stabilized vaccine

Abstract

The present disclosure relates to fusion proteins, and uses thereof, comprising a viral fusion protein ectodomain linked to one or more heptad repeats (HR) from the SARS-COV-2 spike (S) protein or the respiratory syncytial virus (RSV) F protein. The viral fusion proteins are suitable for use as vaccines.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 63 / 383,041, entitled "Stabilized vaccines," filed November 9, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to vaccines against viruses and uses thereof. [Background technology]

[0004] Viral fusion proteins and the RNAs encoding them are vaccine candidates because they are the primary targets of protective neutralizing antibody responses against many medically important viruses, such as enveloped viruses. However, recent evidence indicates that broadly cross-reactive and potent neutralizing antibodies elicited during natural infection react primarily with the pre-fusion form, not the post-fusion form, and the inherent metastable nature of fusion proteins poses an obstacle to the design of effective subunit vaccines. In addition, the pre-fusion form of viral fusion proteins contains epitopes not present in the post-fusion form (e.g., Magro et al., 2012. Proc. Natl. Acad. Sci. USA 109(8):3089-3094). Therefore, for vaccines, stabilized pre-fusion forms are generally considered more antigenically desirable. However, conventional approaches to recombinant expression of these proteins typically result in a premature conformational shift to the structurally more stable post-fusion form.

[0005] Consequently, there is an urgent need for new approaches to generate stabilized recombinant fusion proteins that remain substantially in their pre-fusion form to stimulate more effective immune responses against enveloped viruses. Summary of the Invention

[0006] In the work leading to the present invention, the inventors sought to generate a viral protein ectodomain stabilized in a pre-fusion conformation suitable for use as a vaccine. To achieve this goal, the inventors fused the viral protein ectodomain to at least one heptad repeat (HR) from the SARS-COV-2 spike (S) protein or at least one HR from the respiratory syncytial virus (RSV) fusion (F) protein. For example, the inventors generated a fusion protein comprising the RSV F protein ectodomain fused to two HRs, i.e., HR1 and HR2, of the SARS-COV-2 S protein. The inventors also generated a fusion protein comprising the SARS-COV-2 S protein ectodomain fused to one HR (HR1) of the SARS-COV-2 S protein, and a fusion protein comprising the RSV F protein ectodomain fused to two RSV HRs (HR1 and HR2). The present inventors have shown that proteins can be expressed and trimerized to form structures that, in the case of viral components, can induce an immune response against viral proteins, and that the immune response is protective against infection. These findings provide the basis for reagents and methods useful for immunizing subjects against viral infections, e.g., for vaccines.

[0007] By using the SARS-COV-2 HR or RSV HR sequence, the inventors have used a stabilized structure that a large portion of the population has previously been exposed to, either through vaccination or infection with SARS-COV-2 or RSV. In addition, neither antibodies against the SARS-COV-2 S protein nor antibodies against the RSV F protein are generally used as diagnostic targets, meaning that vaccination with the fusion proteins of the present disclosure is unlikely to induce an immune response that could interfere with diagnostic testing.

[0008] In one example, the present disclosure provides a fusion protein comprising an ectodomain of a viral fusion protein linked to a HR from the SARS-COV-2 S protein or a HR from the RSV F protein.

[0009] In one example, the present disclosure provides a fusion protein comprising an ectodomain of a viral fusion protein linked to the HR from the RSV F protein.

[0010] In one example, the present disclosure provides a fusion protein comprising the ectodomain of a viral fusion protein linked to the HR from the SARS-COV-2 S protein.

[0011] For example, the ectodomain of a viral fusion protein lacks the transmembrane and cytoplasmic domains of the protein.

[0012] In one example, the HR is additional to any HR(s) present in the ectodomain.

[0013] In one example, the HR is HR1 from the SARS-COV-2 S protein.

[0014] In one example, the HR is HR1 from the RSV F protein.

[0015] In one example, the ectodomain is linked to two HRs from the SARS-COV-2 S protein, for example, the HRs are HR1 and HR2 from the SARS-COV-2 S protein.

[0016] An exemplary HR1 from SARS-COV-2 comprises the sequence set forth in SEQ ID NO: 1 or 16.

[0017] An exemplary HR2 from SARS-COV-2 comprises the sequence set forth in SEQ ID NO: 2 or 16.

[0018] In one example, the ectodomain is linked to two HRs from the RSV F protein.

[0019] In one example, HR1 and HR2 are from the RSV F protein.

[0020] An exemplary HR1 from RSV comprises the sequence set forth in SEQ ID NO:14.

[0021] An exemplary HR2 from RSV comprises the sequence set forth in SEQ ID NO:15.

[0022] In one exemplary embodiment of the present disclosure, the ectodomain is a respiratory syncytial virus F protein ectodomain.

[0023] In one example, the ectodomain comprises one or more mutations to stabilize the ectodomain in a pre-fusion conformation.

[0024] In one example, the mutation(s) introduce one or more cysteine ​​residues that form disulfide bonds not present in the native ectodomain, and / or mutations that introduce amino acids that fill a hydrophobic cavity present in the native ectodomain.

[0025] In one example, the ectodomain contains the following mutations that stabilize the F protein ectodomain in the pre-fusion conformation: (i) S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3; (ii) N67I and S215P relative to SEQ ID NO: 3; (iii) N67I, S215P, and E487Q relative to SEQ ID NO: 3; (iv) Contains one or more of D486H, E487Q, F488W, and D489H relative to SEQ ID NO: 3.

[0026] For example, the RSV F protein ectodomain includes mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO:3.

[0027] In one example, the RSV F protein ectodomain comprises or consists of the sequence set forth in SEQ ID NO:18.

[0028] In one example, the fusion protein comprises, in amino to carboxy order: (i) the RSV F protein ectodomain, HR1, and HR2, including the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3; or (ii) RSV F protein ectodomain, HR2, and HR1 containing mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3.

[0029] For example, HR1 and HR2 (or HR2 and HR1) are from RSV.

[0030] In one example, the disclosure provides a fusion protein comprising a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, the following mutations relative to SEQ ID NO:3: S155C, S290C, S190F, and V207L; a linker; HR1 from RSV; and HR2 from RSV.

[0031] In one example, the fusion protein includes, in amino to carboxy order, a respiratory syncytial virus F protein ectodomain containing the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3, a linker including the sequence GGSGGSGGGGSGGSGG (SEQ ID NO: 13), HR1 from RSV, and HR2 from RSV.

[0032] In one example, the disclosure includes a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, the sequence set forth in SEQ ID NO: 18, a linker comprising the sequence set forth in any one of SEQ ID NOs: 10-13, HR1 comprising the sequence set forth in SEQ ID NO: 14, a linker, and HR2 comprising the sequence set forth in SEQ ID NO: 15.

[0033] In one example, the disclosure includes a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, the sequence set forth in SEQ ID NO: 18, a linker comprising the sequence set forth in SEQ ID NO: 13, HR1 comprising the sequence set forth in SEQ ID NO: 14, a linker, and HR2 comprising the sequence set forth in SEQ ID NO: 15.

[0034] In one example, the fusion protein comprises, in amino to carboxy order, a respiratory syncytial virus F protein ectodomain comprising the following mutations relative to SEQ ID NO:3: S155C, S290C, S190F, and V207L; a linker comprising the sequence GGSGGSGGGGSGGSGG (SEQ ID NO:13); HR1 from SARS-COV-2; and HR2 from SARS-COV-2.

[0035] In one example, the disclosure includes a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, the sequence set forth in SEQ ID NO: 18, a linker comprising the sequence set forth in any one of SEQ ID NOs: 10-13, HR1 comprising the sequence set forth in SEQ ID NO: 16, a linker, and HR2 comprising the sequence set forth in SEQ ID NO: 17.

[0036] In one example, the disclosure includes a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, the sequence set forth in SEQ ID NO: 18, a linker comprising the sequence set forth in SEQ ID NO: 13, HR1 comprising the sequence set forth in SEQ ID NO: 16, a linker, and HR2 comprising the sequence set forth in SEQ ID NO: 17.

[0037] In one example, the fusion protein comprises a sequence set forth in any one of SEQ ID NOs: 5-9 or 19-23, and optionally comprises a carboxy-terminal hexa-HIS tag and / or a streptavidin tag.

[0038] In one example, the fusion protein comprises a sequence set forth in any one of SEQ ID NOs: 19-23.

[0039] In one example, the fusion protein comprises a sequence set forth in any one of SEQ ID NOs: 24-28.

[0040] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO:19.

[0041] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO:20.

[0042] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO:21.

[0043] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO:22.

[0044] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO:23.

[0045] In one example, the ectodomain is a SARS-COV-2 S protein ectodomain. In this regard, the ectodomain can include a sequence occurring in any variant of SARS-COV-2, or a combination of mutations occurring in such variants.

