Nanostmctured polypeptides and uses thereof

Abstract

This disclosure relates to a polypeptide having a cyclic arrangement of I53-50A nanostructures, wherein the polypeptide comprises an N-terminal polypeptide segment, a linking polypeptide segment, and a C-terminal polypeptide segment in sequence from the N-terminus to the C-terminus.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 601,517, filed November 21, 2023, and U.S. Provisional Patent Application No. 63 / 552,288, filed February 12, 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] sequence list This application contains a sequence list that has been electronically filed in XML format, and the entire sequence list is hereby incorporated by reference. The XML copy was created on November 14, 2024, named 061291-522001WO.xml, and is 160KB in size. Technical Field

[0003] This invention relates to protein nanostructures, computational methods for designing protein nanostructures, and their use in, for example, vaccines. Background Technology

[0004] Protein nanostructures can be used to display vaccine antigens, serve as gene therapy vectors, or for other purposes.

[0005] The nanostructure I53-50, described in US 2016 / 0122392 A1, is an example of a computationally designed nanostructure. To design I53-50, nanostructures derived from *Thermophyton floccosum* (a type of marine bacterium) were used. Thermotoga maritima The trimer 2-keto-3-deoxy-6-phosphoglucate (KDPG) aldolase (protein database entry 1WA3) and from the intermediate rhizobium of Lophatherum sp. Mesorhizobium loti The structures of the pentamer 2,2-dioxane-pteridine synthase RibH2 (2OBX) were computationally docked to align the symmetry axes of the trimer and pentamer components with the shared symmetry elements of the icosahedron; then Computer simulation The protein-protein interface between the trimer and pentamer components was modified to drive the self-assembly of the two components into a nanostructure with icosahedral symmetry, consisting of trimers and pentamers with 3-fold and 5-fold symmetry axes (referred to as the I53 architecture). The resulting peptide sequence was expressed and purified, and experiments showed that, as predicted, the peptides self-assembled into the expected two-component nanostructure with the I53 architecture. This two-component nanostructure (I53-50) contains 60 copies of each peptide component: 20 copies of the 1WA3-designed trimer component (referred to as "I53-50A") and 12 copies of the designed pentamer component (referred to as "I53-50B").

[0006] Another example of a nanostructure based on the same trimer composition and designated I3-01 is described in US2018 / 0030429 A1. To design I3-01, the structure of KDPG (PDB entry 1WA3) was individually docked to itself; then... count Computer simulation The interfaces between the trimer components were modified to drive the self-assembly of the designed nanostructure. The selected interface residues differed from those in the bicomponent nanostructure. The resulting single polypeptide sequence was expressed and purified, and experiments showed that, as predicted, this polypeptide spontaneously self-assembled into a single-component icosahedral nanostructure with subunits aligned to the icosahedral triplet and protein-protein interfaces on the icosahedral doublet symmetry axis (referred to as the I3 architecture). This single-component nanostructure (I3-01) contained 60 copies of the polypeptide, which were 20 copies of the designed trimer components.

[0007] Both of these designed nanostructures (I53-50 and I3-01) and their variants have been used to manufacture vaccine candidates. For example, US 2020 / 0392187 A1 describes a two-component nanostructure consisting of I53-50A fused to the fusion (F) protein of a pneumovirus and self-assembling with I53-50B to form an icosahedral nanostructure exhibiting F protein trimers on each of the three axes. As another example, WO 2019 / 241483 A1 describes a single-component nanostructure consisting of an engineered envelope (Env) protein fused to the C-terminus of HIV-1's I3-01. This type of nanostructure has demonstrated to be an effective vaccine and is currently in human clinical trials. Specifically, I53-50 nanostructures exhibiting the F protein from respiratory syncytial virus (RSV) or human host pneumovirus (hMPV) or the spike (S) protein of SARS-CoV-2 are in clinical trials as vaccines.

[0008] Nevertheless, there remains a need in the art for novel protein nanostructures capable of displaying other antigens. This disclosure addresses that need. Summary of the Invention

[0009] This disclosure provides a polypeptide having a cyclic arrangement of I53-50A, the polypeptide comprising an assembly domain comprising an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment in sequence from N-terminus to C-terminus, wherein the N-terminal polypeptide segment comprises residues 74-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide segment comprises residues 1-73 of SEQ ID NO: 1 or a variant thereof.

[0010] This disclosure provides a polypeptide having a cyclic arrangement of I53-50A, the polypeptide comprising an assembly domain comprising, in order from N-terminus to C-terminus, an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment, wherein the N-terminal polypeptide segment comprises residues 107-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide segment comprises residues 1-106 of SEQ ID NO: 1 or a variant thereof.

[0011] This disclosure provides a polypeptide having a cyclic arrangement of I53-50A, the polypeptide comprising an assembly domain comprising, in order from N-terminus to C-terminus, an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment, wherein the N-terminal polypeptide segment comprises residues 128-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide segment comprises residues 1-127 of SEQ ID NO: 1 or a variant thereof.

[0012] In some implementations, the variant of SEQ ID NO: 1 is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0013] In some embodiments, the N-terminal polypeptide region and the C-terminal polypeptide region comprise polypeptide sequences, each selected from the A, B, or C pairs provided in the sequence listing, or from variants thereof that are identical to the same as at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0014] In some embodiments, the linker polypeptide segment contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same polypeptide sequence as any of SEQ ID NO: 8-21.

[0015] In some embodiments, the assembly domain comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same polypeptide sequence as any of SEQ ID NO: 22-24.

[0016] In some embodiments, the assembly domain contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the first polypeptide sequence in Table 2 or Table 4.

[0017] This disclosure provides a polypeptide, which is a variant of I53-50A having a C-terminal extension, the polypeptide comprising an assembly domain comprising a base polypeptide segment and an extended polypeptide segment in sequence from the N-terminus to the C-terminus, wherein the base polypeptide segment comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to residues 1-201 of SEQ ID NO: 1.

[0018] In some embodiments, the extended polypeptide segment contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the polypeptide sequence in Table 1.

[0019] In some embodiments, the assembly domain comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same polypeptide sequence as any of SEQ ID NO: 22-25.

[0020] In some embodiments, the assembly domain contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the first polypeptide sequence in Table 2 or Table 4.

[0021] In some implementations, the polypeptide contains one or more amino acid residues at the interface site, enabling the polypeptide to self-assemble to form a trimer component of a single-component nanostructure.

[0022] In some implementations, the polypeptide contains one or more amino acid residues at the interface site, enabling the polypeptide to self-assemble to form a trimer component of a two-component nanostructure.

[0023] In some implementations, the peptide self-assembles to form a trimer component of a nanostructure, wherein the C-terminus of the assembly domain is accessible on the surface of the nanostructure.

[0024] In some embodiments, the peptide self-assembles to form a trimer component, optionally wherein the distance from the C-terminus of the self-assembled domain to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

[0025] In some embodiments, the peptide self-assembles to form a soluble trimer, wherein the C-terminus of the assembly domain is accessible on the surface of the soluble trimer.

[0026] In some embodiments, the peptide self-assembles to form a soluble trimer, wherein the C-terminus of the assembly domain is close to the triple axis of the soluble trimer, optionally wherein the distance from the C-terminus to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

[0027] In some embodiments, the polypeptide is a fusion protein that includes an assembly domain, an optional polypeptide linker, and a heteropeptide in sequence from the N-terminus to the C-terminus.

[0028] In some implementations, the heterologous polypeptide is used as an antigen.

[0029] In some implementations, the antigen is an extracellular domain of a surface protein of a pathogenic object (optionally a virus) or an antigenic fragment thereof.

[0030] In some embodiments, the antigen is OspA or an antigenic fragment thereof, preferably Borrelia burgdorferi (generalized). Borrelia burgdorferi sensu lato OspA.

[0031] In some implementations, the antigen is the extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

[0032] In some implementations, the antigen is an extracellular domain of a bacterial protein or an antigenic fragment thereof.

[0033] This disclosure provides a protein nanostructure comprising a first component containing a first polypeptide and optionally a second component containing a second polypeptide, wherein the first polypeptide is a polypeptide according to this disclosure.

[0034] In some implementations, the first component is a trimer component containing three copies of the first polypeptide.

[0035] In some embodiments, the nanostructure includes a second component, and the second component includes a second assembly domain, the second assembly domain containing a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 26 or 27.

[0036] In some implementations, the second component is a pentamer containing five copies of the second polypeptide.

[0037] In some implementations, the nanostructure comprises 20 copies of the first component.

[0038] In some implementations, the nanostructure further comprises 12 copies of the second component.

[0039] In some implementations, the C-terminus of the first polypeptide is accessible on the surface of the nanostructure.

[0040] In some embodiments, the first polypeptide is a fusion protein that comprises, in order from N-terminus to C-terminus, a first assembly domain, an optional polypeptide linker, and a heteropeptide.

[0041] In some implementations, the heterologous polypeptide is used as an antigen.

[0042] In some implementations, the antigen is an extracellular domain of a surface protein of a pathogenic object (optionally a virus) or an antigenic fragment thereof.

[0043] In some embodiments, the antigen is OspA or an antigenic fragment thereof, preferably OspA of Borrelia burgdorferi.

