Self-assembling insect ferritin nanoparticles for display of co-assembled trimeric antigens
Inventors
Kwong, Peter • Georgiev, Ivelin • Joyce, Michael Gordon • Kanekiyo, Masaru • Druz, Aliaksandr • Baxa, Ulrich • Van Galen, Joseph • Cheng, Cheng • Mascola, John • Tsybovsky, Yaroslav • Yang, YongPing • Graham, Barney • Moin, Syed Mohammad • Boyington, Jeffrey
Assignees
US Department of Health and Human Services
Publication Number
US-10961283-B2
Publication Date
2021-03-30
Expiration Date
2037-06-27
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Abstract
Disclosed are recombinant insect ferritin nanoparticles that can be used to display two different trimeric antigens at an equal ratio. Also disclosed are nucleic acids encoding the recombinant insect ferritin nanoparticles and methods of producing the recombinant insect ferritin nanoparticles. Methods for eliciting an immune response in a subject are also provided.
Core Innovation
This disclosure provides novel recombinant insect ferritin nanoparticles engineered for the display of two different trimeric antigens at an equal ratio. Unlike bacterial ferritin nanoparticles, which comprise 24 copies of a single subunit, insect ferritin includes twelve copies each of two different subunits termed heavy and light chains, allowing self-assembly into a globular nanoparticle with tetrahedral symmetry. The insect ferritin heavy chains and light chains each trimerize, forming four trimers of heavy chains and four trimers of light chains that assemble into the nanoparticle.
The recombinant insect ferritin nanoparticles disclosed herein include heavy chain fusion proteins each fused at the N-terminus to a first protein antigen and light chain fusion proteins each fused at the N-terminus to a second protein antigen with a different amino acid sequence. Upon assembly into the globular nanoparticle, the first and second protein antigens co-assemble into their respective antigen trimers projecting radially outward. This presentation allows simultaneous recognition of both antigens by B cells, potentially eliciting an immune response with improved neutralization breadth.
The problem addressed by this invention arises from the limitations of prior ferritin nanoparticle technologies, especially those based on bacterial ferritin, which only allow random co-assembly of diverse trimeric antigens without control of their pattern or ratio on the nanoparticle's surface. This disclosure overcomes that by utilizing the nature of insect ferritin heavy and light chains to co-assemble distinct trimeric antigens in equal proportions in a defined geometric pattern, thereby enhancing the immunogenic properties of nanoparticle-based vaccines targeting viral envelope proteins such as HIV-1 Env, influenza HA, RSV F, and MPV F.
Claims Coverage
The patent contains one independent claim regarding a recombinant insect ferritin nanoparticle and several dependent claims elaborating on its components and applications. The main inventive features relate to the structural composition of the nanoparticle, the fusion of two distinct trimeric antigens to different ferritin subunits, and the methods for producing and using these nanoparticles.
Two-component recombinant insect ferritin nanoparticle with tetrahedral symmetry
The nanoparticle consists of twelve recombinant insect ferritin heavy chain fusion proteins and twelve recombinant insect ferritin light chain fusion proteins self-assembled into a globular form with tetrahedral symmetry.
Co-display of two different trimeric antigens at equal ratio
Eight self-assembled heterologous trimeric antigens extend radially outward from the nanoparticle surface, comprising four trimers of a first protein fused to the heavy chain and four trimers of a second, different protein fused to the light chain.
Recombinant insect ferritin heavy and light chains with specific amino acid composition
The heavy chain fusion proteins comprise 172 to 174 amino acids from the C-terminus of an insect ferritin heavy chain and the light chain fusion proteins comprise 182 to 184, or 177 amino acids from the C-terminus of an insect ferritin light chain, with specific sequences such as SEQ ID NO: 2 and SEQ ID NO: 6 disclosed.
Insect ferritin from Trichoplusia ni
The ferritin heavy and light chains are derived from Trichoplusia ni insect ferritin sequences, supporting their use as a scaffold for antigen display.
Fusion of viral envelope protein ectodomains and peptide linkers
The first and second protein antigens are viral envelope protein ectodomain trimers, such as HIV-1 Env, influenza HA (including HA stems), RSV F, MPV F, Ebola GP, or coronavirus S protein ectodomains, fused to ferritin subunits optionally via peptide linkers.
Stabilization of HIV-1 Env in prefusion mature closed conformation
HIV-1 Env ectodomains include modifications such as cysteine substitutions forming non-natural disulfide bonds (positions 201 and 433, 501 and 605) and proline substitution (position 559) to stabilize the trimer conformation.
Production and expression constructs
Nucleic acid molecules encoding the recombinant insect ferritin fusion proteins operably linked to promoters, vectors comprising these nucleic acids, and host cells transformed with the vectors are provided for nanoparticle production.
Methods of eliciting immune responses and treating viral infections
Administering an effective amount of the recombinant insect ferritin nanoparticle to a subject elicits immune responses to the trimeric antigens and can treat or inhibit infections by viruses such as HIV-1, influenza, RSV, and MPV.
The claims cover a structurally defined recombinant insect ferritin nanoparticle displaying two distinct trimeric antigens in a controlled 1:1 ratio with tetrahedral symmetry, specific ferritin subunit truncations and sequences, fusion to viral envelope proteins including stabilizing modifications, compositions, and immunogenic methods using these nanoparticles.
Stated Advantages
Display of two diverse trimeric antigens on the same ferritin nanoparticle allows B cells to simultaneously recognize both, leading to an immune response with improved neutralization breadth.
Use of insect ferritin heavy and light chains allows controlled co-assembly of antigens in a defined geometric pattern and ratio, overcoming limitations of random co-assembly in bacterial ferritin nanoparticles.
Presentation of viral antigens in regular repetitive patterns facilitates B-cell activation and can improve antibody responses.
Recombinant insect ferritin nanoparticles induce broadly neutralizing antibody responses against viral antigens such as HIV-1 and influenza.
Documented Applications
Use as a vaccine platform for multimerized display of trimeric viral antigens to elicit immune responses in subjects.
Eliciting immune responses to HIV-1 through nanoparticles displaying two different HIV-1 Env ectodomains.
Eliciting immune responses to influenza viruses through nanoparticles displaying two different influenza HA ectodomains or HA stem proteins.
Eliciting immune responses to respiratory syncytial virus (RSV) using nanoparticles displaying RSV F ectodomains.
Eliciting immune responses to metapneumovirus (MPV) using nanoparticles displaying MPV F ectodomains.
Methods of treating, inhibiting, or preventing viral infections such as HIV-1, influenza, RSV, and MPV infections by administration of the recombinant insect ferritin nanoparticles.
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