[0046] In one example, the SARS-COV-2 ectodomain is (i) K986P and V987P relative to SEQ ID NO: 4, and / or (ii) containing one or more mutations in the furin cleavage site at positions 682 to 685 of SEQ ID NO: 4.

[0047] In one example, the furin cleavage site is mutated from RRAR to QQAA or GSAS.

[0048] In one example, the fusion protein comprises a SARS-COV-2 S protein ectodomain and one HR of the SARS-COV-2 S protein, for example, the HR is HR1.

[0049] In one example, the fusion protein comprises, in amino to carboxy order, the SARS-COV-2 S protein ectodomain and HR1 of the SARS-COV-2 S protein.

[0050] In one example, the fusion protein comprises the sequence set forth in SEQ ID NO:9.

[0051] In one example, the ectodomain and the HR(s) are connected by a linker.

[0052] In one example, the linker comprises glycine and serine, for example, the linker comprises the sequence (GGGGS)2 or (GGGGS)3.

[0053] In one example, the linker is selected from SEQ ID NOs: 10 to 13. For example, the linker comprises the sequence set forth in SEQ ID NO: 13.

[0054] In one example, HR1 and HR2 are linked via an additional linker.

[0055] In one example, the further linker comprises glycine and serine, for example, the further linker comprises the sequence (GGGGS)2 or (GGGGS)3.

[0056] In one example, the additional linker is selected from SEQ ID NOs: 10 to 13. For example, the additional linker comprises the sequence set forth in SEQ ID NO: 13.

[0057] The present disclosure additionally provides a complex or trimer comprising three of the fusion proteins of the present disclosure. For example, the fusion proteins are associated via HR(s). In one example, the trimer is a homotrimer, or the complex is a homocomplex.

[0058] The present disclosure additionally provides nucleic acids encoding the fusion proteins of the present disclosure. For example, the nucleic acid is DNA, such as a plasmid. For example, the nucleic acid is RNA, such as mRNA or sa-mRNA vaccines.

[0059] The present disclosure additionally provides nanoparticles comprising the nucleic acids described herein.

[0060] The present disclosure also provides a composition comprising a fusion protein described herein, a nucleic acid described herein, or a nanoparticle described herein.

[0061] For example, the composition further comprises an adjuvant, such as an oil-in-water emulsion of squalene, polyoxyethylene sorbitan monooleate, and a sorbitan trioleate compound, such as MF59.

[0062] In one example, the composition comprises a fusion protein comprising a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, the following mutations relative to SEQ ID NO: 3: S155C, S290C, S190F, and V207L; a linker comprising the sequence set forth in SEQ ID NO: 13; HR1 from RSV; and HR2 from RSV; and an adjuvant that is an oil-in-water emulsion of squalene, polyoxyethylene sorbitan monooleate, and sorbitan trioleate compounds.

[0063] The present disclosure additionally provides a method of inducing an immune response in a subject, the method comprising administering to the subject a fusion protein described herein, a nucleic acid described herein, a nanoparticle described herein, or a composition described herein. In one example, the immune response is against a virus. For example, the immune response is an antibody response. In one example, the immune response is a protective immune response.

[0064] The present disclosure also provides a method of immunizing a subject, the method comprising administering to the subject a fusion protein described herein, a nucleic acid described herein, a nanoparticle described herein, or a composition described herein.

[0065] The present disclosure also provides a method described herein for treating or preventing infection by a virus, the method comprising administering to a subject a fusion protein described herein, a nucleic acid described herein, a nanoparticle described herein, or a composition described herein.

[0066] In one example, the fusion protein, composition, nucleic acid, or LNP of the present disclosure is administered in combination with an influenza vaccine. In one example, the fusion protein, composition, nucleic acid, or LNP and the influenza vaccine are in the same composition. In another example, the fusion protein, composition, nucleic acid, or LNP and the influenza vaccine are in separate compositions. In one example, the influenza vaccine is aQIVc grown in cultured cells, an MF59-adjuvanted tetravalent vaccine.

[0067] In one example, a fusion protein comprising the RSV F protein ectodomain described herein is administered in conjunction with an influenza vaccine and / or a vaccine against SARS-COV-2.

[0068] In one example, a fusion protein comprising the RSV F protein ectodomain described herein is administered in conjunction with a vaccine against PIV3 and / or hMPV. [Brief explanation of the drawings]

[0069] [Figure 1] 1 is a graphical representation showing a series of fusion proteins comprising the F protein ectodomain containing mutations at sites S155C, S290C, S190F, and V207L ​​(known as "ds Cav1") fused to the HR1 and HR2 domains of the SARS-COV-2 S protein. [Figure 2A] 1 is a diagram showing a cutaway view of an antigen (RSV F protein ectodomain) linked to a trimerization domain, including HR1 and HR2 from the RSV F protein and various linkers, as used in the examples herein. [Figure 2B] 1 is a diagram showing a cutaway view of an antigen (RSV F protein ectodomain) linked to a trimerization domain, including HR1 and HR2 from the RSV F protein and various linkers, as used in the examples herein. [Figure 3A] 1 is a graphical representation showing antigen-binding IgG titers induced after immunization with a composition comprising a fusion protein comprising a RSV F protein ectodomain linked to HR1 and HR2 from the RSV F protein, with the indicated linker and adjuvant (MF59). Proteins were administered at the indicated amounts. Statistical differences are indicated. *p<0.05, **p<0.01, ***p<0.005. [Figure 3B] 1 is a graphical representation showing the virus neutralization titers induced after immunization with a composition comprising a fusion protein comprising the RSV F protein ectodomain linked to HR1 and HR2 from the RSV F protein, with the indicated linker and adjuvant (MF59). Proteins were administered at the indicated amounts. Statistical differences are indicated. *p<0.05, **p<0.01. [Figure 4] FIG. 1 is a diagrammatic representation showing the design of an RSV challenge experiment in cotton rats using fusion proteins of the present disclosure. [Table 1-1] [Table 1-2] DETAILED DESCRIPTION OF THE INVENTION

[0070] overview Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be interpreted to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0071] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. The present disclosure should be understood to include all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and all combinations of such steps or features, or any two or more of such steps or features.

[0072] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0073] Any example of the present disclosure herein shall apply mutatis mutandis to any other example of the present disclosure unless expressly stated otherwise. In other words, any specific example of the present disclosure may be combined with any other specific example of the present disclosure (except where mutually exclusive).

[0074] Any example of this disclosure disclosing a particular feature or group of features, or method or method step, will be construed as providing explicit support for disclaiming that particular feature or group of features, or method or method step.

[0075] Unless otherwise defined, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0076] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J.E. Coligan et al. al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0077] The term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is interpreted as providing explicit support for both meanings or either meaning.

[0078] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0079] As used herein, the term "derived from" shall be interpreted to indicate that a particular integer may be obtained from a particular source, but not necessarily directly from that source. Similarly, the term "based on" shall be interpreted to indicate that a particular integer may be developed or used from a particular source, but not necessarily directly from that source.

[0080] Selected Definitions As used herein, "ectodomain" refers to a viral protein (e.g., a viral fusion protein) that substantially contains the extracellular portion of a mature viral protein, lacking the transmembrane domain and cytoplasmic tail, with or without a signal peptide. The RSV F ectodomain polypeptide contains the endogenous HRA domain and the endogenous HRB domain. The SARS-COV-2 S protein contains the endogenous HR1 domain and the endogenous HR2 domain. For clarity, terms such as "one HR (HR1)" used throughout this specification refer to one heptad repeat region and do not necessarily refer specifically to the SARS-COV-2 S protein HR1. For example, HR1 can refer to the RSV HRA or simply to the first heptad region, as indicated by the context.

[0081] The term "linker" or "flexible linker," as used herein, refers to a proteinaceous molecule containing at least one amino acid residue, typically at least two amino acid residues, linked by a peptide bond(s), which allows two polypeptides linked thereby to move more freely relative to one another compared to their movement without the flexible linker. In certain instances, a flexible linker provides increased rotational freedom for the two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexible linker. Such relative freedom of movement or rotational freedom allows polypeptides linked by a flexible linker to perform their respective functions or elicit their activities with less structural hindrance. A flexible linker may be characterized by the absence of secondary structure, such as a helix or beta sheet, or by the absence of a maximum secondary structure content of 10%, 20%, 30%, or 40%. Non-limiting examples of flexible linkers include the amino acid sequences GS, GSG, GGS, GGSGG, (GGS)2, GGSG, GSGS, AS, GGGS, (GGS)2GG, ((GGS)2GG)2, G4S, (G4S)2, (G4S)3, (G4S)4, G4SG, GSGG, and GSGGS. Additional flexible linker sequences are known in the art. In various examples, the flexible linker contains or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues. In some examples, the flexible linker contains or consists of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues.In some examples, the flexible linker contains or consists of about 1 to about 30 amino acid residues, about 1 to about 25 amino acid residues, about 1 to about 20 amino acid residues, about 1 to about 15 amino acid residues, about 1 to about 12 amino acid residues, about 1 to about 10 amino acid residues, about 1 to about 8 amino acid residues, about 1 to about 6 amino acid residues, about 1 to about 5 amino acid residues, about 1 to about 4 amino acid residues, or about 1 to about 3 amino acid residues. In some examples, the flexible linker contains or consists of about 2 to about 30 amino acid residues, about 2 to about 25 amino acid residues, about 2 to about 20 amino acid residues, about 2 to about 15 amino acid residues, about 2 to about 12 amino acid residues, about 2 to about 10 amino acid residues, about 2 to about 8 amino acid residues, about 2 to about 6 amino acid residues, about 2 to about 5 amino acid residues, or about 2 to about 4 amino acid residues. In some of the same and other embodiments, the flexible linker contains or consists of about 3 to about 30 amino acid residues, about 3 to about 25 amino acid residues, about 3 to about 20 amino acid residues, about 3 to about 15 amino acid residues, about 3 to about 12 amino acid residues, about 3 to about 10 amino acid residues, about 3 to about 8 amino acid residues, about 3 to about 6 amino acid residues, or about 3 to about 5 amino acid residues. In certain embodiments, the flexible linker contains or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 16 amino acid residues.