[0044] In some implementations, the antigen is the extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

[0045] In some implementations, the antigen is an extracellular domain of a bacterial protein or an antigenic fragment thereof.

[0046] In some implementations, the nanostructure has an I53 architecture and / or a quaternary structure that is substantially similar to I53-50.

[0047] This disclosure provides a polynucleotide encoding the nanostructure or polypeptide disclosed herein.

[0048] This disclosure provides a delivery medium comprising the polynucleotides, optionally viral vectors, or lipid nanoparticles disclosed herein.

[0049] This disclosure provides a pharmaceutical composition comprising the nanostructures, polynucleotides, or delivery media disclosed herein, and a pharmaceutically acceptable carrier.

[0050] This disclosure provides a vaccine comprising the nanostructures disclosed herein, the polynucleotides disclosed herein, or the delivery mediums disclosed herein, a pharmaceutically acceptable carrier, and optionally an adjuvant.

[0051] This disclosure provides a host cell suitable for expressing the nanostructures or polypeptides disclosed herein; and / or containing the polynucleotides disclosed herein.

[0052] This disclosure provides a method for preparing peptides or nanostructures, comprising culturing the host cells disclosed herein under conditions suitable for expressing peptides or nanostructures.

[0053] This disclosure provides a method for generating an immune response to an antigen or pathogenic object in a subject in need, comprising optionally administering a vaccine as disclosed herein to the subject via intramuscular injection or inhalation.

[0054] This disclosure provides a method for immunizing a subject against pathogen infection, which includes optionally administering a vaccine as disclosed herein to the subject via intramuscular injection or inhalation.

[0055] This disclosure provides a composition or method as described herein.

[0056] Any aspect or implementation described herein may be combined with any other aspect or implementation disclosed herein. Attached Figure Description

[0057] Figures 1A to 1E The topology diagrams of CompA and its circular arrangement are shown. Figure 1A WT CompA topology. α-helices are indicated by gray bars, labeled H1-H10. β-chains are indicated by white arrows, labeled E1-E8. Figure 1B : Topology diagram of CompA with H11 extending from the head spiral, indicated by dashed lines. Figure 1C : A ring arrangement of CompA with cleavage points between E3 and H4 (residues 73 and 74). Figure 1D : A ring arrangement of CompA with cleavage points between H5 and E5 (residues 106 and 107). Figure 1E : A circular arrangement of CompA with cleavage points between H6 and E6 (residues 127 and 128).

[0058] Figure 2This illustrates a small-scale immobilized metal affinity chromatography (IMAC) pull-down assay of SDS-PAGE. IMAC pull-down flow-through (FT) and elution (E) samples were used to select the construct, showing two bands of expected size in the elution fraction.

[0059] Figure 3 An example negatively stained electron micrograph of a representative construct (CompA.024) is shown, in which the OspA antigen is genetically fused to the carboxyl terminus, demonstrating assembly into a monodisperse VLP of the expected size.

[0060] Figure 4 An exemplary dynamic light scattering size distribution of a representative construct (CompA.024) is shown, in which the OspA antigen is genetically fused to the carboxyl terminus, demonstrating assembly into a monodisperse VLP of the desired size.

[0061] Figures 5A to 5B This illustrates biolayer interference between the antibody and the antigen fused to the carboxyl terminus of the representative construct (CompA.024) or the amino terminus of I53-50CompA. Binding to LA-2 ( Figure 5A ), which binds to the epitope at the carboxyl terminus of the antigen, or to 221-7 ( Figure 5B ), which binds to epitopes along the central structural domain of the antigen.

[0062] Figures 6A to 6C A structural model of a representative design is shown. The extension of the carboxyl terminus with a gray parallel helical segment is illustrated. Figure 6A The extension of the extended ring occurs between the natural carboxyl terminus and the terminal extended helical segment. Figure 6B ). Circular arrangement design ( Figure 6C ).

[0063] Figure 7 Representative particle size distributions of twenty-four constructs (designed to have improved assembly characteristics) assembled after IMAC purification are shown. I53-50 is provided as a reference in each figure.

[0064] Figure 8 SEC chromatograms of four representative assemblies from the purified components are shown.

[0065] Figure 9 The particle size distribution of a representative purified VLP from SEC is shown compared to that of purified I53-50 VLP. Detailed Implementation

[0066] This disclosure generally relates to peptides for forming nanostructures, nanostructures, and uses thereof. In some embodiments, this disclosure provides peptides having the disclosed sequences. In some embodiments, the peptide forms a nanostructure component, wherein the C-terminus of the peptide is accessible on the surface of the nanostructure. In some embodiments, the peptide is a fusion protein comprising, in order from N-terminus to C-terminus, an assembly domain, an optional linker, and a heteropeptide, such as an antigen or an antigenic fragment thereof.

[0067] Computationally designed protein nanomaterials are useful platforms for polymer delivery and vaccine design. Features that make a particular nanomaterial useful include, but are not limited to, modularity, spontaneous self-assembly within the useful concentration range, stability, accessible ends, and particle size. End availability is limited by the composition used to design the particular nanomaterial and the orientation of components within the designed architecture. Not wishing to be theoretically constrained, the local structure of the ends is a contributing element to ensure the correct orientation of any genetically connected domains relative to the surface of the nanomaterial. This disclosure demonstrates that cyclic arrangement can be an effective method for modifying end accessibility. In some embodiments, well-ordered... From the beginning Designed end extensions can also alter end accessibility. In some embodiments, both techniques are used to modify the end availability of nanostructures (e.g., protein nanomaterials I53-50). In some embodiments, cyclic alignment and / or... From the beginning The designed, terminally extended nanomaterials can exhibit the broadly defined Borrelia burgdorferi antigen OspA. In some embodiments, the techniques described herein for altering terminal availability can be applied to I3-01 protein nanomaterials.

[0068] The nanostructure disclosed herein provides an antigen fused to the C-terminus of a first component, such that the antigen is displayed on the surface of the nanostructure. Not wishing to be bound by theory, fusion to the C-terminus may increase or alter the immune response to the antigen. In some cases, fusion to the C-terminus may promote the induction of protective and / or functional immune responses in a subject. In embodiments, the nanostructure comprises a fusion between the C-terminus and N-terminus of the first component via a novel linker polypeptide sequence as shown in Table 3. In embodiments, the nanostructure comprises sequence breaks that generate novel N-terminal and C-terminal breaks compared to a reference sequence fused to the antigen or antigenic fragment.

[0069] This disclosure provides a polypeptide having a cyclic arrangement of I53-50A, the polypeptide comprising an assembly domain comprising an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment in sequence from N-terminus to C-terminus, wherein the N-terminal polypeptide segment comprises residues 74-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide segment comprises residues 1-73 of SEQ ID NO: 1 or a variant thereof.

[0070] MEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGCTE (I53-50A; SEQ IDNO: 1) This disclosure provides a polypeptide having a cyclic arrangement of I53-50A, the polypeptide comprising an assembly domain comprising, in order from N-terminus to C-terminus, an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment, wherein the N-terminal polypeptide segment comprises residues 107-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide segment comprises residues 1-106 of SEQ ID NO: 1 or a variant thereof.

[0071] This disclosure provides a polypeptide having a cyclic arrangement of I53-50A, the polypeptide comprising an assembly domain comprising, in order from N-terminus to C-terminus, an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment, wherein the N-terminal polypeptide segment comprises residues 128-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide segment comprises residues 1-127 of SEQ ID NO: 1 or a variant thereof.

[0072] In some implementations, the variant of SEQ ID NO: 1 is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0073] In some embodiments, the N-terminal polypeptide region and the C-terminal polypeptide region comprise polypeptide sequences, each selected from the A, B, or C pairs provided in the sequence listing, or from variants thereof that are identical to the same as at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0074] In some embodiments, the N-terminal and C-terminal polypeptide regions comprise polypeptide sequences, each selected from pair A, pair B, or pair C, or from variants thereof that are at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them.

[0075] In some embodiments, the linker polypeptide segment contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same polypeptide sequence as any of SEQ ID NO: 8-21.

[0076] In some embodiments, the assembly domain comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same polypeptide sequence as any of SEQ ID NO: 22-24.

[0077] In some embodiments, the assembly domain contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the first polypeptide sequence in Table 2 or Table 4.

[0078] This disclosure provides a C-terminal extended I53-50A polypeptide comprising an assembly domain comprising an N-terminal polypeptide segment and an extended polypeptide segment in sequence from N-terminus to C-terminus, wherein the N-terminal polypeptide segment comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to residues 1-201 of SEQ ID NO: 1.

[0079] In some embodiments, the extended polypeptide segment contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the polypeptide sequence in Table 1.

[0080] As used herein, the term "linking polypeptide segment" refers to a polypeptide in which the C-terminus of a C-terminal polypeptide segment (e.g., the C-terminal polypeptide segment of I53-50A or a variant thereof) is linked to an N-terminal polypeptide segment to computationally generate a cyclic polypeptide chain during cyclic alignment. After computationally generating the cyclic polypeptide, breakpoints between secondary structural elements are identified to generate the N-terminus of the designed polypeptide, which can then be expressed.

