Prefusion-stabilized CMV GB protein nanostructure

Abstract

Provided herein are compositions and methods relating to the CMV gB protein, in which amino acid substitutions are made to disrupt the post-fusion state and / or stabilize the pre-fusion state.

Description

Technical Field

[0001] Statement of Sequence Listing This application includes an electronically submitted sequence listing, which is hereby incorporated by reference in its entirety. The sequence listing submitted herewith is included in an XML file named "21-1661-WO_Sequence-Listing.xml", created on April 25, 2024, and is 449,423 bytes in size.

Background Art

[0002] Cytomegalovirus (CMV) infections maintain a high morbidity rate. These viruses can infect both fibroblasts and epithelial cells and generally cause persistent latent infections by evading the immune system. CMV is a major cause of congenital diseases worldwide and also poses a higher threat of severe diseases to individuals in an immunocompromised state. There are currently no commercially available vaccines for the prevention of either congenital diseases or systemic infections. Two viral surface glycoprotein complexes, including glycoprotein B (gB) and the pentameric complex, are of most interest regarding the induction of protective antibody responses. gB is a class III viral fusion protein used for entry into all cell types, while the pentameric complex mediates attachment to epithelial cells. gB is metastable and can adopt numerous distinct structures. Recombinant forms are often characterized by a postfusion state that does not structurally represent the functional prefusion state observed on viral particles. As a result, there is a need to alter the structural conformation of the gB antigen for use in vaccines to stabilize those prefusion states and to more effectively prevent the virus from entering cells.

[0003] Therefore, there is an unmet need for CMV gB proteins in which amino acid substitutions have been made to disrupt the postfusion state and / or to stabilize the prefusion state.

Summary of the Invention

[0004] This disclosure generally relates to CMV gB proteins in which mutations are inserted to disrupt the post-fusion state and / or stabilize the pre-fusion state. Furthermore, this disclosure provides nanostructures incorporating such CMV gB proteins.

[0005] In one embodiment, the Disclosure provides a polypeptide comprising an external domain of CMV gB in a prefusion structure, wherein the external domain consists of one, two, three, four or more amino acid substitutions, or substitutions at the same amino acid position, selected from the group consisting of E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R, relative to SEQ ID NO: S367I, T3 The amino acid substitutions include 74F, or amino acid substitutions S367I and T374F, or substitutions at positions 367 and / or 374; one, two, three, four or more amino acid substitutions selected from the group consisting of Q591F, Q591Y, S668A, Y218F, N220S, and V552L, or substitutions at the same amino acid position; amino acid substitution E167T; and / or one, two, three, four or more amino acid substitutions selected from the group consisting of C246S, H157R, I156H, R457S, R460S, W240N, and Y242T.

[0006] In some embodiments, the external domain includes amino acid substitutions D217C and S587C. In some embodiments, the external domain includes amino acid substitutions D217C and Y589C.

[0007] In some embodiments, the external domain contains one, two, three, four or more amino acid substitutions selected from the group consisting of E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R.

[0008] In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, and Y690F. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, K700A, and V702Q. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, D679N, E681N, K695D, L680E, and R685Q. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702Q, D679N, E681N, K695D, L680E, R685Q, and V701L. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, D679N, E681N, K695D, L680E, R685Q, E682S, F678N, N688E, and V677T. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702Q, D679N, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, and D699K. In some embodiments, the external domains include amino acid substitutions E686L, R693V, V694L, Y690F, D679H, E681N, K695D, L680E, R685Q, E682S, F678N, N688E, V677T, F687A, M684S, Q692S, and Y696R. In some embodiments, the external domains include amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R.

[0009] In some embodiments, the external domain includes amino acid substitution S367I or amino acid substitution T374F. In some embodiments, the external domain includes amino acid substitutions S367I and T374F.

[0010] In some embodiments, the external domain includes one, two, three, four or more amino acid substitutions selected from the group consisting of Q591F, Q591Y, S668A, Y218F, N220S, and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591F, S668A, and Y218F. In some embodiments, the external domain includes the amino acid substitutions N220S and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591F, S668A, Y218F, N220S, and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591Y, S668A, Y218F, N220S, and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591Y, N220S, and V552L. In some embodiments, the external domain includes amino acid substitutions Q591F, N220S, and V552L.

[0011] In some embodiments, the external domain includes the amino acid substitution E167T or E167S. In some embodiments, the external domain includes one, two, three, four or more amino acid substitutions selected from the group consisting of C246S, H157R, I156H, R457S, R460S, W240N, and Y242T.

[0012] In some embodiments, the polypeptide comprises one of the amino acid substitutions listed in Table 10.

[0013] In some embodiments, the polypeptide comprises a polypeptide sequence or its antigenic fragment that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequences in Table 10, excluding the signal peptide.

[0014] In some embodiments, the CMV gB protein adopts a prefusion structure in the absence of a fusion inhibitor. In some embodiments, the fusion inhibitor is N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamotiol)amino]phenyl}-1,3-thiazole-4-carboxamide.

[0015] In some embodiments, the polypeptide includes a trimerizing domain as a C-terminal fusion to an external domain. In some embodiments, the polypeptide includes a nanostructure assembly domain as a C-terminal fusion to an external domain.

[0016] In some embodiments, the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53-50A (SEQ ID NO: 19), I53-50A.1 (SEQ ID NO: 21), I53-50A.1NegT2 (SEQ ID NO: 22), or I53-50A.1PosT1 (SEQ ID NO: 23). In some embodiments, the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I3-01 (SEQ ID NO: 8). In some embodiments, the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. In some embodiments, the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53_dn5B (SEQ ID NO: 18).

[0017] In another embodiment, the Disclosure provides nanostructures comprising any polypeptide of the Disclosure.

[0018] In some embodiments, the nanostructure comprises a second polypeptide. In some embodiments, the second polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53-50B (SEQ ID NO: 20), I53-50B.1 (SEQ ID NO: 24), I53-50B.1NegT2 (SEQ ID NO: 25), or I53-50B.4PosT1 (SEQ ID NO: 26). In some embodiments, the second polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53_dn5A* (SEQ ID NO: 15), I53_dn5A.1 (SEQ ID NO: 16), or I53_dn5A.2 (SEQ ID NO: 17).

[0019] In another embodiment, the Disclosure provides polynucleotides that encode any polypeptide or any nanostructure of the Disclosure.

[0020] In some embodiments, the polynucleotide is messenger RNA (mRNA).

[0021] In another embodiment, the Disclosure provides lipid nanoparticles (LNPs) comprising mRNA encoding any polypeptide or any nanostructure of the Disclosure.

[0022] In another embodiment, the Disclosure provides a pharmaceutical composition comprising any polypeptide of the Disclosure, any nanostructure of the Disclosure, any polynucleotide of the Disclosure, or LNP of the Disclosure.

[0023] In another embodiment, the Disclosure provides a method for generating an immune response to the CMV gB protein in a subject requiring it, comprising administering an effective amount of the pharmaceutical composition of the Disclosure to the subject.

[0024] In another aspect, the present disclosure provides a method of treating or preventing CMV infection in a subject that needs it, comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0025] In another aspect, the present disclosure provides an expression vector comprising any polynucleotide of the present disclosure operably linked to a suitable control sequence.

[0026] In another aspect, the present disclosure provides a host cell comprising any polynucleotide of the present disclosure, any expression vector of the present disclosure, and / or any polypeptide of the present disclosure.

[0027] In another aspect, the present disclosure provides a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3, or an antigenic fragment thereof, wherein residues 698-702 are optionally present if they do not contain mutations, residue 456 of SEQ ID NO: 3 is absent, and the polypeptide or its antigenic fragment contains one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 2, Table 3, or Table 4. In an aspect, any polypeptide or its antigenic fragment of the present disclosure further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 5, Table 6, Table 7, or Table 8. In an aspect, the polypeptide or its antigenic fragment comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence listed in Table 9. In an aspect, the polypeptide or its antigenic fragment comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence listed in Table 10. In another aspect, the present disclosure provides a fusion protein comprising the polypeptide or its antigenic fragment of the present disclosure and a multimerization domain.

[0028] In some embodiments, the multimerization domain of the fusion protein of the present disclosure comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of the protein listed in Table 12, wherein the residues in parentheses are optional.

[0029] In another embodiment, the Disclosure provides a composition comprising one or more polypeptides or fusion proteins of the Disclosure linked to a scaffold. In an embodiment, the scaffold is a protein scaffold.

[0030] In another embodiment, the Disclosure provides nucleic acids that encode any polypeptide or fusion protein of the Disclosure.

[0031] In another embodiment, the present disclosure provides an expression vector comprising the nucleic acid of the present disclosure operably ligated to a suitable control sequence.

[0032] In another embodiment, the Disclosure provides a host cell comprising any nucleic acid of the Disclosure, any expression vector of the Disclosure, and / or any polypeptide or fusion protein of the Disclosure.

[0033] In another embodiment, the Disclosure provides a pharmaceutical composition comprising a polypeptide, fusion protein, composition, nucleic acid, expression vector, and / or one or more host cells and a pharmaceutically acceptable carrier.

[0034] In another embodiment, the Disclosure provides a vaccine comprising polypeptides, fusion proteins, compositions, nucleic acids, expression vectors, and / or one or more host cells and a pharmaceutically acceptable carrier.

[0035] In some embodiments, the vaccines of the present disclosure further include any other components suitable for use, including but not limited to CMV proteins gH, gL, UL128, UL130, and UL131, or one or more of their antigenic moieties; or any other CMV antigens, including but not limited to pentameric complexes of CMV proteins gH, gL, UL128, UL130, and UL131 or their antigenic moieties.

[0036] In another embodiment, the Disclosure provides a method for treating or limiting CMV infection, comprising administering an amount of the polypeptides, fusion proteins, compositions, vaccines, nucleic acids, expression vectors, host cells, pharmaceutical compositions, and / or vaccines of the Disclosure effective in treating or limiting the progression of CMV infection to a subject in need thereof.

[0037] In another embodiment, this disclosure provides compositions, methods, or uses described herein. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 shows the structural representation of the targeted region in the prefusion CMV gB structure for the addition of manipulated mutations. A) Prefusion structure of CMV gB showing only the outer domain (PDB 7KDP). The highlighted region contains a set of stabilizing mutations according to the names shown. Without being bound by theory, mutations labeled "Bg-KOs" are used to reduce aggregation, improve expression, and / or prevent furin cleavage. B) Postfusion structure of CMV gB (PDB 7KDD). [Figure 2] Figure 2 shows the addition of glycans at N165. A) N165 (shown as a sphere) is exposed to solvent in the prefusion structure (PDB 7KDP, shown in a transparent frame). Two figures are shown, one of which is further magnified (right). B) N165 (shown as a sphere) is embedded in the postfusion structure. Two figures are shown, one from the side of gB (left) and one from the bottom of gB overlooking the three rotation axes (right). [Figure 3] Figure 3 shows recombinant gB designs containing at least residues 20–698 of the soluble extradomain of gB derived from the Towne strain, with some designs containing additional residues up to residue 704. All tested designs possess an exogenous N-terminal signal peptide and a C-terminal octa-histidine tag, and some designs also possess an exogenous trimerizing domain between the C-terminus of the gB extradomain and the octa-histidine tag. [Figure 4] Figure 4 shows chromatographic elution profiles of multiple gB antigens with designed mutations, exhibiting homogeneous profiles consistent with the predicted elution profile for the trimer gB antigen. An equivalent gB antigen without the designed mutation is shown for reference. [Figure 5] Figure 5 shows DLS of multiple gB antigens with designed mutations that exhibit distinctly different hydrodynamic diameters and polydispersity, suggesting steric changes. An equivalent gB antigen without the designed mutation is shown for reference. [Figure 6] Figure 6 shows nanoDSFs of multiple gB antigens with designed mutations, which are measured using intrinsic tryptophan fluorescence. An equivalent gB antigen without the designed mutation is shown for reference. [Figure 7] Figure 7 shows NS-EM micrographs of multiple gB antigens with the designed mutations, demonstrating the disruption of the post-fusion state. An equivalent gB antigen without the designed mutation is shown as a reference, which exhibits a longer, more monodisperse profile consistent with the post-fusion state. [Figure 8] Figure 8 shows the NS-EM 2D class mean of one gB antigen with the designed mutation and an equivalent gB antigen without the designed mutation. [Modes for carrying out the invention]

[0039] definition Throughout this disclosure, certain features (including method steps) are referenced. It should be understood that this disclosure includes all possible combinations of such features. For example, if a particular feature is disclosed in connection with a particular aspect or embodiment, or with a particular claim, that feature may, to the extent possible, be used in combination with other particular aspects and embodiments, and / or in connection with other particular aspects and embodiments.