[0082] As used herein, the terms "furin cleavage site" and "furin-like cleavage site" are used interchangeably herein and refer to a cleavable bond together with adjacent or non-adjacent recognition elements, or both, sufficient for detectable proteolysis at the cleavable bond by furin under conditions suitable for furin protease activity. Furin cleavage sites are known in the art or can be defined by routine methods. For example, Basak, A. et al.,2001.Biochem.J.353:537-545;Bader,O.et al.,2008.BMC Microbiol.8:116;Schilling,O.et al.,2008.Nat.Biotechnol.26:685-694;Rawlings,NDet al.,2008.Nucleic Acids Res.36(Database issue):D320-D325;Rawlings,NDet al.,2010.Nucleic Acids Res.38(Database issue):D227-D233(2010);Seider,NGet al.,2012.Nat.Rev.Drug Discov.10.1038 / nrd3699;Braun,E.et See al., 2019. Clin. Transl. Immunol., 8: el073; Izaguirre. G., 2019. Viruses 11 (2019), 10.3390 / vll090837. See also Coutard, B. et al., 2020. Antiviral Res. 176: 104742, who identified a furin-like cleavage site in SARS-CoV-2.

[0083] As used herein, the term "post-fusion conformation" of a viral fusion protein refers to the structure of an enveloped viral fusion protein in its terminal conformation (i.e., formed at the end of the fusion process) and in its most energetically favorable state. In the post-fusion conformation, the fusion peptide or loop of the fusion protein is in close proximity to the fusion protein transmembrane domain.

[0084] As used herein, the term "pre-fusion conformation" of a viral fusion protein refers to the structure of the viral fusion protein, which is in a metastable conformation (i.e., a semistable conformation that is not the most energetically favorable terminal conformation) and can undergo conformational rearrangement to a terminal post-fusion conformation upon appropriate triggering. Typically, the pre-fusion conformation of a viral fusion protein contains a hydrophobic sequence called a fusion peptide or fusion loop, which is located internally within the pre-fusion conformation and cannot interact with either the viral or host cell membrane. Upon triggering, this hydrophobic sequence is inserted into the host cell membrane, and the fusion protein collapses into a post-fusion hairpin-like conformation. The pre-fusion conformation of a viral fusion protein varies depending on the class of enveloped fusion protein.

[0085] As used herein, the term "conventional mRNA" or "cRNA" or "unamplified RNA" refers to RNA that is a construct that allows for the expression of heterologous RNA and proteins, but that cannot be amplified within a host cell.

[0086] As used herein, the term "self-replicating RNA" refers to an RNA virus-based construct that has been engineered to allow expression of heterologous mRNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified in a host cell, resulting in expression of a desired gene product in the host cell.

[0087] As used herein, the term "nucleotide sequence" or "nucleic acid sequence" will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. Conventionally, sequences are presented from the 5' to the 3' end unless otherwise specified. To facilitate clear description of nucleic acids, particular sequence components will be referred to as, for example, a "first nucleotide sequence" and a "second nucleotide sequence." It should be understood that the first and second sequences can appear in any desired order or orientation unless otherwise specified, and that no particular order or orientation is intended by the terms "first," "second," etc.

[0088] As used herein, the term "antigen" refers to a molecule or structure containing one or more epitopes that induce, elicit, enhance, or boost a cellular and / or humoral immune response.

[0089] The term "polypeptide" or "polypeptide chain" will be understood to mean a series of consecutive amino acids linked by peptide bonds. For example, a protein shall be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). A series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0090] The term "recombinant" shall be understood to mean the product of artificial genetic recombination.

[0091] The term "adjuvant," as used herein, refers to a compound that, when used in combination with a specific immunogen in a composition (e.g., a modified polypeptide, chimeric polypeptide, polypeptide complex, polynucleotide, or nucleic acid construct of the present disclosure), enhances the resulting immune response, including strengthening or broadening the specificity of either or both antibody and cellular immune responses. In the context of the present disclosure, an adjuvant preferably enhances the specific immunogenic effect of an active agent of the present disclosure. The term "adjuvant" is understood to typically not include an agent that confers immunity itself. An adjuvant nonspecifically assists the immune system to enhance antigen-specific immune responses, for example, by promoting antigen presentation to the immune system or the induction of a nonspecific innate immune response. Furthermore, an adjuvant can modulate an antigen-specific immune response, for example, by shifting a predominantly Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, an adjuvant can advantageously modulate cytokine expression / secretion, antigen presentation, the type of immune response, and the like.

[0092] As used herein, the terms "disease," "disorder," or "pathological condition" refer to a disruption or interference with normal function and include, but are not limited to, any particular condition, including diseases or disorders.

[0093] As used herein, a subject "at risk" of developing a disease or condition may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to treatment according to the present disclosure. "At risk" indicates that the subject has one or more risk factors, which are measurable parameters that correlate with development of a disease or condition, known in the art and / or described herein.

[0094] As used herein, the terms "treating," "treat," or "treatment" include administering a protein, RNA, or composition described herein to thereby reduce or eliminate at least one symptom of a particular disease or condition.

[0095] As used herein, the terms "preventing," "prevent," or "prevention" include providing prophylaxis against the occurrence or recurrence of a particular disease or condition in an individual. An individual may be predisposed to or at risk of developing the disease, but may not yet be diagnosed with the disease.

[0096] As used herein, the phrase "slowing the progression of" includes reducing or slowing the progression of a disease or condition and / or at least one symptom of a disease or condition in an individual.

[0097] An "effective amount" refers to at least an amount effective, at a dosage and for a period of time necessary, to achieve a desired result. For example, the desired result can be a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" refers to the amount necessary to effect treatment for a disease or condition as described herein. In some examples of the present disclosure, the term "effective amount" refers to the amount necessary to bring about a change associated with a disease or condition as described herein. An effective amount can vary depending on the disease or condition being treated or the factor being modified, as well as on the body weight, age, ethnic background, sex, health and / or physical condition, and other factors associated with the mammal being treated. Typically, an effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined by a medical professional through routine testing and experimentation. Therefore, this term should not be construed to limit the present disclosure to a specific amount, e.g., weight or number, of RNA. An effective amount can be administered in a single dose or in one or several repeated doses over a treatment period.

[0098] A "therapeutically effective amount" is the minimum concentration required to bring about at least a clear improvement in a particular disease or condition. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the RNA of the present disclosure to induce a desired response in an individual. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the RNA.

[0099] As used herein, the term "prophylactically effective amount" shall be taken to mean an amount of RNA of the present disclosure sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a disease or disorder described herein.

[0100] As used herein, the term "subject" shall be taken to mean any animal, including a human, e.g., a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0101] As used herein, the term "lipid nanoparticle" or "LNP" shall be understood to refer to a lipid-based particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising a compound of any formula described herein. In embodiments, LNPs are formulated in compositions for delivery of polynucleotides to desired targets, such as cells, tissues, organs, tumors, etc. For example, lipid nanoparticles or LNPs refer to any lipid composition, including, but not limited to, those selected from liposomes or vesicles, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; unilamellar, or multiple; multilamellar) micelle-like lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles, which lack a lipid bilayer.

[0102] RSV F protein The RSV F protein directs viral entry by fusion between the virion envelope and the host cell plasma membrane. It is a type I single-pass integral membrane protein with four common domains: an N-terminal ER translocation signal sequence (SS), an ectodomain (ED), a transmembrane domain (TM), and a cytoplasmic tail (CT). The CT contains a single palmitoylated cysteine ​​residue. The sequence of the F protein is highly conserved among RSV isolates but is constantly evolving (Kim et al. (2007) J Med Virol 79:820-828). Unlike most paramyxoviruses, the F protein in RSV can mediate entry and syncytium formation independently of other viral proteins (UN is usually required in addition to F in other paramyxoviruses).