[0081] As used herein, the term "extended polypeptide segment" refers to a polypeptide whose C-terminus is extended (e.g., polypeptide segment I53-50A or a variant thereof). In embodiments, the extended polypeptide segment may extend the C-terminus to the vicinity of the N-terminus of the polypeptide segment without attaching the C-terminus to the N-terminus.

[0082] In some embodiments, the assembly domain comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the same polypeptide sequence as any of SEQ ID NO: 22-25.

[0083] In some embodiments, the assembly domain contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the first polypeptide sequence in Table 2 or Table 4.

[0084] In some implementations, the polypeptide contains one or more amino acid residues at the interface site, enabling the polypeptide to self-assemble to form a trimer component of a single-component nanostructure.

[0085] In some implementations, the polypeptide contains one or more amino acid residues at the interface site, enabling the polypeptide to self-assemble to form a trimer component of a two-component nanostructure.

[0086] In some implementations, the peptide self-assembles to form a trimer component of a nanostructure, with the C-terminus of the assembly domain accessible on the surface of the nanostructure.

[0087] In some embodiments, the peptide self-assembles to form a trimer component, optionally wherein the distance from the C-terminus of the self-assembled domain to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

[0088] In some implementations, the peptide self-assembles to form a soluble trimer, with the C-terminus of the assembly domain accessible on the surface of the soluble trimer.

[0089] In some embodiments, the peptide self-assembles to form a soluble trimer, with the C-terminus of the assembly domain close to the triple axis of the soluble trimer, optionally wherein the distance from the C-terminus to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

[0090] In some embodiments, the polypeptide is a fusion protein that includes an assembly domain, an optional polypeptide linker, and a heteropeptide in sequence from the N-terminus to the C-terminus.

[0091] In some implementations, the heterologous polypeptide is used as an antigen.

[0092] In some implementations, the antigen is an extracellular domain of a surface protein of a pathogenic object (optionally a virus) or an antigenic fragment thereof.

[0093] In some embodiments, the antigen is OspA or an antigenic fragment thereof, preferably OspA of Borrelia burgdorferi.

[0094] In some implementations, the antigen is the extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

[0095] In some implementations, the antigen is an extracellular domain of a bacterial protein; or an antigenic fragment thereof.

[0096] This disclosure provides a protein nanostructure comprising a first component containing a first polypeptide and optionally a second component containing a second polypeptide, wherein the first polypeptide is a polypeptide according to this disclosure.

[0097] In some implementations, the first component is a trimer component containing three copies of the first polypeptide.

[0098] In some embodiments, the nanostructure includes a second component, and the second component includes a second assembly domain, the second assembly domain containing a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 26 or 27.

[0099] In some implementations, the second component is a pentamer containing five copies of the second polypeptide.

[0100] In some implementations, the nanostructure comprises 20 copies of the first component.

[0101] In some implementations, the nanostructure further comprises 12 copies of the second component.

[0102] In some implementations, the C-terminus of the first polypeptide is accessible on the surface of the nanostructure.

[0103] In some embodiments, the first polypeptide is a fusion protein that comprises, in order from N-terminus to C-terminus, a first assembly domain, an optional polypeptide linker, and a heteropeptide.

[0104] In some implementations, the heterologous polypeptide is used as an antigen.

[0105] In some implementations, the antigen is an extracellular domain of a surface protein of a pathogenic object (optionally a virus) or an antigenic fragment thereof.

[0106] In some embodiments, the antigen is OspA or an antigenic fragment thereof, preferably OspA of Borrelia burgdorferi.

[0107] In some implementations, the antigen is the extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

[0108] In some implementations, the antigen is an extracellular domain of a bacterial protein or an antigenic fragment thereof.

[0109] In some implementations, the nanostructure has an I53 architecture and / or a quaternary structure that is substantially similar to I53-50.

[0110] This disclosure provides a polynucleotide encoding the nanostructure or polypeptide disclosed herein.

[0111] This disclosure provides a delivery medium comprising the polynucleotides, optionally viral vectors, or lipid nanoparticles disclosed herein.

[0112] This disclosure provides a pharmaceutical composition comprising the nanostructures, polynucleotides, or delivery media disclosed herein, and a pharmaceutically acceptable carrier.

[0113] This disclosure provides a vaccine comprising the nanostructures disclosed herein, the polynucleotides disclosed herein, or the delivery mediums disclosed herein, a pharmaceutically acceptable carrier, and optionally an adjuvant.

[0114] This disclosure provides a host cell suitable for expressing the nanostructures or polypeptides disclosed herein; and / or containing the polynucleotides disclosed herein.

[0115] This disclosure provides a method for preparing peptides or nanostructures, comprising culturing the host cells disclosed herein under conditions suitable for expressing peptides or nanostructures.

[0116] This disclosure provides a method for generating an immune response to an antigen or pathogenic object in a subject in need, comprising optionally administering a vaccine as disclosed herein to the subject via intramuscular injection or inhalation.

[0117] This disclosure provides a method for immunizing a subject against pathogen infection, which includes optionally administering a vaccine as disclosed herein to the subject via intramuscular injection or inhalation.

[0118] This disclosure provides a composition or method as described herein.

[0119] polypeptide Patent publication US 2015 / 0356240 A1 describes various methods for designing protein assemblies. As described in US Patent Publication US 2016 / 0122392 A1 and International Patent Publication WO 2014 / 124301 A1, isolated peptides are designed for their ability to self-assemble in pairs to form protein nanostructures (such as icosahedral particles). This design involves designing suitable interface residues for each member of the peptide pair, which can assemble to form the protein nanostructure. The protein nanostructures thus formed include symmetrically repeating, non-natural, non-covalent peptide-peptide interfaces that orient a first assembly domain and a second assembly domain toward the protein nanostructure, such as a protein nanostructure with icosahedral symmetry.

[0120] Therefore, in one embodiment, the first and second assembly domains of the components are selected from the sequence listing. In each case, N-terminal methionine residues present in the full-length protein are included, but may be removed to prepare a fusion not included in the sequence. Residues identified in the sequence listing are numbered starting from the N-terminal methionine. In various embodiments, one or more additional residues are deleted from the N-terminus and / or additional residues are added to the N-terminus. In some embodiments, the interface residues of the first assembly domain of I53-50A (SEQ ID NO: 1) are 25, 29, 33, 54, and 57. In some embodiments, the interface residues of the second assembly domain of I53-50B (SEQ ID NO: 27) or I53-50B.4PosT1 (SEQ ID NO: 26) are 24, 28, 36, 124, 125, 127, 128, 129, 131, 132, 133, 135, and 139.

[0121] The sequence pairs together form an I53 polymer with icosahedral symmetry. The identified interface residues are the residue numbers identified in each exemplary polypeptide as present at the interface of the resulting assembled protein nanostructure. Right now, (“identified interface residues”). As can be seen, the number of interface residues in the exemplary polypeptides of SEQ ID NO: 1 and (26 or 27) is in the range of 4-13. In various embodiments, the first assembly domain and the second assembly domain comprise amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in length, and identical at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface positions (depending on the number of interface residues in the given polypeptide) to the amino acid sequence of the polypeptide selected from the group consisting of SEQ ID NO: 1 and (26 or 27).

[0122] In some embodiments, the polypeptide used to form the nanostructure includes a first assembly domain. In some embodiments, the polypeptide used to form the nanostructure includes a first assembly domain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the first polypeptide sequence in Table 2 or Table 4.

[0123] In some embodiments, the protein nanostructure comprises a first component and an optional second component. In some embodiments, the first component comprises a first polypeptide containing a first assembly domain. In some embodiments, the protein nanostructure comprises a first component and an optional second component, wherein the first component comprises a first polypeptide containing a first assembly domain, the first assembly domain comprising a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide sequences in Table 2 or Table 4.

[0124] In some implementations, the first component is a trimer component containing three copies of the first polypeptide.

[0125] In some embodiments, the nanostructure comprises a second component. In some embodiments, the second component comprises a second polypeptide containing a second assembly domain. In some embodiments, the nanostructure comprises a second component containing a second polypeptide containing a second assembly domain, the second assembly domain comprising a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following sequence: NQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVINGMMNVQLNTGVPVLSAVLTPHNYDKSKAHTLLFLALFAVKGMEAARACVEILAAREKIAA (I53-50B.4PosT1; SEQ ID NO: 26); or NQHSHKDYETVRIAVVRARWHAEIVDACVSAFEAAMADIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHRYRDSDAHTLLFLALFAVKGMEAARACVEILAAREKIAA (I53-50B; SEQ ID NO: 27).

[0126] In some implementations, the second component is a pentamer containing five copies of the second polypeptide.

[0127] In some embodiments, the first component comprises a first polypeptide containing a first assembly domain, the first assembly domain comprising at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide sequences in Table 2 or Table 4, and the second component comprises a second polypeptide containing a second assembly domain, the second assembly domain comprising at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 100% identical to the sequences listed in Table 2 or Table 4. 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical polypeptide sequence: NQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVINGMMNVQLNTGVPVLSAVLTPHNYDKSKAHTLLFLALFAVKGMEAARACVEILAAREKIAA (I53-50B.4PosT1; SEQ ID NO: 26); or NQHSHKDYETVRIAVVRARWHAEIVDACVSAFEAAMADIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHRYRDSDAHTLLFLALFAVKGMEAARACVEILAAREKIAA (I53-50B; SEQ ID NO: 27).