[0040] Where this specification refers to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes such possibility), and the method may include one or more other steps performed before any of the defined steps, between two defined steps, or after all of the defined steps (unless the context excludes such possibility).

[0041] The implementation of this technique employs conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the scope of the art, unless otherwise indicated to the contrary, many of which are described below for illustrative purposes. Such techniques are well described in the literature. It should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described, for these may vary depending on the circumstances in which they are used by those skilled in the art.

[0042] All publications and patents described herein are incorporated herein by reference in whole, to the extent that it is specifically and individually suggested that each individual publication or patent is incorporated by reference. In the event of any conflict, this application shall prevail, including all definitions herein. However, no reference to any reference, article, publication, patent, patent gazette, or patent application cited herein shall be, and should not be, considered in any form of acknowledgment or suggestion that it constitutes prior art in effect in any country worldwide, or forms part of universal and general knowledge.

[0043] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described. Specifically, features described in one section may be combined with features in any other section of the specification.

[0044] While illustrative embodiments are described and illustrated, it will be recognized that various modifications can be made to these illustrative embodiments without departing from the spirit and scope of the invention.

[0045] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure pertains. Various scientific dictionaries containing the terms included herein are well known to and available to those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but some preferred methods and materials are described. Thus, the terms defined immediately below are further explained by referring to the entirety of this disclosure.

[0046] The singular forms "a," "an," and "the" are intended to include the plural form unless the context clearly suggests otherwise.

[0047] As used herein, the term “about” means a range of values ​​including the specified value, which a person skilled in the art would reasonably consider to be similar to the specified value. In some embodiments, “about” means within a standard deviation using generally acceptable measurements in the art. In some embodiments, “about” means a range extending to + / -10%, + / -5%, + / -3%, or + / -1% of the specified value.

[0048] The term “at least,” followed by a number, is used herein to indicate the beginning of a range that starts at that number (which may be a range with an upper limit or a range without an upper limit, depending on the variable being defined). For example, “at least 1” means 1 or greater than 1.

[0049] The term “at most” followed by a number is used herein to indicate the end of a range that ends at that number (which may be a range with a lower limit of 1 or 0, or a range with no lower limit, depending on the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. In this specification, when a range is given as “(a first number) to (a second number)” or “(a first number) - (a second number),” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 - 100 mm means a range whose lower limit is 25 mm and whose upper limit is 100 mm.

[0050] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” shall be understood to imply that they include the step or element or group of steps or elements described, but not to exclude any other step or element or group of steps or elements. For example, a composition “comprising” (or “which comprises”) components A, B, and C may consist of (i.e., contain only) components A, B, and C, or it may contain not only components A, B, and C, but also one or more other components.

[0051] As used herein, the term "consisting of" means including and being limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" suggests that the listed elements are required or essential, and that other elements are absent. "Consisting essentially of" means including any elements listed after the phrase, and being limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as expressly provided in this disclosure. Thus, the phrase "consisting essentially of" suggests that the listed elements are required or essential, but other elements are not optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0052] The term "protein nanostructure," as used herein, refers to a symmetric protein assembly in which subunits self-assemble in aqueous solution without requiring lipids or macromolecules other than the protein nanostructure for assembly. Examples of protein nanostructures are described in Hsia et al. Nature 35:136-9 (2016) and Bale et al. Science 353:389-394 (2016). In some embodiments, the protein nanostructure is a one-component protein nanostructure, where a single polypeptide type provides the building blocks for self-assembly to form the protein nanostructure. In some embodiments, the protein nanostructure is a two-component protein nanostructure, where two polypeptide types provide the building blocks for self-assembly to form the protein nanostructure. In some embodiments, the polypeptide type includes an assembly domain, which causes the polypeptide to form a symmetric dimeric, trimer, tetramer, hexamer, or other multimeric component. In a two-component nanostructure, the two components differ in their choice of assembly domain. In some embodiments, the assembly domain of a first polypeptide type causes the polypeptide to form a trimer, and the assembly domain of a second polypeptide type causes the polypeptide to form a pentamer.

[0053] In some embodiments of one-component nanostructures, or some embodiments related thereto, two or more copies of a component further symmetrically self-assemble to form a nanostructure. In some embodiments of two-component nanostructures, or some embodiments related thereto, each of two or more of two different components symmetrically self-assembles to form a nanostructure.

[0054] The protein nanostructures disclosed herein can present potentially antigenic polypeptides intended to induce an immune response against a virus and can be administered as vaccines. In some embodiments, the components comprise trimers of antigenic polypeptides. The vaccines of this disclosure are useful for preventing viral infection and / or reducing the severity of viral infection.

[0055] The term “ferritin protein nanostructure” refers, as used herein, to the use of ferritin to generate symmetrical protein-based protein nanostructures using naturally occurring ferritin sequences or engineered variants thereof. Ferritin-based protein nanostructures are prepared by fusing antigens to ferritin molecules. Examples of ferritin protein nanostructures are described in U.S. Patent Application Publications US2018 / 0021258A1 and US2019 / 033027A1 (using the term nanoparticles rather than protein nanostructures), and their contents are incorporated herein by reference. Furthermore, the molecular structure of ferritin, consisting of 24 subunits that assemble into an octahedral cage with 432 symmetries, has the potential to present multimeric antigens on its surface.

[0056] As used herein, the term “assembly domain” refers to a portion of a component’s subunit that is involved in forming a protein nanostructure through intracomponent interactions and interactions with other copies of the same component (in one-component nanostructures) or other components (e.g., in two-component nanostructures).

[0057] The term "icosahedral particle" refers to a protein nanostructure that has a core with icosahedral symmetry. I53 refers to icosahedral particles constructed from pentamers and trimers. I52 refers to icosahedral particles constructed from pentamers and dimers. T33 refers to tetrahedral particles constructed from two sets of trimers. T32 refers to tetrahedral particles constructed from trimers and dimers.

[0058] Potentially antigenic polypeptides may be attached non-covalently or covalently to the core of a protein nanostructure as a fusion protein or by other means disclosed herein. Multimeric polypeptides may optionally be displayed along the axis of symmetry of the protein nanostructure. Also provided are proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use.

[0059] The terms “antigen” or “immunogen” refer to a compound or composition that, when administered to or expressed in an immune subject, induces a cellular or humoral immune response, such as a cytotoxic T lymphocyte (CTL) response, a B cell response (e.g., the production of antibodies that specifically bind to an epitope), an NK cell response, or any combination thereof. Antigens may include polypeptides (including glycoproteins). In some embodiments, an antigen is a polypeptide or polypeptide complex that induces an immune response. For example, an antigen may include one or more immunogenic epitopes associated with a viral pathogen. As used herein, the term antigen is not limited to the portion of a polypeptide or polypeptide complex containing an antigenic epitope. “Epitope” or “antigenic determinant” refer to a compound or composition that, when administered to or expressed in an immune system, is part of an antigen recognized by the immune system, particularly by antibodies, B cells, or T cells.

[0060] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues and optionally one or more post-translational modifications (e.g., glycosylation) and / or other modifications.

[0061] The term "isolated," when applied to polynucleotides or polypeptides, means that the polynucleotide or polypeptide essentially does not contain other cellular components that would naturally be associated with it. This can be, for example, homogeneous and may be in either a dry or aqueous solution state. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The polynucleotide or polypeptide, which is the dominant species present in the preparation, is substantially purified.

[0062] The terms “virus” or “virus particle” are used according to their obvious and common meaning in virology and refer to a virus particle that includes the viral genome (e.g., DNA, RNA, single-stranded, double-stranded), the viral capsid and associated proteins, and, in the case of enveloped viruses (e.g., herpesviruses), the envelope containing lipids and optionally components of the host cell membrane and / or viral proteins.

[0063] The term "viral infection" or "viral disease" refers to a disease or condition caused by a virus, such as cytomegalovirus (CMV).

[0064] The term "ectodomain" refers to the portion of a transmembrane protein or glycoprotein that lies outside the cell membrane or viral membrane in its native state.

[0065] The term "variant" refers to a polypeptide or polynucleotide that has one or more insertions, deletions, or amino acid substitutions relative to a reference polypeptide or polynucleotide.

[0066] The term "antigenic variant" refers to a variant that has one or more epitopes in common with a reference polypeptide and / or, when administered to a subject, produces the same or similar immune response as the reference polypeptide.

[0067] The term "functional variant" refers to a variant that exhibits at least some activity as a reference polypeptide. For example, a functional variant of an assembly domain can promote multimerization and self-assembly to the same or similar extent as the reference assembly domain, and / or can multimerize and assemble with the same congeneral assembly domain as the reference assembly domain.

[0068] As used herein, the term “bound” means that two parts are bound together, and the bond or multiple bond connecting the two parts may be covalent or non-covalent. In some embodiments, the two parts are covalently bonded to each other (e.g., directly or via a covalently bonded intermediate part ("linker")). In some embodiments, the two parts are non-covalently bonded (e.g., via an ionic bond, van der Waals bond / interaction, hydrogen bond, polar bond, or a combination or mixture thereof). For fusion polypeptides linked at the N-terminus and C-terminus, the linker may be a peptide bond or a peptide of any length.

[0069] The term "domain" refers to any part of a polypeptide that has a tertiary structure.

[0070] The terms "multimerization domain" and "multimerize" refer to the ability of a polypeptide, or a polypeptide domain, to form a tertiary structure with another polypeptide domain. In some embodiments, a multimerization domain can form dimers, trimers, tetramers, pentamers, or hexamers, and / or heteromers with other multimerization domains.

[0071] The term "trimerization domain" refers to a multimerization domain that forms a trimer.

[0072] The term "fragment" refers to a polypeptide that has one or more shortened N-terminus or C-terminus compared to a reference polypeptide.

[0073] The term "functional fragment" refers to a fragment that retains at least one function of its reference polypeptide.

[0074] The term "helix" or "helical" refers to an α-helix secondary structure in a polypeptide that is known to occur or is predicted to occur. For example, a sequence may be described as helical if computational modeling suggests that the sequence is likely to adopt a helical structure.

[0075] The term "component" refers to a protein or protein complex (e.g., a fusion protein containing an assembly domain) that can assemble into a protein nanostructure under appropriate conditions.

[0076] The term "vaccine" refers to a composition that can provide active acquired immunity against a particular disease or pathogen and / or a therapeutic effect (e.g., treatment) against a particular disease or pathogen. Vaccines typically contain one or more agents capable of inducing an immune response in a subject to a pathogen or disease, i.e., a target pathogen or disease. Immunogenic agents stimulate the body's immune system to recognize the agent as a threat or indication of the presence of a target pathogen or disease, thereby inducing immunological memory, which in turn allows the immune system to more easily recognize and destroy any pathogen upon subsequent exposure. Vaccines can be prophylactic (e.g., to prevent or mitigate the effects of natural infection or future infection by a pathogen, or the expected development of cancer in an affected subject) or therapeutic (e.g., to treat cancer in a subject diagnosed with cancer). The administration of a vaccine is called vaccination.