[0103] hRSV F mRNA is translated into a 574-amino acid precursor protein called F0, which contains a signal peptide sequence at its N-terminus that is removed by signal peptidases in the endoplasmic reticulum. F0 is cleaved at two sites (aa 109 / 110 and 136 / 137) by cellular proteases (specifically furin) in the trans-Golgi to remove a short glycosylated intervening sequence and generate two subunits called F1 (approximately 50 kDa; C-terminus; residues 137-574) and F2 (approximately 20 kDa; N-terminus; residues 1-109). F1 also contains a hydrophobic fusion peptide and two hydrophobic heptad repeat regions (HRA and HRB, herein referred to as HR1 and HR2, respectively) at its N-terminus. HRA / HR1 is located near the fusion peptide, and HRB / HR2 is located near the transmembrane domain. The Fi-F2 heterodimer is organized as a homotrimer in the virion.

[0104] The present disclosure can use any desired RSV F ectodomain amino acid sequence, such as the amino acid sequence of SEQ ID NO: 3 or a sequence having identity to SEQ ID NO: 3. Typically, this has at least 75% identity to SEQ ID NO: 3, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 or 2. The sequence can be naturally found in RSV.

[0105] The amino acid sequences in the fusion proteins of the present disclosure can be naturally found in RSV F ectodomain proteins (e.g., soluble RSV F proteins lacking the transmembrane and cytoplasmic domains) and / or can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) single amino acid mutations (insertion, deletion, or substitution) relative to the native RSV sequence. For example, it is known to mutate the F protein to remove its furin cleavage sequence, thereby preventing intracellular processing.

[0106] The RSV F polypeptide or protein may contain one or more mutations that prevent cleavage at one or both of the furin cleavage sites. RSV F ectodomain polypeptides containing such mutations are not cleaved in vivo by cells that produce the polypeptide and are produced as monomers. Examples of suitable furin cleavage mutations that disrupt furin cleavage include replacing amino acid residues 106-109 of SEQ ID NO: 3 with RARK, RARQ, QAQN, or IEGR. Alternatively or additionally, amino acid residues 133-136 of SEQ ID NO: 3 can be replaced with RKKK, ΔΔΔR, QNQN, QQQR, or IEGR. (Δ indicates a deleted amino acid residue.)

[0107] In one example, the RSV F protein ectodomain contains one or more mutations, e.g., amino acid substitutions, to stabilize the ectodomain in its pre-fusion conformation.

[0108] In some instances, the complexes are characterized by a rounded (pre-fusion) shape when viewed in negative stained electron micrographs.

[0109] In some instances, the complex is characterized by its ability to bind to the D25 antibody, which has been characterized to bind to the pre-fusion structure of the RSV-F protein (McClellan et al., Science. 340(6136):1113-1117 2013).

[0110] In another example, the complex comprises a pre-fusion epitope that is not present in the post-fusion form of the RSV F protein.

[0111] In one example, cysteine ​​residues can be inserted or substituted into the HRB / HR2 region to form disulfide bonds and stabilize the RSV F ectodomain.

[0112] In some embodiments, the RSV F ectodomain polypeptide includes an S155C mutation and an S290C mutation.

[0113] In some instances, the RSV F ectodomain polypeptide includes a mutation at amino acid 190 or amino acid 207. For example, the RSV F ectodomain includes the S190F mutation and / or the V207F mutation.

[0114] In some embodiments, the RSV F ectodomain polypeptide includes an S155C mutation, an S290C mutation, an S190F mutation, and a V207F mutation.

[0115] In some instances, the RSV F ecto-domain polypeptide optionally further comprises an internal deletion of all or part of the p27 sequence, including a corresponding deletion of one or more furin sites, For example, the RSV F ecto-domain comprises an internal deletion of about amino acid 103 to about amino acid 136, or about amino acid 103 to 161.

[0116] In one specific example, the RSV F ectodomain polypeptide comprises the RSV F sequence of DS-CAV1 (McClellan et al., Science. 340(6136):1113-1117 2013). For example, the RSV F protein ectodomain comprises or consists of the sequence set forth in SEQ ID NO: 18.

[0117] In some instances, the RSV F complex may be further stabilized in a pre-fusion form using interchain disulfides, including those disclosed in WO2012 / 158613, or peptides conjugated to oligomerization agents, including but not limited to virus-like particles (VLPs), albumin, or RSV G, or other mutations that further stabilize the monomer and allow it to retain its pre-fusion conformation during formulation and immunization.

[0118] In some instances, the RSV F ectodomain can be further stabilized in the pre-fusion form using disulfide bonds or cavity-filling mutations as disclosed in RSV F McLellan, et al., Science, 342 (6158): 592-8 (2013).

[0119] In one example, the RSV F polypeptide, such as the ectodomain polypeptide, can include all amino acid changes of P102A, I379V, M447V, or a combination thereof, for example, P102A, I379V, and M447V, relative to SEQ ID NO: 3.

[0120] SARS-COV-2 S protein In another example, the ectodomain is from the SARS-CoV-2 S protein. As discussed above, the present disclosure contemplates the ectodomain of the S protein from any SARS-CoV-2 variant. Thus, in one example, the S protein is a mutant S protein.

[0121] Suitable mutant S proteins include those that occur in variants of SARS-COV-2.

[0122] In one example, the mutant S protein comprises a mutation in the receptor binding domain, for example, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, In one example, the mutant S protein comprises a mutation in the receptor binding domain selected from the group consisting of N439K, N439L, L452R, S477N, T478I, V483A, and E484D.

[0123] In one example, the mutant S protein comprises a mutation in the receptor binding domain, for example, the mutations include R346K, K417N, K417T, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, selected from the group consisting of T478I, T478K, P479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, and Y508H. In one example, the mutant S protein comprises a mutation in the receptor binding domain selected from the group consisting of R346K, K417N, K417T, N439K, N439L, L452R, S477N, T478I, V483A, E484D, E484K, and N501Y.

[0124] In one example, the mutant S protein is P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V395I, F400C, R403K, R403S, D404Y, V405Y, V406Y, V407Y, V408Y, V409Y, V410Y, V411Y, V412Y, V413Y, V414Y, V415Y, V416Y, V417Y, V418Y, V419Y, V420Y, V421Y, V422Y, V423Y, V424Y, V425Y, V426Y, V427Y, V428Y, V429Y, V430Y, V431Y, V432Y, V433Y, V434Y, V435Y, V436Y, V437Y, V438Y, V439Y, V440Y, V441Y, V442Y, V443Y, V444Y, V445Y, V445Y, V446Y, V447Y, V448Y, V449Y, V450Y, V451Y, V452Y, V453Y, V454Y, V455Y, V456Y, V457Y, V458 and a mutation selected from the group consisting of: 05V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417R, K417N, I418V, Y421S, Y423C, Y423F, Y423S, D427Y, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, and D614G.

[0125] In one example, the mutant S protein is L18F, D80A, T95I, Y144S, Y145N, D215G, P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V39 5I, F400C, R403K, R403S, D405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417N, K417T, K417R, I418V, Y421S, Y423 C, Y423F, Y423S, D427Y, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, T478K, P 479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F49 0L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, Y508H, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, A570D, D614G, P680H, P681H, A701V, T716I, and D950N.

[0126] In one example, the mutant S protein comprises a mutation selected from the group consisting of A67V, ΔHV69-70, T95I, G142D, ΔVYY143-145, ΔN211, L212I, R214_D215insEPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

[0127] In one example, the mutant S protein includes the following mutations: A67V, ΔHV69-70, T95I, G142D, ΔVYY143-145, ΔN211, L212I, R214_D215insEPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

[0128] In one example, the mutant S protein includes at least D614G.

[0129] In one example, the mutant S protein comprises at least a substitution of two proline residues between residues corresponding to amino acids 986 and 987 of SEQ ID NO:4.

[0130] In one example, the mutant S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains a mutation from RRAR to QQAA at residues corresponding to nucleotides 682-685 of SEQ ID NO:4, and / or (ii) lacks a furin cleavage site at the S2' site, and / or (iii) contains a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO:4, and / or (iv) contains a substitution of two proline residues between residues corresponding to amino acids 986 and 987 of SEQ ID NO:4.

[0131] In one example, the S protein lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:4.

[0132] In one example, the S protein lacks a furin cleavage site at the S2' site.

[0133] In one example, the S protein contains a D to G mutation at the residue corresponding to nucleotide 614 of SEQ ID NO:37.

[0134] In one example, the S protein contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:4.

[0135] Exemplary mutant S proteins are known in the art and are described, for example, in WO2022 / 008438, WO2022 / 008438, WO2021 / 213924, WO2022 / 155530, WO2022 / 155524, WO2023 / 089071, WO2023 / 094713, WO2023 / 147091, and / or WO2023147092.