[0128] In some implementations, the nanostructure comprises 20 copies of the first component.

[0129] In some implementations, the nanostructure further comprises 12 copies of the second component.

[0130] In some implementations, the C-terminus of the first polypeptide is accessible on the surface of the nanostructure.

[0131] In some implementations, the first polypeptide contains one or more amino acid residues at the interface site, enabling the polypeptide to self-assemble to form a trimer component of a single-component nanostructure.

[0132] In some implementations, the first polypeptide contains one or more amino acid residues at the interface site, enabling the polypeptide to self-assemble to form a trimer component of a two-component nanostructure.

[0133] In some implementations, when the first polypeptide self-assembles to form a trimer component of a nanostructure, the C-terminus of the assembly domain is accessible on the surface of the nanostructure.

[0134] In some embodiments, when the first polypeptide self-assembles to form a trimer component, the C-terminus of the assembly domain is close to the triple axis of the trimer component. In some embodiments, the distance from the C-terminus to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

[0135] antigen In some embodiments, the protein nanostructure comprises a first component and an optional second component. In such embodiments, the first component comprises a first polypeptide containing a first assembly domain, and optionally, the second component comprises a second polypeptide containing a second assembly domain.

[0136] In some embodiments, the first polypeptide is a fusion protein that comprises, in order from the N-terminus to the C-terminus, a first assembly domain, an optional linker, and a heterologous polypeptide sequence, preferably an antigen.

[0137] In some embodiments, the antigen is an extracellular domain of a surface protein of a pathogenic organism or an antigenic fragment thereof. In some embodiments, the antigen is an extracellular domain of a surface protein of a virus or an antigenic fragment thereof.

[0138] Unwilling to be bound by theory, antigens can naturally anchor at or near their N-terminus.

[0139] Compared to antigens fused to the N-terminus of the first assembly domain, antigens with N-terminal attachments in their native orientation may exhibit improved stability or antigenicity when fused to the C-terminus of the first assembly domain.

[0140] In some implementations, the antigen is fused to the C-terminus of the first assembly domain.

[0141] Exemplary bacterial surface proteins naturally anchored to their N-terminus include, but are not limited to, OspA of *Borrelia generalis*, OspB of *Borrelia generalis*, and *Neisseria meningitidis*. (N. Meningitidis) fHbp, asymmetric or C3-symmetric oligomers of bacterial type II membrane proteins, and asymmetric or C3-symmetric oligomers of bacterial proteins with lipidation sites facing the N-terminus. Exemplary nonbacterial surface proteins naturally anchored to their N-terminus include, but are not limited to, paramyxovirus and / or pneumovirus G proteins.

[0142] In some embodiments, the antigen is OspA or an antigenic fragment thereof. In some embodiments, the antigen is OspA of Borrelia burgdorferi (SEQ ID NO: 28). In some embodiments, the antigen is OspB or an antigenic fragment thereof. In some embodiments, the antigen is OspB of Borrelia burgdorferi (SEQ ID NO: 29).

[0143] In some implementations, the antigen is the fHbp (SEQ ID NO: 30) of Neisseria meningitidis or an antigenic fragment thereof.

[0144] In some implementations, the antigen is derived from a bacterial pathogen exhibiting an asymmetric type II membrane geometry.

[0145] In some implementations, the antigen is an antigen derived from a bacterial pathogen exhibiting a C3-symmetric oligomer geometry.

[0146] In some embodiments, the antigen is an antigen derived from a bacterial pathogen that contains lipidation sites on the N-terminal portion of a protein and exhibits an asymmetric type II membrane geometry.

[0147] In some embodiments, the antigen is an antigen derived from a bacterial pathogen that contains lipidation sites on the N-terminal portion of a protein and exhibits a C3-symmetric oligomer geometry.

[0148] In some embodiments, the antigen is RSV G protein (SEQ ID NO: 31) or an antigenic fragment thereof. In some embodiments, the antigen is hMPV G protein (SEQ ID NO: 32) or an antigenic fragment thereof.

[0149] In some implementations, the antigen is the paramyxovirus and / or pneumonia virus G protein or an antigenic fragment thereof.

[0150] In some implementations, the antigen is the S1 C-terminal domain of coronaviruses, the RBD of paramyxovirus G, H, or HN proteins (e.g., Nipah / Hendra G, PIV3 HN, measles H, mumps HN), the HA head domain of influenza, the fusion domain of class III fusion proteins (e.g., CMV, EBV, HSV, VZV, rabies), gp120 of HIV Env, engineered antigens (e.g., eOD), the VP8 domain of rotavirus, a segment / domain of Plasmodium falciparum CSP, or a segment / domain (e.g., the C-terminal domain) of Borrelia generalis OspA.

[0151] In some implementations, the antigen is the extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

[0152] In some embodiments, the antigen is an extracellular domain of a parasitic protein or an antigenic fragment thereof. In some embodiments, the parasitic protein is derived from Plasmodium parasite.

[0153] In some implementations, the antigen is an extracellular domain of a bacterial protein or an antigenic fragment thereof.

[0154] Polynucleotides, vectors and host cells This disclosure provides a polynucleotide that encodes a nanostructure of any of the embodiments herein or a polypeptide of any of the embodiments herein.

[0155] This disclosure provides a carrier comprising a polynucleotide encoding a nanostructure of any one of the embodiments herein or a polypeptide of any one of the embodiments herein.

[0156] This disclosure provides a host cell suitable for expressing a nanostructure of any of the embodiments herein or a polypeptide of any of the embodiments herein; and / or containing a polynucleotide of any of the embodiments herein.

[0157] This disclosure provides a method for preparing peptides or nanostructures, comprising culturing host cells of any of the embodiments herein under conditions suitable for expressing the peptides or nanostructures of any of the embodiments herein.

[0158] In another aspect, this disclosure provides a polynucleotide encoding any of the aforementioned polypeptides. The polynucleotide may be mRNA, such as modified mRNA. This disclosure further provides a vector comprising any of these polynucleotides. The vector may be a viral vector, such as an adenovirus vector, or a non-viral vector, such as lipid nanoparticles (LNPs). This disclosure further provides host cells transfected or transformed with any of the aforementioned polynucleotides.

[0159] In one aspect, this disclosure provides a method for preparing protein nanostructures, which involves culturing host cells under conditions suitable for the expression of one or more components, individually or separately, that induce the nanostructure; purifying the components individually or separately; contacting a solution of the purified components; and / or incubating the components under conditions suitable for the self-assembly of the components to form nanostructures.

[0160] Pharmaceutical compositions and vaccines This disclosure also provides pharmaceutical compositions. Such pharmaceutical compositions can be used to induce an immune response against an infectious disease in a subject. The pharmaceutical compositions of this disclosure may comprise a pharmaceutically acceptable carrier. A detailed discussion of such carriers can be found in [link to relevant documentation]. Remington: The Science and Practice of PharmacyIn Chapter 30 of (23rd edition, 2021).

[0161] In some embodiments, the pharmaceutical composition may also comprise excipients and / or additives. Examples of such excipients and / or additives are surfactants, stabilizers, complexing agents, antioxidants, or preservatives that prolong the use duration of the final pharmaceutical formulation, flavoring agents, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or its salts (such as disodium EDTA), citric acid, hypozinotriacetic acid, and their salts. In some embodiments, preservatives include, but are not limited to, those that protect the solution from contamination by pathogenic particles, including benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, their salts, or esters.

[0162] Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose, or starch), fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments. Such preparations can be sterilized and, if desired, mixed with excipients such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, and / or aromatic substances, and analogs that do not adversely react with the compounds of this disclosure. Those skilled in the art will recognize that other pharmaceutical excipients may be used in this disclosure.

[0163] In some implementations, one or more tonic agents may be added to provide the desired ionic strength. Tonic agents as used herein include those that do not exhibit pharmacological activity or only exhibit negligible pharmacological activity after application. Both inorganic and organic tonic modifiers may be used.

[0164] In another aspect, this disclosure provides a pharmaceutical composition comprising a polypeptide, protein complex, or nanostructure of the present disclosure.

[0165] This disclosure provides a pharmaceutical composition comprising a nanostructure of any of the embodiments described herein.

[0166] This disclosure provides a vaccine comprising any of the nanostructures described in the embodiments herein.

[0167] In another aspect, this disclosure provides pharmaceutical compositions or vaccines. In embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the nanostructures as described herein. In embodiments, the vaccine comprises an amount of nanostructures that effectively elicit an immune response in a subject. The nanostructures used in the vaccine may be complexed, conjugated, or fused with an antigen. The antigen may be a polypeptide derived from a pathogenic organism or an antigenic fragment thereof.

[0168] In other embodiments, the pharmaceutical composition or vaccine comprises a polynucleotide (such as mRNA) encoding a nanostructure as disclosed herein or a carrier (such as LNP) as disclosed herein (in each case, a therapeutically effective amount or an amount that effectively generates an immune response).