[0077] The terms “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to substances that assist in the administration and absorption of an activator by a subject and may be included in the compositions of this disclosure without causing significantly harmful toxicological effects to the patient, and may mean excipients approved for use in animals, more specifically in humans, by a federal or state regulatory agency or a regulatory agency listed in the United States Pharmacopeia or other generally accepted pharmacopoeia. Non-limited examples of pharmaceutically acceptable excipients include water, NaCl, saline solutions, Ringer’s lactate solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salines (e.g., Ringer’s solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants. Such preparations may be sterilized and, if necessary, may be mixed with additives such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, and / or fragrances, which do not react adversely with the compounds of the Disclosure. Those skilled in the art will recognize that other pharmaceutical additives may be useful in the Disclosure.

[0078] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when administered co-administered with the antigen, or when administered before, during, or after the administration of the antigen to the target. In some cases, LNP polynucleotides (e.g., mRNA) can function as adjuvants.

[0079] The term "TLR4 immunostimulant" refers to an adjuvant that modulates the immune response by stimulating Toll-like receptor 4 (TLR4) in target immune cells. Typical TLR4 immunostimulants include, but are not limited to, monophosphoryl lipid A (MPL), glucopyranosyl lipid A (GLA), and / or synthetic lipid A (SLA). In some embodiments, the antigen is the TLR4 immunostimulant.

[0080] The term “effective amount” refers to the amount of a composition that, when administered to a patient to treat a condition, disorder, or pathology, is sufficient to perform such treatment, or, when administered to a patient to induce an immune response, is sufficient to induce such an immune response. The exact amount is determined in accordance with the active ingredient, the condition, disorder, or pathology being treated and its severity, as well as the age, weight, health status, and responsiveness of the person being treated, and is determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0081] The terms “immunization” and “immunizing” refer to administering a composition to a subject in an amount sufficient to induce a desired immune response after one or more administration steps. Immunization may include 1 to 10 or more doses (e.g., injections) of the composition, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. The initial dose may not induce a detectable immune response because, generally, each subsequent dose enhances the immune response produced by the previous dose. As used herein, the term “immunizing” includes post-exposure prophylaxis.

[0082] The term "protective immune response" refers to an immune response that prevents infection by a pathogen and / or reduces the severity of infection when an object is later exposed to that pathogen, or an immune response that produces a level of immune response that correlates with protection. For example, vaccination may produce a protective immune response if it results in the production of neutralizing antibodies that are present in the plasma or serum of the object (e.g., human, pet, or farm animal) in amounts deemed to protect the object against subsequent infection and / or confer protection to the subject (e.g., New Zealand White (NZW) rabbit).

[0083] The term "polyclonal antibody response" refers to an antibody reaction that involves antibodies possessing multiple specificities and / or diversity in their antibody sequences.

[0084] The term "neutralizing" (e.g., "neutralizing antibody response") refers to an antibody that prevents infection by a pathogen and / or reduces the level of infection by a pathogen. A neutralizing antibody response can be measured either in an in vitro assay (e.g., infection of cultured cells by a pathogen in the presence of an antibody) or in a vivo assay (e.g., by determining the protective amount of an antibody by administering the antibody to a subject before exposure to an infectious dose of the pathogen).

[0085] Antibodies that "bind to," "specific to," or "specifically bind" (as used interchangeably herein) to a target (e.g., a viral protein) are well-known terms in the art, and methods for determining such specific or preferential binding are also well-known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts to or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does with another cell or substance.

[0086] The term "post-exposure prophylaxis" refers to administering an antigen (e.g., a vaccine) to a person who has been previously exposed to and / or infected with a pathogen in order to induce an immune response that protects against infection by the pathogen and / or reduces the severity of one or more symptoms of infection by the pathogen.

[0087] The term "administering" refers to providing a composition to a subject in a manner that enables the composition to have its intended effect. Administration for vaccination or post-exposure prophylaxis may be carried out by intramuscular injection, intravenous injection, intraperitoneal injection, or any other appropriate route.

[0088] "Co-administer" means that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapies. The compositions provided herein may be administered alone or co-administered to a subject. Co-administration means administering compounds individually or in combination (one or more compounds) simultaneously or sequentially. Accordingly, the formulations may also be combined with other active substances (e.g., to reduce metabolic degradation) as desired.

[0089] The term "subject" refers to a human or non-human animal to which the composition may be administered for vaccination, treatment, or other purposes. In some embodiments, non-human animals include non-human primates, rabbits, hamsters, gerbils, pigs, cattle, sheep, goats, guinea pigs, rats, mice, squirrels, wolves, foxes, horses, zebras, giraffes, elephants, cats, dogs, llamas, or ferrets.

[0090] The term “manufacturing” refers to the production of recombinant polynucleotides, polypeptides, delivery carriers, or protein nanostructures on any scale, including at least 25 mL, 50 mL, 1 L, 1,000 L, 50,000 L, or larger.

[0091] The terms "culturing" and "culture medium" refer to standard cell culture and recombinant protein expression techniques.

[0092] The term "host cell" refers to any cell that can be used in the expression of recombinant polypeptides or polynucleotides.

[0093] The term "secrete" refers to the ability of a host cell to release polypeptides expressed in the medium in which they are cultured.

[0094] The term "signal sequence" refers to a polypeptide sequence, usually at the N-terminus of a polypeptide, expressed in a host cell, that directs a polypeptide to a specific intracellular compartment. A signal sequence may be a secretory signal that causes the host cell to secrete the polypeptide into the medium in which the host cell is cultured. A signal sequence can be a "native" signal sequence, which occurs naturally as part of a polypeptide. A signal sequence can also be a non-spontaneous sequence having a polypeptide similar to those found in nature. A variety of signal sequences are known, and selecting an appropriate signal sequence is within the scope of the art.

[0095] The term "purify" refers to separating molecules from other substances present in a composition. Polypeptides may be purified by affinity (e.g., against antibodies or against tags, e.g., using His-tagged resins), by charge (e.g., ion-exchange chromatography), by size (e.g., preparative ultracentrifugation, size exclusion chromatography), or by other methods.

[0096] The terms “polynucleotide” and “nucleic acid,” as used interchangeably herein, refer to polymeric forms of ribonucleotides or deoxyribonucleotides with more than about 100 nucleotides. Therefore, the term includes, but is not limited to, polymers of single-stranded, double-stranded, or multi-stranded DNA or RNA, mRNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases or other native types, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. “Oligonucleotide” generally refers to polynucleotides of about 5 to about 100 nucleotides in single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of oligonucleotides. Oligonucleotides are also known as “oligomers” or “oligos” and may be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides, where applicable to the embodiments described.

[0097] In the context of two or more nucleic acid or polypeptide sequences, the terms “identity,” “identical,” and “sequence identity” refer to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides (i.e., approximately 60% identity across the entire specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity). Such sequences are therefore said to be “substantially identical.” This definition may also refer to or apply to the complementarity of a test sequence. This definition also includes sequences with deletions and / or additions, as well as those with substitutions. The term “amino acid substitution” refers to replacing a single amino acid in a sequence with another amino acid residue. Standard forms of amino acid substitution abbreviations are used. For example, V94R refers to the substitution of valine (V) with arginine (R) in the reference sequence. The abbreviation Arg94 refers to any sequence in which the 94th residue relative to the reference sequence is arginine (Arg). Preferred algorithms can account for gaps, etc., as described below. Preferably, identity exists throughout a region that is at least about 25 amino acids or nucleotides in length, more preferably throughout a region that is 50 to 100 amino acids or nucleotides in length. For sequence comparison, typically one sequence functions as the reference sequence, against which the test sequence is compared. The term "reference sequence" refers to the molecule against which the test sequence is compared.

[0098] Methods for sequence alignment for comparison and determination of sequence identity percentages are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the Needleman-Unsch homology alignment algorithm, J. Mol. Biol. 48:443-53 (1970).

[0099] The "position" of an amino acid or nucleotide base is indicated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, shortenings, and fusions that should be considered when determining optimal alignment, the amino acid residue numbers in a test sequence, which are generally determined by simply counting from the N-terminus, are not necessarily the same as the numbers of their corresponding positions in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, there is no amino acid in the variant that corresponds to the position in the reference sequence at the site of the deletion. If there is an insertion in the aligned reference sequence, that insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of shortening or fusion, there may be stretches of amino acids in either the reference sequence or the aligned sequence that do not correspond to any of the amino acids in the corresponding sequence.

[0100] The terms "numbered with reference to" or "corresponding to," when used in the context of numbering specific amino acid or polynucleotide sequences, refer to the numbering of residues in a specified reference sequence when a given amino acid or polynucleotide sequence is compared to that reference sequence.

[0101] The term “treating” means one or more of the following: alleviating, reducing, delaying, decreasing, reversing, improving, or managing at least one symptom of a condition in a subject. The term “treating” may also mean one or more of the following: preventing or delaying the onset of the disease (i.e., the period before the clinical signs of the condition appear), or reducing the risk of developing or worsening the condition.

[0102] Embodiment Polynucleotides are provided that encode polypeptide monomers for trimer components of a one-component protein nanostructure. When assembled, such protein nanostructures present a viral protein trimer as an antigen.

[0103] GB protein In some embodiments, the protein nanostructure comprises a cytomegalovirus gB protein monomer or a variant thereof. Examples of gB proteins are provided in Table 1. The signal peptide is in parentheses, and the N-terminal residue, near-membrane region, transmembrane region, and cytoplasmic residue are underlined. In each case, the underlined portion may be cleaved to leave an external domain; the same or different signal peptide may be used. In some embodiments, the cytomegalovirus gB viral protein monomer comprises a polypeptide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to any one of the polypeptide sequences in Table 1, or its external domain. In some embodiments, the cytomegalovirus gB viral protein monomer comprises a polypeptide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of the polypeptide sequences disclosed herein.

[0104] [Table 1] TIFF2026517784000002.tif242159TIFF2026517784000003.tif62159

[0105] Sequence ID 1 is the amino acid sequence of glycoprotein B (gB) of the cytomegalovirus (CMV) Towne strain. Sequence ID 2 is the amino acid sequence of gB of the CMV Merlin strain. Sequence ID 3 is the amino acid sequence of gB of the CMV AD169 strain, which shares approximately 95% identity with Sequence ID 1 and has 456 deletions compared to Sequence IDs 1 and 2.

[0106] Amino acid substitutions While not wishing to be bound by theory, sequences of recombinant CMV gB proteins are provided in which amino acid substitutions have been added to disrupt the post-fusion state and / or stabilize the pre-fusion state.

[0107] In some embodiments, the cytomegalovirus gB protein contains one of the following combinations of mutations.

[0108] [Table 2]

[0109] [Table 3]

[0110] [Table 4] TIFF2026517784000007.tif237159TIFF2026517784000008.tif181159

[0111] Without adhering to theory, the above combinations of mutations function to disrupt the post-fusion state and / or stabilize the pre-fusion state, thus making the polypeptide particularly useful as an immunogen to induce an immune response to CMV and / or for vaccination against CMV. In some embodiments, residues 698-702 are optional if none of the mutations in the combination of mutations contain mutations at residues 698-702.

[0112] Tables 2-4 list useful stabilizer gB mutations.

[0113] Table 5 lists additional mutations that are useful in combination with the mutations listed in Tables 2-4.

[0114] [Table 5]

[0115] Table 6 lists additional mutations that are useful in combination with the mutations listed in Tables 2-5.

[0116] [Table 6]

[0117] Table 7 lists additional mutations that are useful in combination with the mutations listed in Tables 2-6.

[0118] [Table 7] TIFF2026517784000012.tif207159

[0119] The mutations listed in Table 8 are known to knock out unstable elements in the gB protein that make it difficult to express, produce, or isolate the protein.

[0120] [Table 8]

[0121] In one embodiment, the Disclosure provides a polypeptide comprising an external domain of CMV gB in a prefusion structure, wherein the external domain consists of one, two, three, four or more amino acid substitutions, or substitutions at the same amino acid position, selected from the group consisting of E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R, relative to SEQ ID NO: S367I, T3 The amino acid substitutions include 74F, or amino acid substitutions S367I and T374F, or substitutions at positions 367 and / or 374; one, two, three, four or more amino acid substitutions selected from the group consisting of Q591F, Q591Y, S668A, Y218F, N220S, and V552L, or substitutions at the same amino acid position; amino acid substitution E167T; and / or one, two, three, four or more amino acid substitutions selected from the group consisting of C246S, H157R, I156H, R457S, R460S, W240N, and Y242T.