[0136] Heptad repeat As described herein, exemplary fusion proteins of the present disclosure comprise complementary first and second heptad repeats (HR1 and HR2) that associate with each other under conditions suitable for their association (e.g., in aqueous solution) to form an antiparallel two-helix bundle.

[0137] In some examples of F protein fusion proteins as described herein, HR1 and HR2 preferably lack complementarity to the HRs of the F protein ectodomain and therefore preferentially form an antiparallel two-helix bundle without the structural elements of the ectodomain.

[0138] In some examples of F protein fusion proteins as described herein, HR1 and HR2 are from the F protein ectodomain and form an antiparallel two-helix bundle without the structural elements of the ectodomain.

[0139] In the case of SARS-COV-2 S protein, HR1 can be included in the fusion protein, and HR1 forms an antiparallel two-helix bundle with HR2 in the ectodomain.

[0140] In some examples, each of the HR1 and HR2 regions independently comprises (abcdefg-) n or (defgabc-) n wherein pattern elements "a" through "g" indicate the conventional heptad positions at which the amino acid types are located, and n is a number greater than or equal to 2, and wherein at least 50% (or at least 51% to at least 99%, and all integer percentages therebetween) of conventional heptad positions "a" and "d" are occupied by hydrophobic amino acid types, and at least 50% (or at least 51% to at least 99%, and all integer percentages therebetween) of conventional heptad positions "b," "c," "e," "f," and "g" are occupied by hydrophilic amino acid types, and the resulting distribution of hydrophobic and hydrophilic amino acid types allows for the identification of heptad repeat regions.

[0141] The HR1 / HRA region present in the F polypeptide is the same as or substantially the same as the HRA region in the amino acid sequence of the F0 form of the naturally occurring F protein from which the truncated F1 and F2 peptides are derived. In the case of an RSV F protein, such as an RSV F ecto-domain polypeptide or a recombinant RSV F ecto-domain polypeptide, the endogenous HR1 / HRA region is from about amino acid 158 to about amino acid 196 of the amino acid sequence set forth in SEQ ID NO: 3. In some examples, the endogenous HRA is a full-length HRA. However, deletions and truncations of the HRA are within the scope of the present disclosure, as long as such deletions and truncations do not substantially alter the ability of RSV F to fold into an acceptable conformation (either pre-fusion or post-fusion) for its intended purpose. The HRA can include one or more mutations, such as those discussed herein.

[0142] The HRB region present in the F polypeptide is the same as or substantially the same as the HRB region in the amino acid sequence of the F0 form of a naturally occurring F protein. In the case of an RSV F protein, such as an RSV F ecto-domain polypeptide or a recombinant RSV F ecto-domain polypeptide, the endogenous HRB region is from about amino acid 488 to about amino acid 513 relative to SEQ ID NO: 3. In some instances, the HRB is a full-length HRB. However, deletions and truncations of the HRB region are within the scope of the present disclosure, as long as such deletions and truncations do not substantially alter the ability of RSV F to fold into an acceptable conformation (either pre-fusion or post-fusion) for its intended purpose. The HRB may include one or more mutations, such as those discussed herein.

[0143] In particular examples, HR1 and HR2 are from SARS-COV-2. For example, HR1 comprises the sequence set forth in SEQ ID NO: 1 or 16. For example, HR2 comprises the sequence set forth in SEQ ID NO: 2 or 17.

[0144] For example, HR1 comprises the sequence set forth in SEQ ID NO: 1. For example, HR2 comprises the sequence set forth in SEQ ID NO: 2. For example, in a construct comprising HR1 and HR2 from SARS-COV-2, HR1 comprises the sequence set forth in SEQ ID NO: 1 and HR2 comprises the sequence set forth in SEQ ID NO: 2.

[0145] For example, HR1 comprises the sequence set forth in SEQ ID NO: 16. For example, HR2 comprises the sequence set forth in SEQ ID NO: 17. For example, in a construct comprising HR1 and HR2 from SARS-COV-2, HR1 comprises the sequence set forth in SEQ ID NO: 16 and HR2 comprises the sequence set forth in SEQ ID NO: 17.

[0146] In particular examples, HR1 and HR2 are from RSV. For example, HR1 comprises the sequence set forth in SEQ ID NO: 14 or 29. For example, HR2 comprises the sequence set forth in SEQ ID NO: 15 or 30.

[0147] For example, HR1 comprises the sequence set forth in SEQ ID NO: 14. For example, HR2 comprises the sequence set forth in SEQ ID NO: 15. For example, in a construct comprising HR1 and HR2 from RSV, HR1 comprises the sequence set forth in SEQ ID NO: 14 and HR2 comprises the sequence set forth in SEQ ID NO: 15.

[0148] For example, HR1 comprises the sequence set forth in SEQ ID NO: 29. For example, HR2 comprises the sequence set forth in SEQ ID NO: 30. For example, in a construct comprising HR1 and HR2 from RSV, HR1 comprises the sequence set forth in SEQ ID NO: 29 and HR2 comprises the sequence set forth in SEQ ID NO: 30.

[0149] In one example, the fusion includes only one heptad repeat (HR1), for example, HR1 includes the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 14.

[0150] Under conditions favorable for the association of the complementary heptad repeats of the structure-stabilizing moieties (e.g., in aqueous solution), their association with each other results in the formation of an antiparallel two-helix bundle that inhibits rearrangement of the modified polypeptide to the post-fusion conformation. This two-helix bundle of the structure-stabilizing moieties can trimerize to form a highly stable six-helix bundle, thus enabling the fusion proteins to self-assemble into a complex. The complex thus assembled can mimic the pre-fusion conformation of a wild-type fusion protein, e.g., the F protein or the SARS-CoV-2 S protein complex, and includes three fusion proteins characterized by six-helix bundles formed by the coiled-coil structure of each structure-stabilizing moiety of the chimeric polypeptide.

[0151] As discussed above, the HR1 and HR2 regions can together form oligomers, typically hexamers composed of three HR1 and three HR2 regions, which are thermodynamically stable and represent the post-fusion conformation of class I viral fusion proteins. HR1 and HR2 regions that have a strong tendency to oligomerize are referred to herein as "complementary" heptad repeat regions.

[0152] Linker The fusion protein of the present disclosure preferably includes a linker separating the heptad repeat regions (also referred to herein as HR1 and HR2). Linkers generally comprise any amino acid residue that cannot be clearly assigned to a heptad repeat sequence. Linkers are frequently used in the field of protein engineering to interconnect different functional units. Linkers are generally conformationally flexible in solution and are preferably and primarily composed of polar amino acid residue types. Typical (frequently used) amino acids in flexible linkers are serine and glycine. Although less preferred, flexible linkers may also include alanine, threonine, and proline. Thus, the intervening linker of the structure-stabilizing moiety is preferably conformationally flexible to ensure relaxed (undisturbed) association of HR1 and HR2 as a two-helix bundle that preferably adopts a helical coiled-coil structure. Linkers suitable for use in the polypeptides contemplated herein will be apparent to those skilled in the art and may generally be any linker used in the art to link amino acid sequences, so long as the linker is structurally flexible in the sense that it allows, and preferably does not disrupt, the characteristic two-helix bundle structure of the structure-stabilizing moieties to be organized.

[0153] The intervening linker is preferably an amino acid sequence generally consisting of at least one amino acid residue, usually at least two amino acid residues, with a non-critical upper limit of about 100 amino acid residues selected for convenience. In certain embodiments, the linker consists of about 1 to about 50 amino acid residues, or about 50 to about 100 amino acid residues, usually about 1 to about 40 amino acid residues, typically about 1 to about 30 amino acid residues. In a non-limiting example, the linker has about the same number of amino acids as the number of amino acids connecting the complementary HR1 and HR2 of SARS-COV-2. In a non-limiting example, the linker has about the same number of amino acids as the number of amino acids connecting the complementary HR1 and HR2 of RSV.

[0154] In a particular, non-limiting example, at least 50% of the amino acid residues in the linker sequence are selected from the group consisting of proline, glycine, and serine. In a further non-limiting example, at least 60%, for example, at least 70%, for example, 80%, and more particularly 90% of the amino acid residues in the linker sequence are selected from the group consisting of proline, glycine, and serine. In another particular example, the linker sequence consists essentially of polar amino acid residues, and in such particular embodiments, at least 50%, for example, at least 60%, for example, 70% or 80%, and more particularly 90% or up to 100% of the amino acid residues in the linker sequence are selected from the group consisting of glycine, serine, threonine, alanine, proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, and arginine. In a particular example, the linker sequence is [GGSG] n GG, [GGGGS] n , [GGGGGG] n , [GGGKGGGG] n , [GGGNGGGG] n , [GGGCGGGG] n where n is an integer from 1 to 10, preferably from 1 to 5, and more preferably from 1 to 3. In one example, the linker is selected from SEQ ID NOs: 10 to 13. For example, the linker comprises the sequence set forth in SEQ ID NO: 13.