[0169] In another aspect, this disclosure provides methods for treating and / or preventing diseases or conditions in subjects in need, as well as methods for inducing an immune response against pathogenic objects in subjects. Such methods may include administering a nanostructure, pharmaceutical composition, or vaccine according to this disclosure to a subject via intramuscular, intravenous, or intranasal route.

[0170] Furthermore, a medicine box and a pre-filled syringe containing any of the aforementioned compositions are provided.

[0171] vaccine In another aspect, this disclosure provides a vaccine comprising a polypeptide, protein complex, or nanostructure as disclosed herein.

[0172] In some implementations, the vaccine contains an adjuvant.

[0173] adjuvant Adjuvants or immunostimulants may also be administered co-administered or in combination with lipid nanoparticle compositions. The advantages of adjuvants include, but are not limited to, enhancing the immunogenicity of antigens, modifying the nature of the immune response, reducing the amount of antigen required for successful immunization, reducing the frequency of required booster immunizations, and improving immune responses in older adults and immunocompromised vaccine recipients. These adjuvants can be co-administered via any route, such as intramuscular, subcutaneous, intravenous, or intradermal injection.

[0174] Adjuvants may include, but are not limited to, natural or synthetic adjuvants. Adjuvants may be organic or inorganic.

[0175] The adjuvant may be selected from any of the following categories: (1) mineral salts, such as aluminum hydroxide and aluminum phosphate or calcium phosphate gels; (2) emulsions, including oil emulsions and surfactant-based formulations, such as microfluidic cleaner-stabilized oil-in-water emulsions, purified saponins, oil-in-water emulsions, and stabilized water-in-oil emulsions; (3) particulate adjuvants, such as virions (monolithic liposomes incorporating influenza hemagglutinin), structured complexes of saponins and lipids, and polylactic acid co-glycolic acid (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; (6) inert mediators, such as gold particles; (7) microbial-derived adjuvants; (8) tonicotinic compounds; (9) carbohydrates; or combinations thereof.

[0176] Adjuvants for nucleic acid vaccines (DNA) have been disclosed, for example, in Kobiyama et al., Vaccines, 2013, 1(3), 278-292, the contents of which are incorporated herein by reference in their entirety. (via Kobiyama et al.) et al. Any publicly disclosed adjuvant may be used in vaccines as described herein.

[0177] Other available adjuvants include those listed in web-based vaccine adjuvant databases, available on the World Wide Web violinet.org / vaxjo / , and described by, for example, Sayers et al. J. Biomedicine and Biotechnology The adjuvants mentioned in Volume 2012 (2012), Article ID 831486, total 13 pages, are incorporated herein by reference in their entirety.

[0178] Specific adjuvants may include cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, potassium aluminum sulfate adjuvant, hydrogel, ISCOM(s)™, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA vaccine adjuvant, cholera toxin, cholera toxin B subunit, liposome, saponin vaccine adjuvant, DDA adjuvant, squalene-based adjuvant, Etx B subunit adjuvant, IL-12 vaccine adjuvant, LTK63 vaccine mutant adjuvant, TiterMax Gold adjuvant, Ribi vaccine adjuvant, Montanide ISA 720 adjuvant, Corynebacterium-derived P40 vaccine adjuvant, MPL™ adjuvant, AS04, AS02, AS01. ELipopolysaccharide vaccine adjuvant, muramyl dipeptide adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetella pertussis component vaccine adjuvant, cationic liposome vaccine adjuvant, adamantamide dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, ADJUMER™, algal dextran, Bay R1005, Theramide®, Stearoyl Tyrosine, Spol, Algammulin, AVRIDINE®, Calcium Phosphate Gel, CTA1-DD Gene Fusion Protein, DOC / Alum Complex, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Recombinant hIFN-γ / Interferon-g, Interleukin-1β, Interleukin-2, Interleukin-7, Sclavo Peptide, Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE Liposomes, Murametide, Murapamitine, D-Murapamitine, NAGO, Nonionic Surfactant Vesicles, PMMA, Protein Cochleate, QS-21, SPT (Antigen preparations), Nanoemulsion vaccine adjuvants, AS03, Quil-A vaccine adjuvants, RC529 vaccine adjuvants, LTR192G vaccine adjuvants, Escherichia coli heat-labile toxin, LT, Amorphous aluminum hydroxyphosphate sulfate adjuvants, Calcium phosphate vaccine adjuvants, Montanide incomplete Seppic adjuvants, Imiquimod, Resiquimod, AF03, Flagellin, Poly(I:C), ISCMATRIX®, Abisco-100 vaccine adjuvants, Albumin-Heparin microparticle vaccine adjuvants, AS-2 vaccine adjuvants, B7-2 vaccine adjuvants, DHEA vaccine adjuvants, Immunoliposomes containing co-stimulatory molecule antibodies, SAF-1, Sendai Proteoliposome, Sendai lipid matrix, Threonyl muramyl dipeptide (TMDP), Ty particle vaccine adjuvants, Bupivacaine vaccine adjuvants, DL-PGL (Poly(DL-polylactic acid-co-glycolic acid)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant E112K of cholera toxin mCT-E112K and / or matrix-S.

[0179] In some embodiments, the adjuvant includes squalene. In some embodiments, the adjuvant includes aluminum hydroxide. In some embodiments, the adjuvant includes AS01. E .

[0180] How to use This disclosure provides a method for preparing peptides or nanostructures, comprising culturing the host cells disclosed herein under conditions suitable for expressing peptides or nanostructures.

[0181] This disclosure provides a method for inducing an immune response to an antigen or pathogenic material in a subject in need, comprising administering the nanostructure of this disclosure to the subject.

[0182] This disclosure provides a method for generating an immune response to an antigen or pathogenic object in a subject in need, comprising optionally administering a vaccine as disclosed herein to the subject via intramuscular injection or inhalation.

[0183] This disclosure provides a method for generating high-titer functional antibodies against antigens or against pathogenic organisms.

[0184] This disclosure provides a method for immunizing a subject against pathogen infection, which includes optionally administering a vaccine as disclosed herein to the subject via intramuscular injection or inhalation.

[0185] In another aspect, this disclosure provides methods of administering the compositions, pharmaceutical compositions, or vaccines described herein.

[0186] In another aspect, this disclosure provides a method for vaccinating a subject, comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method for generating an immune response in a subject, comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a method for treating or preventing a disease in a subject, comprising administering the composition described herein to the subject. In another aspect, this disclosure provides a composition of this disclosure for use in vaccinating, generating an immune response, or treating or preventing a disease. In another aspect, this disclosure provides a composition, method, or use as described herein. In another aspect, this disclosure provides a method for preparing a composition comprising culturing host cells modified to express one or more polypeptides as described herein.

[0187] In some embodiments, the method includes administering the vaccine described herein. In some embodiments, the vaccine is administered via subcutaneous injection. In some embodiments, the vaccine is administered via intramuscular injection. In some embodiments, the vaccine is administered via intradermal injection. In some embodiments, the vaccine is administered intranasally. In one aspect, this disclosure provides a pre-filled syringe containing the vaccine described herein. In another aspect, this disclosure provides a kit comprising either the vaccine described herein or the pre-filled syringe described herein.

[0188] In some embodiments, the unit dose of the pharmaceutical composition comprises about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 70 μg to about 75 μg, about 100 μg to about 125 μg, about 100 μg to about 150 μg, about 125 μg to about 175 μg, about 200 μg to about 250 μg, about 225 μg to about 300 μg, or about 250 μg to about 350 μg of protein nanostructures.

[0189] In some embodiments, the unit dose of the pharmaceutical composition comprises about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 25 μg to about 50 μg, about 50 μg to about 70 μg, about 70 μg to about 75 μg, about 75 μg to about 100 μg, about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, about 175 μg to about 200 μg, about 200 μg to about 250 μg, or about 250 μg to about 300 μg of protein nanostructures.

[0190] definition Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” are also intended to include the plural forms.

[0191] As used herein, the term "about" means a range of values ​​that include a specified value and that a person skilled in the art would consider reasonably similar to the specified value. For example, using measurements generally acceptable in the art, about means within the standard deviation. For example, about means a range extending to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0192] The term “at least” followed by a number is used in this document to indicate the beginning of a range that begins with said number (which may be a range with or without an upper limit, depending on the defined variable). For example, “at least 1” means 1 or greater than 1.

[0193] The term "at most" followed by a number is used herein to indicate the end of a range that ends with said number (which may be a range with an upper limit of 1 or 0 or an unbounded range, depending on the defined variable). For example, "at most 4" means 4 or less, and "at most 40%" means 40% or less. When a range is given in this specification as "(first number) to (second number)" or "(first number) - (second number)", this means a range with the lower limit of the first number and the upper limit of the second number. For example, 25 to 100 mm means a range with the lower limit of 25 mm and the upper limit of 100 mm.