[0122] In some embodiments, the external domain includes amino acid substitutions D217C and S587C. In some embodiments, the external domain includes amino acid substitutions D217C and Y589C.

[0123] In some embodiments, the external domain contains one, two, three, four or more amino acid substitutions selected from the group consisting of E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R.

[0124] In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, and Y690F. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, K700A, and V702Q. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, D679N, E681N, K695D, L680E, and R685Q. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702Q, D679N, E681N, K695D, L680E, R685Q, and V701L. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, D679N, E681N, K695D, L680E, R685Q, E682S, F678N, N688E, and V677T. In some embodiments, the external domain includes amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702Q, D679N, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, and D699K. In some embodiments, the external domains include amino acid substitutions E686L, R693V, V694L, Y690F, D679H, E681N, K695D, L680E, R685Q, E682S, F678N, N688E, V677T, F687A, M684S, Q692S, and Y696R. In some embodiments, the external domains include amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R.

[0125] In some embodiments, the external domain includes amino acid substitution S367I or amino acid substitution T374F. In some embodiments, the external domain includes amino acid substitutions S367I and T374F.

[0126] In some embodiments, the external domain includes one, two, three, four or more amino acid substitutions selected from the group consisting of Q591F, Q591Y, S668A, Y218F, N220S, and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591F, S668A, and Y218F. In some embodiments, the external domain includes the amino acid substitutions N220S and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591F, S668A, Y218F, N220S, and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591Y, S668A, Y218F, N220S, and V552L. In some embodiments, the external domain includes the amino acid substitutions Q591Y, N220S, and V552L. In some embodiments, the external domain includes amino acid substitutions Q591F, N220S, and V552L.

[0127] In some embodiments, the external domain includes the amino acid substitution E167T or E167S. In some embodiments, the external domain includes one, two, three, four or more amino acid substitutions selected from the group consisting of C246S, H157R, I156H, R457S, R460S, W240N, and Y242T.

[0128] In some embodiments, the polypeptide comprises one of the amino acid substitutions listed in Table 10.

[0129] In some embodiments, the polypeptide comprises a polypeptide sequence or its antigenic fragment that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequences in Table 10, excluding the signal peptide.

[0130] In some embodiments, the CMV gB protein adopts a prefusion structure in the absence of a fusion inhibitor, optionally N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamotiol)amino]phenyl}-1,3-thiazole-4-carboxamide.

[0131] In another embodiment, the Disclosure provides a polypeptide comprising an amino acid sequence or antigenic fragment thereof that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3, wherein residues 698-702 are optional if they do not contain mutations, residue 456 of SEQ ID NO: 3 is missing, and the polypeptide or its antigenic fragment comprises one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 as listed in Table 2, Table 3, or Table 4.

[0132] In some embodiments, the polypeptide or its antigenic fragment comprises one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 2 or Table 3. In some embodiments, the polypeptide or its antigenic fragment comprises one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 2. In some embodiments, any polypeptide or its antigenic fragment of the present disclosure further comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 5. In some embodiments, any polypeptide or antigenic fragment of the Disclosure further comprises one, two, three, four, five, six, seven, eight, nine, or a total of ten mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3, as listed in Table 6. In some embodiments, any polypeptide or antigenic fragment of the Disclosure further comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or a total of twelve mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3, as listed in Table 7. In some embodiments, any polypeptide or antigenic fragment of the Disclosure further comprises combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3, as listed in Table 8.

[0133] In some embodiments, any polypeptide of the Disclosure comprises an amino acid sequence or antigenic fragment thereof that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the sequences listed in Table 9. In some embodiments, any polypeptide of the Disclosure comprises an amino acid sequence or antigenic fragment thereof that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the sequences listed in Table 10. In some embodiments, any polypeptide of the Disclosure further comprises any other functional domains depending on the intended use, including but not limited to a secretory signal located at the N-terminus of the polypeptide, and the signal sequence may be any suitable signal sequence depending on the intended use.

[0134] Signal peptide In some embodiments, the encoded polypeptide includes a peptide region that is a signal peptide. Signal peptides are well known in the art. The signal peptide may be a native signal peptide or may be replaced by another signal peptide. Signal peptides function to prompt cells to transfer proteins, typically to the cell membrane. The core of a signal peptide often contains a long stretch of hydrophobic amino acids (about 5-16 residues long) that tends to form a single alpha-helix and is also called the “h-region”. Furthermore, many signal peptides begin with a short stretch of positively charged amino acids, which may help reinforce the proper topology of the polypeptide in transit by what is known as the positive-inside rule. Due to its proximity to the N-terminus, it is called the “n-region”. The ends of a signal peptide typically contain a stretch of amino acids that are recognized and cleaved by a signal peptidase.

[0135] In some embodiments, the polypeptide does not contain a signal peptide.

[0136] Non-limiting examples of signal peptides are provided below. MELLILKANAITTILTAVTFCFASG (Sequence ID 32) MSWKVMIIISLLITPQHGL (Sequence ID 33) MKAILVVLLYTFTTANA (Sequence ID 34) MPISILLIITTMIMASHC (Sequence ID 35) MFVFLVLLPLVSSQC (Sequence ID 36) MVPQVLLFVPLLGFSLCFG (Sequence ID 37)

[0137] Table 9 provides example sequences of fusions containing signal peptides. In each case, the fusion protein is shown along with the signal peptide that is cleaved during the secretion of the fusion protein. The signal peptide is shown in parentheses to suggest that sequence identity for each sequence should be calculated without the signal peptide. Another signal peptide may be substituted using conventional methods known in the art.

[0138] [Table 9] TIFF2026517784000015.tif223159TIFF2026517784000016.tif223159TIFF2026517784000017.tif223159TIFF2026517784000018.tif222159TIFF2026517784000019.tif222159TIFF2026517784000020.tif222159TIFF2026517784000021.tif221159TIFF2026517784000022.tif222159TIFF2 026517784000023.tif223159TIFF2026517784000024.tif222159TIFF2026517784000025.tif222159TIFF2026517784000026.tif222159TIFF2026517784000027.tif223159TIFF2026517784000028.tif224159TIFF2026517784000029.tif222159TIFF2026517784000030.tif222159TIFF202651 7784000031.tif223159TIFF2026517784000032.tif224159TIFF2026517784000033.tif222159TIFF2026517784000034.tif223159TIFF2026517784000035.tif222159TIFF2026517784000036.tif223159TIFF2026517784000037.tif222159TIFF2026517784000038.tif223159TIFF20265177840 00039.tif222159TIFF2026517784000040.tif223159TIFF2026517784000041.tif223159TIFF2026517784000042.tif224159TIFF20265177840000 43.tif222159TIFF2026517784000044.tif223159TIFF2026517784000045.tif222159TIFF2026517784000046.tif223159TIFF2026517784000047.tif222159TIFF2026517784000048.tif222159TIFF2026517784000049.tif224159T IFF2026517784000050.tif222159TIFF2026517784000051.tif223159TIFF2026517 784000052.tif223159TIFF2026517784000053.tif223159TIFF2026517784000054. tif222159TIFF2026517784000055.tif223159TIFF2026517784000056.tif166159.

[0139] Trimerization domain In some embodiments, the encoded polypeptide includes a heterologous trimerizing domain. In some embodiments, the trimerizing domain is located at the C-terminus of the viral protein monomer. In some embodiments, the trimerizing domain is Foldon. A typical Foldon sequence is as follows: GYIPEAPRDGQAYVRKDGEWVLLSTF (Sequence ID 308)

[0140] In some embodiments, the trimerizing domain is a GCN4 coiled-coil domain. A typical sequence of a GCN4 coiled-coil domain is as follows: IEDKIEEILSKIYHIENEIARIKKLI (Sequence No. 309)

[0141] In some embodiments, the assembly domain is expressed as part of a fusion protein comprising a viral protein monomer, a linker, and the assembly domain. Examples of fusion protein sequences are provided in Table 10. In each case, in further embodiments, the assembly domain is either one of the assembly domains provided in Table 10 or another assembly domain.

[0142] [Table 10] TIFF2026517784000058.tif223159TIFF2026517784000059.tif222159TIFF2026517784000060.tif221159TIFF2026517784000061.tif220159TIFF2026517784000062.tif225159TIFF2026517784000063.tif221159TIFF2026517784000064.tif220159TIFF2026517784000065.tif221159TIFF2026517784000066.tif222159TIFF2026517784000067.tif221159TIFF2026517784000068.tif221159TIFF2026517784000069.tif221159TIFF2026517784000070.tif222159TIFF2026517784000071.tif220159TIFF2026517784000072.tif221159TIFF2026517784000073.tif222159TIFF2026517784000074.tif221159TIFF2026517784000075.tif221159TIFF2026517784000076.tif221159TIFF2026517784000077.tif221159TIFF2026517784000078.tif221159TIFF2026517784000079.tif220159TIFF2026517784000080.tif221159TIFF2026517784000081.tif224159TIFF2026517784000082.tif224159TIFF2026517784000083.tif221159TIFF2026517784000084.tif221159TIFF2026517784000085.tif223159TIFF2026517784000086.tif224159TIFF2026517784000087.tif224159TIFF2026517784000088.tif221159TIFF2026517784000089.tif222159TIFF2026517784000090.tif225159TIFF2026517784000091.tif222159TIFF2026517784000092.tif225159TIFF2026517784000093.tif220159TIFF202651 7784000094.tif221159TIFF2026517784000095.tif222159TIFF2026517784000096.tif223159TIFF2026517784000097.tif22159.

[0143] In some embodiments, the polypeptide includes a trimerizing domain as a C-terminal fusion to an external domain. In some embodiments, the polypeptide includes a nanostructure assembly domain as a C-terminal fusion to an external domain.

[0144] In another embodiment, the Disclosure provides a fusion protein comprising the polypeptide or its antigenic fragment and polymerizing domain.

[0145] In some embodiments, the multimerization domain of the fusion protein of this disclosure comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the protein sequences listed in Table 12, where the residues in parentheses are optional.

[0146] Protein nanostructures and assembly domains The gB protein sequences disclosed herein may be presented on nanostructures as fusion proteins with components of the nanostructure or by conjugation to the nanostructure. Any polypeptide having the amino acid substitutions disclosed herein may be used with any nanostructure described herein, known in the art, or to be developed in the future.

[0147] Various protein nanostructures are known in the art and are described, for example, in U.S. Patent Publications US2015 / 0356240A1; US2016 / 0122392A1, US20180030429A1, US20190341124A1, and US2022 / 0072120A1, the contents of which are incorporated herein by reference. In some embodiments, the protein nanostructure includes, as an assembly domain, a variant of KDPG aldolase (protein databank code 1WA3) that has been manipulated to self-assemble into a protein nanostructure.

[0148] One-component nanostructure In its native form, 1WA3 non-covalently assembles to form trimers via a first interface (trimer interface). When 20 copies of the trimer (60 monomers) are computationally joined to form a one-component icosahedral protein nanostructure, the five monomer sets of 1WA3 come into contact with each other via a second interface (pentamer interface). By introducing amino acid substitutions, the pentamer interface is stabilized, thereby allowing the protein nanostructure to spontaneously self-assemble, for example, in expression cells, or when isolated trimers (or monomers) are mixed under appropriate conditions.