[0155] Purification Tag In one example, the fusion protein of the present disclosure comprises a purification tag. Purification tags typically comprise an amino acid stretch that allows for the recovery of the chimeric polypeptide through affinity binding. Numerous purification tags are known in the art, illustrative examples of which include biotin carboxyl carrier protein-tag (BCCP-tag), Myc-tag (c-myc-tag), calmodulin-tag, FLAG-tag, HA-tag, His-tag (hexahistidine-tag, His6, 6H), maltose-binding protein-tag (MBP-tag), Nus-tag, chitin-binding protein-tag (CBP-tag), glutathione-S-transferase-tag (GST-tag), green fluorescent protein-tag (GFP-tag), polyglutamate-tag, amyloid beta-tag, thioredoxin-tag, S-tag, Softag 1, Softag 3, and Strep-tag.

[0156] Complex The present disclosure additionally provides a complex of the fusion proteins described herein, the complex containing an oligomer of the fusion proteins described herein. The term "oligomer" refers to a molecule consisting of two or more, but a limited number of, monomeric units, as opposed to a polymer, which, at least in principle, consists of an unlimited number of monomers. Oligomers include, but are not limited to, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, and the like. An oligomer can be a macromolecular complex formed by the noncovalent association of macromolecules, such as proteins. In this sense, homooligomers are formed by identical molecules, whereas heterooligomers are made from at least two different molecules. In a specific example, an oligomer of the present disclosure is a trimeric polypeptide complex consisting of three polypeptide subunits. In these examples, the trimeric polypeptide can be a "homotrimeric polypeptide complex" consisting of three identical polypeptide subunits, or a "heterotrimeric polypeptide complex" consisting of three polypeptide subunits, in which at least one subunit polypeptide is not identical. The "polypeptide subunit" is a fusion protein of the present disclosure.

[0157] The fusion proteins of the present disclosure can self-assemble under suitable conditions to form a complex. Thus, the present disclosure further encompasses a method of producing a complex, comprising combining fusion proteins of the present disclosure under conditions suitable for complex formation (e.g., in an aqueous solution), thereby producing a complex containing three fusion proteins. The combined fusion proteins may or may not be identical, thereby forming homotrimers and heterotrimers, respectively.

[0158] Generally, fusion proteins self-assemble in a buffered aqueous solution (e.g., a pH of about 5 to about 9). If necessary, mild denaturing conditions can be used, for example, by including urea, a small amount of organic solvent, or heat, to gently denature the fusion protein to facilitate refolding and self-assembly.

[0159] Any suitable preparation of the fusion protein can be used in the method. For example, conditioned cell culture medium containing the desired fusion protein can be used in the method. However, it is desirable to use purified fusion protein in the method. Methods for detecting oligomer formation are well known in the art and include, for example, Western blots using non-boiled, non-reduced samples, and size-exclusion chromatography, including size-exclusion chromatography in conjunction with antibody binding. While such methods do not allow direct observation of six-helix bundles, the formation of oligomers without significant formation of higher-order structure, as determined by Western blot, can be understood to be indicative of six-helix bundle formation. In size-exclusion chromatography, the formation of a complex of appropriate mobility for the oligomer of interest will peak at the appropriate position without significant flow indicative of higher-order structure and without a significant shoulder. Suitable controls for determining the mobility of trimers (e.g., DS-CAV1 with a foldon trimerization domain as provided in McClellan et al., 2013) and monomers (e.g., delta p23 furdel) can be readily identified by those skilled in the art. In one example, at least 60% (by weight) of the fusion protein in a sample is present in the trimer form. In another example, at least 70% (by weight) of the fusion protein in a sample is present in the trimer form. In another example, at least 75% (by weight) of the fusion protein in the sample is present as a trimer. In a further example, at least 80% (by weight) of the fusion protein in the sample is present as a trimer. In another example, at least 85% (by weight) of the fusion protein in the sample is present as a trimer. In a further example, at least 90% (by weight) of the fusion protein in the sample is present as a trimer. In a still further example, at least 95% (by weight) of the fusion protein in the sample is present as a trimer. Methods such as Western blot can be used to measure the amount of protein present in monomeric and trimeric forms. Those skilled in the art will understand the use of appropriate control samples and standards to provide quantitative results or results from Western blots that allow for the determination of the relative portions of protein present in monomeric, trimeric, or other forms. Similarly, chromatographic methods can be performed with appropriate controls to determine at least the relative amounts of protein in monomeric, trimeric, and other forms.

[0160] Compositions and vaccines The present disclosure additionally provides compositions comprising the fusion proteins disclosed herein. The compositions are suitable for administration to mammalian subjects, such as humans, and may include one or more pharmaceutically acceptable carriers and / or excipients, including adjuvants. A thorough discussion of such components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. The compositions are generally in aqueous form. If the composition is an immunogenic composition or a vaccine, the composition elicits an immune response when administered to a mammal, such as a human. In some examples, in the case of a vaccine, the immune response is a neutralizing immune response or a protective immune response.

[0161] The composition may contain a single active ingredient, such as a fusion protein, or several active ingredients. For example, the composition may contain an RSV-F fusion protein and a SARS-COV-2 S fusion protein. In another example, the composition may contain a SARS-COV-2 S fusion protein, where the S protein ectodomain is from a different SARS-COV-2 variant. For example, the composition may contain one or more immunogens from other pathogens, such as influenza.

[0162] The composition may contain a preservative such as thiomersal or 2-phenoxyethanol.

[0163] To control tension, the composition can include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is exemplary and can be present at 1-20 mg / ml. Other salts that can be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, calcium chloride, etc.

[0164] The composition will generally have an osmolality of, for example, within the range of 290-310 mOsm / kg, such as 200 mOsm / kg to 400 mOsm / kg, for example 240-360 mOsm / kg.

[0165] The composition may contain one or more buffers. Typical buffers include phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer (especially with aluminum hydroxide adjuvant), or citrate buffer. Buffers are typically present in the range of 5-20 mM.

[0166] The pH of the composition will generally be from 5.0 to 8.1, and more typically from 6.0 to 8.0, for example, from 6.5 to 7.5, or from 7.0 to 7.8. In one example, the composition is sterile. The composition is preferably non-pyrogenic, for example, containing less than 1 EU (endotoxin unit, a standard measure) per dose, preferably less than 0.1 EU per dose. The composition is preferably gluten-free. Human vaccines are typically administered in a dose of about 0.5 ml, although a half dose (i.e., about 0.25 ml) can be administered to children.

[0167] In one example, the composition includes an adjuvant. According to the present disclosure, the adjuvant may be, but is not limited to, an organic, inorganic, oil-based adjuvant, or a virosome.

[0168] Inorganic adjuvants include, but are not limited to, mineral adjuvants such as aluminum or calcium salts, e.g., aluminum phosphate, aluminum hydroxide (also referred to herein as Al(OH)), potassium aluminum sulfate (also referred to as alum), and calcium phosphate. Such adjuvants may be used with or without other adjuvants. Organic adjuvants include, but are not limited to, squalene.

[0169] Further examples of adjuvants according to the present disclosure include, but are not limited to, MPL (monophosphoryl lipid A), AS03 (developed by GSK, Prepandrix), AS04 (developed by GSK; a combination of MPL and aluminum hydroxide; Fendrix; Cervarix), QS21 (a purified plant extract of saponin from the soapbark tree (Quillaja saponaria) containing triterpene glucosides), AS01 (developed by GSK; liposomes; QS21 and MPL), AS02 (developed by GSK; QS21 and MPL), LT (heat-labile enterotoxin from Escherichia coli), CpG (oligonucleotides containing unmethylated CpG sequences), and MF59 (manufactured by Novartis). MF59 is a submicron oil-in-water emulsion of squalene, polyoxyethylene sorbitan monooleate, and sorbitan trioleate compounds.

[0170] Suitable adjuvants for the present disclosure include, for example, mineral adjuvants or squalene-containing adjuvants, such as an emulsion of squalene, e.g., MF59. In one example, a composition of the present disclosure includes a fusion protein of the present disclosure and either (i) MF59 or (ii) an aluminum salt (such as aluminum hydroxide). In one example, a composition of the present disclosure includes a fusion protein of the present disclosure and MF59.

[0171] The choice of adjuvant depends on the efficiency of the adjuvant in enhancing the immune response, the stability of the composition containing the adjuvant, for example, a vaccine containing the adjuvant, the route of administration, the administration regimen, and the species to be vaccinated.

[0172] Two or more adjuvants can be combined, for example, an aluminum salt can be combined with MPL, QS21, and / or MF59.

[0173] nucleic acid The present disclosure additionally provides nucleic acids encoding the fusion proteins of the present disclosure.