[0194] In the context of two or more nucleic acid or peptide sequences, the term "identical" or percentage "identity" means that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned to obtain maximum correspondence. Sequence alignment methods used for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions in both sequences where the same nucleotide or amino acid residue is present. Percentage sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence and then multiplying the resulting value by 100. For example, when aligned to a reference sequence with 1554 amino acids, a peptide sequence with 1166 matches is 75.0% identical to the test sequence (1166 ÷ 1554 * 100 = 75.0). When used herein, vacancies in the alignment do not reduce percentage sequence identity. Unless otherwise stated, the best sequence alignment used for comparison is the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) implemented by EMBOSS Needle (available at ebi.ac.uk / Tools / psa / emboss_needle / on the World Wide Web) (Madeira et al.) Nucleic Acids Res. The alignment can be performed using 50(W1):W276-W279 (2022)). Other alignment methods may be used, including but not limited to those described in the following literature: Devereux et al., Nucleic Acids Res. 12:387-95 (1984); Atschul et al. J. Mo. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Appl. Math.48(5)(1988); Computational Molecular Biology (Lesk, AM, ed., 1989); BiocomputingInformatics and Genome Projects, (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, ed., 1994); Sequence Analysis in Molecular Biology (von Heinje, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., ed., 1993). Sequence identity was computed using an implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (available at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0195] For example, sequence identity can be determined using standard methods commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using computer programs (such as EMBOSS Needle or BLAST), two polypeptide or two polynucleotide sequences are aligned to achieve optimal matching of their respective residues (along the full length of one or both sequences, or along a predetermined portion of one or both sequences). These programs provide default open penalties and default vacancy penalties, as well as scoring matrices such as PAM 250 (a standard scoring matrix) that can be used in conjunction with the computer program. See Dayhoff et al., in Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3 (1978).

[0196] The term "substantially similar" means that two polypeptides, proteins, assemblies, nanostructures, or other physical embodiments of the present invention may differ in architecture, sequence, conformation, association, and similarity, but provide substantially the same or similar properties, structures, activities, and / or functions. For example, a nanostructure having an I53 architecture and / or quaternary structure provides substantially the same or similar properties, activities, and / or functions as a nanostructure as a nanostructure having an I53-50 architecture and / or quaternary structure. In other words, embodiments of the present disclosure are interchangeable but still achieve the desired results, such as in terms of properties, structures, activities, and / or functions.

[0197] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises / comprising”, “has / having”, and “includes / including” shall be understood to imply inclusion of the stated element or step or group of elements or steps, but not to exclude any other element or step or group of elements or steps. “consisting essentially of / consists essentially” indicates the exclusion of elements or steps that substantially affect the basic and novel features of the claimed invention.

[0198] Any aspect or implementation described herein may be combined with any other aspect or implementation disclosed herein.

[0199] Example Features that make certain nanomaterials useful include modularity, spontaneous self-assembly within the useful concentration range, stability, accessible ends, and particle size. Ring alignment is one method for altering end accessibility. Alternatively, well-ordered de novo-designed end extensions can also alter end accessibility. Here, we have used two techniques to alter the end availability of the protein nanomaterial I53-50 and demonstrated the utility of these novel constructs by exhibiting the generalized Borrelia burgdorferi antigen OspA.

[0200] Example 1: Extension of the C-terminus of I53-50A to make the C-terminal surface accessible This embodiment demonstrates a computational design for an I53-50A variant (referred to as “CompAext”) where the C-terminus of the protein is accessible on the surface of the trimer components, enabling the display of peptide fusions, such as antigens, at the N-terminus of the protein. After modeling this extension, the protein is remodeled to improve contact between the extension and adjacent segments. This results in extensive remodeling of the N-terminal residues of I53-50A, as well as modifications to residues scattered throughout the primary structure (sequence) of I53-50A.

[0201] Use RFDesign to repair the design from the C-end. From the beginning Spiral sections. The loop length between adjacent sections is preserved, but design flexibility is still maintained. This allows for optimization with any... From the beginning The original loop in the contact segment. Between amino acids 1 and 30. From the beginning Segment lengths are sampled. Rfdiffusion is used to construct the final spiral segment. Allowed. From the beginning Spread across sections and adjacent areas. (Introduction) From the beginning In the case of the element, a series of lengths were sampled around the length identified by repair. The highest-scoring design from RFdiffusion was selected for sequence design in ProteinMPNN. The structure of the top sequence was predicted using ColabFold and compared with the design model. The results of the design process are peptide sequences as disclosed in Table 1. A flexible linker sequence was included at the C-terminus of each design to further facilitate the fusion of other peptides (e.g., antigens or purification tags) with the C-terminus. This C-terminal linker and N-terminal leader sequence are underlined; the underlined sequence is optional as it can be replaced by other linkers and leader sequences.

[0202] The resulting constructs are the sequences in Table 2 below, which have "None" in the sort column.

[0203] Extensions of each design are listed in Table 1.

[0204] Table 1: Elongation peptide segments used for C-terminal elongation

[0205] Example 2: Arrangement of CompAext This embodiment describes the cyclic arrangement of I53-50. Various arrangements were modeled computationally. Preferred results pertain to the cyclic arrangement of CompAext. Conceptually, firstly, the C-terminus of I53-50Aext was linked to its N-terminus using an extended polypeptide segment to generate a cyclic polypeptide chain. Secondly, breakpoints between secondary structural elements were identified to generate the N-terminus. Three preferred breakpoints were identified at approximately residues 73, 106, and 127. These arrangements are referred to as Arrangement 1, Arrangement 2, and Arrangement 3, respectively. Finally, computational modeling was applied to design novel contacts between secondary structural elements within the tertiary structure of Arrangements 1, 2, and 3. The resulting sequences are provided in Table 2.

[0206] Arrangement of structural components: I53-50 CompA (or “I53-50A”) is a TIM barrel fold derived from protein 1WA3. A TIM barrel is an approximately circular repeating protein composed of eight pairs of β-chains and α-helices. The β-chains are parallel and oriented around the lumen of the solvation center, with the helices on the outer surface. The lumen is typically capped at one or both ends with additional terminal helical segments. This is also the case for CompA with an N-terminal capped helix (H1). Therefore, the secondary structural elements of CompA are, in order from N-terminus to C-terminus, H1, E1, H2, E2, ..., H8, E8, H9. Due to the structure of the TIM barrel, the protein can be divided into pairs of secondary structural elements and reassembled in any order simply by designing new linking loops. However, some linking pairs are more likely to successfully fold into the desired structure than others. 1WA3 is also a C3-symmetric homotrimer, and most of the interface is formed by loops between the chains and helices, which further limits the number of possible links. Applying those restrictions, the order of helices and chains within the peptide sequence is arranged under the further restriction that helices and chains must alternate, resulting in 3474 possible arrangements.

[0207] To evaluate these permutations, closed loops were repaired using RFDesign. Loop lengths were preserved while maintaining the adjacency of the permutation segments with their original adjacent segments, allowing for design optimization. This enabled the optimization of original loops with any de novo contact loops. Loop lengths between 1 and 30 amino acids were sampled in cases where the sequence differed from the original sequence.

[0208] Selection of Arrangement Elements Most permutations result in poor-quality loop closures or make it impossible to find a viable solution. Among closed permutations, the simplest permutation is ( Right now, Connecting the C-terminus to the N-terminus and then introducing cutoff points elsewhere in the sequence yields the highest score. The permutation with the minimum lddt > 0.75 is selected. Some of these permutations introduce irreconcilable conflicts with the symmetric copies of CompB or CompA in the I53-50 assembly and are discarded.

[0209] Ring and end design To construct the final loop, Rfdiffusion was used. Diffuse of the loop and adjacent regions was allowed. With de novo elements introduced, a range of lengths were sampled around the length identified by repair. The highest-scoring design from Rfdiffusion was selected for sequence design in ProteinMPNN. The structure of the top sequence was predicted using ColabFold and compared with the design model. The results of the design process were peptide sequences as disclosed in Table 2. A linker was included at the C-terminus of each design to further facilitate the fusion of other peptides (e.g., antigens) with the C-terminus. This C-terminal linker and N-terminal leader sequence are underlined; the underlined sequence is optional as it can be replaced with other linkers and leader sequences.

[0210] Table 2: Circular arrangement and C-terminal extension

[0211] The connecting polypeptide segments of each in the arrangement design are listed in Table 3.

[0212] Table 3: Linking polypeptide segments used for circular arrangement

[0213] Example 3: Experimental Verification Designs with pLDDT > 0.90 were prioritized for use in Escherichia coli ( E. coliBicistronic plasmids expressed in the cytoplasm of *E. coli* were used. In one set of constructs, one open reading frame (OPF) encodes CompB (I53-50B); another OSpA encodes each of the CompA (I53-50A variants) listed in Table 2 above, with a 6xHis tag at the C-terminus of CompA. In another set of experiments, one OSpA encodes a full-length wild-type OspA fused to the C-terminus of each of the CompA (I53-50A variants) in Table 2 (in 5' to 3' order, I53-50A variant, peptide linker, and OspA); and another OSpA encodes CompB (I53-50B.4PosT1) with a 6xHis tag at the C-terminus. When expressed in *E. coli* cytoplasm, successful designs were expected to assemble into I53-50-derived nanostructures. Table 2 lists the selected designs. In each sequence in Table 2, the start codon used to link to each sequence beginning with OspA and the peptide linker sequence at the C-terminus are underlined.

