibody presence.
Use in determining an individual's immune response to a vaccine.
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