[0174] In some examples, the nucleic acid is an mRNA or sa-mRNA encoding a fusion protein nucleic acid useful in the present disclosure and may include a first region (e.g., a coding region) of linked nucleosides encoding a polypeptide of interest, a first flanking region (e.g., a 5'-UTR) located at the 5'-end of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3'-end of the first region, at least one 5'-cap region, and a 3'-stabilization region. In some examples, the nucleic acid further includes a polyA region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the nucleic acid may contain one or more intron sequences that can be excised from the nucleic acid. In some examples, the nucleic acid (e.g., mRNA) may include a 5'-cap structure, a chain-terminating nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal. Any one of the regions of the nucleic acid may include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilization region can contain alternative nucleosides, such as L-nucleosides, inverted thymidines, or 2'-O-methyl nucleosides, and / or the coding region, 5'-UTR, 3'-UTR, or cap region can include alternative nucleosides, such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidines (e.g., 5-methyl-cytidine).

[0175] Nucleic acids suitable for use with the present LNPs may contain one or more naturally occurring components, including any of the standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the poly-A tail, and any combination (of a, b, c, or d above) contain the naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0176] In some instances, the nucleic acid may contain one or more surrogate components, as described herein, that confer useful properties, including improved stability of the cell into which the nucleic acid is introduced and / or a lack of substantial induction of an innate immune response. For example, the surrogate nucleic acid exhibits reduced degradation in the cell into which the nucleic acid is introduced, compared to the corresponding unmodified nucleic acid. These surrogate species may enhance the efficiency of protein production, intracellular retention of the nucleic acid, and / or viability of the contacted cell, and may also have reduced immunogenicity.

[0177] Nucleic acids can be naturally occurring or non-naturally occurring. Nucleic acids can contain one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. Nucleic acids can contain any useful modification or alteration, such as a nucleobase, sugar, or internucleoside linkage (e.g., to a phosphate linkage / phosphodiester linkage / phosphodiester backbone). In some embodiments, one or more modifications are present in each of the nucleobase, sugar, and internucleoside linkage.

[0178] Nucleic acids may be uniformly modified or unmodified along the entire length of the molecule, for example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may be uniformly modified or unmodified in a nucleic acid or a given predetermined sequence region thereof.

[0179] Different sugar modifications and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a nucleic acid. One skilled in the art will understand that nucleotide analogs or other modification(s) can be placed at any position(s) of a nucleic acid so as not to substantially reduce the function of the nucleic acid. Modifications can be 5'- or 3'-terminal modifications. In some embodiments, the nucleic acid comprises a modification at the 3'-terminus.

[0180] lipid nanoparticles The present disclosure provides LNPs for delivery of nucleic acids, such as RNA or sa-mRNA, encoding the fusion proteins of the present disclosure.

[0181] In examples, LNPs are approximately 30nm to approximately 160nm, approximately 40nm to approximately 160nm, approximately 50nm to approximately 160nm, approximately 60nm to approximately 160nm, approximately 70nm to approximately 160nm, approximately 50nm. ~about 140nm, about 60nm to about 130nm, about 70nm to about 120nm, about 80nm to about 120nm, about 90nm to about 120nm, about 70 to about 110nm, about 80nm to about 1 The LNPs have an average diameter of 10 nm, or about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, or 160 nm. The diameter of the LNPs can be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), or other methods as known in the art.

[0182] In some examples, LNPs can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of LNPs. A small polydispersity index, e.g., less than 0.3 or less than 0.2, generally indicates a narrow particle size distribution. LNP compositions described herein can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of an LNP composition can be about 0 to about 0.20 or 0.05 to 0.20.

[0183] The LNPs may comprise cationic and / or ionic lipids, neutral lipids, PEG-lipids, and sterols.

[0184] The LNPs may comprise cationic and / or ionic lipids selected from the non-limiting group consisting of: ●3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), ● 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), ●1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), ●2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), ●2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), ●1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and ●8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester.

[0185] It will be clear to those skilled in the art that the reference to PEGylated lipids refers to lipids modified with polyethylene glycol. Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. In embodiments, PEG lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0186] For example, suitable neutral or zwitterionic lipids for use in the present disclosure will be apparent to those of skill in the art, and in embodiments include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids can be saturated or unsaturated.

[0187] Exemplary structural lipids or sterols include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.

[0188] In one example, the structured lipid is a sterol. In an embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is campesterol.

[0189] Methods of Treatment and Administration The compositions of the present disclosure are suitable for administration to mammals, e.g., humans, and the present invention provides a method of inducing an immune response in a mammal, comprising administering a composition (e.g., an immunogenic composition) or a fusion protein or a nucleic acid encoding the same of the present disclosure to the mammal. In certain examples, the immune response is a neutralizing immune response. The composition (e.g., the immunogenic composition) can be used to generate a vaccine formulation for immunizing a mammal. The mammal is typically a human.

[0190] The present disclosure also provides compositions for use as a pharmaceutical, e.g., for use in immunizing a patient against a viral infection, e.g., a RSV infection or a SARS-COV-2 infection, e.g., for use in raising a neutralizing immune response in a patient.

[0191] The immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response (ie, a neutralizing response).

[0192] Methods for assessing antibody responses following vaccination are known in the art. The compositions of the present invention can be administered in a number of suitable ways, such as intramuscular injection (e.g., into the arm or leg), subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, etc. The appropriate route of administration will depend on the age, health, and other characteristics of the mammal. The clinician can determine the appropriate route of administration based on these and other factors.

[0193] The immunogenic compositions and vaccine formulations can be used to treat children and adults, including pregnant women. Thus, subjects can be under 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred subjects for receiving the vaccine are elderly people (e.g., over 50 years old, over 60 years old, and preferably over 65 years old) and pregnant women. However, the vaccine is not suitable only for these groups and can be used more generally in the population.

[0194] Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses can be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple dose schedule, various doses can be given by the same or different routes, e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive patients.

[0195] Multiple doses are typically administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0196] The present disclosure includes the following non-limiting examples. [Example]

[0197] Example 1: RSV-F protein ectodomain-SARS-COV-2 HR fusion protein Fusion proteins containing DS Cav1 variants of the RSV F protein ectodomain (including the S155C, S290C, S190F, and V207L ​​mutations) linked to SARS-COV-2 HR1 and HR2 (in either order) were generated (Figure 1). mRNA encoding the fusion proteins was also generated and encapsulated in LNPs. The proteins were administered with MF59 adjuvant. Administration of the fusion proteins or RNA to mice induced a dose-dependent anti-F protein antibody response that was shown to be neutralizing.

[0198] Example 2: SARS-COV-2 S protein ectodomain-SARS-COV-2 HR fusion protein Fusion proteins containing the SARS-COV-2 S protein ectodomain fused to HR1 of the SARS-COV-2 S protein or to both HR1 and HR2 of the SARS-COV-2 S protein were generated (Figure 1). The SARS-COV-2 S protein ectodomain contained proline substitutions at positions K986P and V987P relative to SEQ ID NO: 4 and a mutation in the furin cleavage site at positions 682-685 from RRAR to QQAA or GSAS. Fusion proteins containing only HR1 comprise the sequence set forth in SEQ ID NO: 9. mRNAs encoding the fusion proteins were also generated.

[0199] We found that fusion proteins containing only HR1 were produced at higher levels and were more stable than fusion proteins containing HR1 and HR2, which was somewhat surprising because the HR1 domain in the fusion protein likely interacts with HR2 in the ectodomain.

[0200] The present inventors further found that administration of the fusion protein or its encoding sa-mRNA (optionally with MF59 adjuvant) induced antibody responses against various SARS-COV-2 variants. Furthermore, the present inventors found that a fusion protein containing only HR1 was more immunogenic than a fusion protein containing HR1 and HR2.

[0201] Example 3: Linker Trimeric recombinant protein antigens containing the RSV-F protein ectodomain linked to SARS-COV-2 HR1 and HR2 were generated with various linker lengths between the antigenic region of the protein and the trimerization domain (Figure 2). The linkers ranged from 0 to 16 amino acids in length. Trimeric recombinant protein antigens containing the RSV-F protein ectodomain linked to RSV HR1 and HR2 with the same linker combinations ranging from 0 to 16 amino acids in length can also be generated. Some examples of linkers tested are listed in Table 1 below. [Table 2]

[0202] Example 4: Linker length affects immunogenicity. Trimeric recombinant protein antigens (Table 1) with different linker lengths were administered to mice (optionally with MF59 adjuvant). Fusion proteins were administered at doses ranging from 1 μg to 0.1 μg. ELISA was performed to confirm IgG binding. The induction of binding IgG was observed to vary depending on the linker length (Figure 3A). Neutralization titers were also observed to vary depending on the linker length (Figure 3B). 0.1 μg was the preferred dose, advantageously providing good binding and neutralization, especially when linker 4 (GGSGGSGGGGSGGSGG, SEQ ID NO: 13) was used in the fusion protein.