[0214] Screening and characterization Through E. coli middle Constructs were screened using 2 ml scale expression and dissolved using sonication. The clarified solutions were purified using Ni-NTA MagBeads. Purification was characterized by SDS-PAGE. Constructs were considered pass if both components were present in the elution fraction, fail if only CompB was present, and indeterminate if only CompA was observed in the elution fraction or no component was observed.

[0215] The constructs were further characterized by expression in *E. coli* at a scale of 500 mL and dissolved using sonication or microfluidization, as open reading frames encoding either a bicistronic construct with CompB or a monocistronic construct encoding only CompA. The clarified solutions were purified by Ni-NTA gravity column chromatography and further purified by SEC. The purified VLPs were characterized by DLS and negative stained EM. Figure 3 The purified CompA was mixed with the purified CompB, further purified by SEC to remove excess components, and characterized by DLS. Figure 4 The effect of circular arrangement on antigen accessibility was assessed by BLI (Figure 5). Antibodies bound to the C-terminus of the antigen were blocked when the genetic fusion from the C-terminus of the antigen to the N-terminus of CompA was displayed on I53-50CompA. Significantly higher antibody binding was observed when the genetic fusion from the N-terminus of the antigen to the C-terminus of CompA was displayed on CompA.024. Figure 5AThis demonstrates improved epitope accessibility. When displayed on I53-50 CompA or CompA.024, antibodies binding to the epitope in the middle of the antigen also bind well to the antigen. Figure 5B Provides representative end extensions ( Figure 6A , Figure 6B ) and circular arrangement ( Figure 6C ) structural model.

[0216] In vitro assembly enhancement To improve in vitro assembly, a second set of designs obtained using multiple methods was sorted (Table 4). These designs were expressed as monocistronic constructs in *E. coli* and purified by IMAC. CompB was mixed with IMAC eluent, and VLP assemblies were measured by DLS. Figure 7 The selection of constructs having the same size distribution as the I53-50 assembly was further purified by SEC. The SEC-purified constructs were mixed in excess with CompB, and the resulting assembly was purified by SEC using a Superose 6 column. A small excess of CompA peak was observed in the SEC chromatogram, eluting at approximately 17.5 ml, but no excess of CompB was observed. Figure 8 The major fractions from the VLP peak (approximately 12 ml elution volume) were collected and analyzed by DLS. Figure 9 ).

[0217] Table 4: Extra Circular Arrangement and C-Terminal Extension

[0218] List of implementation plans Clause 1. A polypeptide for forming a nanostructure comprising an assembly domain comprising at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the first polypeptide sequence in Table 2.

[0219] Clause 2. A protein nanostructure comprising a first component and an optional second component, wherein the first component comprises a first polypeptide containing a first assembly domain, the first assembly domain comprising a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide sequences in Table 2.

[0220] Clause 3. The nanostructure as described in Clause 2, wherein the first component is a trimer component comprising three copies of the first polypeptide.

[0221] Clause 4. A nanostructure as described in Clause 1 or Clause 2, wherein the nanostructure comprises a second component and wherein the second component comprises a second assembly domain comprising a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following sequence: NQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVINGMMNVQLNTGVPVLSAVLTPHNYDKSKAHTLLFLALFAVKGMEAARACVEILAAREKIAA (SEQ ID NO: 26); or NQHSHKDYETVRIAVVRARWHAEIVDACVSAFEAAMADIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHRYRDSDAHTLLFLALFAVKGMEAARACVEILAAREKIAA (SEQ ID NO: 27).

[0222] Clause 5. The nanostructure as described in Clause 4, wherein the second component is a pentamer comprising five copies of the second polypeptide.

[0223] Clause 6. The nanostructure as described in any one of Clauses 2-5, wherein the nanostructure comprises 20 copies of the first component.

[0224] Clause 7. The nanostructure as described in any one of Clauses 2-6, wherein the nanostructure further comprises 12 copies of the second component.

[0225] Clause 8. The nanostructure as described in any one of Clauses 2-7, wherein the C-terminus of the first polypeptide is accessible on the surface of the nanostructure.

[0226] Clause 9. The nanostructure as described in any one of Clauses 2-8, wherein the first polypeptide is a fusion protein comprising, in order from N-terminus to C-terminus, a first assembly domain, an optional linker, and a heterologous polypeptide sequence, preferably an antigen.

[0227] Clause 10. The nanostructure as described in Clause 9, wherein the antigen is an extracellular domain of a surface protein of a pathogenic object (optionally a virus); or an antigenic fragment thereof.

[0228] Clause 11. The nanostructure as described in Clause 10, wherein the antigen is OspA or an antigenic fragment thereof, preferably OspA of Borrelia burgdorferi.

[0229] Clause 12. The nanostructure as described in Clause 9, wherein the antigen is an extracellular domain of a viral glycoprotein; or an antigenic fragment thereof.

[0230] Clause 13. The nanostructure as described in Clause 9, wherein the antigen is an extracellular domain of a bacterial protein; or an antigenic fragment thereof.

[0231] Clause 14. A method for inducing an immune response to an antigen or a pathogenic object in a subject in need, comprising administering to the subject any of the nanostructures described in any of Clauses 2-13.

[0232] Clause 15. A pharmaceutical composition comprising any one of the nanostructures described in Clauses 2-13.

[0233] Clause 16. A vaccine comprising any one of the nanostructures described in Clauses 2-13.

[0234] Clause 17. A polynucleotide encoding a nanostructure as described in any one of Clauses 2-13 or a polypeptide as described in Clause 1.

[0235] Clause 18. A host cell suitable for expressing any one of the nanostructures described in Clauses 2-13 or the polypeptide described in Clause 1; and / or comprising the polynucleotide described in Clause 17.

[0236] Clause 19. A method for preparing a polypeptide or nanostructure, comprising culturing the host cells described in Clause 18 under conditions suitable for expressing the polypeptide or nanostructure.

[0237] sequence list

[0238] Merging by reference The full disclosure of each of the patents and scientific documents mentioned in this article is incorporated herein by reference for all purposes.

[0239] References to any previously published (or derived) information or any known matters in this specification are not and should not be construed as an admission or endorsement or in any way imply that previously published (or derived) information or known matters form part of the general knowledge in the field of effort covered by this specification.

[0240] equivalent The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the foregoing embodiments should be considered illustrative in all respects and not limiting of the invention described herein. Consequently, the scope of the invention is indicated by the appended claims rather than the foregoing description, and all variations within the equivalent meaning and scope of the claims are intended to be included therein.

Claims

1. A polypeptide having a cyclic arrangement of I53-50A, said polypeptide comprising an assembly domain, The assembly domain comprises, in order from N-terminus to C-terminus, an N-terminal polypeptide segment, a linker polypeptide segment, and a C-terminal polypeptide segment, wherein: a) The N-terminal polypeptide region comprises residues 74-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide region comprises residues 1-73 of SEQ ID NO: 1 or a variant thereof; b) The N-terminal polypeptide region comprises residues 107-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide region comprises residues 1-106 of SEQ ID NO: 1 or a variant thereof; or c) The N-terminal polypeptide region comprises residues 128-201 of SEQ ID NO: 1 or a variant thereof, and the C-terminal polypeptide region comprises residues 1-127 of SEQ ID NO: 1 or a variant thereof; Among them, its variants are at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the reference sequence.

2. The polypeptide as described in claim 1, The N-terminal polypeptide region and the C-terminal polypeptide region comprise polypeptide sequences, each selected from pair A, pair B, or pair C, or from variants thereof that are at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to them. 。 3. The polypeptide of any one of claims 1 to 2, wherein the linking polypeptide segment comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the following polypeptide sequences: KKVGEELIKLVTRPEDR (SEQ ID NO: 8); KEVGKKLLLLVTDPADKK (SEQ ID NO: 9); DKESEKLIKLETDPAMLRM (SEQ ID NO: 10); EKEGKKALKLETDPAMKKMV (SEQ ID NO: 11); VQAVHKKALAVAPKLTEAQALM (SEQ ID NO: 12); ENKKVGEELIKLVTRPED (SEQ ID NO: 13); VNNEVGKKLLLLVTDPAD (SEQ ID NO: 14); ENEKEGKKALKLETDPAM (SEQ ID NO: 15); EAKKSEKLIKLETDPAM (SEQ ID NO: 16); PEVEAVHEKALAVAPKLTEAQ (SEQ ID NO: 17); ENKKVGEELIKLVTRPED (SEQ ID NO: 18); ENEKEGKKALKLETDPAM (SEQ ID NO: 19); EAKKSEKLIKLETDPAM (SEQ ID NO: 20); or PEVEAVHEKALAVAPKLTEAQ (SEQ ID NO: 21).

4. The polypeptide of any one of claims 1 to 3, wherein the assembly domain comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the following: a) XXXXXVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHDILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCKWFKAGVLAVGVGKAL VKGKPDEVREKAKKFVKKIRGCTEGTXXXXXXXXXXXXXXXXMKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTS (SEQ ID NO: 22); b) XXXXXXYMPGVMTPTELVKAMKLGHDILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCKWFKAGVLAVGVGKALVKGKPDEVREKAKKFVKKIRGCTEXXXXXXXXX XXXXXXXXXMKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVF (SEQ ID NO: 23); or c) XXXXXXXXLKLFPGEVVGPQFVKAMKGPFPNVKFVPTGVNLDNVCKWFKAGVLAVGVGKALVKGKPDEVREKAKKFVKKIRGCTEXXXXXXXXXXXXXXXXXXMKMEELFKKHK IVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHDI (SEQ ID NO: 24).