[0149] In some embodiments, the pentamer interface contains one, two, three, four or more interface residues, such as amino acid residues, at positions 33, 61, 187, and 190, numbered according to SEQ ID NO: 8. In some embodiments, the assembly domain contains a polypeptide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 8. In some embodiments, the assembly domain contains one, two, three, or four amino acid substitutions at positions 33, 61, 187, and 190 compared to SEQ ID NO: 8. In some embodiments, multiple amino acid substitutions are substitutions of polar residues with nonpolar residues (e.g., A, L, I, M, V, F, or W). In some embodiments, some or all of the amino acid substitutions are substitutions of polar residues with small nonpolar residues (e.g., A, L, I, M, or V). In some embodiments, the protein nanostructure includes amino acid substitutions E33L or E33V; K61L or K61M; D187A or D187V; and / or R190A. In some embodiments, the protein nanostructure includes amino acid substitutions E33L, K61M, D187V, and R190A. In some embodiments, the protein nanostructure includes amino acid substitutions E33V, K61L, D187A, and R190A. In some embodiments, the assembly domain includes amino acid substitutions that inactivate the enzymatic activity of the assembly domain (e.g., K129A). In some embodiments, the assembly domain may further include amino acid substitutions (e.g., MI3; E56M or E56K; P186I; E191A; and / or K194A).

[0150] In some embodiments, the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I3-01 (SEQ ID NO: 8).

[0151] Sequence ID 8 is shown below, with the actual pentamer interface positions highlighted in bold.

[0152] [ka]

[0153] In some embodiments, the assembly domain includes an amino acid substitution that removes a cysteine ​​residue. In some embodiments, the assembly domain includes a C76A and / or C100A substitution. In some embodiments, the assembly domain includes a C76A, C100A, C165A, and / or C203A substitution.

[0154] Examples of assembly domain sequences are provided in Table 11. In each case, the N-terminal MK is optional and is not included when calculating sequence identity, but is shown only for numbering purposes; that is, the MK is included in the reference sequence but not necessarily in the assembly domain of the nanostructure.

[0155] [Table 11]

[0156] Two-component nanostructure In some embodiments, the assembly domain includes amino acid substitutions that remove hydrophobic residues and / or add polar residues. Examples of assembly domains having such substitutions are provided in International Patent Application No. WO2021163481A1, the entire contents of which are incorporated herein by reference. In some embodiments, such amino acid substitutions increase the secretion of the assembly domain when the polynucleotide encoding it is expressed in a cell.

[0157] Sequence ID 9 is shown below, with the example position highlighted in bold.

[0158] [ka]

[0159] In some embodiments, the assembly domain includes amino acid substitutions of F32Y;H37D / E / K / N / Q / R;F43Q;F168D / E / K / N / Q / R / S / T / Y;K169D / E / N / Q;L173D / E / N / Q / S;A174S;S179D / E;K183D / E, and / or T185D / E / K / N / Q / S.

[0160] In some embodiments, the assembly domain includes one, two, three, or four substitutions of H37D, L173Q, S179E, and V182N. In some embodiments, the assembly domain includes amino acid substitutions of H37D, L173Q, S179E, and V182N, as in, for example, SEQ ID NO: 13.

[0161] [ka]

[0162] In some embodiments, the multimerization domain comprises a polypeptide (Table 12) having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the following protein amino acid sequence, where the residues in parentheses are optional. In some embodiments, the multimerization domain is covalently bound to the polypeptide of the CMV gB protein or its antigenic fragment.

[0163] [Table 12]

[0164] In another embodiment, the Disclosure provides nanostructures comprising any polypeptide of the Disclosure. In some embodiments, the assembly domain comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53-50A (SEQ ID NO: 19), I53-50A.1 (SEQ ID NO: 21), I53-50A.1NegT2 (SEQ ID NO: 22), or I53-50A.1PosT1 (SEQ ID NO: 23). In some embodiments, the assembly domain comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53_dn5B (SEQ ID NO: 18). In some embodiments, the nanostructure comprises a second polypeptide. In some embodiments, the second polypeptide contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53-50B (SEQ ID NO: 20), I53-50B.1 (SEQ ID NO: 24), I53-50B.1NegT2 (SEQ ID NO: 25), or I53-50B.4PosT1 (SEQ ID NO: 26). In some embodiments, the second polypeptide contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53_dn5A* (SEQ ID NO: 15), I53_dn5A.1 (SEQ ID NO: 16), or I53_dn5A.2 (SEQ ID NO: 17).

[0165] Ferritin-based nanostructures In some embodiments, the assembly domain is a ferritin polypeptide. In some embodiments, the assembly domain of the ferritin protein nanostructure contains a polypeptide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% identical to one of the following amino acid sequences. MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKVELIGNENHGLYLADQYVKGIAKSRKS(Sequence ID 27) MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDAQN (Sequence ID 28) NFHQDCEAGLNRTVNLKFHSSYVYLSMASYFNRDDVALSNFAKFFRERSEEEKEHAEKLIEYQNQRGGRVFLQSVEKPERDDWANGLEALQTALKLQKSVNQALLDLHAVAADKSDPHMTDFLESPYLSESVETIKKLGDHITSLKKLWSSHPGMAEYLFNKHTLG(Sequence ID 29) QFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS(Sequence ID 30) SGESQVRQNFKPEMEEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDGS(Sequence ID 31)

[0166] In some embodiments, the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31.

[0167] Linker In some embodiments, the encoded polypeptide includes linkers between regions. A wide variety of polypeptide sequences are available and well known in the art. In some embodiments, the linker includes a Gly-Ser linker of any suitable length (i.e., a linker consisting of glycine and serine residues). In some embodiments, the Gly-Ser linker has an amino acid length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. Non-limiting examples of Gly-Ser linkers are shown below. GS (Sequence ID 298) GSS (Sequence ID 299) GSGS (Sequence ID 300) GSGGSGSGSGGS (Sequence No. 301) GSGGSGSGSGGS (Sequence No. 302) GSEKAAKAEEAARK (Sequence ID 303)

[0168] In another embodiment, the Disclosure provides a composition comprising one or more polypeptides or fusion proteins of the Disclosure linked to a scaffold. The scaffold can be any surface to which polypeptides or fusion proteins can be linked, and includes, but is not limited to, beads, glass, polystyrene, nanoparticles, protein scaffolds, and the like.

[0169] In some embodiments, the scaffold includes a protein scaffold.

[0170] In some embodiments, polypeptides are covalently bonded to protein subunits of a protein scaffold to form a fusion protein.

[0171] In some embodiments, the protein subunits of the protein scaffold comprise polypeptides having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the protein sequences listed in Table 12, where the residues in parentheses are optional.

[0172] Polynucleotides In another embodiment, the Disclosure provides polynucleotides encoding any polypeptide or any nanostructure of the Disclosure. The polynucleotide sequences may include RNA or DNA. An isolated polynucleotide refers to a genomic or cDNA sequence that has been removed from its normal surrounding nucleic acid sequence. Such polynucleotide sequences may include additional sequences useful for promoting the expression and / or purification of the encoded protein, and may include, but are not limited to, poly-A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be apparent to those skilled in the art which polynucleotide sequences encode the proteins of the Disclosure.

[0173] In some embodiments, the polynucleotide is messenger RNA (mRNA).

[0174] In another embodiment, the Disclosure provides lipid nanoparticles (LNPs) comprising mRNA encoding any polypeptide or any nanostructure of the Disclosure.

[0175] In another embodiment, the Disclosure provides an expression vector comprising any polynucleotide of the Disclosure operably linked to a suitable control sequence. A “recombinant expression vector” includes a vector that operably links a nucleic acid coding region or gene to a control sequence that can influence the expression of a gene product. An “operably linked control sequence” to a nucleic acid sequence of the Disclosure is a nucleic acid sequence that can influence the expression of a nucleic acid molecule. The control sequences do not need to be contiguous with the nucleic acid sequence insofar as they function to direct its expression. For example, the interposition of an untranslated but transcribed sequence may be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors may be of any kind known in the Art, including, but are not limited to, plasmid and virus-based expression vectors. The regulatory sequences used to drive the expression of disclosed nucleic acid sequences in mammalian systems can be constitutive (driven by one of a wide variety of promoters, including but not limited to CMV, SV40, RSV, actin, and EF) or inductive (driven by one of a number of inductive promoters, including but not limited to tetracycline, ecdysone, and steroid-responsive). The construction of expression vectors for use when transfecting prokaryotic cells is also well known in the art and can therefore be achieved via standard techniques.(See, for example, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Sambrook, Fritsch and Maniatis; Gene Transfer and Expression Protocols, pp. 109-128, edited by E.J. Murray, The Humana Press Inc., Clifton, NJ) and the Ambion 1998 Catalog (Ambion, Austin, TX). Expression vectors may be replicated in a host organism as episomes or by integration into the host's chromosomal DNA. Suitable expression vectors and hosts are well known in the art. In some embodiments, the expression vector comprises a plasmid. However, this disclosure is intended to include other expression vectors that perform equivalent functions, such as viral vectors.

[0176] In another embodiment, the Disclosure provides a host cell comprising any polynucleotide of the Disclosure, any expression vector of the Disclosure, and / or any polypeptide of the Disclosure. The cell may be transiently or stably transfected or transduced. Such transfection or transduction of expression vectors into prokaryotic and eukaryotic cells may be achieved by any technique known in the Art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE-dextran-mediated, polycation-mediated, or viral-mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd edition (RI Freshney. 1987. Liss, Inc. New York, NY)).

[0177] In some embodiments, the polynucleotide is messenger RNA (mRNA). Methods for producing polynucleotides, either by chemical synthesis or in vitro transcription (IVT) (in the case of mRNA), are well known in the art.

[0178] The basic structure of encoding mRNA may be similar to that of “mature” eukaryotic mRNA, and may include some or all of the following features, including (ii) 5' and 3' untranslated regions (UTRs) on both sides, (iii) 7-methylguanosine 5' cap structures and (iv) 3' poly(A) tails at both ends, and (i) a protein-coding open reading frame (ORF). Non-coding structural features can be individually optimized to modulate mRNA stability, translation efficiency, and immunogenicity. By incorporating modified nucleosides, mRNA transcripts called “nucleoside-modified mRNA” can be produced with reduced immunostimulatory activity, thus obtaining an improved safety profile. Furthermore, modified nucleosides enable the design of mRNA vaccines with significantly improved stability and translational capacity because they can circumvent the direct antiviral pathway, which is IFN-induced and programmed to degrade and block invading mRNA. For example, uridine substitution with pseudouridine reduces the activity of 2'-5'-oligoadenylate synthase, which modulates mRNA cleavage by RNase L. Furthermore, even lower activity is measured for protein kinase R, an enzyme involved in the inhibition of mRNA translation processes.

[0179] The polynucleotides of this disclosure may comprise one or more modified (e.g., altered or alternative) nucleic acid bases, nucleosides, nucleotides, or combinations thereof. The polynucleotides may comprise any useful modifications or alterations to, for example, nucleic acid bases, sugars, or nucleoside-to-nucleoside bonds (e.g., to binding phosphates / phosphodiester bonds / phosphodiester backbones). In certain specific embodiments, the alterations (e.g., one or more alterations) are present in each of the nucleic acid bases, sugars, and nucleoside-to-nucleoside bonds. The alterations according to this disclosure may be alterations of the polynucleotide, e.g., substitution of the 2'-H of the 2'-OH of ribofuranosyl in an RNA ring, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), roch nucleic acid (LNA), or hybrids thereof.

[0180] The polynucleotide may contain a 5'-cap structure. The 5'-cap structure of the polynucleotide is involved in nuclear export, enhances the stability of the polynucleotide, and binds mRNA cap-binding proteins (CBPs). The 5'-UTR may be given as the flanking region to the mRNA. In some embodiments, the polynucleotide sequence is codon-optimized. The polynucleotide may contain a "polyA sequence" or a "polyadenylation signal," and the terms are used interchangeably.

[0181] Pharmaceutical composition In another embodiment, the Disclosure provides a pharmaceutical composition comprising one or more polypeptides, fusion proteins, protein nanostructures, compositions, nucleic acids, LNPs, expression vectors, and / or host cells of the Disclosure, and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the Disclosure may be used in the methods of the Disclosure, for example, as described below. In addition to the polypeptides of the Disclosure, the pharmaceutical composition may comprise, for example, (a) lyophilization protectants; (b) surfactants; (c) fillers; (d) isotonic agents; (e) stabilizers; (f) preservatives and / or (g) buffers.