[0203] Example 5: Cotton Rat RSV Challenge Study Design Preclinical studies involve cotton rat studies (Figure 4). After acclimation, 6-week-old female cotton rats (CR) are immunized twice, 3 weeks apart, with vaccine or saline control. Four weeks after the booster vaccination, all CRs are immunized with 10 6Animals are challenged intranasally with plaque-forming units (PFU) of RSV A2 virus. Nasal swabs are collected daily before and for 5 days after virus challenge for viral load quantification by real-time quantitative polymerase chain reaction (RTqPCR). Serum is collected the day before immunization (vaccine or saline), 20 days after immunization (vaccine or saline), 48 days after immunization (vaccine or saline), and 54 days after immunization (vaccine or saline) for immunogenicity testing. On study day 54 (day 5 post-challenge), animals are euthanized and blood is collected for serological assays. Lung and nasal tissues are collected for PFU analysis and viral load by RTqPCR. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11]

Claims

1. A fusion protein comprising the ectodomain of a viral fusion protein linked to the heptad repeat (HR) from the SARS-COV-2 spike (S) protein or the HR from the respiratory syncytial virus (RSV) F protein.

2. A fusion protein comprising the ectodomain of a viral fusion protein linked to the heptad repeat (HR) from the respiratory syncytial virus (RSV) F protein.

3. 3. The fusion protein of claim 1, wherein the HR is HR1 from the RSV F protein.

4. The fusion protein of claim 1 or 2, comprising the ectodomain linked to two HRs from an RSV F protein.

5. The fusion protein of claim 4 , comprising HR1 and HR2 from the RSV F protein.

6. 6. The fusion protein of claim 1, wherein the HR(s) are additional to any HR in the ectodomain.

7. The fusion protein of any one of claims 1 to 6, wherein the ectodomain lacks a transmembrane region and a cytoplasmic domain.

8. The fusion protein of any one of claims 1 to 7, wherein the ectodomain is a respiratory syncytial virus F protein ectodomain.

9. 9. The fusion protein of claim 1, wherein the ectodomain comprises one or more mutations to stabilize the ectodomain in a pre-fusion conformation.

10. 10. The fusion protein of claim 9, wherein the mutation(s) introduce one or more cysteine ​​residues that form disulfide bonds not present in the native ectodomain and / or mutations that introduce amino acids that fill a hydrophobic cavity present in the native ectodomain.

11. The following mutations stabilize the F protein ectodomain in the pre-fusion conformation: (i) S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3; (ii) N67I and S215P relative to SEQ ID NO: 3; (iii) N67I, S215P, and E487Q relative to SEQ ID NO: 3; (iv) The fusion protein of claim 9 or 10, comprising one or more of D486H, E487Q, F488W, and D489H relative to SEQ ID NO:

3.

12. 12. The fusion protein of claim 11, comprising a respiratory syncytial virus F protein ectodomain comprising the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3, or comprising or consisting of the sequence set forth in SEQ ID NO:

18.

13. In order from amino to carboxy, (i) a respiratory syncytial virus F protein ectodomain, HR1 from RSV, and HR2 from RSV, comprising the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3; or (ii) the fusion protein of claim 12, comprising a respiratory syncytial virus F protein ectodomain, HR2 from RSV, and HR1 from RSV, the fusion protein comprising S155C, S290C, S190F, and V207L ​​mutations relative to SEQ ID NO:

3.

14. The fusion protein of any one of claims 1 to 13, comprising a linker positioned between the ectodomain and the HR.

15. The fusion protein of claim 14 , wherein the linker comprises glycine and serine.

16. The fusion protein according to claim 14 or 15, wherein the linker is selected from the sequences set forth in any one of SEQ ID NOs: 10 to 13.

17. 17. The fusion protein of any one of claims 1 to 16, comprising a respiratory syncytial virus F protein ectodomain, a linker, an HR1 from RSV, and an HR2 from RSV, which comprise, in order from amino to carboxy, the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3, or which comprise or consist of the sequence set forth in SEQ ID NO:

18.

18. 17. The fusion protein of any one of claims 1 to 16, comprising a respiratory syncytial virus F protein ectodomain comprising, in amino to carboxy order, S155C, S290C, S190F, and V207L ​​mutations relative to SEQ ID NO:3, or comprising or consisting of the sequence set forth in SEQ ID NO:18, a linker comprising the sequence set forth in SEQ ID NO:13, HR1 from RSV, and HR2 from RSV.

19. A fusion protein comprising the ectodomain of a viral fusion protein linked to the heptad repeat (HR) from the SARS-COV-2 spike (S) protein.

20. 20. The fusion protein of claim 19, wherein the HR is HR1 from SARS-COV-2 S protein.

21. 21. The fusion protein of claim 20, comprising the ectodomain linked to two HRs from the SARS-COV-2 S protein.

22. 22. The fusion protein of claim 21, comprising HR1 and HR2 from the SARS-COV-2 S protein.

23. 23. The fusion protein of any one of claims 19 to 22, wherein the HR(s) are additional to any HR in the ectodomain.

24. The fusion protein of any one of claims 19 to 23, wherein the ectodomain lacks a transmembrane region and a cytoplasmic domain.

25. 25. The fusion protein of any one of claims 19 to 24, wherein the ectodomain comprises one or more mutations to stabilize the ectodomain in a pre-fusion conformation.

26. 26. The fusion protein of any one of claims 19 to 25, wherein the ectodomain is a respiratory syncytial virus F protein ectodomain.

27. The following mutations stabilize the F protein ectodomain in the pre-fusion conformation: (i) S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3; (ii) N67I and S215P relative to SEQ ID NO: 3; (iii) N67I, S215P, and E487Q relative to SEQ ID NO: 3; (iv) The fusion protein of claim 26, comprising one or more of D486H, E487Q, F488W, and D489H relative to SEQ ID NO:

3.

28. 27. The fusion protein of claim 26, comprising a respiratory syncytial virus F protein ectodomain comprising the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3, or comprising or consisting of the sequence set forth in SEQ ID NO:

18.

29. In order from amino to carboxy, (i) a respiratory syncytial virus F protein ectodomain, HR1, and HR2 comprising the mutations S155C, S290C, S190F, and V207L ​​relative to SEQ ID NO: 3, or comprising or consisting of the sequence set forth in SEQ ID NO: 18; or (ii) the fusion protein of claim 28, comprising a respiratory syncytial virus F protein ectodomain, HR2, and HR1, which comprise S155C, S290C, S190F, and V207L ​​mutations relative to SEQ ID NO: 3, or which comprise or consist of the sequence set forth in SEQ ID NO:

18.

30. The fusion protein of any one of claims 19 to 25, wherein the ectodomain is a SARS-COV-2 S protein ectodomain.

31. (i) K986P and V987P relative to SEQ ID NO: 4, and / or (ii) one or more mutations in the furin cleavage site at positions 682 to 685 of SEQ ID NO:

4.

32. The fusion protein of any one of claims 19 to 25, comprising the SARS-COV-2 S protein ectodomain and one HR of SARS-COV-2 S protein.

33. 33. The fusion protein of claim 32, wherein the HR is HR1.

34. The fusion protein of any one of claims 30 to 33, comprising, in order from amino to carboxy terminus, the SARS-COV-2 S protein ectodomain and HR1 of SARS-COV-2 S protein.

35. 35. The fusion protein of any one of claims 1 to 34, wherein the ectodomain and the HR(s) are linked by a linker.

36. The linker has the sequence (GGGGS) ２ or (GGGGS) ３ 36. The fusion protein of claim 35, comprising:

37. A nucleic acid encoding the fusion protein of any one of claims 1 to 36.

38. 38. The nucleic acid of claim 37, which is RNA.

39. 39. The nucleic acid of claim 38, in an mRNA or sa-mRNA vaccine.

40. A nanoparticle comprising the nucleic acid of any one of claims 37 to 39.

41. A composition comprising a fusion protein according to any one of claims 1 to 36, a nucleic acid according to any one of claims 37 to 39, or a nanoparticle according to claim 40.

42. 42. The composition of claim 41, additionally comprising an adjuvant.

43. 43. The composition of claim 42, wherein the adjuvant comprises an oil-in-water emulsion of squalene, polyoxyethylene sorbitan monooleate, and sorbitan trioleate compounds.

44. 41. A method of inducing an immune response in a subject, the method comprising administering to the subject a fusion protein according to any one of claims 1 to 36, a nucleic acid according to any one of claims 37 to 39, or a nanoparticle according to claim 40, or a composition according to any one of claims 41 to 43.

45. 41. A method of immunizing a subject, the method comprising administering to the subject a fusion protein according to any one of claims 1 to 36, a nucleic acid according to any one of claims 37 to 39, a nanoparticle according to claim 40, or a composition according to any one of claims 41 to 43.

46. 41. A method for treating or preventing infection by a virus, comprising administering to said subject a fusion protein according to any one of claims 1 to 36, a nucleic acid according to any one of claims 37 to 39, a nanoparticle according to claim 40, or a composition according to any one of claims 41 to 43.

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