5. The polypeptide of any one of claims 1 to 4, wherein the assembly domain comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the first polypeptide sequence in Table 2 or Table 4.

6. A polypeptide, which is a variant of I53-50A having a C-terminal extension, said polypeptide comprising an assembly domain comprising, in order from N-terminus to C-terminus, a base polypeptide segment and an extended polypeptide segment. The base polypeptide segment therein comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 1-201 of SEQ ID NO:

1.

7. The polypeptide of claim 6, wherein the extended polypeptide segment comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical polypeptide sequences to any of the following: ENHARFAALRAELAGT (SEQ ID NO: 33); ENPELTKEVAAFLAGT (SEQ ID NO: 34); EYSEQFEARKKKLEGT (SEQ ID NO: 35); RIDEEYQKRLEKLRGT (SEQ ID NO: 36); KYKEQLDKQLKLQLGT (SEQ ID NO: 37); KEKEYFEEQLEKLKGT (SEQ ID NO: 38); YSKARLAEIKKALAGT (SEQ ID NO: 39); AADEHMAAIMAALKGT (SEQ ID NO: 40); VGDKLLAELKAQLAGT (SEQ ID NO: 41); YLKANAEKLHKLLAGT (SEQ ID NO: 42); ILAKLKAKILAKLKGT (SEQ ID NO: 43); YSQATLKEILKALAGT (SEQ ID NO: 44); FSKEACKKAILETKDGGSGSGT (SEQ ID NO: 45); PEVQAVHKKALAVAPKGT (SEQ ID NO: 46); EEVKAVQQKALALAPKLTGSGSGT (SEQ ID NO: 47); AEVAANQAKALSLAPPEAGGT (SEQ ID NO: 48); AEVEANQAKALSLAPAPAGSGSGT (SEQ ID NO: 49); EEVEAVQKKALSIAEELEGSGSGT (SEQ ID NO: 50); EEVAAVQKKALSLAPKEPSGT (SEQ ID NO: 51); DTLALLKERKGT (SEQ ID NO: 52); DSMAILEKVKGT (SEQ ID NO: 53); NQMELVKKVFGT (SEQ ID NO: 54); NAMELVKKALGT (SEQ ID NO: 55); NQMELVKKAEGT (SEQ ID NO: 56); DSMELFKKAEGT (SEQ ID NO: 57); DAMELLKEAEAIMGT (SEQ ID NO: 58); DPMKLVEEVEKLLGT (SEQ ID NO: 59); DPMAEVEKAKALEGT (SEQ ID NO: 60); or DPMALVDKVLALFGT (SEQ ID NO: 61).

8. The polypeptide of any one of claims 6 to 7, wherein the assembly domain comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the following: XXXXXXXMKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFY MPGVMTPTELVKAMKLGHDILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCKWFKAGVLAVGVGKALVKGKPDEVREKAKKFVKKIRGCTEXXXXXXXXXXXXXXXX (SEQ ID NO: 25).

9. The polypeptide of any one of claims 6 to 8, wherein the assembly domain comprises a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the first polypeptide sequence in Table 2 or Table 4.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide comprises one or more amino acid residues at the interface site, such that the polypeptide is capable of self-assembling to form a trimer component of a single-component nanostructure.

11. The polypeptide of any one of claims 1 to 9, wherein the polypeptide comprises one or more amino acid residues at the interface site, such that the polypeptide is capable of self-assembling to form a trimer component of a two-component nanostructure.

12. The polypeptide of any one of claims 1 to 11, wherein the polypeptide self-assembles to form a trimer component of a nanostructure, and the C-terminus of the assembled structural domain is accessible on the surface of the nanostructure.

13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide self-assembles to form a trimer component, optionally wherein the distance from the C-terminus of the assembled domain to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

14. The polypeptide of any one of claims 1 to 9, wherein the polypeptide self-assembles to form a soluble trimer, and the C-terminus of the assembly domain is accessible on the surface of the soluble trimer.

15. The polypeptide of any one of claims 1 to 9, wherein the polypeptide self-assembles to form a soluble trimer, the C-terminus of the assembly domain being close to the triple axis of the soluble trimer, optionally wherein the distance from the C-terminus to the triple axis is less than 30 Å, less than 25 Å, or less than 20 Å, or between 10 Å and 30 Å, between 15 Å and 30 Å, between 15 Å and 25 Å, or between 20 Å and 25 Å.

16. The polypeptide of any one of claims 1 to 15, wherein the polypeptide is a fusion protein comprising, in order from N-terminus to C-terminus, the assembly domain, optional polypeptide linker, and heteropeptide.

17. The polypeptide of claim 16, wherein the heterologous polypeptide is an antigen.

18. The polypeptide of claim 17, wherein the antigen is a pathogenic object, optionally an extracellular domain of a viral surface protein or an antigenic fragment thereof.

19. The polypeptide of claim 17, wherein the antigen is OspA or an antigenic fragment thereof, preferably OspA of Borrelia burgdorferi.

20. The polypeptide of claim 17, wherein the antigen is an extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

21. The polypeptide of claim 17, wherein the antigen is an extracellular domain of a bacterial protein or an antigenic fragment thereof.

22. A protein nanostructure comprising a first component containing a first polypeptide and optionally a second component containing a second polypeptide, wherein the first polypeptide is the polypeptide of any one of claims 1 to 21.

23. The nanostructure of claim 22, wherein the first component is a trimer component comprising three copies of the first polypeptide.

24. The nanostructure of claim 22 or claim 23, wherein the nanostructure comprises the second component and wherein the second component comprises a second assembly domain comprising a polypeptide sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following sequence: NQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVINGMMNVQLNTGVPVLSAVLTPHNYDKSKAHTLLFLALFAVKGMEAARACVEILAAREKIAA (SEQ ID NO: 26); or NQHSHKDYETVRIAVVRARWHAEIVDACVSAFEAAMADIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHRYRDSDAHTLLFLALFAVKGMEAARACVEILAAREKIAA (SEQ ID NO: 27).

25. The nanostructure of claim 24, wherein the second component is a pentamer comprising five copies of the second polypeptide.

26. The nanostructure of any one of claims 22 to 25, wherein the nanostructure comprises 20 copies of the first component.

27. The nanostructure of any one of claims 22 to 26, wherein the nanostructure further comprises 12 copies of the second component.

28. The nanostructure of any one of claims 22 to 27, wherein the C-terminus of the first polypeptide is accessible on the surface of the nanostructure.

29. The nanostructure of any one of claims 22 to 28, wherein the first polypeptide is a fusion protein comprising, in order from N-terminus to C-terminus, the first assembly domain, an optional polypeptide linker, and a heteropeptide.

30. The nanostructure according to any one of claims 22 to 28, wherein the heterologous polypeptide is an antigen.

31. The nanostructure of claim 30, wherein the antigen is a pathogenic object, optionally an extracellular domain of a viral surface protein or an antigenic fragment thereof.

32. The nanostructure of claim 30, wherein the antigen is OspA or an antigenic fragment thereof, preferably OspA of Borrelia burgdorferi.

33. The nanostructure of claim 30, wherein the antigen is an extracellular domain of a viral glycoprotein or an antigenic fragment thereof.

34. The nanostructure of claim 30, wherein the antigen is an extracellular domain of a bacterial protein or an antigenic fragment thereof.

35. The nanostructure according to any one of claims 22 to 34, wherein the nanostructure has an I53 architecture and / or a quaternary structure substantially similar to I53-50.

36. A polynucleotide encoding a nanostructure according to any one of claims 22 to 35 or a polypeptide according to any one of claims 1 to 21.

37. A delivery medium comprising the polynucleotide of claim 36, optionally a viral vector or lipid nanoparticles.

38. A pharmaceutical composition comprising the nanostructure of any one of claims 22 to 35, the polynucleotide of claim 36 or the delivery medium of claim 37, and a pharmaceutically acceptable carrier.

39. A vaccine comprising any one of claims 22 to 35, a polynucleotide of claim 36 or a delivery medium of claim 37, a pharmaceutically acceptable carrier, and optionally an adjuvant.

40. A host cell suitable for expressing a nanostructure of any one of claims 22 to 35 or a polypeptide of any one of claims 1 to 21; and / or comprising a polynucleotide of claim 36.

41. A method for preparing a polypeptide or nanostructure, comprising culturing the host cell of claim 40 under conditions suitable for expressing the polypeptide or the nanostructure.

42. A method for inducing an immune response to an antigen or to a pathogenic object in a subject in need, comprising optionally administering the vaccine of claim 39 to the subject via intramuscular injection or inhalation.

43. A method of immunizing a subject against pathogen infection, comprising optionally administering the vaccine of claim 39 to the subject via intramuscular injection or inhalation.

44. A composition or method as described herein.

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