[0182] In some embodiments, the buffer is Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The pharmaceutical composition may also contain a lyophilization protective agent, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition contains preservatives, such as benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercury nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition contains a bulking agent such as glycine. In further embodiments, the pharmaceutical composition includes surfactants, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or combinations thereof. The pharmaceutical composition also includes isotonic agents, such as compounds that make the formulation substantially isotonic or isotonic with human blood. Typical isotonic agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises stabilizers, for example, molecules that, when combined with the protein of interest, substantially prevent or reduce the chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Typical stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0183] Polypeptides, fusion proteins, protein nanostructures, compositions, nucleic acids, expression vectors, and / or host cells may be the sole activator in a pharmaceutical composition, or the composition or vaccine may further contain one or more other activators suitable for the intended use.

[0184] The polypeptides, fusion proteins, protein nanostructures, compositions, pharmaceutical compositions, nucleic acids, expression vectors, and / or host cells of this disclosure may be used for any appropriate purpose, including but not limited to treating or suppressing the development of CMV infection. For example, polypeptides, fusion proteins, protein nanostructures, compositions, pharmaceutical compositions, nucleic acids, expression vectors, and / or host cells may be used to induce an immune response to CMV. One type of immune response is a B-cell response, which results in the production of antibodies against the antigen that triggered the immune response.

[0185] The protective antibodies induced by the methods of this disclosure can provide protection against viral infections by influencing every stage of the viral life cycle. For example, the protective antibodies may prevent CMV from attaching to cells, entering cells, releasing viral ribonucleoproteins into the cytoplasm, forming new viral particles within infected cells, and / or germinating new viral particles from the infected host cell membrane. Preferably, the antibodies induced by the methods of this disclosure prevent CMV from attaching to or entering host cells, prevent fusion of the viral membrane with the endosomal membrane, or prevent the release of newly formed viruses from the infected host cell.

[0186] One aspect of this disclosure is a vaccine composition (vaccine) comprising any polypeptide, fusion protein, or composition disclosed herein for protection against infection by CMV. The vaccines of this disclosure may also contain other components, such as adjuvants, buffers, etc. Typical adjuvants include aluminum phosphate, benzalkonium chloride, ubenimex, and QS21; gene adjuvants such as the IL-2 gene or its fragment, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene or its fragment, IL-18 gene or its fragment, chemokine (CC motif) ligand 21 (CCL21) gene or its fragment, IL-6 gene or its fragment, CpG, LPS, TLR agonists, and other immunostimulatory genes; IL-2 or its fragment, granulocyte-macrophage colony-stimulating factor (GM-CSF) Protein adjuvants such as IL-18 or its fragments, IL-18 or its fragments, chemokine (CC motif) ligand 21 (CCL21) or its fragments, IL-6 or its fragments, CpG, LPS, TLR agonists and other immunostimulatory cytokines or their fragments; lipid adjuvants such as cationic liposomes, N3 (cationic lipid), monophosphoryl lipid A (MPL1); and other adjuvants including cholera toxin, enterotoxin, Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine, marcaine and levamisole.

[0187] Instructions for use and handling This disclosure provides a method for vaccinating a subject against CMV, the method comprising administering polypeptides, fusion proteins, protein nanostructures, compositions, pharmaceutical compositions, nucleic acids, LNPs, expression vectors, and / or host cells to the subject so that an immune response to CMV occurs in the subject.

[0188] The subjects may include, but are not limited to, any suitable subjects including humans and non-human primates, such as chimpanzees and other ape and monkey species; domesticated animals such as cattle, sheep, pigs, seals, goats and horses; domestic mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats and guinea pigs; birds such as chickens, turkeys and other Galliformes birds, tame birds such as ducks and geese, wild birds and game birds, etc. In some embodiments, the subjects are newborns. In some embodiments, the subjects are infants. In some embodiments, the subjects are pregnant. In some embodiments, the subjects are immunocompromised or at risk of becoming immunocompromised, such as candidates for organ transplantation, stem cell transplantation or bone marrow transplantation.

[0189] In the vaccination methods of this disclosure, the subjects to be vaccinated may have been exposed to CMV. As used herein, the term “exposed” implies that the subject has been in contact with a person or animal known to be infected with CMV. The vaccines of this disclosure may be administered by any appropriate technique, including but not limited to conventional syringes, needleless injectors, or microprojectile guns. Appropriate routes of administration include, but are not limited to, parenteral administration, intramuscular injection, intradermal injection, subcutaneous injection, or intrathecal injection, as well as intraspinal injection, direct intraventricular injection, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection.

[0190] The compositions provided herein may be co-administered with other treatments, such as other vaccines. In some embodiments, subjects treated according to the methods provided herein may be administered one or more seasonal or pandemic vaccines, such as an influenza vaccine or a SARS-CoV-2 vaccine. In some embodiments, subjects treated according to the methods provided herein may also be administered a pneumococcal vaccine, a recombinant herpes zoster (shingles) vaccine, or a Tdap vaccine. One, two, or three vaccines may be co-administered with the immunogenic compositions provided herein. "Co-administration" includes both simultaneous and sequential administration. For example, one, two, or three vaccines and the immunogenic compositions provided herein may be administered on the same day. In some embodiments, one, two, or three vaccines and the immunogenic compositions provided herein may be administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, or 12 hours.

[0191] In another embodiment, a method for treating a subject suffering from a viral infection is provided. As used herein, “treat” or “treating” includes, but is not limited to, achieving one or more of the following: (a) reducing the viral titer in the subject; (b) limiting any increase in the viral titer in the subject; (c) reducing the severity of symptoms of a viral infection; (d) limiting or preventing the onset of symptoms after a viral infection; (e) preventing the worsening of symptoms of a viral infection; (f) suppressing or preventing the recurrence of symptoms of a viral infection in a subject that has previously had symptoms of a viral infection; and / or (g) increasing the survival rate. In some embodiments, the vaccination method reduces the risk to a subject that is infected with a virus. In some embodiments, the vaccination method limits the progression of a viral infection. In some embodiments, the vaccination method reduces the severity of symptoms of a viral infection.

[0192] In some embodiments, the methods provided herein may be used to prevent viral infection or disease (e.g., pneumonia or acute respiratory illness) in a subject. As used herein, “prevent” or “preventing” includes, but is not limited to, achieving one or more of the following: (a) producing an immune response (antibodies and / or cell-based, e.g., CD4 T cells, memory B cells, and / or CD8 T cells) in a subject that is expected to confer protection against infection caused by or associated with cytomegalovirus in the subject; (b) producing neutralizing antibodies against cytomegalovirus in the subject that is expected to reduce the severity of symptoms caused by one or more cytomegaloviruses in the subject; (c) preventing cytomegalovirus infection in the subject, as detected by an increase in the titer of the virus in the subject or by an increase in one or more symptoms of a viral infection; (d) reducing the risk of cytomegalovirus infection in a population of the subject; or (e) causing an antibody response (or seroconversion) in the subject, e.g., producing neutralizing antibodies against cytomegalovirus at least four times higher than baseline antibody levels in the subject. Prevention can be assessed in vaccinated subjects by comparing their immune response, particularly immune response indicators, with that of the same subjects before administration (referred to as baseline), subjects who received a placebo, or subjects who received a control vaccine.

[0193] As used herein, “limiting” the development of a viral infection means achieving one or more of the following: (a) producing an immune response (antibodies and / or cell-based, e.g., CD4 T cells, memory B cells, and / or CD8 T cells) in a subject that is expected to limit an increase in viral titer or an increase in symptoms in the subject; (b) producing neutralizing antibodies against cytomegalovirus in the subject to a level that is expected to limit an increase in viral titer or an increase in symptoms in the subject; (c) reducing the viral titer in the subject after exposure to cytomegalovirus compared to a subject not administered the protein complex; and (d) reducing the incidence or severity of symptoms after cytomegalovirus infection. Symptoms that are characteristic of cytomegalovirus infection include, but are not limited to, fever, fatigue, enlarged glands, sore throat, and muscle pain.

[0194] Furthermore, the methods provided herein may be used to prevent or limit the progression of infection by the original strain of cytomegalovirus and / or by variant strains of cytomegalovirus.

[0195] The clinical efficacy of cytomegalovirus vaccines can be evaluated by various means known in the art, including, but not limited to, placebo-controlled clinical efficacy studies that measure the viral load or symptoms of viral disease in vaccinated versus control groups. Immunological indicators correlating with protection, such as neutralizing antibody titers (usually expressed as geometric mean titers), multiplications above baseline (usually expressed as geometric increase multiplications), and antibody response rates (the proportion of subjects achieving a multiplication of neutralizing antibody titers above a given threshold), may also be defined. Guidance on direct and surrogate measurements of clinical efficacy for respiratory diseases is available, for example, in Industry Guidance: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines. US Food & Drug Administration (May 2007) and Respiratory Syncytial Virus Infection: Developing Antiviral Drugs for Prophylaxis and Treatment Guidance for Industry. US Food & Drug Administration (October 2017).

[0196] In some embodiments, the methods described herein induce an immune response in subjects not known to be infected with a virus, and this immune response functions to limit the progression of the infection and the symptoms of the viral infection. In some embodiments, the immune response includes the production of neutralizing antibodies and / or a cell-based response to the virus. In some embodiments, the immune response comprises at least 1 × 10⁻¹⁶ 3 , at least 1 × 10 4 , at least 1 × 10 5 , at least 1 × 10 6 , at least 1 × 10 7 , at least 1 × 10 8 , or at least 1 × 10 9This includes generating a gB protein-specific or other trimer-specific response at a geometric mean antibody titer. In a further embodiment, the immune response includes the production of antibodies against multiple antigenic epitopes on gB protein-specific or other viral protein trimers.

[0197] In another embodiment, the Disclosure provides a method for generating an immune response to the CMV gB protein in a subject requiring it, comprising administering an effective amount of the pharmaceutical composition of the Disclosure to the subject.

[0198] In one embodiment, the methods provided herein may result in an increase in antibody titers in a subject, for example, an increase in virus-specific neutralizing antibodies or virus-specific binding antibodies. Antibody titers may be determined using any suitable assay known in the art or described herein, including but not limited to enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunoabsorption spot (ELISpot), competitive ELISA, immunoprecipitation, immunoblotting, and agglutination tests.

[0199] In some embodiments, neutralization assays or microneutralization (MN) assays may be used to measure the increase in neutralizing antibodies in a subject after administration of the protein complexes described herein. Microneutralization refers to a neutralization assay performed in a miniaturized form, such as a 96-well plate. (Micro)neutralization assays are used to test the inhibition of a virus by an antibody (e.g., purified antibody, serum, or plasma). This assay measures the level of antibody present in a sample that can neutralize the virus in vitro. Generally, microneutralization assays for clinical samples are performed using serial dilutions of serum mixed with a fixed concentration of virus. Methods for performing (micro)neutralization assays are well known. Illustrative microneutralization assays are described; see, for example, van Baalen et al. Vaccine 35 (2017) 46-52.

[0200] If cytomegalovirus-specific neutralizing antibodies are present in the sample, the virus is neutralized, and infection of cells (e.g., HEp-2 cells) is inhibited. Immunofluorescence levels suggestive of viral infection can be analyzed, for example, using a CTL ImmunoSpot® UV analyzer with BioSpot® analysis software for automated counting of infected cells. Results are generally reported in international units per milliliter (IU / mL).

[0201] "Baseline" means the antibody measurement before administration of the first dose of the immunogenic composition provided herein.

[0202] In another embodiment, the methods provided herein may result in an increase in immune cells in a subject (e.g., an increase in virus-specific memory B cells and / or virus-specific T cells). The number of immune cells in a subject may be determined using any suitable assay known in the Art or described herein, including but not limited to FACS and flow cytometry.

[0203] In some embodiments, the composition is a dosage unit formulation comprising a conventional pharmaceutically acceptable carrier, adjuvant, and vehicle, administered via any suitable route, including intranasal, sublingual, oral, parenteral, inhalation spray, rectal, or topical routes. As used herein, the term parenteral includes subcutaneous, intravenous, intra-arterial, intramuscular, intracisional, intratendinous, intrathecal, intracranial, intrathoracic, intra-abdominal, intra-peritoneal, or intra-abdominal.

[0204] In another embodiment, the present disclosure provides a method for treating or preventing CMV infection in a subject requiring such treatment, comprising administering an effective amount of the pharmaceutical composition of the present disclosure to the subject.

[0205] In another embodiment, the Disclosure provides a vaccine comprising one or more polypeptides, fusion proteins, protein nanostructures, compositions, nucleic acids, LNP expression vectors, and / or host cells and pharmaceutically acceptable carriers of the Disclosure.

[0206] In some embodiments, the vaccines of the present disclosure further include CMV proteins gH, gL, UL128, UL130, and UL131, or one or more of their antigenic moieties; or any other CMV antigens, including but not limited to a pentamer complex of CMV proteins gH, gL, UL128, UL130, and UL131 or their antigenic moieties, or any other components as used in the present invention.

[0207] In another embodiment, the Disclosure provides a method for treating or limiting the progression of CMV infection, comprising administering an amount of the polypeptides, fusion proteins, compositions, vaccines, nucleic acids, expression vectors, host cells, pharmaceutical compositions, and / or vaccines of the Disclosure effective in treating or limiting the progression of CMV infection to a subject in need thereof.

[0208] kit This disclosure further provides kits which may be used to prepare and administer the compositions of this disclosure. In some embodiments, the kits provided herein include the compositions disclosed herein and instructions for use in the methods of this disclosure. In some embodiments, the kits include one or more unit doses disclosed herein and instructions for use in the methods of this disclosure. In some embodiments, the kits include vials containing a single dose of the pharmaceutical composition provided herein. In some embodiments, the kits include vials containing multiple doses provided herein. In some embodiments, the kits further include instructions for use of the pharmaceutical composition. In some embodiments, the kits further include diluents for preparing dilutions of the pharmaceutical composition before administration. In some embodiments, the pharmaceutical composition includes an adjuvant. In some embodiments, the kits include a pharmaceutical composition and an adjuvant which may be mixed before administration. In another embodiment, this disclosure provides compositions, methods, or uses described herein.

[0209] Examples While we do not wish to be bound by theory, we are provided with sequences of recombinant CMV gB protein in which mutations are inserted to disrupt the post-fusion state of the gB protein and / or stabilize the pre-fusion state. Multiple regions of gB (Figure 1A) are identified as having suboptimal local stability for the pre-fusion state, and specific mutations are provided that are intended to both improve these structural features and / or destabilize the post-fusion state. For example, the insertion of a glycosylation site at a site exposed in the post-fusion structure is shown in Figure 2B (compared to the pre-fusion, Figure 2A). The designed gB antigen was characterized as a trimer by size exclusion chromatography (SEC), but exhibits a distinctly different biophysical profile as assessed by nano-differential scanning fluorescence (nano-DSF) and dynamic light scattering (DLS). Furthermore, negative staining electron microscopy (NS-EM) confirms that the designed CMV antigen does not favor the post-fusion state as much, instead exhibiting a minor structure that is more similar to the pre-fusion state. In summary, the mutations provided herein are potentially useful for vaccines containing various forms of recombinant gB antigen, including protein-based vaccines, nucleic acid-based vaccines, or vector vaccines.

[0210] Example 1: Materials and Method Expression and protein purification: Genes encoding the mutant CMV gB antigen (corresponding to residues 20-698, 20-702, or 20-704 of SEQ ID NO: 1) were cloned into the pCMV / R vector using XbaI and AvrII restriction sites. In addition to various designed mutants, the whole sequence also contained mutants I156H, H157R, W240N, Y242T, C246S, R457S, and R460S to make the fusion loop more polar, remove unpaired cysteine, and remove polybasic cleavage sites. All sequences were C-terminated with one of the following: the "GGSHHHHHHHH" (SEQ ID NO: 305) sequence, which is preceded by the secretion signal "MGILPSPGMPALLSLVSLLSVLLMGCVAETGT" (SEQ ID NO: 304) and enables purification; the "GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHHH" (SEQ ID NO: 306) histidine-tagged T4 fibrinfoldon sequence, which enables improvement in both trimerization and purification; or the "GSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGHHHHHHHH" (SEQ ID NO: 307) histidine-tagged GCN4 sequence, which enables improvement in both trimerization and purification.

[0211] All proteins were produced in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 37°C, 70% humidity, and 8% CO2 while rotating at 150 rpm. Cell cultures were transfected using PEI-MAX (Polyscience) with cells grown to a cell density of 3 million cells per mL and cultured for 3 days. The supernatant was clarified by centrifugation at 4000 rcf, PDADMAC was added to a final concentration of 0.0375% (Sigma-Aldrich), and the mixture was spun again at 4000 rcf.

[0212] His-tagged proteins were purified from supernatants clarified by batch binding. Each clarified supernatant was supplemented with 1 M Tris-HCl pH8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of approximately 310 mM. Talon cobalt affinity resin (Takara Corporation) was added to the treated supernatants and incubated for 15 minutes with gentle shaking. The resin was collected using vacuum filtration with a 0.2 μm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH8.0 and 300 mM NaCl, and the proteins were eluted using three column volumes of 20 mM Tris pH8.0, 300 mM NaCl, and 300 mM imidazole. Protein purity after affinity purification was evaluated by SDS-PAGE (both reducing and non-reducing). The eluate was concentrated and applied to a Superose6 Increase 10 / 300 column (Cytiva) pre-equilibrated with 25 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol for preparative size exclusion chromatography (SEC). Peaks corresponding to the trimer species were identified based on the elution volume. The purified protein was stored at 4°C.

[0213] Thermal fusion: All proteins were diluted to 1 mg / mL in 25 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol before thermal measurement. Melting temperatures were determined from thermal denaturation and fusion curves using UNcle® (UNchained Labs), based on the centroid-mean (BCM) of intrinsic tryptophan fluorescence, using data collected at 20–95°C with a temperature lamp at 1°C / min in a background of 25 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol. Melting temperatures were calculated using UNcle® analysis software.

[0214] Negative staining electron microscopy: Proteins were adsorbed onto a glow-discharge-treated carbon-coated copper grid for 1 minute, then washed three times with water and stained with 2% uranyl formate. Micrographs were recorded using Leginon software on a 100kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k×4k CCD camera at a nominal magnification of 67,000x. Focus blur ranged from 1.0 to 2.0 mm, and the pixel size was 1.6 Å. Particles were automatically selected using a reference-free method with DogPicker. Contrast transfer function (CTF) evaluation was performed using GCTF. Class mean values ​​were generated using Relion2.1.

[0215] Example 2: Computer-aided design of gB proteins Using a rational structure-based design approach based on the crystal structure of the CMV gB protein (PDB 7KDP), we identified amino acid substitutions that stabilize the prefusion structure or destabilize the postfusion structure of the gB protein. In short, we analyzed the prefusion (PDB 7KDP) and postfusion (PDB 7KDD) structures of CMV gB using PyMol software to identify regions with different structural conformations in either state. For the "C-terminus" and "domain-domain" groups (Table 13), multiple residues within these regions were selected for design using a computational protocol from Rosetta software (Leman et al. Nat. Methods 17(7):665-680 (2020)). The selected residues were then simultaneously mutated using Rosetta to improve non-covalent interactions within the prefusion structure. Output sequences from Rosetta with various types and / or numbers of mutations were scored using Rosetta in both prefusion and postfusion structures to predict the energy effects toward both states (Table 13, "Energy Difference"). Sets of mutations predicted to stabilize the prefusion state and / or destabilize the postfusion state were selected for experimental analysis. For the "disulfide" group (Table 13), pairs of residues that are far apart in the postfusion structure but close enough in the prefusion structure to form a disulfide bond were selected when both were mutated to cysteine. For the "glycosylation" group (Table 13), the E167T mutation was performed to glycosylate N165. N165 is rigidly embedded in the postfusion structure and cannot accommodate additional glycans without introducing structural collisions, but in the prefusion structure it is exposed to the solvent, allowing sufficient space for additional glycans.

[0216] Table 13 shows the computationally predicted stabilization of the CMV gB protein. Representative wild-type sequences (SEQ ID NOs. 1-3) were modified to include amino acid substitutions at I156H, H157R, W240N, Y242T, C246S, R457S, and R460S. These representative sequences were further modified as shown in Table 13.

[0217] [Table 13] TIFF2026517784000104.tif242159TIFF2026517784000105.tif207159

[0218] The first value in Table 13 is the energy difference between the prefusion trimer and the prefusion monomer (pre-trimer-pre-monomer). The second value in Table 13 is the difference between the above and the equivalent difference in the post-fusion state ((pre-trimer-pre-monomer)-(post-trimer-post-monomer)). The last value in Table 13 is the difference in energy change between the prefusion state and the post-fusion state due to the addition of mutation ((mutPre-wtPre)-(mutPost-wtPost)). "REU" stands for "Rosetta energy unit". Methods for calculating these parameters are provided in Park et al. J. Chem. Theory & Computation 12(12) 6201-12 (2016). Monomer calculations are obtained from a model that simulates the dissociation state by moving each chain in the trimer 1000 angstroms apart.

[0219] Example 3: Results Referring to Figure 3, all recombinant gB designs tested contained at least 20–698 residues of the soluble extracellular domain of gB derived from the Towne strain, with some designs containing up to 704 additional residues. All tested designs possessed an exogenous N-terminal signal peptide and a C-terminal octa-histidine tag, and some designs also possessed an exogenous trimerizing domain between the C-terminus of the gB extracellular domain and the octa-histidine tag. All tested designs also featured polar mutations in the fusion loop supporting protein solubility (I156H, H157R, W240N, Y242T), mutations against unpaired cysteine ​​(C246S), and mutations against polybasic cleavage sites (R457S, R460S). Collectively, these are referred to as "Bg-KO". Without being bound by theory, these mutations affect expression levels but not the prefusion:postfusion ratio. Therefore, constructs lacking these amino acid substitutions are expected to still possess the desired prefusion selectivity induced by the designed mutations described and tested in Example 2. In other words, the Bg-KO substitution is an optional feature of the construct.

[0220] Figure 4 shows size exclusion chromatography (SEC) (Superose6 Increase10 / 300GL, Cytiva) of multiple gB antigens with the designed mutations. The designed constructs had homogeneous elution profiles consistent with the trimer gB antigen. An equivalent post-fusion gB antigen without the designed mutation is shown for reference. The dimensions of the post-fusion structure of gB (PDB 7KDD) are particularly characterized by a longer length (approximately 17 nm) and a relatively smaller width (approximately 7 nm). Since SEC elution is influenced by the radius of gyration and hydrodynamic diameter of the molecule, pre-fusion gB should elute later than post-fusion gB. Five different designs with only the Cpack1-long mutation or additional mutations showed slower elution, which is consistent with samples containing an increased ratio of pre-fusion to post-fusion gB.

[0221] Figure 5 shows dynamic light scattering (DLS) of multiple gB antigens with designed mutations that exhibit distinctly different hydrodynamic diameters and polydispersity, suggesting steric changes. Equivalent gB antigens without the designed mutations are shown for reference. Pre-fusion gB should have a longer hydrodynamic diameter than post-fusion gB. Cpack1-long, in combination with Disulf1, dII-dIIIpack1 and GCN4, or Disulf1 and dI-dIV-dV-pack1, exhibits a longer DLS diameter than the post-fusion control.

[0222] Figure 6 shows nano-differential scanning fluorescence (nano-DSF) measurements of multiple gB antigens with the designed mutations, where nano-DSF is measured using intrinsic tryptophan fluorescence. An equivalent gB antigen without the designed mutation is shown for reference. Pre-fusion gB should be less thermally stable than post-fusion gB because the post-fusion state energy required for membrane fusion is lower. Compared to a post-fusion control that showed two melt transitions, the addition of the designed mutation resulted in a different thermal profile with one or more reduced melt temperatures.

[0223] Figure 7 shows negative-stained electron micrographs (NS-EM) of several gB antigens with the designed mutations. An equivalent gB antigen without the designed mutation is shown as a reference, which exhibits a longer profile more monodispersively and consistent with the post-fusion state. All constructs with the designed mutations exhibited less post-fusion characteristics compared to the post-fusion control. Combinations of Cpack1-long and other designed features resulted in a high frequency of structures different from the post-fusion structure. Specifically, particles with more spherical characteristics were observed in combinations of Cpack1-long, Disulf1, and dI-dIV-dV-pack1 mutations, which were more similar to the pre-fusion structure.

[0224] Figure 8 shows the NS-EM 2D class mean of one gB antigen with the designed mutation and an equivalent gB antigen without the designed mutation.

[0225] The foregoing description of embodiments of the present invention is presented for illustrative purposes only; it is not intended to be exhaustive or to limit the invention to any specific form disclosed. Those familiar with the relevant art will recognize that many modifications and variations are possible in light of the above disclosure. Therefore, the scope of the present invention is limited only by the following claims.

Claims

1. A polypeptide comprising an external domain of CMV gB in a prefusion structure, wherein the external domain is located at a position relative to Sequence ID No.

1. a. One, two, three, four or more amino acid substitutions, or substitutions at the same amino acid position, selected from the group consisting of E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R; b. Amino acid substitution S367I, amino acid substitution T374F, or amino acid substitution S367I and T374F, or substitutions at position 367 and / or 374; c. One, two, three, four or more amino acid substitutions, or substitutions at the same amino acid position, selected from the group consisting of Q591F, Q591Y, S668A, Y218F, N220S, and V552L; d. Amino acid substitution E167T; and / or e. One, two, three, four or more amino acid substitutions selected from the group consisting of C246S, H157R, I156H, R457S, R460S, W240N, and Y242T. polypeptides, including

2. The polypeptide according to claim 1, wherein the external domain comprises amino acid substitutions D217C and S587C.

3. The polypeptide according to claim 1, wherein the external domain comprises amino acid substitutions D217C and Y589C.

4. The polypeptide according to any one of claims 1 to 3, wherein the external domain comprises one, two, three, four or more amino acid substitutions selected from the group consisting of E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R.

5. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, and Y690F.

6. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, K700A, and V702Q.

7. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, D679N, E681N, K695D, L680E, and R685Q.

8. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702Q, D679N, E681N, K695D, L680E, R685Q, and V701L.

9. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, D679N, E681N, K695D, L680E, R685Q, E682S, F678N, N688E, and V677T.

10. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702Q, D679N, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, and D699K.

11. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, D679H, E681N, K695D, L680E, R685Q, E682S, F678N, N688E, V677T, F687A, M684S, Q692S, and Y696R.

12. The polypeptide according to claim 4, wherein the external domain comprises amino acid substitutions E686L, R693V, V694L, Y690F, K700A, V702E, D679H, E681N, K695D, L680E, R685Q, V701L, E682S, F678N, N688E, V677T, D699K, F687A, M684S, Q692S, and Y696R.

13. The polypeptide according to any one of claims 1 to 12, wherein the external domain comprises an amino acid substitution S367I or an amino acid substitution T374F.

14. The polypeptide according to any one of claims 1 to 12, wherein the external domain comprises amino acid substitutions S367I and T374F.

15. The polypeptide according to any one of claims 1 to 14, wherein the external domain comprises one, two, three, four or more amino acid substitutions selected from the group consisting of Q591F, Q591Y, S668A, Y218F, N220S, and V552L.

16. The polypeptide according to claim 15, wherein the external domain comprises amino acid substitutions Q591F, S668A, and Y218F.

17. The polypeptide according to claim 15, wherein the external domain comprises amino acid substitutions N220S and V552L.

18. The polypeptide according to claim 15, wherein the external domain comprises amino acid substitutions Q591F, S668A, Y218F, N220S, and V552L.

19. The polypeptide according to claim 15, wherein the external domain comprises amino acid substitutions Q591Y, S668A, Y218F, N220S, and V552L.

20. The polypeptide according to claim 15, wherein the external domain comprises amino acid substitutions Q591Y, N220S, and V552L.

21. The polypeptide according to claim 15, wherein the external domain comprises amino acid substitutions Q591F, N220S, and V552L.

22. The polypeptide according to any one of claims 1 to 21, wherein the external domain comprises an amino acid substitution E167T or E167S.

23. The polypeptide according to any one of claims 1 to 22, wherein the external domain comprises one, two, three, four or more amino acid substitutions selected from the group consisting of C246S, H157R, I156H, R457S, R460S, W240N, and Y242T.

24. A polypeptide according to any one of claims 1 to 23, comprising any one combination of amino acid substitutions listed in Table 10.

25. The polypeptide according to any one of claims 1 to 24, comprising a polypeptide sequence or antigenic fragment thereof that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequences in Table 10, excluding the signal peptide.

26. The polypeptide according to claim 1, wherein the CMV gB protein adopts a prefusion structure in the absence of a fusion inhibitor, optionally N-{4-[({(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}carbamotioil)amino]phenyl}-1,3-thiazole-4-carboxamide.

27. The polypeptide according to any one of claims 1 to 23, comprising a trimerizing domain as the C-terminal fusion to the external domain.

28. The polypeptide according to any one of claims 1 to 23, comprising a nanostructure assembly domain as a C-terminal fusion to the external domain.

29. The polypeptide according to claim 28, wherein the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53-50A (SEQ ID NO: 19), I53-50A.1 (SEQ ID NO: 21), I53-50A.1NegT2 (SEQ ID NO: 22), or I53-50A.1PosT1 (SEQ ID NO: 23).

30. The polypeptide according to claim 28, wherein the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I3-01 (SEQ ID NO: 8).

31. The polypeptide according to claim 28, wherein the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:

31.

32. The polypeptide according to claim 28, wherein the assembly domain contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53_dn5B (SEQ ID NO: 18).

33. A nanostructure comprising the polypeptide according to any one of claims 29 to 32.

34. The nanostructure according to claim 33, comprising a second polypeptide.

35. The nanostructure according to claim 33, wherein the second polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53-50B (SEQ ID NO: 20), I53-50B.1 (SEQ ID NO: 24), I53-50B.1NegT2 (SEQ ID NO: 25), or I53-50B.4PosT1 (SEQ ID NO: 26).

36. The nanostructure according to claim 33, wherein the second polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to I53_dn5A* (SEQ ID NO: 15), I53_dn5A.1 (SEQ ID NO: 16), or I53_dn5A.2 (SEQ ID NO: 17).

37. Polynucleotide encoding a polypeptide according to any one of claims 1 to 32 or a nanostructure according to any one of claims 33 to 36.

38. The polynucleotide according to claim 37, which is messenger RNA (mRNA).

39. Lipid nanoparticles (LNPs) comprising a polypeptide according to any one of claims 1 to 32 or mRNA encoding a nanostructure according to any one of claims 33 to 36.

40. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 32, a nanostructure according to any one of claims 33 to 36, a polynucleotide according to any one of claims 37 to 38, or an LNP according to claim 39.

41. A method for generating an immune response to CMV gB protein in a subject requiring it, comprising administering an effective amount of the pharmaceutical composition described in claim 40 to the subject.

42. A method for treating or preventing CMV infection in a subject requiring such treatment, comprising administering an effective amount of the pharmaceutical composition according to claim 40 to the subject.

43. An expression vector comprising a polynucleotide according to any one of claims 37 to 38, operably linked to a suitable control sequence.

44. A host cell comprising a polynucleotide according to any one of claims 37 to 38, an expression vector according to claim 43, and / or a polypeptide according to any one of claims 1 to 32.

45. A polypeptide comprising an amino acid sequence or antigenic fragment thereof that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3, (a) Residues 698–702 are optional if they do not contain mutations; (b) Residue 456 of Sequence ID No. 3 is missing, and (c) The polypeptide or its antigenic fragment comprises one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 as listed in Table 2, Table 3, or Table 4. Polypeptide.

46. The polypeptide according to claim 45, wherein the polypeptide or its antigenic fragment comprises one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 2 or Table 3.

47. The polypeptide according to claim 45, wherein the polypeptide or its antigenic fragment comprises one or more combinations of mutations to residues 91-702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 2.

48. The polypeptide according to any one of claims 45 to 47, wherein the polypeptide or its antigenic fragment further comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more mutations in residues 91 to 702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 5.

49. The polypeptide according to any one of claims 45 to 48, wherein the polypeptide or its antigenic fragment further comprises one, two, three, four, five, six, seven, eight, nine, or all ten mutations in residues 91 to 702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 6.

50. The polypeptide according to any one of claims 45 to 48, wherein the polypeptide or its antigenic fragment further comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or all twelve of residues 91 to 702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 7.

51. The polypeptide according to any one of claims 45 to 50, further comprising a combination of mutations to residues 91 to 702 of SEQ ID NO: 1 or SEQ ID NO: 3 listed in Table 8.

52. A polypeptide according to any one of claims 45 to 51, comprising an amino acid sequence or antigenic fragment thereof that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the sequences listed in Table 9.

53. A polypeptide according to any one of claims 45 to 52, comprising an amino acid sequence or antigenic fragment thereof that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the sequences listed in Table 10.

54. The polypeptide according to any one of claims 45 to 53, further comprising any other functional domains depending on the intended use, which include but are not limited to a secretory signal located at the N-terminus of the polypeptide, wherein the signal sequence may be any suitable signal sequence depending on the intended use.

55. (a) the polypeptide or antigenic fragment thereof according to any one of claims 45 to 54; and (b) Multimerization domain A fusion protein containing [the specified ingredient].

56. The fusion protein according to claim 55, wherein the polymerizing domain comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the protein sequences listed in Table 12, and the residues in parentheses are optional.

57. A composition comprising one or more polypeptides or fusion proteins according to any one of claims 1 to 56, connected to a scaffold.

58. The composition according to claim 57, wherein the scaffold includes a protein scaffold.

59. The composition according to claim 58, wherein the polypeptide is covalently linked to protein subunits of a protein scaffold to form a fusion protein.

60. The composition according to claim 59, wherein the protein subunit of the protein scaffold comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the protein sequences listed in Table 12, and the residues in parentheses are optional.

61. A nucleic acid encoding a polypeptide or fusion protein according to any one of claims 1 to 56.

62. An expression vector comprising the nucleic acid according to claim 61, which is operably linked to an appropriate control sequence.

63. A host cell comprising the nucleic acid according to claim 61, the expression vector according to claim 62, and / or the polypeptide or fusion protein according to any one of claims 1 to 56.

64. (a) one or more polypeptides, fusion proteins, compositions, nucleic acids, expression vectors, and / or host cells according to any one of claims 1 to 63; and (b) Pharmacologically acceptable carriers A pharmaceutical composition containing the above.

65. (a) one or more polypeptides, fusion proteins, compositions, nucleic acids, expression vectors, and / or host cells according to any one of claims 1 to 63; and (b) Pharmacologically acceptable carriers A vaccine containing [this ingredient].

66. The vaccine according to claim 65, further comprising any other component depending on the intended use, which includes, but is not limited to, any other CMV antigen, including, a pentamer complex of CMV proteins gH, gL, UL128, UL130, and UL131 or its antigenic portion; or any other component depending on the intended use, which includes, but is not limited to, any other CMV antigen.

67. A method for treating or limiting the progression of a CMV infection, comprising administering to a subject in need of it a polypeptide, fusion protein, composition, vaccine, nucleic acid, expression vector, host cell, pharmaceutical composition, and / or vaccine according to any one of claims 1 to 66 in an amount effective for treating or limiting the progression of a CMV infection.

68. The compositions, methods, or uses described herein.

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